WO2022251166A2 - Bacterial compositions comprising soy hemoglobin - Google Patents

Bacterial compositions comprising soy hemoglobin Download PDF

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Publication number
WO2022251166A2
WO2022251166A2 PCT/US2022/030656 US2022030656W WO2022251166A2 WO 2022251166 A2 WO2022251166 A2 WO 2022251166A2 US 2022030656 W US2022030656 W US 2022030656W WO 2022251166 A2 WO2022251166 A2 WO 2022251166A2
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WIPO (PCT)
Prior art keywords
pharmaceutical composition
leghemoglobin
bacteria
soy
prevotella
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PCT/US2022/030656
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French (fr)
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WO2022251166A3 (en
Inventor
Alexander BANK
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Evelo Biosciences, Inc.
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Publication of WO2022251166A2 publication Critical patent/WO2022251166A2/en
Publication of WO2022251166A3 publication Critical patent/WO2022251166A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • composition of a person’s microbiome can play an important role in their health and well-being. Indeed, disruption of an individual’s microbiome has been implicated in numerous diseases, including inflammatory bowel diseases, immune disorders, type 2 diabetes, neurodegenerative disorders, cardiovascular diseases, and cancers. Thus, microbiome modulation is an attractive therapeutic strategy for such diseases.
  • One way to modulate a person’s microbiome is by orally administering to them one or more strains of beneficial bacteria, e.g., formulated for therapeutic use, e.g., in a bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement).
  • a bacterial composition e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement.
  • a bacterial composition e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement.
  • a bacterial composition e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or
  • Hemoglobin is an iron-containing metalloprotein in red blood cells that captures atmospheric oxygen in the lungs and carries it to the rest of the body. Iron is an essential nutrient for almost all forms of life, including bacteria. As hemoglobin is the most abundant reservoir of iron within humans, much of the bacteria that make up the human microbiome use hemoglobin or its derivatives as their primary source of iron. Often, such hemoglobin-dependent bacteria require the presence of hemoglobin or hemin for optimal in vitro growth. However, commercial hemoglobin and its derivatives are typically purified from animal sources, such as from porcine blood, which, inter alia, results in purified hemoglobin being costly. Moreover, GMP (good manufacturing practice)-grade hemoglobin is not easily sourced, making the large-scale manufacture and/or GMP grade manufacture of hemoglobin-dependent bacteria for pharmaceutical purposes particularly challenging.
  • GMP good manufacturing practice
  • soy leghemoglobin and/or fragments thereof are present in compositions and/or solid dosage forms comprising bacteria grown in growth media comprising soy leghemoglobin and/or comprising agents (e.g., microbial extracellular vesicles (mEVs)) derived from bacteria grown in growth media comprising soy leghemoglobin.
  • agents e.g., microbial extracellular vesicles (mEVs)
  • This disclosure is also based, in part, on the discovery that components of soy leghemoglobin are present in pharmaceutical agents comprising bacteria grown in growth media comprising soy leghemoglobin and/or comprising agents (e.g., microbial extracellular vesicles, or mEVs) derived from bacteria grown in growth media comprising soy leghemoglobin.
  • agents e.g., microbial extracellular vesicles, or mEVs
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • bacteria or components thereof, such as mEVs
  • soy leghemoglobin e.g., a nucleic acid soy leghemoglobin component and/or a protein soy leghemoglobin component.
  • methods of making and/or using such bacterial compositions are provided herein.
  • kits for determining the presence and/or amount of at least one component of soy leghemoglobin in bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements.
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • a pharmaceutical agent comprising: a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin.
  • mEVs microbial extracellular vesicles
  • methods of making and/or using such compositions are provided herein.
  • compositions are provided herein.
  • a pharmaceutical composition comprising: a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin.
  • the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin.
  • provided herein are methods of determining the presence and/or amount of soy leghemoglobin in such a pharmaceutical composition.
  • a pharmaceutical agent comprising (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin.
  • mEVs extracellular vesicles
  • methods of making and/or using such pharmaceutical agents are provided herein.
  • provided herein are methods of determining the presence and/or amount of soy leghemoglobin in such a pharmaceutical agent.
  • a solid dosage form comprising: a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin.
  • mEVs microbial extracellular vesicles
  • the at least one component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid.
  • the soy leghemoglobin nucleic acid is soy leghemoglobin DNA.
  • the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin A (LB A).
  • the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin C2 (LGB2).
  • the at least one component of soy leghemoglobin comprises a soy leghemoglobin protein.
  • the soy leghemoglobin protein is soy leghemoglobin A (LB A).
  • the soy leghemoglobin protein is soy leghemoglobin C2 (LGB2).
  • the bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement comprises bacteria.
  • the bacteria are hemoglobin-dependent bacteria (e.g., a species and/or strain of hemoglobin- dependent bacteria provided herein).
  • the bacteria are live, attenuated, or dead.
  • the bacteria are lyophilized bacteria.
  • the bacteria are irradiated (e.g., gamma irradiated).
  • the bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement comprises mEVs.
  • the mEVs are secreted mEVs (smEVs).
  • the mEVs are processed mEVs (pmEVs).
  • the mEVs are from hemoglobin-dependent bacteria (e.g., a species and/or strain of hemoglobin-dependent bacteria provided herein).
  • the mEVs are lyophilized mEVs.
  • the bacteria e.g., the bacteria in the bacterial composition and/or the bacteria from which the mEVs were derived
  • the bacteria are hemoglobin-dependent bacteria.
  • the hemoglobin-dependent bacteria can be any bacteria that require the presence of hemoglobin or a hemoglobin derivative for optimal growth (i.e.. for optimal growth in the absence of soy leghemoglobin or a component thereof provided herein).
  • the hemoglobin-dependent bacteria are bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
  • the hemoglobin-dependent bacteria are of the genus Prevotella.
  • the hemoglobin-dependent bacteria are bacteria of the species Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea,
  • the hemoglobin-dependent bacteria are bacteria of the species Alistipes indistinctus, Alistipes shahii, Alistipes timonensis, Bacillus coagulans, Bacteroides acidifaciens, Bacteroides cellulosilyticus, Bacteroides eggerthii, Bacteroides intestinalis, Bacteroides uniformis, Collinsella aerofaciens, Cloacibacillus evryensis, Clostridium cadaveris, Clostridium cocleatum, Cutihacterium acnes, Eisenhergiella sp., Erysipelotrichaceae sp., Euhacterium hallii/Anaerohutyricum halii, Euhacterium infirmum, Megasphaera micronuciformis, Parahacteroides distasonis, Peptoniphilus lacrimalis, Rarimicrobium hominis, Shuttleworthia sadid,
  • bacteria are of the species Prevotella histicola.
  • the Prevotella histicola is Prevotella histicola Strain B (NRRL accession number B 50329).
  • the Prevotella histicola is Prevotella histicola Strain C (ATCC Deposit Number PTA-126140).
  • the hemoglobin-dependent bacteria are a strain of the species Prevotella histicola.
  • the Prevotella histicola strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g, at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain B 50329.
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.
  • the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g, at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
  • sequence identity e.g, at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity
  • the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
  • the Prevotella histicola strain is Prevotella Strain B 50329 (NRRL accession number B 50329).
  • the Prevotella histicola strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain C (ATCC Deposit Number PTA-126140, deposited on September 10, 2019).
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%
  • the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Prevotella Strain C (PTA-126140).
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity
  • the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Prevotella Strain C (PTA-126140).
  • the Prevotella histicola strain is Prevotella Strain C (PTA-126140).
  • the hemoglobin-dependent bacteria are of the genus Fournierella. In some embodiments, the hemoglobin-dependent bacteria are Fournierella Strain A.
  • the hemoglobin-dependent Fournierella strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Fournierella Strain B (ATCC Deposit Number PTA- 126696, deposited on March 5, 2020).
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%
  • the Fournierella strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Fournierella Strain B (PTA- 126696).
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity
  • the Fournierella strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Fournierella Strain B (PTA- 126696).
  • the Fournierella strain is Fournierella Strain B (PTA- 126696).
  • the hemoglobin-dependent bacteria are of the genus Parabacteroides . In some embodiments, the hemoglobin-dependent bacteria are Parabacteroides Strain A. In some embodiments, the hemoglobin-dependent bacteria are Parabacteroides Strain B.
  • the hemoglobin-dependent bacteria are of the genus Bacteroides. In some embodiments, the hemoglobin-dependent bacteria are Bacteroides Strain A.
  • the hemoglobin-dependent bacteria are of the genus Alii stipes. In some embodiments, the hemoglobin-dependent bacteria are Allistipes Strain A.
  • a solid dosage form comprising: (a) a pharmaceutical agent described herein (e.g., a pharmaceutical agent comprising at least one component of soy leghemoglobin); and (b) at least one diluent, at least one lubricant, at least one glidant, and/or at least one disintegration agent.
  • a pharmaceutical agent described herein e.g., a pharmaceutical agent comprising at least one component of soy leghemoglobin
  • at least one diluent, at least one lubricant, at least one glidant, and/or at least one disintegration agent e.g., a pharmaceutical agent comprising at least one component of soy leghemoglobin.
  • the solid dosage form described herein comprises at least one diluent that has a total mass that is at least, about, or no more than, 1%, 2%, 3%,
  • the at least one diluent has a total mass that is at least 10% and no more than 80% of the total mass of the solid dosage form. In other embodiments, the at least one diluent has a total mass that is at least 20% and no more than 40% of the total mass of the solid dosage form. In some embodiments, the at least one diluent comprises mannitol.
  • the percent of mass of a solid dosage form is on a percent weight:weight basis (%w:w).
  • the solid dosage form described herein comprises at least one lubricant that has a total mass that is at least, about, or no more than, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the total mass of the solid dosage form.
  • the at least one lubricant has a total mass that is at least 0.1% and no more than 5% of the total mass of the solid dosage form.
  • the at least one lubricant comprises magnesium stearate.
  • the solid dosage form described herein comprises at least one glidant that has a total mass that is at least, about, or no more than, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the total mass of the solid dosage form.
  • the at least one glidant has a total mass that is at least 0.01% and no more than 2% of the total mass of the solid dosage form.
  • the at least one glidant comprises colloidal silicon dioxide.
  • the solid dosage form described herein comprises at least one disintegration agents, or certain combinations and/or amounts of disintegration agents, resulting in a decrease in the disintegration time of the composition (e.g., 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold) as compared to conventional solid dosage forms (e.g., solid dosage forms containing conventional amounts of disintegration agents).
  • the solid dosage forms provided herein result in an increase in therapeutic efficacy and/or physiological effect as compared to a pharmaceutical product having conventional solid dosage forms.
  • the solid dosage form comprises a pharmaceutical agent (e.g., bacteria and/or an agent of bacterial origin, such as mEVs, a powder comprising bacteria and/or an agent of bacterial origin, such as mEVs) and one or more disintegration agents (e.g., one, two or three disintegration agents).
  • a pharmaceutical agent e.g., bacteria and/or an agent of bacterial origin, such as mEVs, a powder comprising bacteria and/or an agent of bacterial origin, such as mEVs
  • three disintegration agents e.g., bacteria and/or an agent of bacterial origin, such as mEVs
  • the solid dosage form described herein comprises at least one disintegrant that has a total mass that is at least, about, or no more than, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,
  • the at least one disintegration agent has a total mass that is at least 40% of the total mass of the solid dosage form.
  • the at least one disintegration agent comprises low- substituted hydroxypropyl cellulose (L-HPC, e.g., LH-B1), croscarmellose sodium (Ac-Di- Sol, e.g., Ac-Di-Sol SD-711), and/or crospovidone (PVPP, e.g., Kollidon, e.g., Kollidon CL-F).
  • L-HPC low- substituted hydroxypropyl cellulose
  • Ac-Di- Sol e.g., Ac-Di-Sol SD-711
  • PVPP crospovidone
  • the at least one disintegration agent comprises low- substituted hydroxypropyl cellulose (L-HPC, e.g., LH-B1), croscarmellose sodium (Ac-Di- Sol, e.g., Ac-Di-Sol SD-711), and crospovidone (PVPP, e.g., Kollidon, e.g., Kollidon CL-
  • L-HPC low- substituted hydroxypropyl cellulose
  • Ac-Di- Sol e.g., Ac-Di-Sol SD-711
  • PVPP crospovidone
  • Kollidon e.g., Kollidon CL-
  • the solid dosage forms provided herein comprise L- HPC.
  • the L-HPC is of grade LH-B1.
  • the total L-HPC mass is at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the solid dosage form.
  • the total L-HPC mass is no more than 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the solid dosage form. In certain embodiments, the total L-HPC mass is about 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the solid dosage form.
  • the L-HPC has a total L-HPC mass that is at least 22% and no more than 42% of the total mass of the solid dosage form. In certain embodiments, the total L-HPC mass is about 29% to about 35% of the total mass of the solid dosage form. In certain embodiments, the total L-HPC mass is about 32% of the total mass of the solid dosage form. In some embodiments, wherein the L-HPC is L-HPC of grade LH-B 1.
  • the solid dosage forms provided herein comprise Ac-Di-Sol.
  • the Ac-Di-Sol is of grade SD-711.
  • the total Ac-Di-Sol mass is at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the solid dosage form.
  • the total Ac-Di-Sol mass is no more than 1%, 2%,
  • the total Ac-Di-Sol mass is about 1%
  • the Ac-Di-Sol has a total Ac-Di-Sol mass that is at least 0.01% and no more than 16% of the total mass of the solid dosage form. In certain embodiments, the total Ac-Di-Sol mass is about 3% to about 9 % of the total mass of the solid dosage form. In certain embodiments, the total Ac-Di-Sol (e.g., Ac- Di-Sol SD-711) mass is about 6 % of the total mass of the solid dosage form.
  • the solid dosage forms provided herein comprise PVPP (crospovidone, e.g., Kollidon, e.g., Kollidon CL-F).
  • the total PVPP mass is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the solid dosage form.
  • the total PVPP mass is no more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the solid dosage form. In certain embodiments, the total PVPP mass is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
  • the PVPP has a total PVPP mass that is at least 5% and no more than 25% of the total mass of the solid dosage form. In certain embodiments, the total PVPP mass is about 12% to about 18% of the total mass of the solid dosage form. In certain embodiments, the total PVPP mass is about 15% of the total mass of the solid dosage form.
  • the total L-HPC mass plus the total Ac-Di-Sol mass plus the total PVPP mass is at least 35%, 40%, 45%, or 50% of the total mass of the solid dosage form. In certain embodiments, the total L-HPC mass plus the total Ac-Di-Sol mass plus the total PVPP mass is at least 40% of the total mass of the solid dosage form.
  • the solid dosage forms provided herein comprise:
  • L-HPC (e.g., L-HPC of grade LH-B 1) having a total L-HPC mass that is at least 22% (e.g, at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42%) and no more than 42% (e.g., no more than 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,
  • L-HPC e.g., L-HPC of grade LH-B 1 having a total L-HPC mass that is at least 22% (e.g, at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,
  • the solid dosage form comprises: a total L-HPC mass is about 32% of the total mass of the solid dosage form; a total Ac-Di-Sol mass is about 6% of the total mass of the solid dosage form; and a total PVPP mass is about 15% of the total mass of the solid dosage form.
  • the solid dosage forms provided herein comprise a pharmaceutical agent (e.g. , bacteria and/or mEV) having a total mass that is at least, about, or no more than, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
  • a pharmaceutical agent e.g. , bacteria and/or mEV
  • the pharmaceutical agent has a total pharmaceutical agent mass that is at least 5% and no more than 65% of the total mass of the solid dosage form. In some embodiments, the pharmaceutical agent has a total pharmaceutical agent mass that is at least 5% and no more than 35% of the total mass of the solid dosage form. In some embodiments, the total pharmaceutical agent mass is about 25% of the total mass of the solid dosage form.
  • the solid dosage forms described herein comprise tablets, capsules and/or minitablets (e.g., minitablets in capsules).
  • the solid dosage form comprises a tablet.
  • the tablet is a 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm,
  • the solid dosage form comprises a minitablet.
  • the minitablet is a 1mm minitablet, 1.5 mm minitablet, 2mm minitablet, 3mm minitablet, or 4mm minitablet.
  • a plurality of minitablets are contained in a capsule (e.g., a size 0 capsule can contain about 31 to about 35 (e.g., 33) minitablets, wherein the minitablets are 3mm in size).
  • the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule.
  • the capsule comprises HPMC (hydroxyl propyl methyl cellulose) or gelatin.
  • the solid dosage form is enterically coated (e.g., comprises an enteric coating; e.g., is coated with an enteric coating).
  • the enteric coating is a single enteric coating or more than one enteric coating.
  • the tablets or minitablets are coated with one layer of enteric coating or with two layers of enteric coatings (e.g., an inner enteric coating and an outer enteric coating).
  • the enteric coating comprises an inner enteric coating and an outer enteric coating, and the inner and outer enteric coatings are not identical.
  • the enteric coating comprises a methacrylic acid ethyl acrylate (MAE) copolymer (1:1).
  • MAE methacrylic acid ethyl acrylate
  • the one enteric coating comprises methacrylic acid ethyl acrylate (MAE) copolymer (1:1) (such as Kollicoat MAE 100P).
  • MAE methacrylic acid ethyl acrylate
  • the one enteric coating comprises a Eudragit copolymer, e.g., a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).
  • a Eudragit copolymer e.g., a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).
  • the enteric coating comprises cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), a fatty acid, a wax, shellac (esters of aleurtic acid), a plastic, a plant fiber, zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch, a starch derivative, a dextrin, a methyl acrylate- methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), a methyl methacrylate-methacrylic acid copolymer, or sodium alginate.
  • CAP cellulose acetate phthalate
  • CAT cellulose acetate trimellitate
  • PVAP poly(vinyl acetate phthalate)
  • HPPMCP
  • the enteric coating comprises an anionic polymeric material.
  • solid dosage form comprising: (a) a pharmaceutical agent described herein (e.g., a pharmaceutical agent comprising at least one component of soy leghemoglobin); and (b) at least one diluent, at least one lubricant, and/or at least one glidant.
  • a pharmaceutical agent described herein e.g., a pharmaceutical agent comprising at least one component of soy leghemoglobin
  • at least one diluent, at least one lubricant, and/or at least one glidant e.g., a pharmaceutical agent comprising at least one component of soy leghemoglobin
  • a solid dosage form comprising (a) a pharmaceutical agent described herein (e.g., a pharmaceutical agent comprising at least one component of soy leghemoglobin); and (b) a diluent.
  • the total pharmaceutical agent mass is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total mass of the solid dosage form.
  • the total pharmaceutical agent mass is no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,
  • the total mass of the diluent is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
  • the total mass of the diluent is no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the total mass of the solid dosage form.
  • the diluent comprises mannitol.
  • the solid dosage form provided herein comprises a lubricant.
  • the total lubricant mass is at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the solid dosage form. In certain embodiments, the total lubricant mass is no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the solid dosage form. In certain embodiments, the total lubricant mass is about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the solid dosage form. In certain embodiments, the total lubricant mass is about 0.5% to about 1.5% of the total mass of the solid dosage form. In certain embodiments, the total lubricant mass is about 1% of the total mass of the solid dosage form. In some embodiments, the lubricant comprises magnesium stearate.
  • the solid dosage forms provided herein comprise a glidant.
  • the glidant is colloidal silicon dioxide.
  • the total glidant mass is at least 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,
  • the total glidant mass is no more than 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the solid dosage form. In certain embodiments, the total glidant mass is about 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the solid dosage form. In certain embodiments, the total glidant mass is about 0.25% to about 0.75% of the total mass of the solid dosage form. In certain embodiments, the total glidant mass is about 0.5% of the total mass of the solid dosage form.
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is at least 20% and no more than 55% of the total mass of the solid dosage form;
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is about 20% to about 50% of the total mass of the solid dosage form;
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is at least 5% and no more than 95% of the total mass of the solid dosage form;
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is about 8% to about 92% of the total mass of the solid dosage form;
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is about 30% to about 50% of the total mass of the solid dosage form;
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise:
  • the solid dosage forms provided herein comprise: (i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 13.51% of the total mass of the solid dosage form;
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is about 90.22% of the total mass of the solid dosage form;
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms of a pharmaceutical agent as described herein comprise capsules.
  • the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule.
  • the capsule is a size 0 capsule.
  • the capsule comprises HPMC (hydroxyl propyl methyl cellulose) or gelatin.
  • the capsule comprises HPMC (hydroxyl propyl methyl cellulose).
  • the capsule is banded.
  • the capsule is banded with an HPMC-based banding solution.
  • the solid dosage form is enterically coated (e.g., comprises an enteric coating; e.g., is coated with an enteric coating).
  • the solid dosage form is enteric coated to dissolve at pH 5.5.
  • the enteric coating comprises a polymethacrylate- based copolymer. In some embodiments, the enteric coating comprises poly(methacrylic acid-co-ethyl acrylate).
  • the enteric coating comprises a methacrylic acid ethyl acrylate (MAE) copolymer (1:1).
  • MAE methacrylic acid ethyl acrylate
  • the enteric coating comprises methacrylic acid ethyl acrylate (MAE) copolymer (1:1) (such as Kollicoat MAE 100P).
  • MAE methacrylic acid ethyl acrylate
  • the enteric coating comprises a Eudragit copolymer, e.g., a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).
  • a Eudragit copolymer e.g., a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).
  • the enteric coating comprises cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), a fatty acid, a wax, shellac (esters of aleurtic acid), a plastic, a plant fiber, zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch, a starch derivative, a dextrin, a methyl acrylate- methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), a methyl methacrylate-methacrylic acid copolymer, or sodium alginate.
  • CAP cellulose acetate phthalate
  • CAT cellulose acetate trimellitate
  • PVAP poly(vinyl acetate phthalate)
  • HPPMCP
  • the enteric coating comprises an anionic polymeric material.
  • the pharmaceutical agent can be a powder that comprises the bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component, and, can comprise additional agents such as, e.g., cryoprotectant.
  • the pharmaceutical agent is a lyophilized powder of bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component that optionally, further comprises additional agents, such as a cryoprotectant.
  • the pharmaceutical agent comprises bacteria and the dose of bacteria is about 1 x 10 7 to about 2 x 10 12 (e.g., about 3 x 10 10 or about 1.5 x 10 11 ) cells (e.g., wherein cell number is determined by total cell count, which is determined by Coulter counter), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the dose is about 1 x 10 7 to about 2 x 10 12 (e.g., about 3 x 10 10 or about 1.5 x 10 11 ) cells (e.g., wherein cell number is determined by total cell count, which is determined by Coulter counter), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the pharmaceutical agent comprises bacteria and the dose of bacteria is about 1 x 10 7 to about 1 x 10 13 , wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the pharmaceutical agent comprises bacteria and the dose of bacteria is about 1 x 10 9 , about 3 x 10 9 , about 5 x 10 9 , about 1.5 x 10 10 , or about 5 x 10 10 cells, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the pharmaceutical agent comprises bacteria and the dose of bacteria is about 8 x 10 10 cells, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the pharmaceutical agent comprises bacteria and the dose of bacteria is about 1.6 x 10 11 cells, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the pharmaceutical agent comprises mEVs and the dose of mEVs is about 1 x 10 5 to about 2 x 10 12 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the pharmaceutical agent comprises bacteria and the dose of bacteria is about 3.2 x 10 11 cells, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the pharmaceutical agent comprises a powder comprising bacteria and/or mEVs and the dose of the pharmaceutical agent (e.g., a powder comprising bacteria and/or mEVs) is about 10 mg to about 3500 mg, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the dose of the pharmaceutical agent e.g., a powder comprising bacteria and/or mEVs
  • the pharmaceutical agent comprises a powder comprising bacteria and/or mEVs and the dose of the pharmaceutical agent (e.g., a powder comprising bacteria and/or mEVs) is about 30 mg to about 1300 mg (by weight of bacteria and/or mEVs powder) (about 25, about 30, about 35, about 50, about 75, about 100, about 120, about 150, about 250, about 300, about 350, about 400, about 500, about 600, about 700, about 750, about 800, about 900, about 1000, about 1100, about 1200, about 1250, about 1300, about 2000, about 2500, about 3000, or about 3500 mg wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the dose of the pharmaceutical agent e.g., a powder comprising bacteria and/or mEVs
  • the dose of the pharmaceutical agent is about 30 mg to about 1300 mg (by weight of bacteria and/or mEVs powder) (about 25, about 30, about 35, about 50, about 75, about 100
  • the pharmaceutical agent comprises bacteria and/or mEVs and the dose of pharmaceutical agent (e.g., bacteria and/or mEVs) is about 2x10 6 to about 2x10 16 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • pharmaceutical agent e.g., bacteria and/or mEVs
  • the dose of pharmaceutical agent is about 2x10 6 to about 2x10 16 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)
  • the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the pharmaceutical agent comprises bacteria and/or mEVs and the dose of pharmaceutical agent (e.g., bacteria and/or mEVs) is about 5 mg to about 900 mg total protein (e.g., wherein total protein is determined by Bradford assay or BCA), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • pharmaceutical agent e.g., bacteria and/or mEVs
  • the dose of pharmaceutical agent is about 5 mg to about 900 mg total protein (e.g., wherein total protein is determined by Bradford assay or BCA), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the disclosure provides a method of preventing or treating a subject (e.g., human) (e.g., a subject in need of treatment), the method comprising administering to the subject a bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement)provided herein.
  • a bacterial composition e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement
  • the disclosure provides a method of preventing or treating a subject (e.g. , human) (e.g. , a subject in need of treatment), the method comprising administering to the subject a solid dosage form provided herein.
  • the disclosure provides use of a bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement)for the treatment or prevention of a disease of a subject.
  • a bacterial composition e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement
  • a subject e.g. , human
  • a subject in need of treatment e.g., a subject in need of treatment.
  • the bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • the bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • treats bacterial septic shock, cytokine storm and/or viral infection such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection.
  • the bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • decreases inflammatory cytokine expression e.g., decreased IL-8, IL-6, IL-Ib, and/or TNFa expression levels.
  • kits for preparing a solid dosage form comprising (a) combining (i) a pharmaceutical agent provided herein (e.g., bacteria disclosed herein and/or an agent of bacterial origin, such as mEVs disclosed herein) (e.g., comprising at least one component of soy leghemoglobin), and (ii) at least one diluent, at least one lubricant, at least one glidant, and/or at least one (e.g., one, two or three) disintegration agent to prepare a pharmaceutical composition, and (b) compressing the pharmaceutical composition into a solid dosage form.
  • the method further comprises the step of enterically coating the solid dosage form to obtain an enterically coated solid dosage form.
  • the solid dosage form is a tablet.
  • the solid dosage form is a minitablet.
  • a pharmaceutical agent e.g., bacteria disclosed herein and/or an agent of bacterial origin, such as mEVs disclosed herein
  • a diluent, lubricant, and/or glidant e.g., into a pharmaceutical composition.
  • the method comprises blending.
  • the method further comprises loading the pharmaceutical composition into a capsule.
  • the capsule comprises HPMC.
  • the method further comprises banding the capsule.
  • the capsule is banded with an HPMC-based banding solution.
  • the method further comprises the step of enterically coating the solid dosage form to obtain an enterically coated solid dosage form.
  • the solid dosage form is a capsule.
  • provided herein is a method of testing a pharmaceutical agent comprising bacteria (e.g., bacteria provided herein) and/or mEVs (e.g., mEVs provided herein) (e.g., a pharmaceutical agent provided herein), the method comprising performing an assay to detect the presence of soy leghemoglobin in the pharmaceutical agent.
  • bacteria e.g., bacteria provided herein
  • mEVs e.g., mEVs provided herein
  • a pharmaceutical agent provided herein e.g., a pharmaceutical agent provided herein
  • a method of testing a bacterial composition comprising performing an assay to detect the presence of a component of soy leghemoglobin in the bacterial composition.
  • a bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • bacteria e.g., bacteria provided herein
  • mEVs e.g., mEVs provided herein
  • the component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid.
  • the soy leghemoglobin nucleic acid is soy leghemoglobin DNA.
  • the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin A (LB A).
  • the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin C2 (LGB2).
  • the assay to detect the presence of a component of soy leghemoglobin is a nucleic acid amplification assay, a sequencing assay, and/or a microarray assay.
  • the assay to detect the presence of a component of soy leghemoglobin is a polymerase chain reaction (PCR) assay, such as a quantitative polymerase chain reaction (qPCR) assay or digital PCR.
  • PCR polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • the component of soy leghemoglobin is a soy leghemoglobin protein.
  • the soy leghemoglobin protein is soy leghemoglobin A (LB A).
  • the soy leghemoglobin protein is a sequence encoding soy leghemoglobin C2 (LGB2).
  • the soy leghemoglobin protein is detected using an antibody specific for the soy leghemoglobin protein, HPLC or UPLC.
  • soy leghemoglobins that can be used in growth media to facilitate the in vitro culturing of hemoglobin-dependent bacteria, including bacteria of the genus Prevotella, such as Prevotella histicola.
  • the soy leghemoglobin provided herein can allow for large-scale manufacture and/or GMP grade manufacture of hemoglobin-dependent bacteria, e.g., for therapeutic use and/or in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement).
  • soy leghemoglobin is purified from soy roots or soy root nodules.
  • soy leghemoglobin provided herein are recombinantly expressed.
  • recombinant expression of the disclosed soy leghemoglobin facilitates, inter alia, the cost-effective, vegetarian, kosher, and GMP-grade production of soy leghemoglobin; it is an attractive way to produce soy leghemoglobin for use in bacterial cell culture applications.
  • hemoglobin-dependent bacteria e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use). Growth of hemoglobin-dependent bacteria is accomplished through the inclusion in the cell culture media of a soy leghemoglobin provided herein.
  • a bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • growth of hemoglobin-dependent bacteria is accomplished through the inclusion in the cell culture media of a soy leghemoglobin provided herein.
  • hemoglobin-dependent bacteria e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) in growth media that includes a soy leghemoglobin provided herein.
  • a bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • growth media that includes a soy leghemoglobin provided herein.
  • compositions comprising a soy leghemoglobin provided herein that are useful for culturing hemoglobin-dependent bacteria in conditions free from conventional hemoglobin (e.g., soy leghemoglobin) (e.g., conditions not comprising a hemoglobin sourced from an animal) or a derivative thereof, as well as methods of making and/or using such compositions.
  • a soy leghemoglobin provided herein that are useful for culturing hemoglobin-dependent bacteria in conditions free from conventional hemoglobin (e.g., soy leghemoglobin) (e.g., conditions not comprising a hemoglobin sourced from an animal) or a derivative thereof, as well as methods of making and/or using such compositions.
  • the heme -containing polypeptide comprises an amino acid sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity to an amino acid sequence of SEQ ID NO: 4, 5, 114, or 115.
  • the heme-containing polypeptide comprises the amino acid sequence of SEQ ID NO: 4, 5, 114, or 115.
  • the soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • the soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • the soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • the component of soy leghemoglobin comprises a nucleic acid that encodes an amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • the component of soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
  • the component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
  • the component of soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
  • the component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 5 or GenBank:
  • the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
  • the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
  • the soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN) .
  • the soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN) .
  • the soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
  • the component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN) .
  • the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _SOYBN).
  • the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _SOYBN).
  • the soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
  • the soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
  • the soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
  • the component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN ).
  • the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN ).
  • the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN ).
  • the soy leghemoglobin provided herein is recombinantly expressed.
  • the recombinant soy leghemoglobin provided herein further comprises a heterologous polypeptide, such as a histidine tag, TAP (tandem affinity purification) tag, TEV cleavage site, a FLAG tag, a GST tag, and/or an immunoglobulin domain.
  • a heterologous polypeptide enhances or facilitates purification and/or detection of the heme-containing polypeptide.
  • the recombinant polypeptide is expressed in a host cell (e.g., from an exogenous nucleic acid, such as an expression vector present in the host cell).
  • the host cells are bacteria cells, yeast cells, insect cells, or mammalian cells (e.g., a mammalian cell line). In some embodiments, the host cells are yeast cells. In some embodiments, the host cell is Pichia Pastoris. It will be understood that Pichia pastoris has been reclassified as Komagataella species, such as Komagataella phaffii, Komagataella pastoris, or Komagataella pseudopastoris, though the term “ Pichia pastoris ” is still in use and may refer to any appropriate Komagataella species. Representative recombinant heme- containing polypeptides using Pichia Pastoris host cells are disclosed in U.S. Patent Nos. 9,938,327; 10,273,492; 10,798,958, which are hereby incorporated by reference herein in their entireties. In some embodiments, the host cell is Escherichia coli.
  • a growth medium for use in culturing hemoglobin-dependent bacteria e.g., for use in abacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use
  • the growth medium comprising a soy leghemoglobin provided herein.
  • the growth medium comprises hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use).
  • a soy leghemoglobin for use as a substitute for conventional hemoglobin or a derivative thereof in a growth medium for hemoglobin-dependent bacteria.
  • hemoglobin-dependent bacteria e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use
  • a bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • the method comprising incubating the hemoglobin-dependent bacteria in a growth medium that comprises a soy leghemoglobin provided herein.
  • hemoglobin-dependent bacteria e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use
  • a bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • the method comprising (a) adding a soy leghemoglobin provided herein and hemoglobin- dependent bacteria to a growth medium; and (b) incubating the hemoglobin-dependent bacteria in the growth medium.
  • composition comprising a growth medium comprising a soy leghemoglobin provided herein and hemoglobin-dependent bacteria.
  • hemoglobin- dependent bacteria e.g, for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) in a growth medium comprising a soy leghemoglobin provided herein.
  • a bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • a growth medium comprising a soy leghemoglobin provided herein.
  • hemoglobin-dependent bacteria e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use
  • the method comprising comprises incubating the hemoglobin-dependent bacteria in a bioreactor provided herein.
  • the growth medium comprises at least 0.001 g/L, at least 0.005 g/L, at least 0.01 g/L, at least 0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, at least 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L, at least 0.1 g/L, at least 0.2 g/L, at least 0.3 g/L, at least 0.4 g/L, at least 0.5 g/L, at least 0.75 g/L, at least 1 g/L, at least 1.25 g/L, at least 1.5 g/L, at least 1.75 g/L, at least 2 g/L, at least 2.25 g/L, at least 2.5 g/L, at least 2.75 g/L, at least 3 g/L, at least 3.25 g/L, at least
  • the growth medium comprises at least 0.005 g/L and no more than 1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.02 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.05 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.2 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.5 g/L of a soy leghemoglobin.
  • the growth medium comprises about 1 g/L of a soy leghemoglobin.
  • the growth medium comprises yeast extract, soy peptone A2SC 19649, Soy peptone El 10 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, glucidex 21 D, and/or glucose.
  • the growth media comprises about 5 g/L glucose, about 10 g/L yeast extract 19512, about 10 g/L soy peptone A2 SC 19649, about 10 g/L soypeptone El 10 19885, about 2.5 g/L dipotassium phosphate K2HP04, and about 0.5 g/L L-cysteine-HCl.
  • the growth medium is at a pH of 5.5 to 7.5. In certain embodiments, the growth medium is at a pH of about 6.5.
  • the growth medium does not comprise hemoglobin or a derivative thereof. In certain embodiments, the growth medium does not comprise animal products. In certain embodiments, the growth medium does not comprise a heme-containing polypeptide sourced from an animal.
  • the hemoglobin-dependent bacteria e.g ., for use in abacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use
  • a bacteria composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • for therapeutic use can be any bacteria that require the presence of hemoglobin or a hemoglobin derivative for optimal growth (i.e. for optimal growth in the absence of a soy leghemoglobin provided herein).
  • the hemoglobin- dependent bacteria are bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
  • the hemoglobin- dependent bacteria are of the genus Prevotella. In some embodiments, the hemoglobin- dependent bacteria are of the species Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella
  • the hemoglobin- dependent bacteria are of the species Alistipes indistinctus, Alistipes shahii, Alistipes timonensis, Bacillus coagulans, Bacteroides acidifaciens, Bacteroides cellulosilyticus, Bacteroides eggerthii, Bacteroides intestinalis, Bacteroides uniformis, Collinsella aerofaciens, Cloacibacillus evryensis, Clostridium cadaveris, Clostridium cocleatum, Cutibacterium acnes, Eisenbergiella sp., Erysipelotrichaceae sp., Eubacterium hallii/Anaerobutyricum halii, Eubacterium infirmum, Megasphaera micronuciformis, Parabacteroides distasonis, Peptoniphilus lacrimalis, Rarimicrobium hominis, Shuttleworthia sacetate, or Turicibacter sanguinis.
  • the hemoglobin-dependent bacteria e.g ., for use in bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use
  • bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • for therapeutic use are a strain of the species Prevotella histicola.
  • the Prevotella histicola strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g, at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain B 50329.
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
  • the Prevotella histicola strain is a strain that comprises at least at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity
  • the Prevotella histicola strain is a strain that comprises at least at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
  • the Prevotella histicola strain is Prevotella Strain B 50329 (NRRL accession number B 50329).
  • the Prevotella histicola strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain C (ATCC Deposit Number PTA-126140, deposited on September 10, 2019).
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%
  • the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Prevotella Strain C (PTA-126140).
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity
  • the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Prevotella Strain C (PTA-126140).
  • the Prevotella histicola strain is Prevotella Strain C (PTA-126140).
  • the hemoglobin-dependent bacteria are a strain of Prevotella bacteria comprising one or more proteins listed in Table 1.
  • the hemoglobin-dependent bacteria e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) are from a strain of Prevotella substantially free of one or more of the proteins listed in Table 2.
  • the growth medium comprises at least 0.001 g/L, at least 0.005 g/L, at least 0.01 g/L, at least 0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, at least 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L, at least 0.1 g/L, at least 0.2 g/L, at least 0.3 g/L, at least 0.4 g/L, at least 0.5 g/L, at least 0.75 g/L, at least 1 g/L, at least 1.25 g/L, at least 1.5 g/L, at least 1.75 g/L, at least 2 g/L, at least 2.25 g/L, at least 2.5 g/L, at least 2.75 g/L, at least 3 g/L, at least 3.25 g/L, at least
  • the growth medium comprises at least 0.005 g/L and no more than 1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.05 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.02 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.2 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.5 g/L of a soy leghemoglobin.
  • the growth medium comprises about 1 g/L of a soy leghemoglobin provided herein.
  • the growth medium comprises yeast extract, soy peptone A2SC 19649, Soy peptone El 10 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, glucidex 21 D, and/or glucose.
  • the growth media comprises about 5 g/L glucose, about 10 g/L yeast extract 19512, about 10 g/L soy peptone A2 SC 19649, about 10 g/L soypeptone El 10 19885, about 2.5 g/L dipotassium phosphate K2HP04, and about 0.5 g/L L-cysteine-HCl.
  • the growth medium is at a pH of 5.5 to 7.5. In certain embodiments, the growth medium is at a pH of about 6.5.
  • the growth medium does not comprise conventional hemoglobin (e.g., comprises soy leghemoglobin) (e.g., not comprising a hemoglobin sourced from an animal) or a derivative thereof. In certain embodiments, the growth medium does not comprise animal products. In certain embodiments, the growth medium does not comprise a heme-containing polypeptide sourced from an animal.
  • conventional hemoglobin e.g., comprises soy leghemoglobin
  • the growth medium does not comprise animal products.
  • the growth medium does not comprise a heme-containing polypeptide sourced from an animal.
  • the hemoglobin-dependent bacteria (e.g., for use in abacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) grow at an increased rate in the growth medium comprising a soy leghemoglobin provided herein compared to the rate at which the hemoglobin-dependent bacteria grow in the same growth medium but without the soy leghemoglobin .
  • a bacteria composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • the rate at which the hemoglobin-dependent bacteria grow in the growth medium comprising a soy leghemoglobin provided herein is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least
  • the growth rate is increased by 200% to 400%.
  • the hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) grow to a higher cell density in the growth medium comprising a soy leghemoglobin provided herein, compared to the cell density to which the hemoglobin-dependent bacteria grow in the same growth medium but without the soy leghemoglobin.
  • a bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • the hemoglobin-dependent bacteria grow to a cell density in the growth medium comprising a soy leghemoglobin provided herein that is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, or at least 400% higher than the cell density to which the hemoglobin-dependent bacteria grow in the same growth medium but
  • hemoglobin- dependent bacteria e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) disclosed herein and a heme-containing polypeptide soy leghemoglobin disclosed herein.
  • a bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • heme-containing polypeptide soy leghemoglobin disclosed herein.
  • FIG. 1 shows the growth curves for Strain A ( Prevotella histicola strain B 50329 (NRRL accession number B 50329)) with various sources of hemoglobin.
  • FIG. 2 shows the growth curves for Strain A ( Prevotella histicola strain B 50329 (NRRL accession number B 50329)) with various sources and concentrations of hemoglobin.
  • FIG. 3 shows the growth curves for Parahacteroides Strain A with various sources and concentrations of hemoglobin.
  • FIG. 4 shows the growth curves for Bacteroides Strain B with various sources and concentrations of hemoglobin.
  • FIG. 5 shows the growth curves for Alistipes Strain C with various sources and concentrations of hemoglobin.
  • FIG. 6 shows an exemplary qPCR standard curve for the detection of soy leghemoglobin with TaqMan qPCR assay.
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • bacteria or components thereof, such as mEVs
  • soy leghemoglobin e.g., a nucleic acid soy leghemoglobin component and/or a protein soy leghemoglobin component
  • methods of making and/or using such compositions e.g., provided herein are methods of determining the presence and/or amount of at least one component of soy leghemoglobin in such a composition.
  • hemoglobin-dependent bacteria e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use).
  • a soy leghemoglobin that can be added to culture media to facilitate the growth of hemoglobin-dependent bacteria.
  • These heme -containing polypeptides may be purified directly from various organisms, or a processed form thereof (e.g., flour, protein hydrolysate, meals, etc.).
  • the soy leghemoglobin may be recombinantly produced, e.g., recombinantly expressed in a host cell.
  • compositions for culturing hemoglobin-dependent bacteria in growth media that includes a soy leghemoglobin provided herein.
  • compositions e.g., growth media
  • a soy leghemoglobin provided herein that are useful for culturing hemoglobin-dependent bacteria, as well as methods of making and/or using such compositions.
  • anaerobic conditions are conditions with reduced levels of oxygen compared to normal atmospheric conditions.
  • anaerobic conditions are conditions wherein the oxygen levels are partial pressure of oxygen (pCh) no more than 8%.
  • anaerobic conditions are conditions wherein the pO 2 is no more than 2%.
  • anaerobic conditions are conditions wherein the pO 2 is no more than 0.5%.
  • anaerobic conditions may be achieved by purging a bioreactor and/or a culture flask with a gas other than oxygen such as, for example, nitrogen and/or carbon dioxide (CO 2 ).
  • the term “bacterial composition” includes compositions comprising bacteria and/or components thereof, such as mEVs.
  • the bacterial composition can be a pharmaceutical composition, a pharmaceutical agent, and/or a solid dosage form.
  • the bacterial composition can be a medicinal product, a medical food, a food product, and/or a dietary supplement.
  • disclosure related to pharmaceutical agents, solid dosage forms, and/or pharmaceutical compositions and methods of preparing and/or evaluating the same also relate to other types of bacterial compositions as well as of preparing and/or evaluating bacterial composition generally.
  • derivatives of hemoglobin include compounds that are derived from hemoglobin that can facilitate growth of hemoglobin-dependent bacteria.
  • Examples of derivatives of hemoglobin include hemin and protoporphyrin.
  • genomic is used broadly to refer to any nucleic acid associated with a biological function.
  • genomic sequence is used broadly to refer to any nucleic acid associated with a biological function.
  • gene applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
  • heme-containing polypeptide refers to a polypeptide that covalently or noncovalently binds to a heme moiety.
  • “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, T, et al, Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al, J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J.
  • Microbiome broadly refers to the microbes residing on or in body site of a subject or patient.
  • Microbes in a microbiome may include bacteria, viruses, eukaryotic microorganisms, and/or viruses.
  • Individual microbes in a microbiome may be metabolically active, dormant, latent, or exist as spores, may exist planktonically or in biofdms, or may be present in the microbiome in sustainable or transient manner.
  • the microbiome may be a commensal or healthy-state microbiome or a disease-state microbiome.
  • the microbiome may be native to the subject or patient, or components of the microbiome may be modulated, introduced, or depleted due to changes in health state (e.g., precancerous or cancerous state) or treatment conditions (e.g., antibiotic treatment, exposure to different microbes).
  • the microbiome occurs at a mucosal surface.
  • the microbiome is a gut microbiome.
  • the microbiome is a tumor microbiome.
  • Non-animal-derived polypeptide refers to a polypeptide that is not purified from an animal source.
  • a non-animal -derived polypeptide includes both polypeptides purified from non-animal sources and polypeptides that are recombinantly expressed.
  • the term “non-animal-derived polypeptide” would include a polypeptide that is recombinantly expressed from a sequence that encodes an animal polypeptide, e.g., corresponds to a polypeptide encoded by an animal gene.
  • strain refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species.
  • the genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof.
  • regulatory region e.g., a promoter, a terminator,
  • strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome.
  • one strain compared with another of the same species
  • a biosynthetic capability such as an auxotrophic strain
  • strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.
  • hemoglobin-dependent bacteria may be for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use (e.g., in a subject, e.g., human).
  • a bacterial composition e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement
  • a subject e.g., human
  • hemoglobin dependent bacteria refers to bacteria for which growth rate is slowed and/or maximum cell density is reduced when cultured in growth media lacking conventional hemoglobin (e.g., soy leghemoglobin), a hemoglobin derivative or a heme-containing polypeptide disclosed herein when compared to the same growth media containing conventional hemoglobin (e.g., soy leghemoglobinf), a hemoglobin derivative or a heme-containing polypeptide disclosed herein.
  • conventional hemoglobin e.g., soy leghemoglobin
  • soy leghemoglobinf a hemoglobin derivative or a heme-containing polypeptide disclosed herein.
  • the hemoglobin-dependent bacteria are bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
  • the hemoglobin-dependent bacteria are of the genus Prevotella.
  • the hemoglobin-dependent bacteria are of the species Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola,
  • the hemoglobin-dependent bacteria are of the species Alistipes indistinctus, Alistipes shahii, Alistipes timonensis, Bacillus coagulans, Bacteroides acidifaciens, Bacteroides cellulosilyticus, Bacteroides eggerthii, Bacteroides intestinalis, Bacteroides uniformis, Collinsella aerofaciens, Cloacibacillus evryensis, Clostridium cadaveris, Clostridium cocleatum, Cutibacterium acnes, Eisenbergiella sp., Erysipelotrichaceae sp., Eubacterium hallii/Anaerobutyricum halii, Eubacterium infirmum, Megasphaera micronuciformis, Parabacteroides distasonis, Peptoniphilus lacrimalis, Rarimicrobium hominis, Shuttleworthia sacetate, or Turicibacter sanguinis.
  • the hemoglobin-dependent Prevotella strain is Prevotella Strain B 50329 (NRRL accession number B 50329).
  • the hemoglobin-dependent P evotella strain is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g ., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain B 50329.
  • sequence identity e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
  • the Prevotella histicola strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain C (ATCC Deposit Number PTA-126140, deposited on September 10, 2019).
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%
  • the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Prevotella Strain C (PTA-126140).
  • sequence identity e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity
  • the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Prevotella Strain C (PTA-126140).
  • the Prevotella histicola strain is Prevotella Strain C (PTA-126140).
  • the hemoglobin-dependent Prevotella strain is a strain of Prevotella bacteria comprising one or more (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more) proteins listed in Table 1 and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more) genes encoding proteins listed in Table 1.
  • the hemoglobin- dependent Prevotella strain comprises all of the proteins listed in Table 1 and/or all of the genes encoding the proteins listed in Table 1.
  • the Prevotella bacteria is a strain of Prevotella bacteria free or substantially free of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) proteins listed in Table 2 and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) genes encoding proteins listed in Table 2.
  • Prevotella bacteria is free of all of the proteins listed in Table 2 and/or all of the genes encoding the proteins listed in Table 2.
  • the hemoglobin-dependent Prevotella strain is a strain of Prevotella bacteria comprising one or more of the proteins listed in Table 1 and that is free or substantially free of one or more proteins listed in Table 2.
  • the hemoglobin-dependent Prevotella strain is a strain of Prevotella bacteria that comprises all of the proteins listed in Table 1 and/or all of the genes encoding the proteins listed in Table 1 and that is free of all of the proteins listed in Table 2 and/or all of the genes encoding the proteins listed in Table 2.
  • ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122.
  • the deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited plasmid, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicant acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
  • Soy leghemoglobin can be used in growth media to facilitate the in vitro culturing of bacteria.
  • culturing bacteria in growth media comprising soy leghemoglobin results in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) comprising said bacteria or agents therefrom (e.g., mEVs), and comprising soy leghemoglobin or components thereof.
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • said bacteria or agents therefrom e.g., mEVs
  • soy leghemoglobin Various components of soy leghemoglobin are present and can be detected in the bacterial compositions described herein, including soy leghemoglobin nucleic acids and/ soy leghemoglobin proteins.
  • the component of soy leghemoglobin is a soy leghemoglobin nucleic acid.
  • the soy leghemoglobin nucleic acid is intended to include DNA (e.g., genomic DNA, cDNA) and RNA (e.g, mRNA, tRNA, rRNA, coding RNA, non-coding RNA, small RNA, etc.).
  • the nucleic acid molecule can be single- stranded or double-stranded.
  • the soy leghemoglobin nucleic acid is soy leghemoglobin genomic DNA.
  • the soy leghemoglobin nucleic acid can comprise a coding sequence (e.g., a sequence encoding a soy leghemoglobin protein). In some embodiments, the soy leghemoglobin nucleic acid comprises only a portion of a sequence encoding a soy leghemoglobin protein. In some embodiments, the soy leghemoglobin nucleic acid comprises a non-coding sequence.
  • the nucleic acid is soy leghemoglobin DNA.
  • the soy leghemoglobin DNA comprises a sequence encoding a soy leghemoglobin protein.
  • the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin A (LB A).
  • the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin C2 (LGB2).
  • the bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • a soy leghemoglobin protein e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • the soy leghemoglobin protein is soy leghemoglobin A (LB A).
  • the soy leghemoglobin protein is a sequence encoding soy leghemoglobin C2 (LGB2).
  • culturing bacteria in growth media comprising soy leghemoglobin results in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) comprising said bacteria or agents therefrom (e.g., mEVs), and comprising soy leghemoglobin.
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • said bacteria or agents therefrom e.g., mEVs
  • the methods and compositions provided herein relate to methods of testing bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)to detect (e.g., assay for) the presence of a soy leghemoglobin (or fragments thereof) in the composition.
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • detect e.g., assay for
  • samples for detection can be readily prepared by dissolving the composition in an appropriate buffer/medium prior to testing.
  • soy leghemoglobins that are able to be used in culture media to facilitate the growth of otherwise hemoglobin-dependent bacteria (e.g., for use in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) and/or for therapeutic use).
  • Soy leghemoglobin is a polypeptide that is capable of binding covalently and/or noncovalently to a heme moiety.
  • the soy leghemoglobin comprises a heme moiety.
  • soy leghemoglobins used in the methods and/or compositions provided herein are bound by heme.
  • the soy leghemoglobin is a globin and can include a globin fold, which comprises a series of seven to nine alpha helices.
  • Globin type proteins can be of any class (e.g., class I, class II, or class III), and in some embodiments, can transport or store oxygen.
  • a soy leghemoglobin can be a non-symbiotic type of hemoglobin or a leghemoglobin.
  • a soy leghemoglobin can be a monomer, i.e., a single polypeptide chain, or can be a dimer, a trimer, tetramer, and/or higher order oligomers.
  • Non-limiting examples of non-animal hemoglobins can include an androglobin, a cytoglobin, a globin E, a globin X, a globin Y, a hemoglobin, a leghemoglobin, a flavohemoglobin, Hell's gate globin I, a myoglobin, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a cytoglobin, a histoglobin, a neuroglobins, a chlorocruorin, a truncated hemoglobin (e.g., HbN or HbO), a truncated 2/2 globin, a hemoglobin 3 (e.g. , Glb3), a cytochrome, or a peroxidase.
  • an androglobin a cytoglobin, a globin E, a globin X
  • the soy leghemoglobin may be purified from plants (e.g., legumes). In some embodiments, the soy leghemoglobin is that of an organism of the genus Glycine. In some embodiments, soy leghemoglobin is recombinantly expressed. [180] In certain embodiments, soy leghemoglobins can be from a legume such as Glycine max (soybean); sometimes referred to Glycine hispida. In some embodiments, the soy leghemoglobinis recombinantly expressed.
  • soy leghemoglobins can be a non-symbiotic hemoglobin.
  • the non-symbiotic hemoglobin can be from any plant.
  • a non-symbiotic hemoglobin can be from a plant selected from the group consisting of soybean, sprouted soybean, alfalfa, golden flax, black bean, black eyed pea, northern bean, tobacco, pea, garbanzo, moong bean, cowpeas, pinto beans, pod peas, quinoa, sesame, sunflower, wheat berries, spelt, barley, wild rice, and rice.
  • the soy leghemoglobin is recombinantly expressed.
  • recombinantly expressed non-animal hemoglobin can be isolated from fungi such as Saccharomyces cerevisiae, Pichia pastoris,
  • the non-animal hemoglobin is recombinantly expressed in Pichia pastoris.
  • recombinantly expressed non-animal hemoglobin can be isolated from one or more bacteria such as Escherichia coli, Bacillus suhtilis, Bacillus megaterium, Synechocistis sp., Bacillus suhtilis, Aquifex aeolicus, Methylacidiphilum infernorum (Hell’s Gate), or thermophilic bacteria (e.g, that grow at temperatures greater than 45°C) such as Thermophilus or Thermohifidafusca.
  • bacteria such as Escherichia coli, Bacillus suhtilis, Bacillus megaterium, Synechocistis sp., Bacillus suhtilis, Aquifex aeolicus, Methylacidiphilum infernorum (Hell’s Gate), or thermophilic bacteria (e.g, that grow at temperatures greater than 45°C) such as Thermophilus or Thermohifid
  • recombinantly expressed non-animal hemoglobin can be isolated from algae such as Chlamydomonas eugametos.
  • recombinantly expressed non-animal hemoglobin can be isolated from protozoans such as Paramecium caudatum or Tetrahymena pyriformis.
  • soy leghemoglobin is symbiotic.
  • the present disclosure also contemplates recombinantly expressed heme- containing polypeptides, such as those found in plants, bacteria, cyanobacteria, fungus, algae, grain, legume, and/or protozoa.
  • the soy leghemoglobin is a polypeptide listed in Table 3.
  • the soy leghemoglobin is a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with an amino acid sequence listed in Table 3.
  • soy leghemoglobin is a polypeptide encoded by a cDNA sequence listed in Table 3. In certain embodiments, the soy leghemoglobinis a polypeptide encoded by a cDNA sequence having at least 70%,
  • soy leghemoglobin is recombinantly expressed.
  • the soy leghemoglobin is a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • the component of soy leghemoglobin is a nucleic acid that encodes the amino acid sequence sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the component of soy leghemoglobin is a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%,
  • the component of soy leghemoglobin is a nucleic acid that encodes the amino acid sequence sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the soy leghemoglobin is a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
  • the soy component of leghemoglobin is a nucleic acid that encodes the amino acid sequence sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the soy leghemoglobin is a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • the component of soy leghemoglobin is a nucleic acid that encodes the amino acid sequence sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate ( ⁇ RTI 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well-known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • SEQ ID NO: 4 Glycine max (soybean) leghemoglobin C2 amino acid sequence (GenBank: NP_001235248.2)
  • the methods and compositions provided herein relate to methods of testing bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) to detect (e.g., assay for) the presence of a component of soy leghemoglobin in the composition
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • detect e.g., assay for
  • samples for detection can be readily prepared by dissolving the composition form in an appropriate buffer/medium prior to testing.
  • the component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid.
  • the soy leghemoglobin nucleic acid is soy leghemoglobin DNA.
  • the soy leghemoglobin DNA comprises a sequence from soy leghemoglobin A (LB A).
  • the soy leghemoglobin DNA comprises a sequence from soy leghemoglobin C2 (LGB2).
  • the soy leghemoglobin nucleic acid e.g., DNA
  • telomere length is detected using a polymerase chain reaction (PCR) assay, such as quantitative polymerase chain reaction (qPCR) assay or digital PCR.
  • PCR polymerase chain reaction
  • the component of soy leghemoglobin is a soy leghemoglobin protein.
  • the soy leghemoglobin protein is Soy Leghemoglobin A.
  • the soy leghemoglobin protein is detected by high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC).
  • the soy leghemoglobin protein is detected by mass spectrometry (e.g., tandem mass spectrometry, MALDI-TOF).
  • the provided herein are methods related to the detection of soy leghemoglobin nucleic acids.
  • the soy leghemoglobin nucleic acid may be isolated, however, it will be appreciated by those skilled in the art that many detection assays (e.g., PCR, e.g., qPCR or digital PCR) can be performed to detect the nucleic acid in a sample without isolating the nucleic acid.
  • a nucleic acid can be isolated using standard molecular biology techniques and the sequence information in the public database records, e.g., NCBI, Uniprot, etc.
  • nucleic acid molecules of the present invention can be isolated using standard hybridization and cloning techniques (e.g ., as described in Green and Sambrook, ed., Molecular Cloning: A Laboratory Manual, Fourth ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2014).
  • amplification-based assays can be used to determine the presence of amount of the soy leghemoglobin DNA.
  • the nucleic acid sequences act as a template in an amplification reaction (e.g., Polymerase Chain Reaction (PCR)).
  • PCR Polymerase Chain Reaction
  • the amount of amplification product will be proportional to the amount of template in the original sample (see e.g., Kralik and Ricchi (2017) Front. Microbiol. 8:108).
  • Comparison to appropriate controls e.g. amplification of a bacterial nucleic acid sequence, provides a measure of the copy number or amount of the soy leghemoglobin nucleic acid.
  • Methods of “quantitative” amplification are well-known to those of skill in the art.
  • quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction.
  • Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis is described in Ginzonger, el al. (2000) Cancer Research 60:5405-5409.
  • Fluorogenic quantitative PCR may also be used in the methods of the present invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and SYBR green.
  • LCR ligase chain reaction
  • Genomics 4 560
  • Barringer et al. (1990) Gene 89: 117 transcription amplification
  • transcription amplification Kwoh, et al. (1989 ) Proc. Natl. Acad. Sci. USA 86: 1173
  • self-sustained sequence replication Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874
  • dot PCR digital PCR
  • linker adapter PCR etc.
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. etal. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • the RNA molecules can first be converted to cDNA by a reverse transcriptase (primer-specific cDNA synthesis), prior to performing the amplification methods.
  • a reverse transcriptase primer-specific cDNA synthesis
  • Various amplification and detection methods can be used.
  • Real time PCR may also be used.
  • the presence of soy leghemoglobin nucleic acids can be detected in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) provided herein by sequencing nucleic acids present in the composition.
  • the sequencing assay used is a next generation sequencing (NGS) assay.
  • Nucleic acid sequencing processes include, but are not limited to chain termination sequencing, sequencing by ligation, sequencing by synthesis, pyrosequencing, ion semiconductor sequencing, single-molecule real-time sequencing, and/or 454 sequencing.
  • the NGS Modality is any of the following: SwabSeq, 1 Amplicon, 384 well plate, 96 Nextera barcode set, UDI’s, NextSeq; SwabSeq - 1 Amplicon, 384 well plate, 384 Truseq UDI barcode set, using NextSeq; or SwabSeq - 1 Amplicon, 384 well plate, 4000 UDI Truseq barcode set, NovaSeq. SwabSeq - Multiplex, 384 well plate, CDI barcode set, NovaSeq.
  • the presence and amount of soy leghemoglobin nucleic acid can be detected on a DNA array, chip or a microarray.
  • Labeled nucleic acids of a test sample (those present in the composition) obtained from a subject may be hybridized to a solid surface comprising the soy leghemoglobin nucleic acid (e.g., DNA or RNA). Positive hybridization signal is obtained with the sample containing the soy leghemoglobin nucleic acid.
  • Methods of preparing DNA arrays and their use are well-known in the art (see, e.g., U.S. Pat.
  • Methods of detecting a soy leghemoglobin nucleic acid include, but are not limited to, hybridization-based assays.
  • Hybridization-based assays include traditional “direct probe” methods, such as Southern blots or dot blots. The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g., membrane or glass) bound methods or array-based approaches.
  • detecting a soy leghemoglobin DNA in a sample involves a Southern Blot.
  • a Southern Blot the genomic DNA or fragments thereof (typically separated on an electrophoretic gel) is hybridized to a probe specific for the target region.
  • Comparison of the intensity of the hybridization signal from the probe for the soy leghemoglobin DNA with control probe signal from analysis of bacterial DNA provides an estimate of the amount of the soy leghemoglobin nucleic acid.
  • a probe hybridizes the soy leghemoglobin DNA under stringent conditions.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%; preferably 85%) identical to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C.
  • SSC sodium chloride/sodium citrate
  • the form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example, 32 P and 35 S. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of suitably labeled bases.
  • a Northern blot may be utilized for evaluating the presence or amount of RNA in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)of the present disclosure.
  • RNA is hybridized to a probe specific for the soy leghemoglobin RNA. Comparison of the intensity of the hybridization signal from the probe for the soy leghemoglobin RNA with control probe signal from analysis of bacterial RNA provides an estimate of the relative amount of the soy leghemoglobin RNA.
  • a simpler version of the Southern blot and the Northern blot may be performed using a dot blot format, in which the sample comprising the soy leghemoglobin DNA is simply spotted (without electrophoretic separation) on a platform and the hybridization method is carried out.
  • the methods and compositions provided herein relate to the detection of soy leghemoglobin proteins.
  • the presence or amount of a soy leghemoglobin protein in bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • Exemplary methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluore scent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn. pp 217-262, 1991 which is incorporated by reference).
  • binder-ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.
  • ELISA and RIA procedures may be conducted such that a desired protein standard (e.g., a known soy leghemoglobin protein, the presence of which is being tested in a composition) is labeled (with a radioisotope such as 125 I or 35 S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample (e.g., the soy leghemoglobin protein present in the composition), brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay).
  • a desired protein standard e.g., a known soy leghemoglobin protein, the presence of which is being tested in a composition
  • a radioisotope such as 125 I or 35 S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase
  • the unlabelled sample
  • soy leghemoglobin protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-biomarker proteinantibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay).
  • radioactivity or the enzyme assayed ELISA-sandwich assay.
  • Other conventional methods may also be employed as suitable.
  • a “one-step” assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody.
  • a “two-step” assay involves washing before contacting, the mixture with labeled antibody.
  • Other conventional methods may also be employed as suitable.
  • Enzymatic and radiolabeling of soy leghemoglobin proteins and/or the antibodies may be effected by conventional means.
  • Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected.
  • some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.
  • Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.
  • Anti-soy leghemoglobin protein antibodies are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including 125 I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
  • a secondary immunological reagent such as labeled protein A or anti-immunoglobulin (suitable labels including 125 I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
  • Antibodies that may be used to detect a soy leghemoglobin protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the protein to be detected.
  • An antibody may have a K d of at most about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M.
  • the phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant.
  • An antibody may bind preferentially to the target soy leghemoglobin protein relative to other proteins, such as related proteins.
  • Antibodies may be prepared according to methods known in the art.
  • agents that specifically bind to a soy leghemoglobin protein other than antibodies are used, such as peptides or small molecules.
  • Peptides or small molecules that specifically bind to a biomarker protein can be identified by any means known in the art.
  • specific peptide binders of a target soy leghemoglobin protein can be screened for using peptide phage display libraries.
  • a soy leghemoglobin protein or fragment thereof may also be detected using mass spectrometry and/or HPLC and/or UPLC as for detection of a small molecule as described below.
  • the methods and compostitions provided herein relate to the detection of soy leghemoglobin small molecules.
  • a chromatography method is used to detect a soy leghemoglobin small molecule (or protein). Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), etc.
  • the small molecule or protein detected using a chromatography method is a pigment
  • any one or combination of the methods decribed herein can be used to detect and quantify the amount of at least one component of soy leghemoglobin present in the bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) provided herein.
  • the chromatography is HPLC or UPLC. These methods provide sensitivity that allows separation and detection of a trace amount of at least one component of soy leghemoglobin present in the composition
  • MS Mass spectrometry
  • MS is an analytical technique that measures the mass-to-charge ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.
  • a mass spectrum is a plot of the ion signal as a function of the mass-to- charge ratio. These spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical identity or structure of molecules and other chemical compounds.
  • mass spectrometry-based methods can be utilized to detect the small molecules, nucleic acids, or proteins or fragments thereof of soy leghemoglobin including, but are not limited to, tandem mass spectrometry (MS/MS), MALDI-TOF (a combination of a matrix-assisted laser desorption/ionization source with a time-of-flight mass analyzer), inductively coupled plasma-mass spectrometry (ICP-MS), accelerator mass spectrometry (AMS), thermal ionization-mass spectrometry (TIMS), isotope ratio mass spectrometry (IRMS), and spark source mass spectrometry (SSMS).
  • tandem mass spectrometry MS/MS
  • MALDI-TOF a combination of a matrix-assisted laser desorption/ionization source with a time-of-flight mass analyzer
  • ICP-MS inductively coupled plasma-mass spectrometry
  • AMS accelerator mass spectrometry
  • TMS thermal ionization-mass spect
  • a tandem mass spectrometer is one capable of multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation.
  • one mass analyzer can isolate one peptide from many entering a mass spectrometer.
  • a second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID).
  • CID collision-induced dissociation
  • a third mass analyzer sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer overtime, as in a quadrupole ion trap.
  • CID collision-induced dissociation
  • ECD electron capture dissociation
  • ETD electron transfer dissociation
  • IRMPD infrared multiphoton dissociation
  • BIRD blackbody infrared radiative dissociation
  • ED electron-detachment dissociation
  • SID surface-induced dissociation
  • Mass spectrometry-based detection of soy leghemoglobin small molecules, nucleic acids, or proteins or fragments thereof, can be enhanced by coupling it with chromatographic and/or other separation techniques. Separation may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip, liquid chromatography, gas chromatography). Electrophoresis is a method which can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip.
  • gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof.
  • a gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient.
  • capillaries used for electrophoresis include capillaries that interface with an electrospray.
  • CE Capillary electrophoresis
  • CZE capillary zone electrophoresis
  • CIEF capillary isoelectric focusing
  • cITP capillary isotachophoresis
  • CEC capillary electrochromatography
  • Capillary isotachophoresis is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities.
  • Capillary zone electrophoresis also known as free-solution CE (FSCE)
  • FSCE free-solution CE
  • CIEF Capillary isoelectric focusing
  • CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.
  • the methods and compositions provided herein relate to the detection of soy leghemoglobin (or fragments thereof).
  • a soy leghemoglobin in bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • Exemplary methods include, but are not limited to, immunodiffusion, Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluore scent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn pp 217-262, 1991 which is incorporated by reference).
  • binder-ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.
  • the soy leghemoglobin is detected by high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC).
  • HPLC high performance liquid chromatography
  • UPLC ultra performance liquid chromatography
  • mass spectrometry e.g., tandem mass spectrometry, MALDI-TOF
  • soy leghemoglobin is detected by enzyme-linked immunosorbent assay (ELISA).
  • ELISA and RIA procedures may be conducted such that a desired protein standard (e.g., a known soy leghemoglobin protein, the presence of which is being tested in a composition) is labeled (with a radioisotope such as 125 I or 35 S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample (e.g., the soy leghemoglobin protein present in the composition), brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay).
  • a desired protein standard e.g., a known soy leghemoglobin protein, the presence of which is being tested in a composition
  • a radioisotope such as 125 I or 35 S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase
  • the unlabelled sample
  • soy leghemoglobin protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-biomarker proteinantibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay).
  • radioactivity or the enzyme assayed ELISA-sandwich assay.
  • Other conventional methods may also be employed as suitable.
  • a “one-step” assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody.
  • a “two-step” assay involves washing before contacting, the mixture with labeled antibody.
  • Other conventional methods may also be employed as suitable.
  • Enzymatic and radiolabeling of soy leghemoglobin proteins and/or the antibodies may be detected by conventional means.
  • Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be detected.
  • some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.
  • Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.
  • Anti- soy leghemoglobin protein antibodies are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including 125 I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
  • a secondary immunological reagent such as labeled protein A or anti-immunoglobulin (suitable labels including 125 I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
  • Antibodies that may be used to detect a soy leghemoglobin protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the biomarker protein to be detected.
  • An antibody may have a Kd of at most about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M.
  • the phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant.
  • An antibody may bind preferentially to the target soy leghemoglobin protein relative to other proteins, such as related proteins.
  • Antibodies may be prepared according to methods known in the art.
  • agents that specifically bind to a soy leghemoglobin protein other than antibodies are used, such as peptides or small molecules.
  • Peptides or small molecules that specifically bind to a biomarker protein can be identified by any means known in the art. For example, specific peptide binders of a target soy leghemoglobin hemoglobin protein can be screened for using peptide phage display libraries.
  • An soy leghemoglobin protein or fragments thereof may also be detected using mass spectrometry and/or HPLC and/or UPLC.
  • a chromatography method is used to detect a soy leghemoglobin (or fragments thereof). Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), etc.
  • LC liquid chromatography
  • GC gas chromatography
  • HPLC high performance liquid chromatography
  • UPLC ultra performance liquid chromatography
  • any one or combination of the methods decribed herein can be used to detect and quantify the amount of soy leghemoglobin present in the bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) provided herein.
  • soy leghemoglobin present in the bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • the chromatography is HPLC or UPLC. These methods provide sensitivity that allows separation and detection of a trace amount of soy leghemoglobin present in the composition.
  • Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.
  • a mass spectrum is a plot of the ion signal as a function of the mass-to- charge ratio. These spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical identity or structure of molecules and other chemical compounds.
  • mass spectrometry-based methods can be utilized to detect the small molecules, or proteins or fragments thereof of soy leghemoglobin including, but are not limited to, tandem mass spectrometry (MS/MS), MALDI-TOF (a combination of a matrix- assisted laser desorption/ionization source with a time-of-flight mass analyzer), inductively coupled plasma-mass spectrometry (ICP-MS), accelerator mass spectrometry (AMS), thermal ionization-mass spectrometry (TIMS), isotope ratio mass spectrometry (IRMS), and spark source mass spectrometry (SSMS).
  • tandem mass spectrometry MS/MS
  • MALDI-TOF a combination of a matrix- assisted laser desorption/ionization source with a time-of-flight mass analyzer
  • ICP-MS inductively coupled plasma-mass spectrometry
  • AMS accelerator mass spectrometry
  • TMS thermal ionization-mass spectrometry
  • IRMS iso
  • a tandem mass spectrometer is one capable of multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation.
  • one mass analyzer can isolate one peptide from many entering a mass spectrometer.
  • a second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID).
  • CID collision-induced dissociation
  • a third mass analyzer sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer overtime, as in a quadrupole ion trap.
  • CID collision-induced dissociation
  • ECD electron capture dissociation
  • ETD electron transfer dissociation
  • IRMPD infrared multiphoton dissociation
  • BIRD blackbody infrared radiative dissociation
  • ED electron-detachment dissociation
  • SID surface-induced dissociation
  • Mass spectrometry-based detection of soy leghemoglobin small molecules, or proteins or fragments thereof, can be enhanced by coupling it with chromatographic and/or other separation techniques. Separation may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip, liquid chromatography, gas chromatography). Electrophoresis is a method which can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip.
  • gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof.
  • a gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient.
  • capillaries used for electrophoresis include capillaries that interface with an electrospray.
  • CE Capillary electrophoresis
  • CZE capillary zone electrophoresis
  • CIEF capillary isoelectric focusing
  • cITP capillary isotachophoresis
  • CEC capillary electrochromatography
  • Capillary isotachophoresis is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities.
  • Capillary zone electrophoresis also known as free-solution CE (FSCE)
  • FSCE free-solution CE
  • CIEF Capillary isoelectric focusing
  • CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.
  • isolated and purified soy leghemoglobins can be derived from non-animal sources such as plants, bacteria, cyanobacteria, fungus, algae, grain, legume, and/or protozoa.
  • the isolated and purified proteins can be obtained from genetically modified organisms such as genetically modified bacteria or yeast.
  • the isolated and purified polypeptides are chemically synthesized or obtained via in vitro synthesis.
  • the non-animal- derived polypeptide is recombinantly expressed.
  • a soy leghemoglobin can be recombinantly produced using polypeptide expression techniques (e.g., cloning and expressing an exogenous nucleic acid encoding a heme -containing polypeptide in host cells, such as bacterial cells (e.g., Escherichia coli), insect cells, fungal cells such as yeast cells (e.g., Pichia Pastoris), plant cells, or mammalian cells).
  • the exogenous nucleic acid may be operably linked to a regulatory sequence (e.g., promoter).
  • the exogenous nucleic acid may be in a vector, such as an expression vector.
  • the exogenous nucleic acid may be in a viral vector suitable for transmission of DNA into host cells (e.g., insect cells, mammalian cells).
  • host cells e.g., insect cells, mammalian cells.
  • the recombinant polypeptides may be expressed and purified as an intracellular protein, a secreted protein, and/or an insoluble protein (e.g., contained in inclusion bodies), which can be solubilized using urea and slowly refolded in a native condition.
  • the recombinant proteins comprise a heterologous sequence (e.g., a histidine tag, TAP (tandem affinity purification) tag, TEV cleavage site, a FLAG tag, a GST tag, and/or an immunoglobulin domain) that may be useful in purification and/or detection of the polypeptide.
  • a heterologous sequence e.g., a histidine tag, TAP (tandem affinity purification) tag, TEV cleavage site, a FLAG tag, a GST tag, and/or an immunoglobulin domain
  • soy leghemoglobin can be purified on the basis of their molecular weight, for example, by size exclusion chromatography, ultrafiltration through membranes, or density centrifugation.
  • the polypeptides can be separated based on their surface charge, for example, by isoelectric precipitation, anion exchange chromatography, or cation exchange chromatography.
  • Polypeptides can also be separated on the basis of their solubility, for example, by ammonium sulfate precipitation, isoelectric precipitation, surfactants, detergents, or solvent extraction.
  • Polypeptides can also be separated by their affinity to another molecule, using, for example, hydrophobic interaction chromatography, reactive dyes, or hydroxyapatite.
  • Affinity chromatography can also use antibodies having specific binding affinity for the protein of interest, nickel NTA for His-tagged recombinant proteins, lectins to bind to sugar moieties on a glycoprotein, or other molecules which specifically bind the polypeptides of interest.
  • soy leghemoglobin can be purified from e.g., whole roots or root nodules (e.g., soy roots or soy root nodules).
  • Whole roots or root nodules can be harvested and lysed, for example in 20 mM potassium phosphate pH 7.4, 100 mM potassium chloride and 5 mM EDTA using a grinder-blender. During this process, leghemoglobin is released into the buffer.
  • Root-nodule lysate containing leghemoglobin can be cleared from cell debris by filtration through 5 ⁇ m filter. In some embodiments, filtration is followed by centrifugation (7000 x g, 20 min).
  • Clarified lysate containing leghemoglobin is then filtered through 200 nm filter and applied to an anion-exchange chromatography column (High Prep Q; High Prep DEAE, GE Healthcare) on a fast protein liquid chromatography machine (GE Healthcare).
  • Leghemoglobin is collected in the flowthrough fractions and concentrated over 3 kDa filtration membrane to a desired concentration.
  • leghemoglobin Purity (partial abundance) of the purified leghemoglobin is analyzed by SDS-PAGE gel: in the lysate, leghemoglobin can be present at 20-40 %, while after the anion-exchange purification, it can be present at 70-80 %.
  • soybean leghemoglobin flowthrough from the anion-exchange chromatography is applied onto size -exclusion chromatography (Sephacryl S-100 HR, GE Healthcare). Soybean leghemoglobin is eluted as two fractions corresponding to dimeric and monomeric species. Purity (partial abundance) of leghemoglobin is analyzed by SDS-PAGE and can be ⁇ 90-100 %.
  • Proteins in legume root-nodule lysates can be transferred to 10 mM sodium carbonate pH 9.5, 50 mM sodium chloride buffer, filtered through 200 nm filter, and applied onto an anion-exchange chromatography column on a fast protein liquid chromatography instrument (GE Healthcare).
  • Leghemoglobin can bind the anion exchange chromatography matrix and the bound leghemoglobin can be eluted using a sodium chloride gradient. Purity (partial abundance) of leghemoglobin can be analyzed by SDS- PAGE and can be ⁇ 60-80 %.
  • leghemoglobin Undesired small molecules from legume roots can be removed from purified leghemoglobin by passing the leghemoglobin solution over the anion-exchange resin.
  • the anion exchange resin is FFQ, DEAE, Amberlite IRA900, Dowex 22, or Dowex 1x4.
  • Leghemoglobin purified either by ammonium sulfate fractionation (60 % wt/v and 90 % wt/v ammonium sulfate) or by anion-exchange chromatography is buffer exchanged into 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride, and the solution is passed over one of the above mentioned anion-exchange resins. Flowthrough that contains leghemoglobin can be collected.
  • standard polypeptide synthesis techniques e.g. , liquid-phase polypeptide synthesis techniques or solid-phase polypeptide synthesis techniques
  • cell-free translation techniques can be used to produce soy leghemoglobinproteins synthetically.
  • each subunit of the tetrameric hemoglobin may be individually expressed and isolated from inclusion bodies in E. coli.
  • the denatured b- subunits can be refolded and reconstituted with hemin in the presence of native reduced ⁇ - chains to produce functional, tetrameric hemoglobin (e.g., Fronticelli etal. (1991) J Protein Chem 10:495-501).
  • the ⁇ and ⁇ subunits can be coexpressed to produce a large amount of intact, soluble tetrameric hemoglobin in E. Coli or P. pastoris (e.g., Hoffman etal. (1990) P roc Natl Acad Sci USA 87:8521-8525; Anwised et al. (2016) Protein J 35:256-268).
  • ⁇ genes can be fused to create a di- ⁇ gene, which is inserted in an operon with a copy of the b-gene to express a tetramer that does not dissociate into ⁇ 1 ⁇ 1 dimers under a physiological condition (e.g., Shen et al. (1993) Proc Natl Acad Sci USA 90:8108-8112).
  • the soy leghemoglobin is sterilized, e.g., prior to combining with other components of a growth media. Sterilization may be by Ultra High Temperature (UHT) processing, autoclaving or filtering. In some embodiments, the soy leghemoglobinis autoclaved.
  • Ultra High Temperature UHT
  • filtering In some embodiments, the soy leghemoglobinis autoclaved.
  • soy leghemoglobin is filtered, e.g., the soy leghemoglobinis sterilized prior to use (e.g., prior to addition to growth media or with sterilization of the growth media) in methods to culture hemoglobin-dependent bacteria (e.g., for use in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) and/or for therapeutic use).
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • soy leghemoglobins provided herein can allow for large-scale manufacture and/or GMP grade manufacture of hemoglobin- dependent bacteria, e.g., for therapeutic use and/or in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements).
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements.
  • growth media comprising a soy leghemoglobin disclosed herein.
  • the growth media comprises an amount of a soy leghemoglobin disclosed herein sufficient to support growth of hemoglobin-dependent bacteria (e.g., for use in a bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) and/or for therapeutic use).
  • a bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • the growth medium comprises at least 0.001 g/L, at least 0.005 g/L, at least 0.01 g/L, at least 0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, at least 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L, at least 0.1 g/L, at least 0.2 g/L, at least 0.3 g/L, at least 0.4 g/L, at least 0.5 g/L, at least 0.75 g/L, at least 1 g/L, at least 1.25 g/L, at least 1.5 g/L, at least 1.75 g/L, at least 2 g/L, at least 2.25 g/L, at least 2.5 g/L, at least 2.75 g/L, at least 3 g/L, at least 3.25 g/L, at least 3.5 g
  • the growth media comprises at least 0.005 g/L and no more than 1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.02 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.05 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.2 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.5 g/L of a soy leghemoglobin.
  • the growth medium comprises about 1 g/L of a soy leghemoglobin disclosed herein. In some embodiments, the growth medium comprises about 0.01 g/L to about 0.2 g/L of a soy leghemoglobin.
  • the growth medium comprises at least 0.001 g/L, at least 0.005 g/L, at least 0.01 g/L, at least 0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, at least 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L, at least 0.1 g/L, at least 0.2 g/L, at least 0.3 g/L, at least 0.4 g/L, at least 0.5 g/L, at least 0.75 g/L, at least 1 g/L, at least 1.25 g/L, at least 1.5 g/L, at least 1.75 g/L, at least 2 g/L, at least 2.25 g/L, at least 2.5 g/L, at least 2.75 g/L, at least 3 g/L, at least 3.25 g/L, at least 3.5 g
  • the growth medium comprises about 0.02 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.05 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.2 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.5 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 1 g/L of a soy leghemoglobin.
  • the growth medium comprises about 0.01 g/L to about 0.2 g/L of a soy leghemoglobin.
  • the growth media does not comprise hemoglobin or a derivative thereof (or other soy leghemoglobin) that is sourced from an animal. In some embodiments, the growth media does not comprise animal products.
  • the growth media may contain sugar, yeast extracts, plant based peptones, buffers, salts, trace elements, surfactants, anti-foaming agents, and/or vitamins.
  • the growth media comprise yeast extract, soy peptone A2SC 19649, Soy peptone El 10 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, glucidex 21 D, and/or glucose.
  • the growth media comprises 5 g/L to 15g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L yeast extract 19512.
  • the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L soy peptone A2SC 19649.
  • the growth media comprises 10 g/L to 15 g/L Soy peptone El 10 19885. In some embodiments, the growth media comprises 12.5 g/L Soy peptone El 10 19885. In some embodiments, the growth media comprises 10 g/L soy peptone El 10 19885.
  • the growth media comprises 1 g/L to 3 g/L dipotassium phosphate. In some embodiments, the growth media comprises 1.59 g/L dipotassium phosphate. In some embodiments, the growth media comprises 2.5 g/L dipotassium phosphate.
  • the growth media comprises 0 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.91 g/L monopotassium phosphate. In some embodiments, the growth media does not comprise monopotassium phosphate.
  • the growth media comprises 0.1 g/L to 1.0 g/L L- cysteine-HCl. In some embodiments, the growth media comprises 0.5 g/L L-cysteine-HCl.
  • the growth media comprises 0 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises 0.5 g/L ammonium chloride. In some embodiments, the growth media does not comprise ammonium chloride.
  • the growth media comprises 0 g/L to 30 g/L glucidex 21 D. In some embodiments, the growth media comprises 25 g/L glucidex 21 D. In some embodiments, the growth media does not comprise glucidex 21 D.
  • the growth media comprises 5 g/L to 15g/L glucose. In some embodiments, the growth media comprises 10 g/L glucose. In some embodiments, the growth media comprises 5 g/L glucose.
  • the growth media comprises a soy leghemoglobin provided herein, about 10 g/L yeast extract 19512, about 12.5 g/L soy peptone A2SC 19649, about 12.5 g/L soy peptone El 10 19885, about 1.59 g/L dipotassium phosphate, about 0.91 g/L monopotassium phosphate, about 0.5 g/L ammonium chloride, about 25 g/L glucidex 21 D, and/or about 10 g/L glucose.
  • the growth medium is the growth medium of Table 5.
  • the growth media comprises a soy leghemoglobin provided herein, about 10 g/L yeast extract 19512, about 10 g/L soy peptone A2SC 19649, about 10 g/L soy peptone El 10 19885, about 2.5 g/L dipotassium phosphate, about 0.5 g/L L-cysteine-HCl, and/or about 5 g/L glucose.
  • the growth medium is the growth medium of Table 6.
  • the growth media is at a pH of 5.5 to 7.5. In some embodiments, the growth media is at a pH of about 6.5.
  • a soy leghemoglobin prior to being added to the growth media, a soy leghemoglobin is prepared as a liquid mixture and sterilized by autoclaving or filtration. In some embodiments, the soy leghemoglobin is added to the growth media, which is then sterilized as described below.
  • the media is sterilized.
  • Sterilization may be by Ultra High Temperature (UHT) processing, autoclaving or filtering.
  • UHT processing is performed at very high temperature for short periods of time.
  • the UHT range may be from 135-180°C.
  • the medium may be sterilized from between 10 to 30 seconds at 135°C.
  • hemoglobin-dependent bacteria e.g., for use in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) and/or for therapeutic use).
  • Such methods may comprise incubating the hemoglobin- dependent bacteria in a growth media provided herein.
  • the methods may comprise maintaining the temperature and pH of the growth media as disclosed herein.
  • the culturing may begin in a relatively small volume of growth media (e.g., 1 L) where bacteria are allowed to reach the log phase of growth.
  • Such culture may be transferred to a larger volume of growth media (e.g. , 20 L) for further growth to reach a larger biomass. Depending on the need of the final amount of biomass, such transfer may be repeated more than once.
  • the methods may comprise the incubation of the hemoglobin-dependent bacteria in bioreactors.
  • the hemoglobin-dependent bacteria are incubated at a temperature of 35°C to 39°C. In some embodiments, the hemoglobin-dependent bacteria are incubated at a temperature of about 37°C.
  • the methods and/or compositions provided herein increase the growth rate of hemoglobin-dependent bacteria such that hemoglobin-dependent bacteria grow at an increased rate in the growth media comprising a soy leghemoglobin disclosed herein, compared to the rate at which the hemoglobin-dependent bacteria grow in the same growth media but without the soy leghemoglobin disclosed herein.
  • the rate at which the hemoglobin-dependent bacteria grow in the growth media comprising a soy leghemoglobin disclosed herein is higher than the rate at which the hemoglobin-dependent bacteria grow in the same growth media but without the soy leghemoglobin disclosed herein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least
  • the growth rate is increased by about 200% to about 400%.
  • the rate may be measured as the cell density (as measured by e.g., optical density at the wavelength of 600 nm (OD600)) reached within a given amount of time. In certain embodiments, such rate is measured and compared during the log phase (or exponential phase) of the bacterial growth, optionally wherein the log phase is early log phase.
  • the methods and/or compositions provided herein increase the bacterial cell density such that the hemoglobin-dependent bacteria grow to a higher bacterial cell density in the growth media comprising a soy leghemoglobin disclosed herein, compared to the cell density to which the hemoglobin-dependent bacteria grow in the same growth media but without the soy leghemoglobin disclosed herein.
  • the hemoglobin-dependent bacteria grow to a cell density in the growth media comprising a soy leghemoglobin disclosed herein is higher than the cell density to which the hemoglobin-dependent bacteria grow in the same growth media but without the soy leghemoglobin disclosed herein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 3
  • the bacterial cell density higher than about 200% to about 400%.
  • the cell density may be measured (e.g. , by OD600 or by cell counting) at the stationary phase of bacterial growth, optionally wherein the stationary phase is early stationary phase.
  • the stationary phase is determined as the phase where the growth rate is retarded followed by an exponential phase of growth (e.g., from a growth curve). In other embodiments, the stationary phase is determined by the low glucose level in the growth media.
  • the methods provided herein comprise incubating the hemoglobin-dependent bacteria under anaerobic atmosphere.
  • provided herein are methods of culturing hemoglobin-dependent bacteria under anaerobic atmosphere comprising CO 2 .
  • the anaerobic atmosphere comprises greater than 1% CO 2 .
  • the anaerobic atmosphere comprises greater than 5% CO 2 .
  • the anaerobic atmosphere comprises at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% CO 2 .
  • the anaerobic atmosphere comprises at least 10% CO 2 .
  • the anaerobic atmosphere comprises at least 20% CO 2 .
  • the anaerobic atmosphere comprises from 10% to 40% CO 2 . In some embodiments, the anaerobic atmosphere comprises from 20% to 30% CO 2 . In some embodiments, the anaerobic atmosphere comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% CO 2 . In some embodiments, the anaerobic atmosphere comprises about 25% CO 2 .
  • the anaerobic atmosphere comprises N 2 . In some embodiments, the anaerobic atmosphere comprises less than 95% N 2 . In some embodiments, the anaerobic atmosphere comprises less than 90% N 2 . In some embodiments, the anaerobic atmosphere comprises less than 95%, less than 92%, less than 90%, less than 87%, less than 85%, less than 82%, less than 80%, less than 77% N 2. In some embodiments, the anaerobic atmosphere comprises less than 85% N 2 . In some embodiments, the anaerobic atmosphere comprises less than 80% N 2 . In some embodiments, the anaerobic atmosphere comprises from 65% to 85% N 2 .
  • the anaerobic atmosphere comprises from 70% to 80% N 2 . In some embodiments, the anaerobic atmosphere comprises about 65%, about 66%, about 67%, about 28%, about 69%, about 70%, about 71%, about 72% about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% N 2 . In some embodiments, the anaerobic atmosphere comprises about 75% N 2 .
  • the anaerobic atmosphere consists essentially of CO 2 and N 2 . In some embodiments, the anaerobic atmosphere comprises about 25% CO 2 and about 75% N 2 . In some embodiments, the anaerobic atmosphere comprises about 20% CO 2 and about 80% N 2 . In some embodiments, the anaerobic atmosphere comprises about 30% CO 2 and about 70% N 2 .
  • kits for culturing hemoglobin-dependent bacteria under anaerobic conditions comprising a greater level of CO 2 compared to conventional anaerobic culture conditions (e.g. , at a level of greater than 1% CO 2 , e.g., at a level of greater than 5% CO 2 , such as at a level of about 25% CO 2 ).
  • bioreactors comprising hemoglobin-dependent bacteria being cultured under conditions comprising a greater level of CO 2 compared to conventional anaerobic culture conditions (e.g., at a level of greater than 1% CO 2 , such as at a level of about 25% CO 2 ).
  • the methods and compositions provided herein result in increased bacterial yield compared to conventional culture conditions.
  • provided herein are methods of culturing hemoglobin- dependent bacteria under anaerobic conditions comprising a lower level of N 2 compared to conventional anaerobic culture conditions (e.g., at a level of less than 95% N 2 , e.g., at a level of less than 90% N 2 , such as at a level of about 75% N 2 ).
  • bioreactors comprising hemoglobin-dependent bacteria being cultured under conditions comprising a lower level of N 2 compared to conventional anaerobic culture conditions (e.g. , at a level of less than 95% N 2 such as at a level of about 75% N 2 ).
  • the methods and compositions provided herein result in increased bacterial yield compared to conventional culture conditions.
  • the method comprises the steps of a) purging a bioreactor with an anaerobic gaseous mixture comprising greater than 1% CO 2 ; and b) culturing the hemoglobin-dependent bacteria in the bioreactor purged in step a).
  • the anaerobic gaseous mixture comprises greater than 1% CO 2 .
  • the anaerobic gaseous mixture comprises at least about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% CO 2 .
  • the anaerobic gaseous mixture comprises at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,
  • the anaerobic gaseous mixture comprises from 5% to 35% CO 2 , 10% to 40% CO 2 , 10% to 30% CO 2 , 15% to 30% CO 2 , 20% to 30% CO 2 , 22% to 28% CO 2 , or 24%, to 26% CO 2 .
  • the anaerobic gaseous mixture comprises greater than 5% CO 2 .
  • the anaerobic gaseous mixture comprises at least 10% CO 2 .
  • the anaerobic gaseous mixture comprises at least 20% CO 2 .
  • the anaerobic gaseous mixture comprises from 10% to 40% CO 2 . In some embodiments, the anaerobic gaseous mixture comprises from 20% to 30% CO 2 . In some embodiments, the anaerobic gaseous mixture comprises about 25% CO 2 .
  • the method comprises the steps of a) purging a bioreactor with an anaerobic gaseous mixture comprising less than 95% N 2 ; and b) culturing the hemoglobin- dependent bacteria in the bioreactor purged in step a).
  • the anaerobic gaseous mixture comprises less than 95% N 2 .
  • the anaerobic gaseous mixture comprises less than 95%, less than 92%, less than 90%, less than 87%, less than 85%, less than 82%, less than 80%, less than 77% N 2 .
  • the anaerobic gaseous mixture comprises about 65%, about 66%, about 67%, about 28%, about 69%, about 70%, about 71%, about 72% about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% N 2 .
  • the anaerobic gaseous mixture comprises less than 95% N 2 .
  • the anaerobic gaseous mixture comprises less than 90% N 2 .
  • the anaerobic gaseous mixture comprises from 65% to 85% N 2 .
  • the anaerobic gaseous mixture comprises from 70% to 80% N 2 CO 2 .
  • the anaerobic gaseous mixture comprises about 75% N 2 .
  • the anaerobic gaseous mixture consists essentially of CO 2 and N 2 . In some embodiments, the anaerobic gaseous mixture comprises about 25% CO 2 and about 75% N 2 . In some embodiments, the anaerobic atmosphere comprises about 20% CO 2 and about 80% N 2 . In some embodiments, the anaerobic atmosphere comprises about 30% CO 2 and about 70% N 2 .
  • the anaerobic gaseous mixture comprises CO 2 and N 2 in a ratio of about 1:99, about 2:98, about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about 9:91, about 10:90, 11:89, about 12:88, about 13:87, about 14:86, about 15:85, about 16:84, about 17:83, about 18:82, about 19:81, about 20:80, 21:79, about 22:78, about 23:77, about 24:76, about 25:75, about 26:74, about 27:73, about 28:72, about 29:71, about 30:70, 31:69, about 32:68, about 33:67, about 34:66, about 35:65, about 36:64, about 37:63, about 38:62, about 39:61, or about 40:50 CO 2 to N 2 .
  • the mixed gas composition provides an atmosphere in the bioreactor comprising CO 2
  • an anaerobic gaseous mixture is continuously added to the bioreactor during culturing.
  • the continuously added anaerobic gaseous mixture is added at a rate of 0.01 to 0.1 vvm.
  • the continuously added anaerobic gaseous mixture is added at a rate of 0.02vvm.
  • the continuously added anaerobic gaseous mixture comprises any one of gaseous mixtures described above.
  • the methods provided herein further comprises the step of inoculating a growth media with the hemoglobin-dependent bacteria, wherein the bacteria are cultured in the growth media according to the methods provided herein.
  • the volume of the inoculated hemoglobin-dependent bacteria is between 0.01% and 10% v/v of the growth media (e.g., about 0.1% v/v of the growth media, about 0.5% v/v of the growth media, about 1% v/v of the growth media, about 5% v/v of the growth media). In some embodiments, the volume of hemoglobin-dependent bacteria is about 1 mL.
  • inoculum can be prepared in flasks or in smaller bioreactors where growth is monitored.
  • the inoculum size may be between approximately 0.1% v/v and 5% v/v of the total bioreactor volume.
  • the inoculum is 0.1-3% v/v, 0.1-1% v/v, 0.1-0.5% v/v, or 0.5-1% v/v of the total bioreactor volume.
  • the inoculum is about 0.1% v/v, about 0.2% v/v, about 0.3% v/v, about 0.4%, v/v, about 0.5% v/v, about 0.6% v/v, about 0.7% v/v, about 0.8% v/v, about 0.9% v/v, about 1% v/v, about 1.5% v/v, about 2% v/v, about 2.5% v/v, about 3% v/v, about 4%, v/v, or about 5% v/v of the total bioreactor volume.
  • the bioreactor before the inoculation, is prepared with growth medium at desired pH and temperature.
  • the initial pH of the culture medium may be different than the process set-point. pH stress may be detrimental at low cell concentration; the initial pH could be between pH 7.5 and the process set-point.
  • pH may be set between 4.5 and 8.0, preferably 6.5.
  • the pH can be controlled through the use of sodium hydroxide, potassium hydroxide, or ammonium hydroxide.
  • the temperature may be controlled from 25°C to 45°C, for example at 37°C.
  • the bioreactor fermentation time can vary.
  • fermentation time can vary from 5 hours to 48 hours.
  • fermentation time may be from 5 hours to 24 hours, 8 hours to 24 hours, 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 24 hours, 10 hours to 18 hours, 10 hours to 16 hours, 10 hours to 14 hours, 10 hours to 12 hours, 12 hours to 24 hours, 12 hours to 18 hours, 12 hours to 16 hours, or 12 hours to 14 hours.
  • culturing the hemoglobin-dependent bacteria comprises agitating the culture at a RPM of 50 to 300. In some embodiments, the hemoglobin-dependent bacteria is agitated at a RPM of about 150.
  • a culturing method comprises culturing the hemoglobin-dependent bacteria for at least 5 hours (e.g., at least 10 hours). In some embodiments, the hemoglobin-dependent bacteria is cultured for 10-24 hours. In some embodiments, the hemoglobin-dependent bacteria is cultured for 14 to 16 hours. In some embodiments, the method further comprises the step of inoculating about 5% v/v of the cultured bacteria in a growth media.
  • the growth media is about 20L in volume. In some embodiments, the hemoglobin-dependent bacteria is cultured for 10-24 hours. In some embodiments, the hemoglobin-dependent bacteria is cultured for 12-14 hours. In some embodiments, the method further comprises the step of inoculating about 0.5 %v/v of the cultured bacteria in a growth medium. In some embodiments, the growth medium is about 3500L in volume. In some embodiments, the hemoglobin-dependent bacteria is cultured for 10-24 hours. In some embodiments, the hemoglobin-dependent bacteria is cultured for 12-14 hours. In some embodiments, the hemoglobin-dependent bacteria is cultured at least until a stationary phase is reached.
  • the culturing method further comprises the step of harvesting the cultured bacteria.
  • the harvest time may be based on either glucose level being below 2 g/L or when stationary phase is reached.
  • the method further comprises the step of centrifuging the cultured bacteria after harvesting (e.g., to produce a cell paste).
  • the method further comprises diluting the cell paste with a stabilizer solution to produce a cell slurry.
  • the method further comprises the step of lyophilizing the cell slurry to produce a powder.
  • the method further comprises irradiating the powder with gamma radiation.
  • the culture is cooled (e.g, to 10°C) and centrifuged collecting the cell paste.
  • a stabilizer may be added to the cell paste and mixed thoroughly.
  • Harvesting may be performed by continuous centrifugation.
  • Product may be resuspended with various excipients to a desired final concentration.
  • Excipients can be added for cryo protection or for protection during lyophilization.
  • Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti -oxidants.
  • droplets of cell pellets Prior to lyophilization, droplets of cell pellets may be mixed with excipients and submerged in liquid nitrogen.
  • the cell slurry may be lyophilized. Lyophilization of material, including live bacteria, may begin with primary drying. During the primary drying phase, the ice is removed. Here, a vacuum is generated and an appropriate amount of heat is supplied to the material for the ice to sublime. During the secondary drying phase, product bound water molecules may be removed. Here, the temperature is raised higher than in the primary drying phase to break any physico-chemical interactions that have formed between the water molecules and the product material. The pressure may also be lowered further to enhance desorption during this stage. After the freeze-drying process is complete, the chamber may be filled with an inert gas, such as nitrogen. The product may be sealed within the freeze dryer under dry conditions, preventing exposure to atmospheric water and contaminants. The lyophilized material may be gamma irradiated (e.g., 17.5 kGy).
  • gamma irradiated e.g., 17.5 kGy
  • bioreactors comprising growth media provided herein (i.e., a growth media comprising a soy leghemoglobin disclosed herein and/or hemoglobin-dependent bacteria (e.g., for use in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) and/or for therapeutic use) provided herein.
  • the hemoglobin-dependent bacteria are Prevotella bacteria (e.g., aPrevotella strain provided herein).
  • provided herein are methods of culturing bacteria in such bioreactors.
  • the bioreactor is under the anaerobic conditions mentioned above.
  • bioreactors comprising hemoglobin-dependent bacteria under an anaerobic atmosphere disclosed above.
  • bioreactors of various sizes are provided herein.
  • the bioreactors are at least 1L in volume, at least 5L in volume, at least 10L in volume, at least 15L in volume, at least 20L in volume, at least 30L in volume, at least 40L in volume, at least 50L in volume, at least 100L in volume, at least 200L in volume, at least 250L in volume, at least 500L in volume, at least 750L in volume, at least 1000L in volume, at least 1500L in volume, at least 2000L in volume, at least 2500L in volume, at least 3000L in volume, at least 3500L in volume, at least 4000L in volume, at least 5000L in volume, at least 7500L in volume, at least 10,000L in volume, at least 15,000L in volume, or at least 20,000L in volume.
  • the bioreactors are about 1L in volume, about 5L in volume, about 10L in volume, about 15L in volume, about 20L in volume, about 30L in volume, about 40L in volume, about 50L in volume, about 100L in volume, about 200L in volume, about 250L in volume, about 500L in volume, about 750L in volume, about 1000L in volume, about 1500L in volume, about 2000L in volume, about 2500L in volume, about 3000L in volume, about 3500L in volume, about 4000L in volume, about 5000L in volume, about 7500L in volume, about 10,000L in volume, about 15,000L in volume, or about 20,000L in volume.
  • the bacteria of the bacterial composition e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement
  • the bacteria of the bacterial composition are modified to reduce toxicity or other adverse effects, to enhance delivery
  • e.g., oral delivery e.g., by improving acid resistance, muco- adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization
  • target desired cell types e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages
  • to enhance their immunomodulatory and/or therapeutic effect of the bacteria and/or mEVs e.g., either alone or in combination with another therapeutic agent
  • to enhance immune activation or suppression by the bacteria and/or mEVs such as smEVs and/or pm
  • the engineered bacteria described herein are modified to improve bacteria and/or mEV (such as smEV and/or pmEV) manufacturing (e.g., higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times).
  • the engineered bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may result in the overexpression and/or underexpression of one or more genes.
  • the engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
  • the bacteria and/or mEVs are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.
  • the therapeutic moiety is a cancer-specific moiety.
  • the cancer-specific moiety has binding specificity for a cancer cell (e.g., has binding specificity for a cancer-specific antigen).
  • the cancer-specific moiety comprises an antibody or antigen binding fragment thereof.
  • the cancer-specific moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
  • the cancer-specific moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof.
  • the cancer-specific moiety is a bipartite fusion protein that has two parts: a first part that binds to and/or is linked to the bacterium and a second part that is capable of binding to a cancer cell (e.g., by having binding specificity for a cancer-specific antigen).
  • the first part is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
  • the first part has binding specificity for the mEV (e.g., by having binding specificity for a bacterial antigen).
  • the first and/or second part comprises an antibody or antigen binding fragment thereof. In some embodiments, the first and/or second part comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the first and/or second part comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In certain embodiments, co-administration of the cancer- specific moiety with the pharmaceutical agent (either in combination or in separate administrations) increases the targeting of the pharmaceutical agent to the cancer cells.
  • the bacteria and/or mEVs described herein can be modified such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (e.g., a magnetic bead).
  • the magnetic and/or paramagnetic moiety is comprised by and/or directly linked to the bacteria.
  • the magnetic and/or paramagnetic moiety is linked to and/or a part of a bacteria- or an mEV-binding moiety that binds to the bacteria or mEV.
  • the bacteria- or mEV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
  • the bacteria- or mEV-binding moiety has binding specificity for the bacteria or mEV (e.g., by having binding specificity for a bacterial antigen).
  • the bacteria- or mEV- binding moiety comprises an antibody or antigen binding fragment thereof.
  • the bacteria- or mEV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
  • the bacteria- or mEV-binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof.
  • co-administration of the magnetic and/or paramagnetic moiety with the bacteria or mEVs can be used to increase the targeting of the mEVs (e.g., to cancer cells and/or a part of a subject where cancer cells are present.
  • the pmEVs described herein can be prepared using any method known in the art.
  • the pmEVs are prepared without a pmEV purification step.
  • bacteria from which the pmEVs described herein are released are killed using a method that leaves the bacterial pmEVs intact, and the resulting bacterial components, including the pmEVs, are used in the methods and compositions described herein.
  • the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein).
  • the bacteria are killed using UV irradiation.
  • the pmEVs described herein are purified from one or more other bacterial components. Methods for purifying pmEVs from bacteria (and optionally, other bacterial components) are known in the art. In some embodiments, pmEVs are prepared from bacterial cultures using methods described in Thein, et al. (./. Proteome Res. 9(12):6135-6147 (2010)) or Sandrini, et al. ( Bio-protocol 4(21): el287 (2014)), each of which is hereby incorporated by reference in its entirety.
  • the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g., at 10,000- 15,000 x g for 10- 15 min at room temperature or 4°C).
  • the supernatants are discarded and cell pellets are frozen at -80°C.
  • cell pellets are thawed on ice and resuspended in 100 mM Tris-HCl, pH 7.5 supplemented with
  • cells are lysed using an Emulsiflex C-3 (Avestin, Inc.) under conditions recommended by the manufacturer.
  • debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min at 4°C.
  • supernatants are then centrifuged at 120,000 x g for 1 hour at 4°C.
  • pellets are resuspended in ice-cold 100 mM sodium carbonate, pH 11, incubated with agitation for 1 hr at 4°C, and then centrifuged at 120,000 x g for 1 hour at 4°C.
  • pellets are resuspended in 100 mM Tris-HCl, pH 7.5, re- centrifuged at 120,000 x g for 20 min at 4°C, and then resuspended in 0.1 M Tris-HCl, pH 7.5 or in PBS. In some embodiments, samples are stored at -20°C.
  • pmEVs are obtained by methods adapted from Sandrini et al, 2014.
  • bacterial cultures are centrifuged at 10,000-15,500 x g for 10-15 min at room temp or at 4°C.
  • cell pellets are frozen at -80°C and supernatants are discarded.
  • cell pellets are thawed on ice and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA supplemented with 0.1 mg/mL lysozyme.
  • samples are incubated with mixing at room temp or at 37°C for 30 min.
  • samples are re-frozen at -80°C and thawed again on ice.
  • DNase I is added to a final concentration of 1.6 mg/mL and MgC12 to a final concentration of 100 mM.
  • samples are sonicated using a QSonica Q500 sonicator with 7 cycles of 30 sec on and 30 sec off.
  • debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min. at 4°C. In some embodiments, supernatants are then centrifuged at 110,000 x g for 15 min at 4°C.
  • pellets are resuspended in 10 mM Tris-HCl, pH 8.0, 2% Triton X-100 and incubated 30-60 min with mixing at room temperature. In some embodiments, samples are centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in PBS and stored at -20°C.
  • a method of forming (e.g., preparing) isolated bacterial pmEVs comprises the steps of: (a) centrifuging a bacterial culture, thereby forming a first pellet and a first supernatant, wherein the first pellet comprises cells; (b) discarding the first supernatant; (c) resuspending the first pellet in a solution; (d) lysing the cells; (e) centrifuging the lysed cells, thereby forming a second pellet and a second supernatant; (f) discarding the second pellet and centrifuging the second supernatant, thereby forming a third pellet and a third supernatant; (g) discarding the third supernatant and resuspending the third pellet in a second solution, thereby forming the isolated bacterial pmEVs.
  • the method further comprises the steps of: (h) centrifuging the solution of step (g), thereby forming a fourth pellet and a fourth supernatant; (i) discarding the fourth supernatant and resuspending the fourth pellet in a third solution. In some embodiments, the method further comprises the steps of: (j) centrifuging the solution of step (i), thereby forming a fifth pellet and a fifth supernatant; and (k) discarding the fifth supernatant and resuspending the fifth pellet in a fourth solution. [321] In some embodiments, the centrifugation of step (a) is at 10,000 x g. In some embodiments the centrifugation of step (a) is for 10-15 minutes.
  • step (b) further comprises freezing the first pellet at -80 °C .
  • the solution in step (c) is 100mM Tris-HCl, pH 7.5 supplemented with lmg/ml DNasel.
  • the solution in step (c) is 10mM Tris-HCl, pH 8.0, ImM EDTA, supplemented with 0.1 mg/ml lysozyme.
  • step (c) further comprises incubating for 30 minutes at 37 °C or room temperature.
  • step (c) further comprises freezing the first pellet at -80 °C .
  • step (c) further comprises adding DNase I to a final concentration of 1.6mg/ml. In some embodiments, step (c) further comprises adding MgCl 2 to a final concentration of lOOmM.
  • the cells are lysed in step (d) via homogenization. In some embodiments, the cells are lysed in step (d) via emulsiflex C3. In some embodiments, the cells are lysed in step (d) via sonication. In some embodiments, the cells are sonicated in 7 cycles, wherein each cycle comprises 30 seconds of sonication and 30 seconds without sonication. In some embodiments, the centrifugation of step (e) is at 10,000 x g. In some embodiments, the centrifugation of step (e) is for 15 minutes. In some embodiments, the centrifugation of step
  • (e) is at 4 °C or room temperature.
  • the centrifugation of step (f) is at 120,000 x g. In some embodiments, the centrifugation of step (f) is at 110,000 x g. In some embodiments, the centrifugation of step (f) is for 1 hour. In some embodiments, the centrifugation of step
  • step (f) is for 15 minutes. In some embodiments, the centrifugation of step (f) is at 4 °C or room temperature. In some embodiments, the second solution in step (g) is 100 mM sodium carbonate, pH 11. In some embodiments, the second solution in step (g) is lOmM Tris-HCl pH 8.0, 2% triton X-100. In some embodiments, step (g) further comprises incubating the solution for 1 hour at 4 °C. In some embodiments, step (g) further comprises incubating the solution for 30-60 minutes at room temperature. In some embodiments, the centrifugation of step (h) is at 120,000 x g. In some embodiments, the centrifugation of step (h) is at
  • the centrifugation of step (h) is for 1 hour. In some embodiments, the centrifugation of step (h) is for 15 minutes. In some embodiments, the centrifugation of step (h) is at 4 °C or room temperature.
  • the third solution in step (i) is 100mM Tris-HCl, pH 7.5. In some embodiments, the third solution in step (i) is PBS. In some embodiments, the centrifugation of step (j) is at 120,000 x g. In some embodiments, the centrifugation of step (j) is for 20 minutes. In some embodiments, the centrifugation of step (j) is at 4 °C or room temperature. In some embodiments, the fourth solution in step (k) is 100mM Tris-HCl, pH 7.5 or PBS.
  • pmEVs obtained by methods provided herein may be further purified by size based column chromatography, by affinity chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column.
  • Samples are applied to a 35- 60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C.
  • pmEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated pmEVs may be DNase or proteinase K treated.
  • the sterility of the pmEV preparations can be confirmed by plating a portion of the pmEVs onto agar medium used for standard culture of the bacteria used in the generation of the pmEVs and incubating using standard conditions.
  • select pmEVs are isolated and enriched by chromatography and binding surface moieties on pmEVs.
  • select pmEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
  • pmEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019). [328] In some embodiments, pmEVs are lyophilized.
  • pmEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).
  • pmEVs are UV irradiated.
  • pmEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
  • pmEVs are acid treated.
  • pmEVs are oxygen sparged (e.g., at 0.1 vvm for two hours).
  • pmEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • the smEVs described herein can be prepared using any method known in the art.
  • the smEVs are prepared without an smEV purification step.
  • bacteria described herein are killed using a method that leaves the smEVs intact and the resulting bacterial components, including the smEVs, are used in the methods and compositions described herein.
  • the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein).
  • the bacteria are killed using UV irradiation.
  • the bacteria are heat-killed.
  • the smEVs described herein are purified from one or more other bacterial components. Methods for purifying smEVs from bacteria are known in the art. In some embodiments, smEVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015) or Jeppesen, et al. Cell 177:428 (2019), each of which is hereby incorporated by reference in its entirety. In some embodiments, the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g.
  • the culture supernatants are then passed through filters to exclude intact bacterial cells (e.g., a 0.22 pm filter).
  • the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS.
  • filtered supernatants are centrifuged to pellet bacterial smEVs (e.g., at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C).
  • the smEVs are further purified by resuspending the resulting smEV pellets (e.g., in PBS), and applying the resuspended smEVs to an Optiprep (iodixanol) gradient or gradient (e.g., a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (e.g., at 200,000 x g for 4-20 hours at 4°C).
  • Optiprep iodixanol gradient or gradient
  • centrifugation e.g., at 200,000 x g for 4-20 hours at 4°C.
  • smEV bands can be collected, diluted with PBS, and centrifuged to pellet the smEVs (e.g. , at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C).
  • the purified smEVs can be stored, for example, at -80°C
  • the smEVs are further purified by treatment with DNase and/or proteinase K.
  • cultures of bacteria can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria.
  • Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells.
  • Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration.
  • ammonium sulfate precipitation 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4°C.
  • Precipitations can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C.
  • the resulting pellets contain bacteria smEVs and other debris.
  • filtered supernatants can be centrifuged at 100,000- 200,000 x g for 1-16 hours at 4°C.
  • the pellet of this centrifugation contains bacteria smEVs and other debris such as large protein complexes.
  • supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
  • smEVs can be obtained from bacteria cultures continuously during growth, or at selected time points during growth, for example, by connecting a bioreactor to an alternating tangential flow (ATF) system (e.g., XCell ATF from Repligen).
  • ATF alternating tangential flow
  • the ATF system retains intact cells (>0.22 um) in the bioreactor, and allows smaller components (e.g., smEVs, free proteins) to pass through a filter for collection.
  • the system may be configured so that the ⁇ 0.22 um filtrate is then passed through a second filter of 100 kDa, allowing species such as smEVs between 0.22 um and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor.
  • the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture. smEVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
  • smEVs obtained by methods provided herein may be further purified by size-based column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0.
  • the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample.
  • the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep.
  • Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, e.g., 4-24 hours at 4°C.
  • smEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated smEVs may be DNase or proteinase K treated.
  • purified smEVs are processed as described previously (G. Norheim, et al. PLoS ONE.
  • bands containing smEVs are resuspended to a final concentration of 50 ⁇ g/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art.
  • This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v).
  • adjuvant for example aluminum hydroxide at a concentration of 0-0.5% (w/v).
  • smEVs in PBS are sterile-filtered to ⁇ 0.22 um.
  • samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using fdtration (e.g., Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
  • fdtration e.g., Amicon Ultra columns
  • dialysis e.g., dialysis
  • ultracentrifugation 200,000 x g, > 3 hours, 4°C
  • the sterility of the smEV preparations can be confirmed by plating a portion of the smEVs onto agar medium used for standard culture of the bacteria used in the generation of the smEVs and incubating using standard conditions.
  • select smEVs are isolated and enriched by chromatography and binding surface moieties on smEVs.
  • select smEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
  • the smEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019).
  • smEVs are lyophilized.
  • smEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).
  • smEVs are UV irradiated.
  • smEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
  • smEVs s are acid treated.
  • smEVs are oxygen sparged (e.g., at 0.1 vvm for two hours).
  • the phase of growth can affect the amount or properties of bacteria and/or smEVs produced by bacteria.
  • smEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • the growth environment e.g., culture conditions
  • the yield of smEVs can be increased by an smEV inducer, as provided in Table 4.
  • Table 4 Culture Techniques to Increase smEV Production
  • the method can optionally include exposing a culture of bacteria to an smEV inducer prior to isolating smEVs from the bacterial culture.
  • the culture of bacteria can be exposed to an smEV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • solid dosage forms e.g., pharmaceutical compositions having a solid dosage form
  • a pharmaceutical agent that contains bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component.
  • the pharmaceutical agent can optionally contain one or more additional components, such as a cryoprotectant.
  • the pharmaceutical agent can be lyophilized (e.g., resulting in a powder).
  • the pharmaceutical agent can be combined with one or more excipients (e.g., pharmaceutically acceptable excipients) in the solid dosage form.
  • the solid dosage form comprises a pharmaceutical agent (e.g., bacteria and/or an agent (e.g., component) of bacterial origin, such as mEVs, a powder comprising bacteria and/or an agent (e.g., component) of bacterial origin, such as mEVs) and one or more disintegration agents.
  • a pharmaceutical agent e.g., bacteria and/or an agent (e.g., component) of bacterial origin, such as mEVs, a powder comprising bacteria and/or an agent (e.g., component) of bacterial origin, such as mEVs) and one or more disintegration agents.
  • the total pharmaceutical agent mass is at least 5%, 10%, 15%, 20% or 25 % of the total mass of the pharmaceutical composition.
  • the total pharmaceutical agent mass is no more than 45%, 40%, 35%, 30%, or 25% of the total mass of the pharmaceutical composition.
  • the total mass of the one or more disintegrating agents is at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total mass of the pharmaceutical composition. In some embodiments, the total mass of the one or more disintegrating agents is no more than 70%, 65%, 60%, or 55% of the total mass of the pharmaceutical composition. In some embodiments, the one or more disintegration agents comprise low-substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac- Di-Sol), and/or crospovidone (PVPP).
  • L-HPC low-substituted hydroxypropyl cellulose
  • Ac- Di-Sol croscarmellose sodium
  • PVPP crospovidone
  • the solid dosage forms provided herein comprise L- HPC.
  • the L-HPC is (or comprises L-HPC) of grade LH-B1.
  • the total L-HPC mass is at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition.
  • the total L-HPC mass is no more than 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition. In certain embodiments, the total L-HPC mass is about 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,
  • the solid dosage forms provided herein comprise Ac-Do-Sol.
  • the Ac -Di-Sol is (or comprises Ac -Di-Sol) of grade SD- 711.
  • the total Ac -Di-Sol mass is at least 0.01%, 0.1%, 1%, 2%,
  • the total Ac-Di-Sol mass is no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition.
  • the total Ac-Di-Sol mass is no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition.
  • the total Ac-Di-Sol mass is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition.
  • the solid dosage forms provided herein comprise PVPP.
  • the total PVPP mass is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition.
  • the total PVPP mass is no more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition.
  • the total PVPP mass is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition.
  • the solid dosage forms provided herein comprise:
  • L-HPC e.g., L- HPC of grade LH-B 1
  • L-HPC mass e.g., at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,
  • Ac-Di-Sol e.g., Ac-Di-Sol of grade SD-711 having a total Ac-Di-Sol mass that is at least 0.01% (e.g., at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16%) and no more than 16% (e.g., no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16%) of the total mass of the pharmaceutical composition; and (iv) PVPP having a total PVPP mass that is at least 5% (e.g., at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16%) of the total mass of the pharmaceutical composition; and (iv) PVPP
  • the total L-HPC mass plus the total Ac-Di-Sol mass plus the total PVPP mass is at least 35%, 40%, 45%, or 50% of the total mass of the pharmaceutical composition.
  • the solid dosage form comprises: a total L-HPC mass is about 32% of the total mass of the pharmaceutical composition; a total Ac-Di-Sol mass is about 6% of the total mass of the pharmaceutical composition; and a total PVPP mass is about 15% of the total mass of the pharmaceutical composition.
  • the solid dosage forms provided herein further comprise mannitol.
  • the mannitol is (or comprises) mannitol SD200.
  • the total mannitol mass is at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% of the total mass of the pharmaceutical composition.
  • the total mannitol mass is no more than 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,
  • the total mannitol mass is about 10%, 11%, 12%, 13%, 14%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%,
  • the solid dosage forms provided herein comprise magnesium stearate.
  • the total magnesium stearate mass is at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11% of the total mass of the pharmaceutical composition.
  • the total magnesium stearate mass is no more than 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11% of the total mass of the pharmaceutical composition.
  • the total magnesium stearate mass is about 0.01%, 0.1%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, or 11% of the total mass of the pharmaceutical composition.
  • the solid dosage forms provided herein comprise colloidal silica.
  • the colloidal silica is (or comprises) Aerosil 200.
  • the total colloidal silica mass is at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11% of the total mass of the pharmaceutical composition.
  • the total colloidal silica mass is no more than 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11% of the total mass of the pharmaceutical composition.
  • the total colloidal silica mass is about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11% of the total mass of the pharmaceutical composition.
  • the solid dosage form comprises a pharmaceutical agent, wherein the pharmaceutical agent comprises bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component and a diluent.
  • the total pharmaceutical agent mass is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total mass of the pharmaceutical composition.
  • the total pharmaceutical agent mass is no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total mass of the pharmaceutical composition.
  • the total mass of the diluent is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
  • the total mass of the diluent is no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the total mass of the pharmaceutical composition.
  • the diluent comprises mannitol.
  • the solid dosage form provided herein comprises a lubricant.
  • the total lubricant mass is at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 0.5% to about 1.5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 1% of the total mass of the pharmaceutical composition. In some embodiments, the lubricant comprises magnesium stearate.
  • the solid dosage forms provided herein comprise a glidant.
  • the glidant is colloidal silicon dioxide.
  • the total glidant mass is at least 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,
  • the total glidant mass is no more than 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.25% to about 0.75% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.5% of the total mass of the pharmaceutical composition.
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is about 20% to about 50% of the total mass of the pharmaceutical composition
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is at least 5% and no more than 95% of the total mass of the pharmaceutical composition
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is about 8% to about 92% of the total mass of the pharmaceutical composition;
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is about 30% to about 50% of the total mass of the pharmaceutical composition;
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is about 50% of the total mass of the pharmaceutical composition;
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the solid dosage forms provided herein comprise: (i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 13.51 % of the total mass of the pharmaceutical composition; (ii) a diluent (e.g., mannitol) having a total mass that is about 84.99% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the pharmaceutical composition.
  • the solid dosage forms provided herein comprise:
  • a pharmaceutical agent having a total pharmaceutical agent mass that is about 90.22% of the total mass of the pharmaceutical composition
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • solid dosage forms comprising a pharmaceutical agent that contains bacteria.
  • the bacteria can be live bacteria (e.g. , powder or biomass thereof); non-live (dead) bacteria (e.g. , powder or biomass thereof); non replicating bacteria (e.g., powder or biomass thereof); gamma irradiated bacteria (e.g., powder or biomass thereof); and/or lyophilized bacteria (e.g., powder or biomass thereof).
  • solid dosage forms comprising a pharmaceutical agent that contains mEVs.
  • the mEVs can be from culture media (e.g., culture supernatant).
  • the mEVs can be from live bacteria (e.g., powder or biomass thereof); the mEVs can be from non-live (dead) bacteria (e.g, powder or biomass thereof); the mEVs can be from non-replicating bacteria (e.g., powder or biomass thereof); the mEVs can be from gamma irradiated bacteria (e.g. , powder or biomass thereof); and/or the mEVs can be from lyophilized bacteria (e.g., powder or biomass thereof).
  • the pharmaceutical agent comprises mEVs substantially or entirely free of bacteria (e.g, whole bacteria), bacteria (e.g., live bacteria, dead (e.g., killed) , non-replicating bacteria, attenuated bacteria.
  • bacteria e.g., live bacteria, dead (e.g., killed)
  • non-replicating bacteria attenuated bacteria.
  • the pharmaceutical agents and/or pharmaceutical compositions comprise both mEVs and bacteria (e.g., whole bacteria) (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • the pharmaceutical agents comprise bacteria and/or mEVs from one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the hemoglobin-dependent bacteria strains.
  • the pharmaceutical agents comprise bacteria and/or mEVs from one of the bacteria strains or species described herein, e.g., from bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttlew orthia, Turicibacter, or Veillonella.
  • the pharmaceutical agents comprise lyophilized bacteria and/or mEVs.
  • the pharmaceutical agent comprises gamma irradiated bacteria and/or mEVs.
  • the mEVs (such as smEVs and/or pmEVs) can be gamma irradiated after the mEVs are isolated (e.g., prepared).
  • mEVs such as smEVs and/or pmEVs
  • electron microscopy e.g., EM of ultrathin frozen sections
  • NTA nanoparticle tracking analysis
  • Coulter counting Coulter counting
  • DLS dynamic light scattering
  • Coulter counting reveals the numbers of bacteria and/or mEVs (such as smEVs and/or pmEVs) in a given sample.
  • Coulter counting reveals the numbers of particles with diameters of 0.7-10 um.
  • the Coulter counter alone can reveal the number of bacteria and/or mEVs (such as smEVs and/or pmEVs) in a sample.
  • pmEVs are 20-600 nm in diameter.
  • a Nanosight instrument can be obtained from Malvern Pananlytical.
  • the NS300 can visualize and measure particles in suspension in the size range 10-2000nm.
  • NTA allows for counting of the numbers of particles that are, for example, 50- 1000 nm in diameter.
  • DLS reveals the distribution of particles of different diameters within an approximate range of 1 nm - 3 um.
  • mEVs can be characterized by analytical methods known in the art (e.g. , Jeppesen, et al. Cell 177:428 (2019)).
  • the bacteria and/or mEVs may be quantified based on particle count.
  • total protein content of a bacteria and/or mEV preparation can be measured using NTA.
  • NTA can be performed using Zetaview.
  • the bacteria and/or mEVs may be quantified based on the amount of protein, lipid, or carbohydrate. For example, total protein content of a bacteria and/or preparation can be measured using the Bradford assay or BCA.
  • mEVs are isolated away from one or more other bacterial components of the source bacteria or bacterial culture. In some embodiments, bacteria are isolated away from one or more other bacterial components of the source bacterial culture. In some embodiments, the pharmaceutical agent further comprises other bacterial components.
  • the mEV preparation obtained from the source bacteria may be fractionated into subpopulations based on the physical properties (e.g., sized, density, protein content, binding affinity) of the subpopulations.
  • One or more of the mEV subpopulations can then be incorporated into the pharmaceutical agents of the invention.
  • compositions and/or solid dosage forms comprising pharmaceutical agents that comprise bacteria and/or mEVs (such as smEVs and/or pmEVs) useful for the treatment and/or prevention of disease (e.g. , a cancer, an autoimmune disease, an inflammatory disease, a metabolic disease, or a dysbiosis), as well as methods of making and/or identifying such bacteria and/or mEVs, and methods of using pharmaceutical agents and pharmaceutical compositions and/or solid dosage forms thereof (e.g. , for the treatment of a cancer, an autoimmune disease, an inflammatory disease, or a metabolic disease, either alone or in combination with other therapeutics).
  • mEVs such as smEVs and/or pmEVs
  • the pharmaceutical agents comprise both mEVs (such as smEVs and/or pmEVs) and bacteria (e.g., whole bacteria) (e.g., live bacteria, dead (e.g., killed) bacteria, non-replicating bacteria, attenuated bacteria).
  • the pharmaceutical agents comprise bacteria in the absence of mEVs (such as smEVs and/or pmEVs).
  • the pharmaceutical agents comprise mEVs (such as smEVs and/or pmEVs) in the absence of bacteria.
  • the pharmaceutical agents comprise mEVs (such as smEVs and/or pmEVs) and/or bacteria from hemoglobin- dependent bacteria.
  • the pharmaceutical agents comprise bacteria and/or mEVs from one of the bacteria strains or species described herein, e.g., from bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides,
  • Cloacibacillus Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propioni bacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
  • a subject e.g., human subject.
  • the pharmaceutical agents are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format.
  • the pharmaceutical agent is combined with an adjuvant such as an immuno- adjuvant (e.g. , a STING agonist, a TLR agonist, or a NOD agonist).
  • the pharmaceutical composition and/or solid dosage form comprises at least one carbohydrate.
  • the pharmaceutical composition and/or solid dosage form comprises at least one lipid.
  • the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22: 1), docosapentaenoic acid (EPA), docosanoic acid (22
  • the pharmaceutical composition and/or solid dosage form comprises at least one mineral or mineral source.
  • minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium.
  • Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
  • the pharmaceutical composition and/or solid dosage form comprises at least one vitamin.
  • the at least one vitamin can be fat-soluble or water- soluble vitamins.
  • Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin.
  • Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
  • the pharmaceutical composition and/or solid dosage form comprises an excipient.
  • Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a glidant, a diluent, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
  • Suitable excipients that can be included in the solid dosage form can be one or more pharmaceutically acceptable excipients known in the art. For example, see Rowe, Sheskey, and Quinn, eds., Handbook of Pharmaceutical Excipients, sixth ed.; 2009; Pharmaceutical Press and American Pharmacists Association.
  • the pharmaceutical agent can be prepared as a powder (e.g., for resuspension).
  • the pharmaceutical composition can be prepared as a powder (e.g., for resuspension).
  • Solid dosage forms can similarly be prepared for other types of bacterial compositions, such as medicinal products, medical foods, food products, and/or dietary supplements.
  • the solid dosage form described herein can be, e.g. , a tablet or a minitablet. Further, a plurality of minitablets can be in (e.g. , loaded into) a capsule.
  • the solid dosage form comprises a tablet (> 4mm) (e.g., 5mm-17mm).
  • the tablet is a 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm or 18mm tablet.
  • the size refers to the diameter of the tablet, as is known in the art.
  • the size of the tablet refers to the size of the tablet prior to application of an enteric coating.
  • the solid dosage form comprises a minitablet.
  • the minitablet can be in the size range of lmm-4 mm range.
  • the minitablet can be a 1mm minitablet, 1.5 mm minitablet, 2mm minitablet, 3mm minitablet, or 4mm minitablet.
  • the size refers to the diameter of the minitablet, as is known in the art.
  • the size of the minitablet refers to the size of the minitablet prior to application of an enteric coating.
  • the minitablets can be in a capsule.
  • the capsule can be a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule.
  • the capsule that contains the minitablets can comprise HPMC (hydroxyl propyl methyl cellulose) or gelatin.
  • HPMC hydroxyl propyl methyl cellulose
  • the minitablets can be inside a capsule: the number of minitablets inside a capsule will depend on the size of the capsule and the size of the minitablets. As an example, a size 0 capsule can contain 31-35 (an average of 33) minitablets that are 3 mm minitablets.
  • the solid dosage form e.g., tablet or minitablet
  • is enterically coated e.g., comprises an enteric coating; e.g., is coated with an enteric coating.
  • the solid dosage form described herein can be a capsule.
  • the solid dosage forms can comprise capsules.
  • the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule.
  • the capsule comprises HPMC (hydroxyl propyl methyl cellulose) or gelatin.
  • the capsule comprises HPMC (hydroxyl propyl methyl cellulose).
  • the capsule is banded.
  • the solid dosage form e.g., capsule
  • is enterically coated e.g., comprises an enteric coating; e.g., is coated with an enteric coating.
  • the solid dosage form (e.g. , tablet or minitablet or capsule) described herein can be enterically coated, e.g., with one enteric coating layer or with two layers of enteric coating, e.g., an inner enteric coating and an outer enteric coating.
  • the inner enteric coating and outer enteric coating are not identical (e.g., the inner enteric coating and outer enteric coating do not contain the same components in the same amounts).
  • the enteric coating can allow for release of the pharmaceutical agent, e.g. , in the small intestine, e.g. , upper small intestine, e.g., duodenum and/or jejunum.
  • EUDRAGIT is the brand name for a diverse range of polymethacrylate- based copolymers. It includes anionic, cationic, and neutral copolymers based on methacrylic acid and methacrylic/acrylic esters or their derivatives.
  • Examples of other materials that can be used in the enteric coating include cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, shellac (esters of aleurtic acid), plastics, plant fibers, zein, AQUA-ZEIN® (an aqueous zein formulation containing no alcohol), amylose starch, starch derivatives, dextrins, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), methyl methacrylate- methacrylic acid copolymers, and/or sodium alginate.
  • CAP cellulose acetate phthalate
  • CAT cellulose acetate trimellitate
  • PVAP poly(vinyl acetate phthalate)
  • the enteric coating (e.g., the one enteric coating or the inner enteric coating and/or the outer enteric coating) can include a methacrylic acid ethyl acrylate (MAE) copolymer (1:1).
  • MAE methacrylic acid ethyl acrylate
  • the one enteric coating can include methacrylic acid ethyl acrylate (MAE) copolymer (1:1) (such as Kollicoat MAE 100P).
  • MAE methacrylic acid ethyl acrylate
  • the one enteric coating can include a Eudragit coplymer, e.g. , a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g. , Eudragit FS 30 D).
  • a Eudragit coplymer e.g. , a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g. , Eudragit FS 30 D).
  • enteric coating e.g., the one enteric coating or the inner enteric coating and/or the outer enteric coating
  • materials that can be used in the enteric coating include those described in, e.g., U.S. 6312728; U.S. 6623759; U.S. 4775536; U.S. 5047258; U.S. 5292522; U.S. 6555124; U.S. 6638534; U.S. 2006/0210631; U.S. 2008/200482; U.S. 2005/0271778; U.S. 2004/0028737; WO 2005/044240.
  • methacrylic acid copolymers include: poly (methacrylic acid, methyl methacrylate) 1:1 sold, for example, under the Eudragit LI 00 trade name; poly(methacrylic acid, ethyl acrylate) 1:1 sold, for example, under the Eudragit L100-55 trade name; partially- neutralized poly(methacrylic acid, ethyl acrylate) 1 : 1 sold, for example, under the Kollicoat MAE-100P trade name; and poly(methacrylic acid, methyl methacrylate) 1:2 sold, for example, under the Eudragit S100 trade name.
  • methacrylic acid copolymers include: poly (methacrylic acid, methyl methacrylate) 1:1 sold, for example, under the Eudragit LI 00 trade name; poly(methacrylic acid, ethyl acrylate) 1:1 sold, for example, under the Eudragit L100-55 trade name; partially- neutralized poly(methacrylic acid, ethyl acrylate)
  • the solid dosage form (e.g. , tablet or minitablet or capsule) described herein further comprises a sub-coating.
  • the solid dosage form comprises a sub-coating, e.g., in addition to the enteric coating, e.g., the sub-coating is beneath the enteric coating (e.g., between the solid dosage form and the enteric coating).
  • the sub-coating comprises Opadry QX, e.g., Opadry QX Blue.
  • the dose of the pharmaceutical agent is the dose per capsule or tablet or per total number of minitablets used in a capsule.
  • total cell count can be determined by Coulter counter.
  • the dose is total cell count of about 1 x 10 7 to about 1 x 10 13 cells (e.g., wherein cell number is determined by total cell count, which is determined by Coulter counter) per capsule or tablet or per total number of minitablets used in a capsule.
  • the dose is about 3 x 10 10 or about 1.5 x 10 11 cells (e.g., wherein cell number is determined by total cell count, which is determined by Coulter counter) per capsule or tablet or total per total number of minitablets used in a capsule.
  • the dose is about 8 x 10 10 or about 1.6 x 10 11 , , or about 3.2 x 10 11 cells (e.g., wherein cell number is determined by total cell count, which is determined by Coulter counter) per capsule or tablet or total per total number of minitablets used in a capsule.
  • the dose of mEVs is about 1 x 10 5 to about 2 x 10 12 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the dose of mEVs is about 2x10 6 to about 2x10 16 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
  • the pharmaceutical agent dose can be a milligram (mg) dose determined by weight the pharmaceutical agent (e.g., a powder comprising bacteria and/or an agent of bacterial origin, such as mEVs).
  • the dose of the pharmaceutical agent is per capsule or tablet or per total number of minitablets, e.g., in a capsule.
  • a 1x dose of the pharmaceutical agent of about 400 mg about 200 mg of the pharmaceutical agent is present per capsule and two capsules are administered, resulting in a dose of about 400 mg.
  • the two capsules can be administered, for example, 1x or 2x daily.
  • minitablet about 0.1 to about 3.5 mg (0.1, 0.35, 1.0, 3.5 mg) of the pharmaceutical agent can be contained per minitablet.
  • the minitablets can be inside a capsule: the number of minitablets inside a capsule will depend on the size of the capsule and the size of the minitablets. For example, an average of 33 (range of 31-35)
  • 3mm minitablets fit inside a size 0 capsule.
  • the dose range will be 3.3 mg- 115.5 mg (for 33 minitablets in size 0 capsule) per capsule (3.1 mg- 108.5 mg for 31 minitablets in size 0 capsule) (3.5 mg- 122.5 mg for 35 minitablets in size 0 capsule).
  • Multiple capsules and/or larger capsule(s) can be administered to increase the administered dose and/or can be administered one or more times per day to increase the administered dose.
  • the dose can be about 3 mg to about 125 mg of the pharmaceutical agent, per capsule or tablet or per total number of minitablets, e.g., in a capsule.
  • the dose can be about 35 mg to about 1200 mg (e.g., about 35 mg, about 125 mg, about 350 mg, or about 1200 mg) of the pharmaceutical agent.
  • the dose of the pharmaceutical agent can be about 30 mg to about 3500 mg (about 25, about 50, about 75, about 100, about 150, about 250, about 300, about 350, about 400, about 500, about 600, about 750, about 1000, about 1250, about 1300, about 2000, about 2500, about 3000, or about 3500 mg).
  • a human dose can be calculated appropriately based on allometric scaling of a dose administered to a model organism (e.g., mouse).
  • a model organism e.g., mouse
  • one or two tablets or capsules can be administered one or two times a day.
  • the pharmaceutical agent contains the bacteria and/or an agent of bacterial origin, such as mEVs, or contains a powder comprising bacteria and/or an agent of bacterial origin, such as mEVs, and can also contain one or more additional components, such as a cryoprotectant etc.
  • the mg (by weight) dose of the pharmaceutical agent is, e.g., about 1 mg to about 500 mg per capsule, or per tablet, or per total number of minitablets, e.g., used in a capsule.
  • compositions e.g., a pharmaceutical composition, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement
  • a pharmaceutical composition e.g., a pharmaceutical composition, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement
  • the bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • bacterial compositions allow, e.g., for oral administration of bacteria (or components thereof, such as mEVs) contained therein.
  • the solid dosage forms having the disclosed combinations and/or amounts of disintegration agents provide a decrease in disintegration times (e.g., 2-fold, 4-fold, 6- fold, 8-fold), which can further result in an increase in therapeutic efficacy and/or physiological effect as compared to the same solid dosage forms that do not have the disclosed combinations of the disintegration agents.
  • the bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • a cancer inflammation, autoimmunity, a metabolic condition, or a dysbiosis.
  • the bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements described herein can be used in the treatment and/or prevention of bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection).
  • bacterial septic shock e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements described herein
  • viral infection such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection.
  • the bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements described herein can be used to decrease inflammatory cytokine expression (e.g., decreased IL-8, IL-6, IL-Ib, and/or TNF ⁇ expression levels).
  • inflammatory cytokine expression e.g., decreased IL-8, IL-6, IL-Ib, and/or TNF ⁇ expression levels.
  • compositions and/or solid dosage form e.g. , for oral administration
  • a pharmaceutical agent e.g., a therapeutically effective amount thereof
  • the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and soy leghemoglobin
  • solid dosage form further comprises the disclosed disintegration agents are described herein.
  • the methods and administered pharmaceutical composition and/or solid dosage forms described herein allow, e.g. , for oral administration of a pharmaceutical agent contained therein.
  • the pharmaceutical composition and/or solid dosage form can be administered to a subject is a fed or fasting state.
  • the pharmaceutical composition and/or solid dosage form can be administered, e.g., on an empty stomach (e.g., one hour before eating or two hours after eating).
  • the pharmaceutical composition and/or solid dosage form can be administered one hour before eating.
  • the pharmaceutical composition and/or solid dosage form can be administered two hours after eating.
  • the methods and administered bacterial composition allow, e.g., for oral administration of bacteria (or components thereof, such as mEVs) contained therein.
  • the bacterial composition can be administered to a subject is a fed or fasting state.
  • the bacterial composition can be administered, e.g., on an empty stomach (e.g., one hour before eating or two hours after eating).
  • the bacterial composition can be administered one hour before eating.
  • the bacterial composition can be administered two hours after eating.
  • Bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • Bacterial compositions for use in the treatment and/or prevention of a cancer, inflammation, autoimmunity, a metabolic condition, or a dysbiosis are provided herein.
  • a bacterial composition e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement
  • a bacterial composition e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement
  • a medicament for the treatment and/or prevention of a cancer, inflammation, autoimmunity, a metabolic condition, or a dysbiosis is provided herein.
  • a bacterial composition e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement
  • a medicament for the treatment and/or prevention of bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection) is provided herein.
  • compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • a medicament for decreasing inflammatory cytokine expression e.g., decreased IL-8, IL-6, IL-Ib, and/or TNF ⁇ expression levels
  • inflammatory cytokine expression e.g., decreased IL-8, IL-6, IL-Ib, and/or TNF ⁇ expression levels
  • a pharmaceutical agent e.g., comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein or a powder comprising bacteria and/or mEVs (such as smEVs and/or pmEVs and comprising at least one component of soy leghemoglobin), and (ii) at least one diluent, at least one lubricant, at least one glidant, and/or at least one (e.g., one, two, or three) disintegration agent.
  • a pharmaceutical agent e.g., comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein or a powder comprising bacteria and/or mEVs (such as smEVs and/or pmEVs and comprising at least one component of soy leghemoglobin
  • at least one diluent at least one
  • the at least one diluent comprises mannitol.
  • the at least one lubricant comprises magnesium stearate.
  • the at least one glidant comprises colloidal silicon dioxide.
  • the at least one disintegration agent comprises low-substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac-Di-Sol), and/or crospovidone (PVPP).
  • the total pharmaceutical agent mass is at least 5%, 10%, 15%, 20% or 25 % of the total mass of the pharmaceutical composition. In some embodiments the total pharmaceutical agent mass is no more than 45%, 40%, 35%, 30%, or 25% of the total mass of the pharmaceutical composition. In some embodiments, the total mass of the one or more disintegrating agents is at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total mass of the pharmaceutical composition. In some embodiments, the total mass of the one or more disintegrating agents is no more than 70%, 65%, 60%, or 55% of the total mass of the pharmaceutical composition.
  • the one or more disintegration agents comprise low- substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac-Di-Sol), and/or crospovidone (PVPP).
  • the solid dosage forms provided herein comprise L-HPC.
  • the L-HPC is of grade LH-B1.
  • the total L-HPC mass is at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition.
  • the total L-HPC mass is no more than 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition. In certain embodiments, the total L-HPC mass is about 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition.
  • the solid dosage forms provided herein comprise Ac -Do-Sol.
  • the Ac-Di-Sol is of grade SD-711.
  • the total Ac -Di- Sol mass is at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition.
  • the total Ac-Di-Sol mass is no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%,
  • the total Ac-Di-Sol mass is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition.
  • the solid dosage forms provided herein comprise PVPP.
  • the total PVPP mass is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition. In certain embodiments, the total PVPP mass is no more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition.
  • the total PVPP mass is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition.
  • the method further comprises compressing the pharmaceutical composition, thereby forming a tablet or a minitablet. In some embodiments, the method further comprises enterically coating the tablet or minitablet, thereby preparing the enterically coated tablet. In certain embodiments, the method further comprises loading the minitablets into a capsule.
  • the methods of preparing a solid dosage form of a pharmaceutical composition can comprise blending, encapsulation, banding, and coating of capsules.
  • methods of preparing a solid dosage form of a pharmaceutical composition comprising combining (e.g., blending) into a pharmaceutical composition a pharmaceutical agent (e.g., comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein or a powder comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein) and one or more additional components described herein.
  • a pharmaceutical agent e.g., comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein
  • a pharmaceutical agent e.g., comprising bacteria and/or mEVs (such as smEVs and/
  • a pharmaceutical agent e.g., comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein or a powder comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein) and a diluent.
  • the total pharmaceutical agent mass is at least 20%, 25%, 30%, 35%, 40%, 45%, 50% or 55% of the total mass of the pharmaceutical composition. In some embodiments the total pharmaceutical agent mass is no more than 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% of the total mass of the pharmaceutical composition.
  • the total mass of the diluent is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the total mass of the pharmaceutical composition. In some embodiments, the total mass of the diluent is no more than 80%, 75%, 70%, 65%, 60%, 55%, 50%, or 45% of the total mass of the pharmaceutical composition. In some embodiments, the diluent comprises mannitol.
  • the method further comprises combining a lubricant.
  • the total lubricant mass is at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 0.5% to about 1.5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 1% of the total mass of the pharmaceutical composition. In some embodiments, the lubricant comprises magnesium stearate.
  • the method further comprises combining a glidant.
  • the glidant is colloidal silicon dioxide.
  • the total glidant mass is at least 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition.
  • the total glidant mass is no more than 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition.
  • the total glidant mass is about 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.25% to about 0.75% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.5% of the total mass of the pharmaceutical composition.
  • the method comprises combining: (i) a pharmaceutical agent having a total pharmaceutical agent mass that is at least 20% and no more than 55% of the total mass of the pharmaceutical composition, (ii) a diluent (e.g., mannitol) having a total mass that is at least 45% and no more than 80% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is at least 0.1% and no more than 5% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is at least 0.01% and no more than 2% of the total mass of the pharmaceutical composition.
  • a pharmaceutical agent having a total pharmaceutical agent mass that is at least 20% and no more than 55% of the total mass of the pharmaceutical composition
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium
  • the method comprises combining: (i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 20% to about 50% of the total mass of the pharmaceutical composition, (ii) a diluent (e.g., mannitol) having a total mass that is about 50% to 80% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the pharmaceutical composition.
  • a pharmaceutical agent having a total pharmaceutical agent mass that is about 20% to about 50% of the total mass of the pharmaceutical composition
  • a diluent e.g., mannitol
  • a lubricant e.g., magnesium stearate
  • a glidant e.g., colloidal silicon dioxide
  • the method further comprises loading the pharmaceutical composition into a capsule (e.g., encapsulation).
  • the method further comprises banding the capsule after loading.
  • the method further comprises enterically coating the capsule. Additional Aspects of the Solid Dosage Forms
  • the solid dosage forms comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin, and wherein the solid dosage form further comprises the described disintegration agents, can provide a therapeutically effective amount of the pharmaceutical agent to a subject, e.g., a human.
  • a pharmaceutical agent e.g., a therapeutically effective amount thereof
  • the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin
  • mEVs extracellular vesicles
  • the solid dosage forms comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin, and wherein the solid dosage form further comprises the described disintegration agents, can provide a non-natural amount of the therapeutically effective components (e.g., present in the pharmaceutical agent) to a subject, e.g. , a human.
  • a pharmaceutical agent e.g., a therapeutically effective amount thereof
  • the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin
  • mEVs extracellular vesicles
  • the solid dosage forms comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin, and wherein the solid dosage form further comprises the described disintegration agents, can provide an unnatural quantity of the therapeutically effective components (e.g. , present in the pharmaceutical agent) to a subject, e.g. , a human.
  • a pharmaceutical agent e.g., a therapeutically effective amount thereof
  • the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin
  • mEVs extracellular vesicles
  • the solid dosage forms comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin, and wherein the solid dosage form further comprises the described disintegration agents, can bring about one or more changes to a subject, e.g. , human, e.g. , to treat or prevent a disease or a health disorder.
  • a pharmaceutical agent e.g., a therapeutically effective amount thereof
  • the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin
  • mEVs extracellular vesicles
  • the solid dosage forms e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin, and wherein the solid dosage form further comprises the described disintegration agents, has potential for significant utility, e.g. , to affect a subject, e.g. , a human, e.g. , to treat or prevent a disease or a health disorder.
  • a pharmaceutical agent e.g., a therapeutically effective amount thereof
  • the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin
  • mEVs extracellular vesicles
  • the solid dosage form further comprises the described disintegration agents
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • a subject e.g., a human milk, a human milk, a human milk, a human milk, a human milk, a human milk, a nursing woman, a nursing woman, a nursing woman, a nursing woman, a nursing woman, a nursing woman, a nursing woman, a nursing woman, nursing woman, and/or dietary supplements.
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • the dosage regimen can be any of a variety of methods and amounts, and can be determined by one skilled in the art according to known clinical factors. As is known in the medical arts, dosages for any one patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the particular microorganism to be administered, duration and route of administration, the kind and stage of the disease, for example, tumor size, and other compounds such as drugs being administered concurrently or near-concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art.
  • appropriate minimum dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate.
  • the dose of a pharmaceutical agent (e.g., in a solid dosage form) described herein may be appropriately set or adjusted in accordance with the dosage form, the route of administration, the degree or stage of a target disease, and the like.
  • the dose administered to a subject is sufficient to prevent disease (e.g., autoimmune disease, inflammatory disease, metabolic disease, or cancer), delay its onset, or slow or stop its progression, or relieve one or more symptoms of the disease.
  • disease e.g., autoimmune disease, inflammatory disease, metabolic disease, or cancer
  • dosage will depend upon a variety of factors including the strength of the particular agent (e.g., pharmaceutical agent) employed, as well as the age, species, condition, and body weight of the subject.
  • the size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular pharmaceutical agent and the desired physiological effect.
  • the dosages of the pharmaceutical agents used in accordance with the invention vary depending on the active agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage.
  • the dose should be sufficient to result in slowing, and preferably regressing, the growth of a tumor and most preferably causing complete regression of the cancer, or reduction in the size or number of metastases
  • the dose should be sufficient to result in slowing of progression of the disease for which the subject is being treated, and preferably amelioration of one or more symptoms of the disease for which the subject is being treated.
  • Separate administrations can include any number of two or more administrations, including two, three, four, five or six administrations.
  • One skilled in the art can readily determine the number of administrations to perform or the desirability of performing one or more additional administrations according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein.
  • the methods provided herein include methods of providing to the subject one or more administrations of a solid dosage form, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results.
  • the time period between administrations can be any of a variety of time periods.
  • the time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response.
  • the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be no more than the time period for a subject to mount an immune response, such as no more than about one week, no more than about ten days, no more than about two weeks, or no more than about a month.
  • the methods and bacterial compositions relate to the treatment or prevention of a disease or disorder associated a pathological immune response, such as an autoimmune disease, an allergic reaction and/or an inflammatory disease.
  • a disease or disorder associated a pathological immune response, such as an autoimmune disease, an allergic reaction and/or an inflammatory disease.
  • the disease or disorder is an inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis).
  • the disease or disorder is psoriasis.
  • the disease or disorder is psoriatic arthritis.
  • the disease or disorder is atopic dermatitis.
  • the disease or disorder is asthma.
  • compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • a “subject in need thereof’ includes any subject that has a disease or disorder associated with a pathological immune response (e.g., an inflammatory bowel disease), as well as any subject with an increased likelihood of acquiring a such a disease or disorder.
  • a pathological immune response e.g., an inflammatory bowel disease
  • the bacterial compositions can be used, for example, as a pharmaceutical composition for preventing or treating (reducing, partially or completely, the adverse effects of) an autoimmune disease, such as chronic inflammatory bowel disease, systemic lupus erythematosus, psoriasis, muckle-wells syndrome, rheumatoid arthritis, multiple sclerosis, or Hashimoto's disease; an allergic disease, such as a food allergy, pollenosis, or asthma; an infectious disease, such as an infection with Clostridium difficile; an inflammatory disease such as a TNF -mediated inflammatory disease (e.g., an inflammatory disease of the gastrointestinal tract, such as pouchitis, a cardiovascular inflammatory condition, such as atherosclerosis, or an inflammatory lung disease, such as chronic obstructive pulmonary disease); a pharmaceutical agent and/or a pharmaceutical composition
  • an autoimmune disease such as chronic inflammatory bowel disease, systemic lupus erythematosus, psoriasis, mu
  • the methods and bacterial compositions are useful for the treatment of inflammation.
  • the inflammation of any tissue and organs of the body including musculoskeletal inflammation, vascular inflammation, neural inflammation, digestive system inflammation, ocular inflammation, inflammation of the reproductive system, and other inflammation, as discussed below.
  • Immune disorders of the musculoskeletal system include, but are not limited, to those conditions affecting skeletal joints, including joints of the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle, and foot, and conditions affecting tissues connecting muscles to bones such as tendons.
  • immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis, tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis, myositis, and osteitis (including, for example, Paget's disease, osteitis pubis, and osteitis fibrosa cystic).
  • arthritis including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis
  • tendonitis synovitis, ten
  • Ocular immune disorders refers to a immune disorder that affects any structure of the eye, including the eye lids.
  • ocular immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, blepharitis, blepharochalasis, conjunctivitis, dacryoadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis.
  • Examples of nervous system immune disorders which may be treated with the methods and solid dosage forms described herein include, but are not limited to, encephalitis, Guillain-Barre syndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis, myelitis and schizophrenia.
  • Examples of inflammation of the vasculature or lymphatic system which may be treated with the methods and compositions described herein include, but are not limited to, arthrosclerosis, arthritis, phlebitis, vasculitis, and lymphangitis.
  • Examples of digestive system immune disorders which may be treated with the methods and solid dosage forms described herein include, but are not limited to, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, and proctitis.
  • Inflammatory bowel diseases include, for example, certain art-recognized forms of a group of related conditions.
  • Crohn's disease regional bowel disease, e.g., inactive and active forms
  • ulcerative colitis e.g., inactive and active forms
  • the inflammatory bowel disease encompasses irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis.
  • Other less common forms of IBD include indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet’s disease, sarcoidosis, scleroderma, IBD-associated dysplasia, dysplasia associated masses or lesions, and primary sclerosing cholangitis.
  • reproductive system immune disorders which may be treated with the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)s described herein include, but are not limited to, cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis, and vulvodynia.
  • compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • Such conditions include, but are not limited to, acute disseminated alopecia universalise, Behcet's disease, Chagas' disease, chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, diabetes mellitus type 1, giant cell arteritis, goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto's disease, Henoch- Schonlein purpura, Kawasaki's disease, lupus erythematosus, microscopic colitis, microscopic polyart
  • compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • T-cell mediated hypersensitivity diseases having an inflammatory component.
  • Such conditions include, but are not limited to, contact hypersensitivity, contact dermatitis (including that due to poison ivy), uticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dustmite allergy) and gluten-sensitive enteropathy (Celiac disease).
  • immune disorders which may be treated with the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, ulceris, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel
  • Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosus, psoriasis, chronic obstructive pulmonary disease, and inflammation accompanying infectious conditions (e.g., sepsis).
  • the methods and bacterial compositions relate to the treatment or prevention of a metabolic disease or disorder a, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), Nonalcoholic Steatohepatitis (NASH) or a related disease.
  • a metabolic disease or disorder a such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • the methods and bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • NAFLD Nonalcoholic Fatty Liver Disease
  • NASH Nonalcoholic Steatohepatitis
  • compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • a “subject in need thereof’ includes any subject that has a metabolic disease or disorder, as well as any subject with an increased likelihood of acquiring a such a disease or disorder.
  • the bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • a metabolic disease such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), Nonalcoholic Steatohepatitis (NASH), or a related disease.
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • the methods and bacterial compositions relate to the treatment of cancer.
  • any cancer can be treated using the methods described herein.
  • cancers that may treated by methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • pharmaceutical compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • Hodgkin's disease Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fimgoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
  • the cancer comprises a solid tumor.
  • the cancer comprises breast cancer (e.g., triple negative breast cancer).
  • the cancer comprises colorectal cancer (e.g., microsatellite stable (MSS) colorectal cancer).
  • MSS microsatellite stable
  • the cancer comprises renal cell carcinoma.
  • the cancer comprises lung cancer (e.g., non small cell lung cancer).
  • the cancer comprises bladder cancer.
  • the cancer comprises gastroesophageal cancer.
  • the methods and bacterial compositions relate to the treatment of a leukemia.
  • leukemia includes broadly progressive, malignant diseases of the hematopoietic organs/systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
  • Non-limiting examples of leukemia diseases include, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leuk
  • the methods and bacterial compositions relate to the treatment of a carcinoma.
  • carcinoma refers to a malignant growth made up of epithelial cells tending to infdtrate the surrounding tissues, and/or resist physiological and non-physiological cell death signals and gives rise to metastases.
  • Non-limiting exemplary types of carcinomas include, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma,
  • the methods and bacterial compositions relate to the treatment of a sarcoma.
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance.
  • Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing' s sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic s
  • Additional exemplary neoplasias that can be treated using the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein include Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, plasmacytoma, colorectal cancer, rectal cancer, and adrenal cortical cancer.
  • the cancer treated is a melanoma.
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • melanomas are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
  • tumors that can be treated using methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above.
  • pharmaceutical compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • lymphoproliferative disorders include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer
  • tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma, hypemephroid adenocarcinoma, bile duct carcinoma, chor
  • Cancers treated in certain embodiments also include precancerous lesions, e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucous fibrosis, actinic (solar) elastosis and cervical dysplasia.
  • precancerous lesions e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen
  • Cancers treated in some embodiments include non-cancerous or benign tumors, e.g., of endodermal, ectodermal or mesenchymal origin, including, but not limited to cholangioma, colonic polyp, adenoma, papilloma, cystadenoma, liver cell adenoma, hydatidiform mole, renal tubular adenoma, squamous cell papilloma, gastric polyp, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.
  • Other Diseases and Disorders e.g., of endodermal, ectodermal or mesenchymal origin, including, but not limited to cholangioma, colonic polyp,
  • the methods and bacterial compositions relate to the treatment of liver diseases.
  • diseases include, but are not limited to, Alagille Syndrome, Alcohol-Related Liver Disease, Alpha- 1 Antitrypsin Deficiency, Autoimmune Hepatitis, Benign Liver Tumors, Biliary Atresia, Cirrhosis, Galactosemia, Gilbert Syndrome, Hemochromatosis, Hepatitis A, Hepatitis B, Hepatitis C, Hepatic Encephalopathy, Intrahepatic Cholestasis of Pregnancy (ICP), Lysosomal Acid Lipase Deficiency (LAL-D), Liver Cysts, Liver Cancer, Newborn Jaundice, Primary Biliary Cholangitis (PBC), Primary Sclerosing Cholangitis (PSC), Reye
  • ICP Pregnancy
  • LAL-D Lysosomal Acid Lipase Deficiency
  • Liver Cancer Newborn Jaundice
  • the methods and bacterial compositions may be used to treat neurodegenerative and neurological diseases.
  • the neurodegenerative and/or neurological disease is Parkinson’s disease, Alzheimer’s disease, prion disease, Huntington’s disease, motor neuron diseases (MND), spinocerebellar ataxia, spinal muscular atrophy, dystonia, idiopathicintracranial hypertension, epilepsy, nervous system disease, central nervous system disease, movement disorders, multiple sclerosis, encephalopathy, peripheral neuropathy or post-operative cognitive dysfunction.
  • gut microbiota also called the “gut microbiota”
  • gut microbiota can have a significant impact on an individual’s health through microbial activity and influence (local and/or distal) on immune and other cells of the host
  • a healthy host-gut microbiome homeostasis is sometimes referred to as a “eubiosis” or “normobiosis,” whereas a detrimental change in the host microbiome composition and/or its diversity can lead to an unhealthy imbalance in the microbiome, or a “dysbiosis” (Hooks and O’Malley. Dysbiosis and its discontents. American Society for Microbiology. Oct 2017. Vol. 8. Issue 5. mBio 8:e01492-17. https://doi.org/10.1128/mBio.01492-17).
  • Dysbiosis, and associated local or distal host inflammatory or immune effects may occur where microbiome homeostasis is lost or diminished, resulting in: increased susceptibility to pathogens; altered host bacterial metabolic activity; induction of host proinflammatory activity and/or reduction of host anti inflammatory activity.
  • Such effects are mediated in part by interactions between host immune cells (e.g., T cells, dendritic cells, mast cells, NK cells, intestinal epithelial lymphocytes (IEC), macrophages and phagocytes) and cytokines, and other substances released by such cells and other host cells.
  • host immune cells e.g., T cells, dendritic cells, mast cells, NK cells, intestinal epithelial lymphocytes (IEC), macrophages and phagocytes
  • a dysbiosis may occur within the gastrointestinal tract (a “gastrointestinal dysbiosis” or “gut dysbiosis”) or may occur outside the lumen of the gastrointestinal tract (a “distal dysbiosis”).
  • Gastrointestinal dysbiosis is often associated with a reduction in integrity of the intestinal epithelial barrier, reduced tight junction integrity and increased intestinal permeability.
  • Citi, S. Intestinal Barriers protect against disease, Science 359: 1098- 99 (2016); Srinivasan et al., TEER measurement techniques for in vitro barrier model systems. J. Lab. Autom. 20: 107-126 (2015).
  • a gastrointestinal dysbiosis can have physiological and immune effects within and outside the gastrointestinal tract.
  • dysbiosis has been associated with a wide variety of diseases and conditions including: infection, cancer, autoimmune disorders (e.g., systemic lupus erythematosus (SLE)) or inflammatory disorders (e.g., functional gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease), neuroinflammatory diseases (e.g., multiple sclerosis), transplant disorders (e.g., graft-versus-host disease), fatty liver disease, type I diabetes, rheumatoid arthritis, Sjogren’s syndrome, celiac disease, cystic fibrosis, chronic obstructive pulmonary disorder (COPD), and other diseases and conditions associated with immune dysfunction.
  • autoimmune disorders e.g., systemic lupus erythematosus (SLE)
  • inflammatory disorders e.g., functional gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease
  • neuroinflammatory diseases e.g.
  • Exemplary bacterial compositions can treat a dysbiosis and its effects by modifying the immune activity present at the site of dysbiosis.
  • such compositions can modify a dysbiosis via effects on host immune cells, resulting in, e.g., an increase in secretion of anti-inflammatory cytokines and/or a decrease in secretion of pro- inflammatory cytokines, reducing inflammation in the subject recipient or via changes in metabolite production.
  • Exemplary bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • exemplary bacterial compositions that are useful for treatment of disorders associated with a dysbiosis contain one or more types of immunomodulatory bacteria and/or mEVs (microbial extracellular vesicles) derived from such bacteria.
  • Such compositions are capable of affecting the recipient host’s immune function, in the gastrointestinal tract, and/or a systemic effect at distal sites outside the subject’s gastrointestinal tract.
  • Exemplary bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • a population of immunomodulatory bacteria of a single bacterial species e.g., a single strain
  • Such compositions are capable of affecting the recipient host’s immune function, in the gastrointestinal tract, and /or a systemic effect at distal sites outside the subject’s gastrointestinal tract.
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • an isolated population of immunomodulatory bacteria or mEVs derived from such bacteria are administered (e.g., orally) to a mammalian recipient in an amount effective to treat a dysbiosis and one or more of its effects in the recipient.
  • the dysbiosis may be a gastrointestinal tract dysbiosis or a distal dysbiosis.
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • the bacterial compositions can treat a gastrointestinal dysbiosis and one or more of its effects by modulating the recipient immune response via cellular and cytokine modulation to reduce gut permeability by increasing the integrity of the intestinal epithelial barrier.
  • the bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • compositions contain one or more types of bacteria or mEVs capable of altering the relative proportions of host immune cell subpopulations, e.g., subpopulations of T cells, immune lymphoid cells, dendritic cells, NK cells and other immune cells, or the function thereof, in the recipient.
  • compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • compositions contain a population of immunomodulatory bacteria or mEVs of a single bacterial species e.g., a single strain) capable of altering the relative proportions of immune cell subpopulations, e.g., T cell subpopulations, immune lymphoid cells, NK cells and other immune cells, or the function thereof, in the recipient subject.
  • immune cell subpopulations e.g., T cell subpopulations, immune lymphoid cells, NK cells and other immune cells, or the function thereof, in the recipient subject.
  • the invention provides methods of treating a gastrointestinal dysbiosis and one or more of its effects by orally administering to a subject in need thereof bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) which alters the microbiome population existing at the site of the dysbiosis.
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • the bacterial compositions can contain one or more types of immunomodulatory bacteria or mEVs or a population of immunomodulatory bacteria or mEVs of a single bacterial species (e.g., a single strain).
  • the invention provides methods of treating a distal dysbiosis and one or more of its effects by orally administering to a subject in need thereof bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) which alters the subject’s immune response outside the gastrointestinal tract.
  • bacterial compositions e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements
  • the bacterial compositions can contain one or more types of immunomodulatory bacteria or mEVs or a population of immunomodulatory bacteria or mEVs of a single bacterial species (e.g., a single strain).
  • bacterial compositions useful for treatment of disorders associated with a dysbiosis stimulate secretion of one or more anti-inflammatory cytokines by host immune cells.
  • Anti-inflammatory cytokines include, but are not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGF , and combinations thereof.
  • bacterial compositions useful for treatment of disorders associated with a dysbiosis that decrease (e.g., inhibit) secretion of one or more pro-inflammatory cytokines by host immune cells.
  • Pro-inflammatory cytokines include, but are not limited to, IFN ⁇ , IL-12p70, IL-1 ⁇ , IL-6, IL-8, MCP1, MIPl ⁇ , MIRIb, TNF ⁇ , and combinations thereof.
  • Other exemplary cytokines are known in the art and are described herein.
  • the invention provides a method of treating or preventing a disorder associated with a dysbiosis in a subject in need thereof, comprising administering (e.g., orally administering) to the subject a composition in the form of a probiotic or medical food comprising bacteria or mEVs in an amount sufficient to alter the microbiome at a site of the dysbiosis, such that the disorder associated with the dysbiosis is treated.
  • a composition of the instant invention in the form of a probiotic or medical food may be used to prevent or delay the onset of a dysbiosis in a subject at risk for developing a dysbiosis.
  • a pharmaceutical composition comprising: a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin.
  • mEVs microbial extracellular vesicles
  • exemplary embodiment 2 Provided in exemplary embodiment 2 is a pharmaceutical composition of embodiment 1, wherein the at least one component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid.
  • exemplary embodiment 3 Provided in exemplary embodiment 3 is a pharmaceutical composition of embodiment 2, wherein the soy leghemoglobin nucleic acid is soy leghemoglobin DNA.
  • exemplary embodiment 4 Provided in exemplary embodiment 4 is a pharmaceutical composition of embodiment 3, wherein the soy leghemoglobin DNA comprises a sequence encoding soybean leghemoglobin A (LB A) or soybean leghemoglobin C2 (LGB2).
  • soy leghemoglobin DNA comprises a sequence encoding soybean leghemoglobin A (LB A) or soybean leghemoglobin C2 (LGB2).
  • exemplary embodiment 5 Provided in exemplary embodiment 5 is a pharmaceutical composition of embodiment 1, wherein the at least one component of soy leghemoglobin comprises a soybean leghemoglobin protein.
  • exemplary embodiment 6 Provided in exemplary embodiment 6 is a pharmaceutical composition of embodiment 5, wherein the soy leghemoglobin protein is soy leghemoglobin A or soybean leghemoglobin C2 (LGB2).
  • soy leghemoglobin protein is soy leghemoglobin A or soybean leghemoglobin C2 (LGB2).
  • exemplary embodiment 7 Provided in exemplary embodiment 7 is a pharmaceutical composition of any one of embodiments 1-6, wherein the at least one component of soy leghemoglobin is purified from soy roots or soy root nodules.
  • exemplary embodiment 8 Provided in exemplary embodiment 8 is a pharmaceutical composition of any one of embodiments 1-7, wherein the at least one component of soy leghemoglobin is recombinantly expressed.
  • exemplary embodiment 9 Provided in exemplary embodiment 9 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • exemplary embodiment 10 Provided in exemplary embodiment 10 is a pharmaceutical composition of embodiment 9, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO:
  • exemplary embodiment 11 Provided in exemplary embodiment 11 is a pharmaceutical composition of embodiment 9 or 10, wherein the at least one component of soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • exemplary embodiment 12 Provided in exemplary embodiment 12 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • exemplary embodiment 13 Provided in exemplary embodiment 13 is a pharmaceutical composition of embodiment 12, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • exemplary embodiment 14 Provided in exemplary embodiment 14 is a pharmaceutical composition of embodiment 12 or 13, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
  • exemplary embodiment 15 Provided in exemplary embodiment 15 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
  • exemplary embodiment 16 Provided in exemplary embodiment 16 is a pharmaceutical composition of embodiment 15, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO:
  • exemplary embodiment 17 Provided in exemplary embodiment 17 is a pharmaceutical composition of embodiment 15 or 16, wherein the at least one component of soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
  • exemplary embodiment 18 Provided in exemplary embodiment 18 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
  • exemplary embodiment 19 Provided in exemplary embodiment 19 is a pharmaceutical composition of embodiment 18, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
  • exemplary embodiment 20 Provided in exemplary embodiment 20 is a pharmaceutical composition of embodiment 18 or 19, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence SEQ ID NO: 5 or GenBank: NP_001235928.1.
  • exemplary embodiment 21 Provided in exemplary embodiment 21 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
  • exemplary embodiment 22 Provided in exemplary embodiment 22 is a pharmaceutical composition of embodiment 21, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO:
  • exemplary embodiment 23 Provided in exemplary embodiment 23 is a pharmaceutical composition of embodiment 21 or 22, wherein the at least one component of soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
  • exemplary embodiment 24 Provided in exemplary embodiment 24 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
  • exemplary embodiment 25 Provided in exemplary embodiment 25 is a pharmaceutical composition of embodiment 24, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
  • exemplary embodiment 26 Provided in exemplary embodiment 26 is a pharmaceutical composition of embodiment 24 or 25, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ _OYBN) .
  • exemplary embodiment 27 Provided in exemplary embodiment 27 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGB A SOYBN) .
  • exemplary embodiment 28 Provided in exemplary embodiment 28 is a pharmaceutical composition of embodiment 27, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO:
  • exemplary embodiment 29 Provided in exemplary embodiment 29 is a pharmaceutical composition of embodiment 27 or 28, wherein the at least one component of soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
  • exemplary embodiment 30 Provided in exemplary embodiment 30 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
  • exemplary embodiment 31 Provided in exemplary embodiment 31 is a pharmaceutical composition of embodiment 30, wherein the at least one component of soy leghemoglobin comprises comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
  • exemplary embodiment 32 Provided in exemplary embodiment 32 is a pharmaceutical composition of embodiment 30 or 31, wherein the at least one component of soy leghemoglobin comprises comprises a nucleic acid that encodes the amino acid sequence SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
  • exemplary embodiment 33 Provided in exemplary embodiment 33 is a pharmaceutical composition of embodiment 8, wherein the recombinantly expressed polypeptide comprises a heterologous polypeptide.
  • exemplary embodiment 34 Provided in exemplary embodiment 34 is a pharmaceutical composition of embodiment 33, wherein the heterologous polypeptide comprises a histidine tag, TAP (tandem affinity purification) tag, TEV cleavage site, a FLAG tag, a GST tag, and/or an immunoglobulin domain.
  • the heterologous polypeptide comprises a histidine tag, TAP (tandem affinity purification) tag, TEV cleavage site, a FLAG tag, a GST tag, and/or an immunoglobulin domain.
  • exemplary embodiment 35 is a pharmaceutical composition of any one of embodiments 8, 33, or 34, wherein the recombinant polypeptide is expressed and purified from an exogenous nucleic acid in a host cell.
  • exemplary embodiment 36 is a pharmaceutical composition of embodiment 35, wherein the exogenous nucleic acid is in a vector.
  • exemplary embodiment 37 Provided in exemplary embodiment 37 is a pharmaceutical composition of embodiment 36, wherein the vector is an expression vector.
  • exemplary embodiment 38 Provided in exemplary embodiment 38 is a pharmaceutical composition of any one of embodiments 35-37, wherein the host cell is a bacteria, yeast, insect, or mammalian cell line.
  • exemplary embodiment 39 Provided in exemplary embodiment 39 is a pharmaceutical composition of embodiment 38, wherein the host cell is Pichia Pastoris.
  • exemplary embodiment 40 Provided in exemplary embodiment 40 is a pharmaceutical composition of any one of embodiments 1-39, wherein the at least one component of soy leghemoglobin is a soy leghemoglobin recombinantly expressed in Pichia pastoris.
  • exemplary embodiment 41 Provided in exemplary embodiment 41 is a pharmaceutical composition of any one of embodiments 1-40, wherein the bacteria are hemoglobin-dependent bacteria.
  • exemplary embodiment 42 Provided in exemplary embodiment 42 is a pharmaceutical composition of any one of embodiments 1-41, wherein the bacteria are of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Foumierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
  • the bacteria are of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Ery
  • exemplary embodiment 43 Provided in exemplary embodiment 43 is a pharmaceutical composition of embodiment 42, wherein the bacteria are of the genus Foumierella.
  • exemplary embodiment 44 Provided in exemplary embodiment 44 is a pharmaceutical composition of embodiment 43, wherein the Foumierella are Foumierella Strain B (ATCC Deposit Number PTA- 126696).
  • exemplary embodiment 45 Provided in exemplary embodiment 45 is a pharmaceutical composition of embodiment 42, wherein the bacteria are of the genus Prevotella.
  • exemplary embodiment 46 Provided in exemplary embodiment 46 is a pharmaceutical composition of embodiment 45, wherein the bacteria are Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotell
  • exemplary embodiment 47 Provided in exemplary embodiment 47 is a pharmaceutical composition of embodiment 45, wherein the bacteria are of the species Prevotella histicola.
  • exemplary embodiment 48 Provided in exemplary embodiment 48 is a pharmaceutical composition of embodiment 45, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
  • exemplary embodiment 49 Provided in exemplary embodiment 49 is a pharmaceutical composition of embodiment 45, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain C (ATTC Deposit Number PTA-126140).
  • exemplary embodiment 50 Provided in exemplary embodiment 50 is a pharmaceutical composition of embodiment 45, wherein the Prevotella are Prevotella Strain B 50329 (NRRL accession number B 50329).
  • exemplary embodiment 51 Provided in exemplary embodiment 51 is a pharmaceutical composition of embodiment 45, wherein the Prevotella are Prevotella Strain C (ATTC Deposit Number PTA-126140).
  • exemplary embodiment 52 Provided in exemplary embodiment 52 is a pharmaceutical composition of embodiment 45, wherein the Prevotella bacteria (i) comprise one or more proteins listed in Table 1, and/or (ii) are substantially free of a protein listed in Table 2.
  • exemplary embodiment 53 Provided in exemplary embodiment 53 is a pharmaceutical composition of any one of embodiments 1 to 52, wherein the bacterial are live, attenuated, or dead.
  • exemplary embodiment 54 Provided in exemplary embodiment 54 is a pharmaceutical composition of any one of embodiments 1 to 53, wherein the bacteria are lyophilized bacteria.
  • exemplary embodiment 55 Provided in exemplary embodiment 55 is a pharmaceutical composition of any one of embodiments 1-54, wherein the pharmaceutical agent comprises mEVs.
  • mEVs are secreted mEVs (smEVs).
  • exemplary embodiment 57 Provided in exemplary embodiment 57 is a pharmaceutical composition of embodiment 55, wherein the mEVs are processed mEVs (pmEVs).
  • exemplary embodiment 58 Provided in exemplary embodiment 58 is a pharmaceutical of any one of embodiments 55 to 57, wherein the mEVs are from hemoglobin-dependent bacteria.
  • mEVs are from bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Foumierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
  • exemplary embodiment 60 Provided in exemplary embodiment 60 is a pharmaceutical composition of embodiment 59, wherein the mEVs are from bacteria of the genus Foumierella.
  • exemplary embodiment 61 Provided in exemplary embodiment 61 is a pharmaceutical composition of embodiment 60, wherein the Foumierella are Foumierella Strain B (ATCC Deposit Number PTA- 126696).
  • exemplary embodiment 62 Provided in exemplary embodiment 62 is a pharmaceutical composition of embodiment 59, wherein the mEVs are from bacteria of the genus Prevotella.
  • exemplary embodiment 63 Provided in exemplary embodiment 63 is a pharmaceutical composition of embodiment 62, wherein the bacteria are Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae,
  • exemplary embodiment 64 Provided in exemplary embodiment 64 is a pharmaceutical composition of embodiment 62, wherein the mEVs are from bacteria of the species Pr votella histicola.
  • exemplary embodiment 65 Provided in exemplary embodiment 65 is a pharmaceutical composition of embodiment 62, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
  • exemplary embodiment 66 Provided in exemplary embodiment 66 is a pharmaceutical composition of embodiment 62, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain C (ATTC Deposit Number PTA-126140).
  • exemplary embodiment 67 Provided in exemplary embodiment 67 is a pharmaceutical composition of embodiment 62, wherein the Prevotella are Prevotella Strain B 50329 (NRRL accession number B 50329).
  • exemplary embodiment 68 Provided in exemplary embodiment 68 is a pharmaceutical composition of embodiment 62, wherein the Prevotella are Prevotella Strain C (ATTC Deposit Number PTA-126140).
  • exemplary embodiment 69 Provided in exemplary embodiment 69 is a pharmaceutical composition of embodiment 62, wherein the Prevotella bacteria (i) comprise one or more proteins listed in Table 1, and/or (ii) are substantially free of a protein listed in Table 2.
  • exemplary embodiment 70 Provided in exemplary embodiment 70 is a pharmaceutical composition of any one of embodiments 55 to 69, wherein the mEVs are lyophilized mEVs.
  • exemplary embodiment 71 Provided in exemplary embodiment 71 is a pharmaceutical composition of any one of embodiments 1 to 70, further comprising a cryoprotectant.
  • a solid dosage form comprising: (i) a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin; and (ii) at least one diluent, at least one lubricant, at least one glidant, and/or at least one disintegration agent.
  • mEVs bacteria and/or microbial extracellular vesicles
  • disintegration agent Provided in exemplary embodiment 73 is a solid dosage form of embodiment 72, wherein the at least one diluent has a total mass that is at least 1% and no more than 95% of the total mass of the solid dosage form.
  • exemplary embodiment 74 Provided in exemplary embodiment 74 is a solid dosage form of embodiment 72, wherein the at least one diluent comprises mannitol.
  • exemplary embodiment 75 Provided in exemplary embodiment 75 is a solid dosage form of any one of embodiments 72 to 74, wherein the at least one lubricant has a total mass that is at least
  • exemplary embodiment 76 is a solid dosage form of any one of embodiments 72 to 75, wherein the at least one lubricant comprises magnesium stearate.
  • exemplary embodiment 77 Provided in exemplary embodiment 77 is a solid dosage form of any one of embodiments 72 to 76, wherein the at least one glidant has a total mass that is at least 0.01% and no more than 2% of the total mass of the solid dosage form.
  • exemplary embodiment 78 Provided in exemplary embodiment 78 is a solid dosage form of any one of embodiments 72 to 77, wherein the at least one glidant comprises colloidal silicon dioxide.
  • exemplary embodiment 79 Provided in exemplary embodiment 79 is a solid dosage form of any one of embodiments 72 to 78, wherein the at least one disintegration agent has a total mass that is at least 40% of the total mass of the solid dosage form.
  • exemplary embodiment 80 is a solid dosage form of any one of embodiments 72 to 79, wherein the at least one disintegration agent comprises low- substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac-Di-Sol), and/or crospovidone (PVPP).
  • L-HPC low- substituted hydroxypropyl cellulose
  • Ac-Di-Sol croscarmellose sodium
  • PVPP crospovidone
  • exemplary embodiment 81 is a solid dosage form of any one of embodiments 72 to 80, wherein the the at least one disintegration agent comprises low- substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac-Di-Sol), and crospovidone (PVPP).
  • L-HPC low- substituted hydroxypropyl cellulose
  • Ac-Di-Sol croscarmellose sodium
  • PVPP crospovidone
  • exemplary embodiment 82 Provided in exemplary embodiment 82 is a solid dosage form of embodiment 80 or 81, wherein the L-HPC has a total L-HPC mass that is at least 22% and no more than 42% of the total mass of the solid dosage form.
  • exemplary embodiment 83 Provided in exemplary embodiment 83 is a solid dosage form of any one of embodiments 80 to 82, wherein the L-HPC is L-HPC of grade LH-B1.
  • exemplary embodiment 84 Provided in exemplary embodiment 84 is a solid dosage form of any one of embodiments 80 to 83, wherein the Ac-Di-Sol has a total Ac -Di-Sol mass that is at least 0.01% and no more than 16% of the total mass of the solid dosage form.
  • exemplary embodiment 85 Provided in exemplary embodiment 85 is a solid dosage form of any one of embodiments 80 to 84, wherein the Ac-Di-Sol is Ac-Di-Sol of grade SD-711.
  • exemplary embodiment 86 Provided in exemplary embodiment 86 is a solid dosage form of any one of embodiments 80 to 85, wherein the PVPP has a total PVPP mass that is at least 5% and no more than 25% of the total mass of the solid dosage form.
  • exemplary embodiment 87 Provided in exemplary embodiment 87 is a solid dosage form of any one of embodiments 80 to 86, wherein the total L-HPC mass plus the total Ac-Di-Sol mass plus the total PVPP mass is at least 40% of the total mass of the solid dosage form.
  • exemplary embodiment 88 Provided in exemplary embodiment 88 is a solid dosage form of any one of embodiments 75 to 82, wherein the total L-HPC mass is at least 22% and no more than 42% of the total mass of the solid dosage form; the total Ac-Di-Sol mass is at least 0.01% and no more than 16% of the total mass of the solid dosage form; and the total PVPP mass is at least 5% and no more than 25% of the total mass of the solid dosage form.
  • a solid dosage form of any one of embodiments 75 to 83 wherein the total L-HPC mass is about 32% of the total mass of the solid dosage form; the total Ac-Di-Sol mass is about 6% of the total mass of the solid dosage form; and the total PVPP mass is about 15% of the total mass of the solid dosage form.
  • exemplary embodiment 90 Provided in exemplary embodiment 90 is a solid dosage form of any one of embodiments 67 to 84, wherein the pharmaceutical agent has a total pharmaceutical agent mass that is at least 5% and no more than 65% of the total mass of the solid dosage form.
  • exemplary embodiment 91 Provided in exemplary embodiment 91 is a solid dosage form of embodiment 85, wherein the pharmaceutical agent has a total pharmaceutical agent mass that is at least 5% and no more than 35% of the total mass of the solid dosage form.
  • exemplary embodiment 92 Provided in exemplary embodiment 92 is a solid dosage form of embodiment 85, wherein the total pharmaceutical agent mass is about 25% of the total mass of the solid dosage form.
  • exemplary embodiment 93 Provided in exemplary embodiment 93 is a solid dosage form of any one of embodiments 67 to 87, wherein the solid dosage form is a tablet.
  • exemplary embodiment 94 Provided in exemplary embodiment 94 is a solid dosage form of embodiment 88, wherein tablet is a 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, or 18mm tablet.
  • exemplary embodiment 95 Provided in exemplary embodiment 95 is a solid dosage form of any one of embodiments 67 to 87, wherein the solid dosage form is a minitablet.
  • exemplary embodiment 96 Provided in exemplary embodiment 96 is a solid dosage form of embodiment 90, wherein the minitablet is a 1mm minitablet, 1.5 mm minitablet, 2mm minitablet, 3mm minitablet, or 4mm minitablet.
  • exemplary embodiment 97 Provided in exemplary embodiment 97 is a solid dosage form of embodiment 91 or 92, wherein a plurality of minitablets are contained in a capsule.
  • exemplary embodiment 98 Provided in exemplary embodiment 98 is a solid dosage form of any one of embodiments 67 to 92, further comprising an enteric coating.
  • exemplary embodiment 99 Provided in exemplary embodiment 99 is a solid dosage form of embodiment 93, wherein the enteric coating is a single enteric coating or more than one enteric coating.
  • exemplary embodiment 100 Provided in exemplary embodiment 100 is a solid dosage form of embodiment 93 or 94, wherein the enteric coating comprises an inner enteric coating and an outer enteric coating, and wherein the inner and outer enteric coatings are not identical.
  • exemplary embodiment 101 Provided in exemplary embodiment 101 is a solid dosage form of embodiment any one of embodiments 93 to 95, wherein the enteric coating comprises a methacrylic acid ethyl acrylate (MAE) copolymer (1:1).
  • MAE methacrylic acid ethyl acrylate
  • exemplary embodiment 102 Provided in exemplary embodiment 102 is a solid dosage form of any one of embodiments 93 to 96, wherein the enteric coating comprises cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), a fatty acid, a wax, shellac (esters of aleurtic acid), a plastic, a plant fiber, zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch, a starch derivative, a dextrin, a methyl acrylate- methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), a methyl methacrylate-methacrylic acid copolymer, or sodium alginate.
  • CAP cellulose acetate phthalate
  • a solid dosage form comprising: (i) a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin; and (ii) at least one diluent, at least one lubricant, and/or at least one glidant.
  • mEVs microbial extracellular vesicles
  • exemplary embodiment 105 Provided in exemplary embodiment 105 is a solid dosage form of embodiment 99, wherein the at least one diluent has a total mass that is at least 1% and no more than 95% of the total mass of the solid dosage form.
  • exemplary embodiment 106 Provided in exemplary embodiment 106 is a solid dosage form of embodiment 99 or 100, wherein the at least one diluent comprises mannitol.
  • exemplary embodiment 107 Provided in exemplary embodiment 107 is a solid dosage form of any one of embodiments 99 to 101, wherein the at least one lubricant has a total mass that is at least
  • exemplary embodiment 108 Provided in exemplary embodiment 108 is a solid dosage form of any one of embodiments 99 to 102, wherein the at least one lubricant comprises magnesium stearate.
  • exemplary embodiment 109 Provided in exemplary embodiment 109 is a solid dosage form of any one of embodiments 99 to 103, wherein the at least one glidant has a total mass that is at least 0.01% and no more than 2% of the total mass of the solid dosage form.
  • exemplary embodiment 110 Provided in exemplary embodiment 110 is a solid dosage form of any one of embodiments 99 to 104, wherein the at least one glidant comprises colloidal silicon dioxide.
  • exemplary embodiment 111 Provided in exemplary embodiment 111 is a solid dosage form of any one of embodiments 99 to 105, wherein the pharmaceutical agent has a total pharmaceutical agent mass that is at least 5% and no more than 95% of the total mass of the solid dosage form.
  • exemplary embodiment 112 Provided in exemplary embodiment 112 is a solid dosage form of embodiment 106, wherein the pharmaceutical agent has a total pharmaceutical agent mass that is at least 20% and no more than 50% of the total mass of the solid dosage form.
  • exemplary embodiment 113 Provided in exemplary embodiment 113 is a solid dosage form of embodiment 106, wherein the total pharmaceutical agent mass is about 30% to about 50% of the total mass of the solid dosage form.
  • exemplary embodiment 114 Provided in exemplary embodiment 114 is a solid dosage form of any one of embodiments 99 to 108, wherein (i) the total pharmaceutical agent mass is at least 5% and no more than 95% of the total mass of the solid dosage form; (ii) the total diluent mass is at least 1% and no more than 95% of the total mass of the solid dosage form; (iii) the total lubricant mass is at least 0.1% and no more than 5% of the total mass of the solid dosage form; and (iv) the total glidant mass is at least 0.01% and no more than 2% of the total mass of the solid dosage form.
  • exemplary embodiment 115 Provided in exemplary embodiment 115 is a solid dosage form of any one of embodiments 99 to 108, wherein (i) the total pharmaceutical agent mass is about 20% to about 50% of the total mass of the solid dosage form; (ii) the total diluent mass is about 50% to 80% of the total mass of the solid dosage form; (iii) the total lubricant mass is about 1% of the total mass of the solid dosage form; and (iv) the total glidant mass is about 0.5% of the total mass of the solid dosage form.
  • exemplary embodiment 116 Provided in exemplary embodiment 116 is a solid dosage form of any one of embodiments 99 to 108, wherein (i) the total pharmaceutical agent mass is about 30% to about 50% of the total mass of the solid dosage form; (ii) the total diluent mass is about 45% to 70% of the total mass of the solid dosage form; (iii) the total lubricant mass is about 1% of the total mass of the solid dosage form; and (iv) the total glidant mass is about 0.5% of the total mass of the solid dosage form.
  • a solid dosage form of any one of embodiments 99 to 108 wherein (i) the total pharmaceutical agent mass is about 50% of the total mass of the solid dosage form; (ii) the total diluent mass is about 48.5% of the total mass of the solid dosage form; (iii) the total lubricant mass is about 1% of the total mass of the solid dosage form; and (iv) the total glidant mass is about 0.5% of the total mass of the solid dosage form.
  • a solid dosage form of any one of embodiments 104 to 113 wherein (i) the total pharmaceutical agent mass is about 13.51% of the total mass of the solid dosage form; (ii) the total diluent mass is about 84.99% of the total mass of the solid dosage form; (iii) the total lubricant mass is about 1% of the total mass of the solid dosage form; and (iv) the total glidant mass is about 0.5% of the total mass of the solid dosage form.
  • a solid dosage form of any one of embodiments 104 to 113 wherein (i) the total pharmaceutical agent mass is about 90.22% of the total mass of the solid dosage form; (ii) the total diluent mass is about 8.28% of the total mass of the solid dosage form; (iii) the total lubricant mass is about 1% of the total mass of the solid dosage form; and (iv) the total glidant mass is about 0.5% of the total mass of the solid dosage form.
  • exemplary embodiment 120 Provided in exemplary embodiment 120 is a solid dosage form of any one of embodiments 104 to 119, wherein the solid dosage form is a capsule.
  • exemplary embodiment 121 Provided in exemplary embodiment 121 is a solid dosage form of embodiment 120, wherein the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule.
  • exemplary embodiment 122 Provided in exemplary embodiment 122 is a solid dosage form of embodiment 120 or 121, wherein the capsule is a size 0 capsule.
  • exemplary embodiment 123 Provided in exemplary embodiment 123 is a solid dosage form of any one of embodiments 104 to 122, further comprising an enteric coating.
  • exemplary embodiment 124 Provided in exemplary embodiment 124 is a solid dosage form of embodiment 123, wherein the solid dosage form is enteric coated to dissolve at pH 5.5.
  • exemplary embodiment 125 Provided in exemplary embodiment 125 is a solid dosage form of embodiment 123 or 124, wherein the enteric coating comprises a polymethacrylate-based copolymer.
  • exemplary embodiment 126 Provided in exemplary embodiment 126 is a solid dosage form of any one of embodiments 123 to 125, wherein the enteric coating comprises poly(methacrylic acid- co-ethyl acrylate).
  • exemplary embodiment 127 Provided in exemplary embodiment 127 is a solid dosage form of any one of embodiments 123 to 126, wherein the enteric coating comprises amethacrylic acid ethyl acrylate (MAE) copolymer (1:1) (e.g., Kollicoat MAE 100P).
  • MAE amethacrylic acid ethyl acrylate
  • a solid dosage form of any one of embodiments 123 to 127 wherein the enteric coating comprises a Eudragit copolymer, e.g., a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).
  • a Eudragit copolymer e.g., a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).
  • Eudragit L e.g., Eudragit L 100-55; Eudragit L 30 D-55
  • Eudragit S e
  • CAP cellulose acetate phthalate
  • CAT cellulose acetate trimellitate
  • PVAP
  • exemplary embodiment 130 Provided in exemplary embodiment 130 is a solid dosage form of any one of embodiments 123 to 129, wherein the enteric coating comprises an anionic polymeric material.
  • exemplary embodiment 131 Provided in exemplary embodiment 131 is a method of preventing or treating a disease of a subject comprising administering to the subject a pharmaceutical composition of any one of embodiments 1 to 71.
  • exemplary embodiment 132 Provided in exemplary embodiment 132 is a method of preventing or treating a disease in a subject comprising administering to the subject a solid dosage form of any one of embodiments 72 to 130.
  • exemplary embodiment 133 Provided in exemplary embodiment 133 is a use of a pharmaceutical composition of any one of embodiments 1 to 71 for the treatment or prevention of a disease of a subject.
  • exemplary embodiment 134 Provided in exemplary embodiment 134 is a use of a solid dosage form of any one of embodiments 72 to 130 for the treatment or prevention of a disease in a subject.
  • exemplary embodiment 135 Provided in exemplary embodiment 135 is a method or use of any one of embodiments 131 to 134, wherein the disease is a cancer, inflammation, autoimmunity, a metabolic condition, or a dysbiosis.
  • exemplary embodiment 136 Provided in exemplary embodiment 136 is a method or use of any one of embodiments 131 to 134, wherein the disease is bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection).
  • the disease is bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection).
  • exemplary embodiment 137 Provided in exemplary embodiment 137 is a method or use of any one of embodiments 131 to 134, wherein the solid dosage form decreases inflammatory cytokine expression (e.g., decreased IL-8, IL-6, IL-Ib, and/or TNF ⁇ expression levels).
  • inflammatory cytokine expression e.g., decreased IL-8, IL-6, IL-Ib, and/or TNF ⁇ expression levels.
  • a method of preparing a solid dosage form comprising: (A) combining into a pharmaceutical composition: (i) a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin; and (ii) at least one diluent, at least one lubricant, at least one glidant, and/or at least one disintegration agent. (B) compressing the pharmaceutical composition into a solid dosage form.
  • mEVs microbial extracellular vesicles
  • exemplary embodiment 139 Provided in exemplary embodiment 139 is a method of embodiment 138, further comprising the step of enterically coating the solid dosage form to obtain an enterically coated solid dosage form.
  • exemplary embodiment 140 Provided in exemplary embodiment 140 is a method of embodiment 138 or 139, wherein the solid dosage form is a tablet.
  • exemplary embodiment 141 Provided in exemplary embodiment 141 is a method of embodiment 138 or 139, wherein the solid dosage form is a minitablet.
  • a method of preparing a solid dosage form comprising combining into a pharmaceutical composition: (i) a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin; and (ii) at least one diluent, at least one lubricant, and/or at least one glidant.
  • a pharmaceutical agent wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin; and (ii) at least one diluent, at least one lubricant, and/or at least one glidant.
  • mEVs microbial extracellular vesicles
  • exemplary embodiment 143 Provided in exemplary embodiment 143 is a method of embodiment 142, further comprising blending and/or loading the pharmaceutical composition into a capsule.
  • exemplary embodiment 144 Provided in exemplary embodiment 144 is a method of embodiment 143, further comprising banding the capsule.
  • exemplary embodiment 145 Provided in exemplary embodiment 145 is a method of embodiment 144, wherein the capsule is banded with an HPMC-based banding solution.
  • exemplary embodiment 146 Provided in exemplary embodiment 146 is a method of any one of embodiments 142 to 145, further comprising the step of enterically coating the solid dosage form to obtain an enterically coated solid dosage form.
  • exemplary embodiment 147 Provided in exemplary embodiment 147 is a method of any one of embodiments 142 to 146, wherein the solid dosage form is a capsule.
  • exemplary embodiment 148 Provided in exemplary embodiment 148 is a method of testing a pharmaceutical composition comprising bacteria and/or microbial extracellular vesicles (mEVs), the method comprising performing an assay to detect the presense of a component of a soy leghemoglobin in the pharmaceutical composition.
  • mEVs microbial extracellular vesicles
  • exemplary embodiment 149 Provided in exemplary embodiment 149 is a method of embodiment 148, wherein the component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid.
  • exemplary embodiment 150 Provided in exemplary embodiment 150 is a method of embodiment 149, wherein the soyn leghemoglobin nucleic acid is soy leghemoglobin DNA.
  • soybean leghemoglobin DNA comprises a sequence encoding soybean leghemoglobin A (LB A) or soy leghemoglobin C2 (LGB2).
  • exemplary embodiment 152 Provided in exemplary embodiment 152 is a method of embodiment 148, wherein the assay to detect the presence of a component of soybean leghemoglobin is a nucleic acid amplification assay, a sequencing assay, and/or a microarray assay.
  • the assay to detect the presence of a component of soybean leghemoglobin is a nucleic acid amplification assay, a sequencing assay, and/or a microarray assay.
  • exemplary embodiment 153 Provided in exemplary embodiment 153 is a method of embodiment 148 or 152, the assay to detect the presence of a component of soybean leghemoglobin is a polymerase chain reaction (PCR) assay.
  • PCR polymerase chain reaction
  • exemplary embodiment 154 Provided in exemplary embodiment 154 is a method of embodiment 148, wherein the component of soy leghemoglobin is a soy leghemoglobin protein.
  • exemplary embodiment 155 Provided in exemplary embodiment 155 is a method of embodiment 154, wherein the soy leghemoglobin protein is soybean leghemoglobin A (LBA) or soy leghemoglobin C2 (LGB2) .
  • LBA soybean leghemoglobin A
  • LGB2 soy leghemoglobin C2
  • exemplary embodiment 156 Provided in exemplary embodiment 156 is a method of embodiment 154 or 155, wherein the soybean leghemoglobin protein is detected using an antibody specific for the soybean leghemoglobin protein, HPLC or UPLC.
  • exemplary embodiment 157 Provided in exemplary embodiment 157 is a method of any one of embodiments 154 to 156, wherein the pharmaceutical composition comprises bacteria.
  • exemplary embodiment 158 Provided in exemplary embodiment 158 is a method of embodiment 157, wherein the bacteria are hemoglobin-dependent bacteria.
  • exemplary embodiment 159 is a method of embodiment 157 or 158, wherein the bacteria are of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Foumierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
  • the bacteria are of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae
  • exemplary embodiment 160 Provided in exemplary embodiment 160 is a method of embodiment 159, wherein the bacteria are of the genus Foumierella.
  • exemplary embodiment 161 Provided in exemplary embodiment 161 is a method of embodiment 160, wherein the Fournierella are Fournierella Strain B (ATCC Deposit Number PTA- 126696).
  • exemplary embodiment 162 Provided in exemplary embodiment 162 is a method of embodiment 159, wherein the bacteria are of the genus Prevotella.
  • exemplary embodiment 163 Provided in exemplary embodiment 163 is a method of embodiment 162, wherein the bacteria are Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevo
  • exemplary embodiment 164 Provided in exemplary embodiment 164 is a method of embodiment 162, wherein the bacteria are of the species Prevotella histicola.
  • exemplary embodiment 165 Provided in exemplary embodiment 165 is a method of embodiment 162, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
  • exemplary embodiment 166 Provided in exemplary embodiment 166 is a method of embodiment 162, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain C (ATTC Deposit Number PTA- 126140).
  • exemplary embodiment 167 Provided in exemplary embodiment 167 is a method of embodiment 162, wherein the Prevotella are Prevotella Strain B 50329 (NRRL accession number B 50329).
  • exemplary embodiment 168 Provided in exemplary embodiment 168 is a method of embodiment 162, wherein the Prevotella are Prevotella Strain C (ATTC Deposit Number PTA- 126140).
  • exemplary embodiment 169 Provided in exemplary embodiment 162, wherein the Prevotella bacteria (i) comprise one or more proteins listed in Table 1, and/or (ii) are substantially free of a protein listed in Table 2.
  • exemplary embodiment 170 Provided in exemplary embodiment 170 is a method of any one of embodiments 157 to 169, wherein the bacterial are live, attenuated, or dead.
  • exemplary embodiment 171 Provided in exemplary embodiment 171 is a method of any one of embodiments 157 to 170, wherein the bacteria are lyophilized bacteria.
  • exemplary embodiment 172 Provided in exemplary embodiment 172 is a method of any one of embodiments 148 to 171, wherein the pharmaceutical composition comprises mEVs.
  • mEVs are secreted mEVs (smEVs).
  • exemplary embodiment 174 Provided in exemplary embodiment 174 is a method of embodiment 172, wherein the mEVs are processed mEVs (pmEVs).
  • exemplary embodiment 175 Provided in exemplary embodiment 175 is a method of any one of embodiments 172 to 174, wherein the mEVs are from hemoglobin-dependent bacteria.
  • exemplary embodiment 176 is a method of any one of embodiments 172 to 175, wherein the mEVs are from bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
  • the mEVs are from bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergi
  • exemplary embodiment 177 Provided in exemplary embodiment 177 is a method of embodiment 176, wherein the mEVs are from bacteria of the genus Fournierella.
  • exemplary embodiment 178 Provided in exemplary embodiment 178 is a method of embodiment 177, wherein the Fournierella are Fournierella Strain B (ATCC Deposit Number PTA- 126696).
  • exemplary embodiment 179 Provided in exemplary embodiment 179 is a method of embodiment 176, wherein the mEVs are from bacteria of the genus Prevotella.
  • exemplary embodiment 180 Provided in exemplary embodiment 180 is a method of embodiment 179, wherein the bacteria are Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotell
  • exemplary embodiment 181 Provided in exemplary embodiment 181 is a method of embodiment 179, wherein the mEVs are from bacteria of the species Prevotella histicola.
  • exemplary embodiment 182 Provided in exemplary embodiment 182 is a method of embodiment 179, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
  • exemplary embodiment 183 Provided in exemplary embodiment 183 is a method of embodiment 179, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain C (ATTC Deposit Number PTA-126140).
  • exemplary embodiment 184 Provided in exemplary embodiment 184 is a method of embodiment 179, wherein the Prevotella are Prevotella Strain B 50329 (NRRL accession number B 50329).
  • exemplary embodiment 185 Provided in exemplary embodiment 185 is a method of embodiment 179, wherein the Prevotella are Prevotella Strain C (ATTC Deposit Number PTA-126140).
  • exemplary embodiment 186 Provided in exemplary embodiment 186 is a method of embodiment 179, wherein the Prevotella bacteria (i) comprise one or more proteins listed in Table 1, and/or (ii) are substantially free of a protein listed in Table 2.
  • the clarified tissue lysates or protein solutions prepared as described below are subjected to ammonium sulfate precipitation. Briefly, the clarified tissue lysates or protein solutions are buffered with 1M HEPES, pH 7.4 or 1 M Tris-HCl, pH 7.4, to a final concentration of 50 mM. Proteins are precipitated by addition of ammonium sulfate in at least two steps at 50% and 80% saturation. The lysate with ammonium sulfate is stirred gently for a minimum of 4 hours and a maximum of 16 hours at 4°C. The solution is centrifuged at 25,000 x g for 1 hour at 4 °C.
  • the protein composition at individual fractionation step is monitored by SDS-PAGE and protein concentrations are measured by standard UV-Vis methods.
  • Hemoglobin, myoglobin, or other heme-containing polypeptide is further separated by size exclusion chromatography (Sephacryl S-100 HR, GE Healthcare) based on the molecular size of the protein.
  • the fraction is optionally further purified using an ion exchange chromatography (High Prep Q; Prep S; High Prep DEAE, GE Healthtcare).
  • the fraction containing the heme -containing polypeptide is determined by SDS-PAGE and/or mass spectrometry.
  • Carps are coarsely chopped and quickly frozen in liquid nitrogen.
  • the frozen tissue is ground in liquid nitrogen with mortar and pestle until fine powder is formed.
  • the powder is stored in -80 °C until further use.
  • Pea total proteins Dry green or yellow pea flour is used to extract total pea proteins. The flour is suspended in 10 volumes of 20 mM potassium phosphate buffer pH 8 and 100 mM sodium chloride and stirred for 1 hour. Soluble protein is separated from pea seed debris by centrifugation. The supernatant is collected and filtered through a 0.2 micron membrane and concentrated using a 10 KDa cutoff PES membrane.
  • Lentil total proteins Air-classified lentil flour is used to extract crude mixture of lentil proteins. Flour is suspended in 5 volumes of 20 mM potassium phosphate buffer pH 7.4 and 0.5 M sodium chloride and stirred for 1 hour. Soluble protein is separated from the unextracted protein and lentil seed debris by centrifugation (8000 g, 20 minutes ). The supernatant is collected and filtered through a 0.2 micron membrane and concentrated using a 10 KDa cutoff PES membrane.
  • Chickpea / Garbanzo bean total proteins Garbanzo bean flour is suspended in 5 volumes of 20 mM potassium phosphate buffer pH 7.4 and 0.5 M sodium chloride and stirred for 1 hour. Soluble protein is separated from the unextracted protein and chickpea seed debris by centrifugation (8000 g, 20 minutes ). The supernatant is collected and filtered through a 0.2 micron membrane and concentrated using a 10 KDa cutoff PES membrane.
  • Soybean proteins are also extracted by suspending the defatted soy flour in 4-15 volumes (e.g., 5 volumes) of 20 mM sodium carbonate, pH 9 (or water, pH adjusted to 9 after addition of the flour) or 20 mM potassium phosphate buffer pH 7.4 and 100 mM sodium chloride. The slurry is stirred for one hour and centrifuged at 8000 x g for 20 minutes. The extracted proteins are ultrafiltered and then processed as above or alternatively, the supernatant is collected and filtered through a 0.2 micron membrane and concentrated using a 10 KDa cutoff PES membrane.
  • 4-15 volumes e.g., 5 volumes
  • 20 mM sodium carbonate pH 9 (or water, pH adjusted to 9 after addition of the flour) or 20 mM potassium phosphate buffer pH 7.4 and 100 mM sodium chloride.
  • the slurry is stirred for one hour and centrifuged at 8000 x g for 20 minutes.
  • the extracted proteins are ultrafiltered and then processed as above or
  • Amaranth flour dehvdrins Amaranth flour is suspended in 5 volumes of 0.5 M sodium chloride, pH 4.0 and stirred for 1 hour. Soluble protein is separated from the unextracted protein and debris by centrifugation (8000 x g, 20 minutes). The supernatant is collected and filtered through a 0.2 micron membrane and concentrated using a 3 KDa cutoff PES membrane. Further enrichment of dehydrins from this fraction is obtained by boiling the concentrated protein material, spinning at 8000 x g for 10 minutes, and collecting the supernatant.
  • Pea globulins Dry green pea flour is used to extract pea globulin proteins. The flour is suspended in 10 volumes of 50 mM potassium phosphate buffer pH 8 and 0.4 M sodium chloride and stirred for 1 hour. Soluble protein is separated from pea seed debris by centrifugation. The supernatant is subjected to ammonium sulfate fractionation in two steps at 50 % and 80 % saturation. The 80 % pellet containing globulins of interest is stored at -20 °C until further use. Protein is recovered from the pellet and prepared for use as described above.
  • Soybean 7S and 11 S globulins Globulins from soybean flour are isolated by first suspending lowfat/defatted soy flour in 4-15 volumes of 20 mM potassium phosphate pH 7.4. The slurry is centrifuged at 8000 x g for 20 mins or clarified by 5 micron filtration and the supernatant is collected. The crude protein extract contains both the 7S and 1 IS globulins. The solution is filtered using a 0.2 micron filter and concentrated using a 10 kDa molecular weight cutoff PES membrane on a Spectrum Labs KrosFlo hollow fiber tangential flow filtration system or by passing over the anion-exchange resin prior to use in experiments.
  • the 1 IS globulins are separated from the 7S proteins by isoelectric precipitation.
  • the pH of the crude protein extract is adjusted to 6.4 with dilute HC1, stirred for 30 min-1 hour and then centrifuged to collect the 1 IS precipitate and 7S proteins in the supernatant.
  • the 1 IS fraction is resuspended with 10 mM potassium phosphate pH 7.4 and the protein fractions are micro-filtered and concentrated prior to use.
  • Mung bean 8S globulins Mung bean flour is used to extract 8S globulins by first suspending the flour in 4 volumes of 50 mM potassium phosphate buffer pH 7, 0.5M NaCl. After centrifugation, proteins in the supernatant are fractionated by addition of ammonium sulfate in 2 steps at 50 % and 90 % saturation respectively. The precipitate from the 90 % fraction contains the 8S globulins, which is saved at -20 °C until further use. 8S globulins are recovered from the pellet and prepared for use as described above.
  • Mung bean globulins are also extracted by suspending the flour in 4 volumes of 20 mM sodium carbonate buffer, pH 9 (or water adjusted to pH 9 after addition of the mung bean flour). The slurry is centrifuged (or filtered) to remove solids, ultrafiltered, and then processed as described above.
  • Leghemoglobin Soy root nodules are suspended and lysed in 20 mM potassium phosphate pH 7.4, 100 mM potassium chloride and 5 mM EDTA using a grinder-blender. During this process, leghemoglobin is released into the buffer. Root- nodule lysate containing leghemoglobin is cleared from cell debris by filtration through 5 micron filter.
  • leghemoglobin is collected in the flowthrough fraction and concentrated over 3 kDa molecular weight cutoff PES membrane on a Spectrum Labs KrosFlo hollow fiber tangential flow filtration system to a desired concentration. Purity (partial abundance) of purified leghemoglobin is analyzed by SDS-PAGE gel.
  • leghemoglobin In lysate, leghemoglobin is typically present at 20-40 %, while after anion-exchange purification, it can be present at 70-80 %.
  • the soybean leghemoglobin flowthrough from anion-exchange chromatography is optionally applied onto size- exclusion chromatography (Sephacryl S-100 HR, GE Healthcare). Soybean leghemoglobin elutes as two fractions corresponding to dimeric and monomeric species. Purity (partial abundance) of leghemoglobin is analyzed by SDS-PAGE and can be in the range of ⁇ 90- 100%. Analysis of UV-VIS spectra (250-700nm) can reveal spectral signature consistent with heme loaded leghemoglobin.
  • Example 3 Exemplary Manufacturing Process of Hemoglobin-dependent Bacteria
  • a hemoglobin solution is also prepared by dissolving a piscine hemoglobin, an avian hemoglobin, a fungal hemoglobin, a plant hemoglobin, or a bacterial hemoglobin in 0.01 M NaOH.
  • the solution is sterilized by autoclaving.
  • a working concentration of 0.02 g/L or 0.2 g/L is used.
  • a myoglobin solution, a leghemoglobin solution, or other heme- containing polypeptide solution e.g., peroxidase, cytochrome c
  • phosphate buffer saline pH 7.4, or 0.01 M NaOH.
  • the solution is sterilized by autoclaving, and is added to the growth media at various working concentrations (e.g., 0.02 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L, or 0.5 g/L).
  • hemoglobin-dependent bacteria e.g., Prevotella histicola
  • the hemoglobin-dependent bacteria are grown in growth media comprising the components listed in Table 5.
  • the media is filter sterilized prior to use.
  • Table 5 Exemplary Growth Media
  • Table 6 Another Exemplary Growth Media
  • a 1L bottle is inoculated with a lmL of a cell bank sample that had been stored at -80 °C.
  • the culture is used to inoculate a 20 L bioreactor at 5 % v/v.
  • the culture is used to inoculate a 3500 L bioreactor at 0.5% v/v.
  • Fermentation culture is continuously mixed with addition of a mixed gas at 0.02 VVM with a composition of 25 % CO 2 and 75 % N 2 . pH is maintained at 6.5 with ammonium hydroxide and temperature is controlled at 37 °C. Harvest time is based on when the stationary phase is reached (after approximately 12 to 14 hours of growth).
  • a nucleic acid encoding Glycine max leghemoglobin C2 (Uniprot KB P02236) with an N-terminal His6 epitope tag and a TEV cleavage site is cloned into the pJexpress401 vector (DNA2.0), and transformed into E. coli BL21.
  • Transformed cells are grown by fed-batch fermentation supplemented with kanamycin, 0.1 mM ferric chloride and 10 ug/ml 5 -aminolevulinic acid. Expression is induced by 0.3 mM isopropyl ⁇ -D-1- thiogalactopyranoside (IPTG) and cells are grown at 30 degrees °C for 24 hr.
  • IPTG isopropyl ⁇ -D-1- thiogalactopyranoside
  • Leghemoglobin concentration is determined by soret peak absorbance and adjusted to 60-70 mg/ml.
  • the final leghemoglobin product is frozen in liquid nitrogen, lyophilized, and stored at -20 degrees C. Purity (partial abundance) of leghemoglobin is analyzed by SDS-PAGE and determined to be about 80%. Analysis of UV-VIS spectra (250-700 nm) reveals spectral signature consistent with heme-loaded leghemoglobin.
  • Glycine max leghemoglobin C2 and eight Pichia pastoris heme biosynthesis genes are cloned into the Pichia pastoris expression vector pJA (BioGrammatics Inc.; Carlsbad, Calif.) under the control of the pAOXl methanol inducible promoter.
  • Pichia pastoris strain Bgl 1 (BioGrammatics, Inc.) is transformed with linearized plasmids, and stable integrants are selected by antibiotic resistance. Table 8
  • Transformed Pichia cells are grown by fed-batch fermentation and leghemoglobin expression is induced with methanol for 120 hours at 30 degrees °C.
  • Cells are concentrated by centrifugation, resuspended in water, and lysed by high pressure homogenization. Solids are removed by treatment with Tramfloc 863 A, centrifugation, and 0.2 micron microfiltration (Koch Membrane Systems). The soluble lysate is concentrated and diafiltered with water using 3 kDa ultrafiltration (Spectrum Laboratories). The formulated lysate is partially purified using HPA25L anion exchange resin (Mitsubishi) to a final purity of about 40%.
  • HPA25L anion exchange resin Mitsubishi
  • leghemoglobin solution is re-formulated by concentration and water diafiltration using 3 kDa ultrafiltration (Spectrum Laboratories) and further purified using Q Fast Flow anion exchange resin (GE Lifesciences).
  • the final leghemoglobin product is concentrated using 3 kD ultrafiltration and frozen at -20 degrees C.
  • the final product is about 80% pure and contains 80 g/L leghemoglobin. See U.S. Patent No. 10,798,958.
  • Example 5 Testing Sov Leghemoglobin as a Substitute for Animal-Sourced Hemoglobin in Growth Media
  • Solutions IM1 and IM2 were prepared by resuspending the ImpossibleTM meat in distilled water and adding 0.01 M NaOH to achieve a concentration similar to the concentration used for the animal-sourced hemoglobin or spirulina stock solutions. After resuspension, solids were removed from the solutions by centrifugation. Solutions IM3 and IM4 were prepared by resuspending the ImpossibleTM meat in either water or 0.01 M NaOH solution followed by heating until boiling. Solids were then removed by double filtration through a gauze filter.
  • Percentage of the solution was calculated based on estimation that the ImpossibleTM Meat contains 2% of soy leghemoglobin (the package states 2% or less). The actual concentration of soy leghemoglobin in the solution is expected to be equal or less than the calculated estimate.
  • Soy hemoglobin is added to cultures of hemoglobin-dependent bacteria to support the growth of the bacteria, as described above.
  • An aliquot of bacterial culture is resuspended in PBS, centrifuged to pellet the microbes, and filtered.
  • the bacterial culture supernatant is collected, serial diluted, and then incubated with antibody-oligonucleotide conjugated probes.
  • the antibodies are specific to soy hemoglobin.
  • the supernatant is incubated with the conjugated probes to allow sufficient time for antibodies to bind to target epitope.
  • Another inbucabtion step is performed to allow for ligation of proximal oligonucleotides.
  • the oligonucleotides can be ligated, serving as the template for PCR amplification and quantification. PCR is performed on the oligonucleotides to detect presence of soy hemoglobin.

Abstract

Methods and compositions related to pharmaceutical agents, pharmaceutical compositions, and solid dosage forms comprising at least one component of soy leghemoglobin and bacteria or agents of bacterial origin are provided herein.

Description

BACTERIAL COMPOSITIONS COMPRISING SOY HEMOGLOBIN
CROSS-REFERENCE TO RELATED APPLICATION
[1] This application claims the benefit of U.S. Provisional Application serial number 63/192,805, filed May 25, 2021, the entire contents of which are incorporated herein by reference.
BACKGROUND
[2] The composition of a person’s microbiome can play an important role in their health and well-being. Indeed, disruption of an individual’s microbiome has been implicated in numerous diseases, including inflammatory bowel diseases, immune disorders, type 2 diabetes, neurodegenerative disorders, cardiovascular diseases, and cancers. Thus, microbiome modulation is an attractive therapeutic strategy for such diseases.
SUMMARY
[3] One way to modulate a person’s microbiome is by orally administering to them one or more strains of beneficial bacteria, e.g., formulated for therapeutic use, e.g., in a bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement). However, development of such therapies has been hindered by the fact that large-scale production of many bacterial strains has proven challenging, particularly for bacterial strains that require hemoglobin (or its derivatives such as hemin) for growth.
[4] Hemoglobin is an iron-containing metalloprotein in red blood cells that captures atmospheric oxygen in the lungs and carries it to the rest of the body. Iron is an essential nutrient for almost all forms of life, including bacteria. As hemoglobin is the most abundant reservoir of iron within humans, much of the bacteria that make up the human microbiome use hemoglobin or its derivatives as their primary source of iron. Often, such hemoglobin-dependent bacteria require the presence of hemoglobin or hemin for optimal in vitro growth. However, commercial hemoglobin and its derivatives are typically purified from animal sources, such as from porcine blood, which, inter alia, results in purified hemoglobin being costly. Moreover, GMP (good manufacturing practice)-grade hemoglobin is not easily sourced, making the large-scale manufacture and/or GMP grade manufacture of hemoglobin-dependent bacteria for pharmaceutical purposes particularly challenging.
[5] Accordingly, there is a great need for improved compositions and methods for growing hemoglobin-dependent bacteria, e.g., without the need for hemoglobin sourced from an animal (e.g., without the need for porcine or bovine hemoglobin).
[6] This disclosure is based, in part, on the discovery that soy leghemoglobin and/or fragments thereof are present in compositions and/or solid dosage forms comprising bacteria grown in growth media comprising soy leghemoglobin and/or comprising agents (e.g., microbial extracellular vesicles (mEVs)) derived from bacteria grown in growth media comprising soy leghemoglobin.
[7] This disclosure is also based, in part, on the discovery that components of soy leghemoglobin are present in pharmaceutical agents comprising bacteria grown in growth media comprising soy leghemoglobin and/or comprising agents (e.g., microbial extracellular vesicles, or mEVs) derived from bacteria grown in growth media comprising soy leghemoglobin.
[8] Thus, in certain aspects, provided herein are bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)comprising bacteria (or components thereof, such as mEVs) and at least one component of soy leghemoglobin (e.g., a nucleic acid soy leghemoglobin component and/or a protein soy leghemoglobin component). In certain aspects, provided herein are methods of making and/or using such bacterial compositions.
[9] In some aspects, provided herein are methods of determining the presence and/or amount of at least one component of soy leghemoglobin in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements).
[10] In certain aspects, provided herein are bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) comprising: a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin. In certain aspects, provided herein are methods of making and/or using such compositions.
[11] In some aspects, provided herein are methods of determining the presence and/or amount of soy leghemoglobin in such compositions.
[12] In certain aspects, provided herein is a pharmaceutical composition comprising: a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin. In certain aspects, provided herein are methods of making and/or using suchpharmaceutical compositions.
[13] In some aspects, provided herein are methods of determining the presence and/or amount of soy leghemoglobin in such a pharmaceutical composition.
[14] In certain aspects, provided herein is a pharmaceutical agent comprising (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin. In certain aspects, provided herein are methods of making and/or using such pharmaceutical agents.
[15] In some aspects, provided herein are methods of determining the presence and/or amount of soy leghemoglobin in such a pharmaceutical agent.
[16] In certain aspects, provided herein is a solid dosage form comprising: a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin.
[17] In some embodiments, the at least one component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid. In some embodiments, the soy leghemoglobin nucleic acid is soy leghemoglobin DNA. In some embodiments, the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin A (LB A). In some embodiments, the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin C2 (LGB2).
[18] In some embodiments, the at least one component of soy leghemoglobin comprises a soy leghemoglobin protein. In some embodiments, the soy leghemoglobin protein is soy leghemoglobin A (LB A). In some embodiments, the soy leghemoglobin protein is soy leghemoglobin C2 (LGB2).
[19] In certain embodiments, the bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement comprises bacteria. In certain embodiments, the bacteria are hemoglobin-dependent bacteria (e.g., a species and/or strain of hemoglobin- dependent bacteria provided herein). In some embodiments, the bacteria are live, attenuated, or dead. In some embodiments, the bacteria are lyophilized bacteria. In some embodiments, the bacteria are irradiated (e.g., gamma irradiated).
[20] In certain embodiments, the bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement comprises mEVs. In some embodiments, the mEVs are secreted mEVs (smEVs). In other embodiments, the mEVs are processed mEVs (pmEVs). In some embodiments, the mEVs are from hemoglobin-dependent bacteria (e.g., a species and/or strain of hemoglobin-dependent bacteria provided herein). In some embodiments, the mEVs are lyophilized mEVs.
[21] In some embodiments, the bacteria (e.g., the bacteria in the bacterial composition and/or the bacteria from which the mEVs were derived) are hemoglobin- dependent bacteria. In some embodiments of the methods and compositions provided herein, the hemoglobin-dependent bacteria can be any bacteria that require the presence of hemoglobin or a hemoglobin derivative for optimal growth (i.e.. for optimal growth in the absence of soy leghemoglobin or a component thereof provided herein). In some embodiments, the hemoglobin-dependent bacteria are bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella. In some embodiments, the hemoglobin-dependent bacteria are of the genus Prevotella. In some embodiments, the hemoglobin-dependent bacteria are bacteria of the species Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopes, Prevotella shahii, Prevotella zoo gleofor mans, or Prevotella veroralis. In some embodiments, the hemoglobin-dependent bacteria are bacteria of the species Alistipes indistinctus, Alistipes shahii, Alistipes timonensis, Bacillus coagulans, Bacteroides acidifaciens, Bacteroides cellulosilyticus, Bacteroides eggerthii, Bacteroides intestinalis, Bacteroides uniformis, Collinsella aerofaciens, Cloacibacillus evryensis, Clostridium cadaveris, Clostridium cocleatum, Cutihacterium acnes, Eisenhergiella sp., Erysipelotrichaceae sp., Euhacterium hallii/Anaerohutyricum halii, Euhacterium infirmum, Megasphaera micronuciformis, Parahacteroides distasonis, Peptoniphilus lacrimalis, Rarimicrobium hominis, Shuttleworthia satelles, or Turicibacter sanguinis.
[22] In some embodiments, bacteria are of the species Prevotella histicola. In some embodiments, the Prevotella histicola is Prevotella histicola Strain B (NRRL accession number B 50329). In some embodiments, the Prevotella histicola is Prevotella histicola Strain C (ATCC Deposit Number PTA-126140).
[23] In some embodiments, the hemoglobin-dependent bacteria are a strain of the species Prevotella histicola. In some embodiments, the Prevotella histicola strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g, at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain B 50329. In certain embodiments, the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g, at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329). In certain embodiments, the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329). In certain embodiments, the Prevotella histicola strain is Prevotella Strain B 50329 (NRRL accession number B 50329).
[24] In some embodiments, the Prevotella histicola strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain C (ATCC Deposit Number PTA-126140, deposited on September 10, 2019). In certain embodiments, the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Prevotella Strain C (PTA-126140). In certain embodiments, the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Prevotella Strain C (PTA-126140). In certain embodiments, the Prevotella histicola strain is Prevotella Strain C (PTA-126140).
[25] In some embodiments, the hemoglobin-dependent bacteria are of the genus Fournierella. In some embodiments, the hemoglobin-dependent bacteria are Fournierella Strain A.
[26] In some embodiments, the hemoglobin-dependent Fournierella strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Fournierella Strain B (ATCC Deposit Number PTA- 126696, deposited on March 5, 2020). In certain embodiments, the Fournierella strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Fournierella Strain B (PTA- 126696). In certain embodiments, the Fournierella strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Fournierella Strain B (PTA- 126696). In certain embodiments, the Fournierella strain is Fournierella Strain B (PTA- 126696).
[27] In some embodiments, the hemoglobin-dependent bacteria are of the genus Parabacteroides . In some embodiments, the hemoglobin-dependent bacteria are Parabacteroides Strain A. In some embodiments, the hemoglobin-dependent bacteria are Parabacteroides Strain B.
[28] In some embodiments, the hemoglobin-dependent bacteria are of the genus Bacteroides. In some embodiments, the hemoglobin-dependent bacteria are Bacteroides Strain A.
[29] In some embodiments, the hemoglobin-dependent bacteria are of the genus Alii stipes. In some embodiments, the hemoglobin-dependent bacteria are Allistipes Strain A.
[30] In certain aspects, provided herein is a solid dosage form comprising: (a) a pharmaceutical agent described herein (e.g., a pharmaceutical agent comprising at least one component of soy leghemoglobin); and (b) at least one diluent, at least one lubricant, at least one glidant, and/or at least one disintegration agent.
[31] In certain embodiments, the solid dosage form described herein comprises at least one diluent that has a total mass that is at least, about, or no more than, 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,
51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% of the total mass of the solid dosage form. In some embodiments, the at least one diluent has a total mass that is at least 10% and no more than 80% of the total mass of the solid dosage form. In other embodiments, the at least one diluent has a total mass that is at least 20% and no more than 40% of the total mass of the solid dosage form. In some embodiments, the at least one diluent comprises mannitol.
[32] As used herein, the percent of mass of a solid dosage form is on a percent weight:weight basis (%w:w).
[33] In certain embodiments, the solid dosage form described herein comprises at least one lubricant that has a total mass that is at least, about, or no more than, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the total mass of the solid dosage form. In some embodiments, the at least one lubricant has a total mass that is at least 0.1% and no more than 5% of the total mass of the solid dosage form. In some embodiments, the at least one lubricant comprises magnesium stearate.
[34] In certain embodiments, the solid dosage form described herein comprises at least one glidant that has a total mass that is at least, about, or no more than, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the total mass of the solid dosage form. In some embodiments, the at least one glidant has a total mass that is at least 0.01% and no more than 2% of the total mass of the solid dosage form. In some embodiments, the at least one glidant comprises colloidal silicon dioxide.
[35] In certain embodiments, the solid dosage form described herein comprises at least one disintegration agents, or certain combinations and/or amounts of disintegration agents, resulting in a decrease in the disintegration time of the composition (e.g., 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold) as compared to conventional solid dosage forms (e.g., solid dosage forms containing conventional amounts of disintegration agents). In certain embodiments, the solid dosage forms provided herein result in an increase in therapeutic efficacy and/or physiological effect as compared to a pharmaceutical product having conventional solid dosage forms.
[36] In certain embodiments, the solid dosage form comprises a pharmaceutical agent (e.g., bacteria and/or an agent of bacterial origin, such as mEVs, a powder comprising bacteria and/or an agent of bacterial origin, such as mEVs) and one or more disintegration agents (e.g., one, two or three disintegration agents). In certain embodiments, the solid dosage form comprises a pharmaceutical agent (e.g., bacteria and/or an agent of bacterial origin, such as mEVs, a powder comprising bacteria and/or an agent of bacterial origin, such as mEVs) and three disintegration agents.
[37] In some embodiments, the solid dosage form described herein comprises at least one disintegrant that has a total mass that is at least, about, or no more than, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,
35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% of the total mass of the solid dosage form. In some embodiments, the at least one disintegration agent has a total mass that is at least 40% of the total mass of the solid dosage form.
[38] In certain embodiments, the at least one disintegration agent comprises low- substituted hydroxypropyl cellulose (L-HPC, e.g., LH-B1), croscarmellose sodium (Ac-Di- Sol, e.g., Ac-Di-Sol SD-711), and/or crospovidone (PVPP, e.g., Kollidon, e.g., Kollidon CL-F). In some embodiments, the at least one disintegration agent comprises low- substituted hydroxypropyl cellulose (L-HPC, e.g., LH-B1), croscarmellose sodium (Ac-Di- Sol, e.g., Ac-Di-Sol SD-711), and crospovidone (PVPP, e.g., Kollidon, e.g., Kollidon CL-
F).
[39] In certain embodiments, the solid dosage forms provided herein comprise L- HPC. In some embodiments, the L-HPC is of grade LH-B1. In certain embodiments, the total L-HPC mass is at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the solid dosage form. In certain embodiments, the total L-HPC mass is no more than 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the solid dosage form. In certain embodiments, the total L-HPC mass is about 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the solid dosage form. In some embodiments, the L-HPC has a total L-HPC mass that is at least 22% and no more than 42% of the total mass of the solid dosage form. In certain embodiments, the total L-HPC mass is about 29% to about 35% of the total mass of the solid dosage form. In certain embodiments, the total L-HPC mass is about 32% of the total mass of the solid dosage form. In some embodiments, wherein the L-HPC is L-HPC of grade LH-B 1.
[40] In certain embodiments, the solid dosage forms provided herein comprise Ac-Di-Sol. In some embodiments, the Ac-Di-Sol is of grade SD-711. In certain embodiments, the total Ac-Di-Sol mass is at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the solid dosage form. In certain embodiments, the total Ac-Di-Sol mass is no more than 1%, 2%,
3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the solid dosage form. In certain embodiments, the total Ac-Di-Sol mass is about 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the solid dosage form. In certain embodiments, the Ac-Di-Sol has a total Ac-Di-Sol mass that is at least 0.01% and no more than 16% of the total mass of the solid dosage form. In certain embodiments, the total Ac-Di-Sol mass is about 3% to about 9 % of the total mass of the solid dosage form. In certain embodiments, the total Ac-Di-Sol (e.g., Ac- Di-Sol SD-711) mass is about 6 % of the total mass of the solid dosage form.
[41] In certain embodiments, the solid dosage forms provided herein comprise PVPP (crospovidone, e.g., Kollidon, e.g., Kollidon CL-F). In certain embodiments, the total PVPP mass is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the solid dosage form. In certain embodiments, the total PVPP mass is no more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the solid dosage form. In certain embodiments, the total PVPP mass is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the solid dosage form. In certain embodiments, the PVPP has a total PVPP mass that is at least 5% and no more than 25% of the total mass of the solid dosage form. In certain embodiments, the total PVPP mass is about 12% to about 18% of the total mass of the solid dosage form. In certain embodiments, the total PVPP mass is about 15% of the total mass of the solid dosage form.
[42] In certain embodiments, the total L-HPC mass plus the total Ac-Di-Sol mass plus the total PVPP mass is at least 35%, 40%, 45%, or 50% of the total mass of the solid dosage form. In certain embodiments, the total L-HPC mass plus the total Ac-Di-Sol mass plus the total PVPP mass is at least 40% of the total mass of the solid dosage form.
[43] In certain embodiments, the solid dosage forms provided herein comprise:
(i) L-HPC (e.g., L-HPC of grade LH-B 1) having a total L-HPC mass that is at least 22% (e.g, at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42%) and no more than 42% (e.g., no more than 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,
37%, 38%, 39%, 40%, 41%, or 42%) of the total mass of the solid dosage form; (ii) Ac-Di- Sol (e.g, Ac-Di-Sol of grade SD-711) having a total Ac-Di-Sol mass that is at least 0.01% (e.g, at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16%) and no more than 16% (e.g., no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16%) of the total mass of the solid dosage form; and (iii) PVPP having a total PVPP mass that is at least 5% (e.g., at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%) and no more than 25% (no more than 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%) of the total mass of the solid dosage form. In some embodiments, the solid dosage form comprises: a total L-HPC mass is about 32% of the total mass of the solid dosage form; a total Ac-Di-Sol mass is about 6% of the total mass of the solid dosage form; and a total PVPP mass is about 15% of the total mass of the solid dosage form.
[44] In some embodiments, the solid dosage forms provided herein comprise a pharmaceutical agent (e.g. , bacteria and/or mEV) having a total mass that is at least, about, or no more than, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,
30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,
45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,
60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total mass of the solid dosage form. In some embodiments, the pharmaceutical agent has a total pharmaceutical agent mass that is at least 5% and no more than 65% of the total mass of the solid dosage form. In some embodiments, the pharmaceutical agent has a total pharmaceutical agent mass that is at least 5% and no more than 35% of the total mass of the solid dosage form. In some embodiments, the total pharmaceutical agent mass is about 25% of the total mass of the solid dosage form. In certain embodiments, the solid dosage forms described herein comprise tablets, capsules and/or minitablets (e.g., minitablets in capsules).
[45] In some embodiments, the solid dosage form comprises a tablet. In some embodiments, the tablet is a 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm,
9mm, 9.5mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, or 18mm tablet.
[46] In some embodiments, the solid dosage form comprises a minitablet. In some embodiments, the minitablet is a 1mm minitablet, 1.5 mm minitablet, 2mm minitablet, 3mm minitablet, or 4mm minitablet. In some embodiments, a plurality of minitablets are contained in a capsule (e.g., a size 0 capsule can contain about 31 to about 35 (e.g., 33) minitablets, wherein the minitablets are 3mm in size). In some embodiments, the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule. In some embodiments, the capsule comprises HPMC (hydroxyl propyl methyl cellulose) or gelatin.
[47] In some embodiments, the solid dosage form is enterically coated (e.g., comprises an enteric coating; e.g., is coated with an enteric coating). In some embodiments, the enteric coating is a single enteric coating or more than one enteric coating. In some embodiments, the tablets or minitablets are coated with one layer of enteric coating or with two layers of enteric coatings (e.g., an inner enteric coating and an outer enteric coating). In some embodiments, the enteric coating comprises an inner enteric coating and an outer enteric coating, and the inner and outer enteric coatings are not identical.
[48] In some embodiments, the enteric coating comprises a methacrylic acid ethyl acrylate (MAE) copolymer (1:1).
[49] In some embodiments, the one enteric coating comprises methacrylic acid ethyl acrylate (MAE) copolymer (1:1) (such as Kollicoat MAE 100P).
[50] In some embodiments, the one enteric coating comprises a Eudragit copolymer, e.g., a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).
[51] In some embodiments, the enteric coating comprises cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), a fatty acid, a wax, shellac (esters of aleurtic acid), a plastic, a plant fiber, zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch, a starch derivative, a dextrin, a methyl acrylate- methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), a methyl methacrylate-methacrylic acid copolymer, or sodium alginate.
[52] In some embodiments, the enteric coating comprises an anionic polymeric material.
[53] In certain aspects, provided herein is solid dosage form comprising: (a) a pharmaceutical agent described herein (e.g., a pharmaceutical agent comprising at least one component of soy leghemoglobin); and (b) at least one diluent, at least one lubricant, and/or at least one glidant.
[54] In certain aspects, provided herein is a solid dosage form comprising (a) a pharmaceutical agent described herein (e.g., a pharmaceutical agent comprising at least one component of soy leghemoglobin); and (b) a diluent. In certain embodiments, the total pharmaceutical agent mass is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total mass of the solid dosage form. In some embodiments, the total pharmaceutical agent mass is no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,
20%, 15%, 10%, or 5% of the total mass of the solid dosage form.
[55] In some embodiments, the total mass of the diluent is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, or 95% of the total mass of the solid dosage form. In some embodiments, the total mass of the diluent is no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the total mass of the solid dosage form. In some embodiments, the diluent comprises mannitol.
[56] In certain embodiments, the solid dosage form provided herein comprises a lubricant. In certain embodiments, the total lubricant mass is at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the solid dosage form. In certain embodiments, the total lubricant mass is no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the solid dosage form. In certain embodiments, the total lubricant mass is about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the solid dosage form. In certain embodiments, the total lubricant mass is about 0.5% to about 1.5% of the total mass of the solid dosage form. In certain embodiments, the total lubricant mass is about 1% of the total mass of the solid dosage form. In some embodiments, the lubricant comprises magnesium stearate.
[57] In certain embodiments, the solid dosage forms provided herein comprise a glidant. In some embodiments, the glidant is colloidal silicon dioxide. In certain embodiments, the total glidant mass is at least 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,
0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the solid dosage form. In certain embodiments, the total glidant mass is no more than 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the solid dosage form. In certain embodiments, the total glidant mass is about 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the solid dosage form. In certain embodiments, the total glidant mass is about 0.25% to about 0.75% of the total mass of the solid dosage form. In certain embodiments, the total glidant mass is about 0.5% of the total mass of the solid dosage form.
[58] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is at least 20% and no more than 55% of the total mass of the solid dosage form; (ii) a diluent (e.g., mannitol) having a total mass that is at least 45% and no more than 80% of the total mass of the solid dosage form; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is at least 0.1% and no more than 5% of the total mass of the solid dosage form; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is at least 0.01% and no more than 2% of the total mass of the solid dosage form.
[59] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 20% to about 50% of the total mass of the solid dosage form; (ii) a diluent (e.g., mannitol) having a total mass that is about 50% to 80% of the total mass of the solid dosage form; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the solid dosage form; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the solid dosage form.
[60] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is at least 5% and no more than 95% of the total mass of the solid dosage form; (ii) a diluent (e.g., mannitol) having a total mass that is at least 1% and no more than 95% of the total mass of the solid dosage form; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is at least 0.1% and no more than 5% of the total mass of the solid dosage form; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is at least 0.01% and no more than 2% of the total mass of the solid dosage form.
[61] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 8% to about 92% of the total mass of the solid dosage form; (ii) a diluent (e.g., mannitol) having a total mass that is about 5% to 90% of the total mass of the solid dosage form; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the solid dosage form; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the solid dosage form.
[62] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 30% to about 50% of the total mass of the solid dosage form; (ii) a diluent (e.g., mannitol) having a total mass that is about 45% to 70% of the total mass of the solid dosage form; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the solid dosage form; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the solid dosage form.
[63] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 50% of the total mass of the solid dosage form; (ii) a diluent (e.g., mannitol) having a total mass that is about 48.5% of the total mass of the solid dosage form; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the solid dosage form; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the solid dosage form. In certain embodiments, the solid dosage forms provided herein comprise: (i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 13.51% of the total mass of the solid dosage form;
(ii) a diluent (e.g., mannitol) having a total mass that is about 84.99% of the total mass of the solid dosage form; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the solid dosage form; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the solid dosage form.
[64] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 90.22% of the total mass of the solid dosage form; (ii) a diluent (e.g., mannitol) having a total mass that is about 8.28% of the total mass of the solid dosage form; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the solid dosage form; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the solid dosage form.
[65] In certain embodiments, the solid dosage forms of a pharmaceutical agent as described herein comprise capsules. In some embodiments, the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule. In some embodiments, the capsule is a size 0 capsule. In some embodiments, the capsule comprises HPMC (hydroxyl propyl methyl cellulose) or gelatin. In some embodiments, the capsule comprises HPMC (hydroxyl propyl methyl cellulose). In some embodiments, the capsule is banded. In some embodiments, the capsule is banded with an HPMC-based banding solution.
[66] In some embodiments, the solid dosage form is enterically coated (e.g., comprises an enteric coating; e.g., is coated with an enteric coating).
[67] In some embodiments, the solid dosage form is enteric coated to dissolve at pH 5.5.
[68] In some embodiments, the enteric coating comprises a polymethacrylate- based copolymer. In some embodiments, the enteric coating comprises poly(methacrylic acid-co-ethyl acrylate).
[69] In some embodiments, the enteric coating comprises a methacrylic acid ethyl acrylate (MAE) copolymer (1:1).
[70] In some embodiments, the enteric coating comprises methacrylic acid ethyl acrylate (MAE) copolymer (1:1) (such as Kollicoat MAE 100P).
[71] In some embodiments, the enteric coating comprises a Eudragit copolymer, e.g., a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).
[72] In some embodiments, the enteric coating comprises cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), a fatty acid, a wax, shellac (esters of aleurtic acid), a plastic, a plant fiber, zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch, a starch derivative, a dextrin, a methyl acrylate- methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), a methyl methacrylate-methacrylic acid copolymer, or sodium alginate.
[73] In some embodiments, the enteric coating comprises an anionic polymeric material.
[74] The pharmaceutical agent can be a powder that comprises the bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component, and, can comprise additional agents such as, e.g., cryoprotectant. For example, in some embodiments, the pharmaceutical agent is a lyophilized powder of bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component that optionally, further comprises additional agents, such as a cryoprotectant.
[75] In some embodiments, the pharmaceutical agent comprises bacteria and the dose of bacteria is about 1 x 107 to about 2 x 1012 (e.g., about 3 x 1010 or about 1.5 x 1011) cells (e.g., wherein cell number is determined by total cell count, which is determined by Coulter counter), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
[76] In some embodiments, the pharmaceutical agent comprises bacteria and the dose of bacteria is about 1 x 107to about 1 x 1013, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule. In some embodiments, the pharmaceutical agent comprises bacteria and the dose of bacteria is about 1 x 109, about 3 x 109, about 5 x 109, about 1.5 x 1010, or about 5 x 1010 cells, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule. In some embodiments, the pharmaceutical agent comprises bacteria and the dose of bacteria is about 8 x 1010 cells, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule. In some embodiments, the pharmaceutical agent comprises bacteria and the dose of bacteria is about 1.6 x 1011 cells, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
[77] In some embodiments, the pharmaceutical agent comprises mEVs and the dose of mEVs is about 1 x 105 to about 2 x 1012 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule. In some embodiments, the pharmaceutical agent comprises bacteria and the dose of bacteria is about 3.2 x 1011 cells, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
[78] In some embodiments, the pharmaceutical agent comprises a powder comprising bacteria and/or mEVs and the dose of the pharmaceutical agent (e.g., a powder comprising bacteria and/or mEVs) is about 10 mg to about 3500 mg, wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
[79] In some embodiments, the pharmaceutical agent comprises a powder comprising bacteria and/or mEVs and the dose of the pharmaceutical agent (e.g., a powder comprising bacteria and/or mEVs) is about 30 mg to about 1300 mg (by weight of bacteria and/or mEVs powder) (about 25, about 30, about 35, about 50, about 75, about 100, about 120, about 150, about 250, about 300, about 350, about 400, about 500, about 600, about 700, about 750, about 800, about 900, about 1000, about 1100, about 1200, about 1250, about 1300, about 2000, about 2500, about 3000, or about 3500 mg wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
[80] In some embodiments, the pharmaceutical agent comprises bacteria and/or mEVs and the dose of pharmaceutical agent (e.g., bacteria and/or mEVs) is about 2x106 to about 2x1016 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
[81] In some embodiments, the pharmaceutical agent comprises bacteria and/or mEVs and the dose of pharmaceutical agent (e.g., bacteria and/or mEVs) is about 5 mg to about 900 mg total protein (e.g., wherein total protein is determined by Bradford assay or BCA), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
[82] In some aspects, the disclosure provides a method of preventing or treating a subject (e.g., human) (e.g., a subject in need of treatment), the method comprising administering to the subject a bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement)provided herein. In some aspects, the disclosure provides a method of preventing or treating a subject (e.g. , human) (e.g. , a subject in need of treatment), the method comprising administering to the subject a solid dosage form provided herein. In some aspects, the disclosure provides use of a bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement)for the treatment or prevention of a disease of a subject. In some aspects, the disclosure provides use of a bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement) provided herein for the preparation of a medicament for treating a subject (e.g. , human) (e.g., a subject in need of treatment).
[83] In some embodiments, the bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) provided herein treats a cancer, inflammation, autoimmunity, a metabolic condition, or a dysbiosis.
[84] In some embodiments, the bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) provided herein treats bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection).
[85] In some embodiments, the bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) provided herein decreases inflammatory cytokine expression (e.g., decreased IL-8, IL-6, IL-Ib, and/or TNFa expression levels).
[86] In certain aspects, provided herein are methods of preparing a solid dosage form, the method comprising (a) combining (i) a pharmaceutical agent provided herein (e.g., bacteria disclosed herein and/or an agent of bacterial origin, such as mEVs disclosed herein) (e.g., comprising at least one component of soy leghemoglobin), and (ii) at least one diluent, at least one lubricant, at least one glidant, and/or at least one (e.g., one, two or three) disintegration agent to prepare a pharmaceutical composition, and (b) compressing the pharmaceutical composition into a solid dosage form. In some embodiments, the method further comprises the step of enterically coating the solid dosage form to obtain an enterically coated solid dosage form. In some embodiments, the solid dosage form is a tablet. In some embodiments, the solid dosage form is a minitablet.
[87] In certain aspects, provided herein are methods of preparing a solid dosage form, the method comprising combining (i) a pharmaceutical agent (e.g., bacteria disclosed herein and/or an agent of bacterial origin, such as mEVs disclosed herein) (e.g., comprising at least one component of soy leghemoglobin), and (ii) a diluent, lubricant, and/or glidant, e.g., into a pharmaceutical composition. In some embodiments, the method comprises blending. In some embodiments, the method further comprises loading the pharmaceutical composition into a capsule. In some embodiments, the capsule comprises HPMC. [88] In some embodiments, the method further comprises banding the capsule. In some embodiments, the capsule is banded with an HPMC-based banding solution.
[89] In some embodiments, the method further comprises the step of enterically coating the solid dosage form to obtain an enterically coated solid dosage form. In some embodiments, the solid dosage form is a capsule.
[90] In certain aspects, provided herein is a method of testing a pharmaceutical agent comprising bacteria (e.g., bacteria provided herein) and/or mEVs (e.g., mEVs provided herein) (e.g., a pharmaceutical agent provided herein), the method comprising performing an assay to detect the presence of soy leghemoglobin in the pharmaceutical agent.
[91] In certain aspects, provided herein is a method of testing a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement)comprising bacteria (e.g., bacteria provided herein) and/or mEVs (e.g., mEVs provided herein), the method comprising performing an assay to detect the presence of a component of soy leghemoglobin in the bacterial composition.
[92] In certain embodiments, the component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid. In some embodiments, the soy leghemoglobin nucleic acid is soy leghemoglobin DNA. In some embodiments, the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin A (LB A). In some embodiments, the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin C2 (LGB2). In some embodiments, the assay to detect the presence of a component of soy leghemoglobin is a nucleic acid amplification assay, a sequencing assay, and/or a microarray assay. In some embodiments, the assay to detect the presence of a component of soy leghemoglobin is a polymerase chain reaction (PCR) assay, such as a quantitative polymerase chain reaction (qPCR) assay or digital PCR.
[93] In certain embodiments, the component of soy leghemoglobin is a soy leghemoglobin protein. In some embodiments, the soy leghemoglobin protein is soy leghemoglobin A (LB A). In some embodiments, the soy leghemoglobin protein is a sequence encoding soy leghemoglobin C2 (LGB2). In some embodiments, the soy leghemoglobin protein is detected using an antibody specific for the soy leghemoglobin protein, HPLC or UPLC. [94] In certain aspects, provided herein are soy leghemoglobins that can be used in growth media to facilitate the in vitro culturing of hemoglobin-dependent bacteria, including bacteria of the genus Prevotella, such as Prevotella histicola. The soy leghemoglobin provided herein can allow for large-scale manufacture and/or GMP grade manufacture of hemoglobin-dependent bacteria, e.g., for therapeutic use and/or in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement). In some embodients, soy leghemoglobin is purified from soy roots or soy root nodules. In some embodiments, soy leghemoglobin provided herein are recombinantly expressed. Notably, recombinant expression of the disclosed soy leghemoglobin facilitates, inter alia, the cost-effective, vegetarian, kosher, and GMP-grade production of soy leghemoglobin; it is an attractive way to produce soy leghemoglobin for use in bacterial cell culture applications.
[95] In certain aspects, provided herein are methods and compositions that facilitate the culturing of hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use). Growth of hemoglobin-dependent bacteria is accomplished through the inclusion in the cell culture media of a soy leghemoglobin provided herein.
[96] Thus, in certain aspects, provided herein are methods and compositions for culturing hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) in growth media that includes a soy leghemoglobin provided herein. In some aspects, provided herein are compositions (e.g., growth media) comprising a soy leghemoglobin provided herein that are useful for culturing hemoglobin-dependent bacteria in conditions free from conventional hemoglobin (e.g., soy leghemoglobin) (e.g., conditions not comprising a hemoglobin sourced from an animal) or a derivative thereof, as well as methods of making and/or using such compositions.
[97] Exemplary heme -containing polypeptide and nucleic acid sequences useful in the methods and compositions provided herein are listed, for example, in Table 3. In some embodiments, the heme -containing polypeptide comprises an amino acid sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity to an amino acid sequence of SEQ ID NO: 4, 5, 114, or 115. In some embodiments, the heme-containing polypeptide comprises the amino acid sequence of SEQ ID NO: 4, 5, 114, or 115.
[98] In some embodiments, the soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
[99] In some embodiments, the soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
[100] In some embodiments, the soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
[101] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes an amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
[102] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
[103] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
[104] In some embodiments, the component of soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
[105] In some embodiments, the component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
[106] In some embodiments, the component of soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
[107] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 5 or GenBank:
NP 001235928.1. [108] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
[109] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
[110] In some embodiments, the soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN) .
[111] In some embodiments, the soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN) .
[112] In some embodiments, the soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
[113] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN) .
[114] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _SOYBN).
[115] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _SOYBN).
[116] In some embodiments, the soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
[117] In some embodiments, the soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
[118] In some embodiments, the soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN). [119] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN ).
[120] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN ).
[121] In some embodiments, the component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN ).
[122] In certain embodiments, the soy leghemoglobin provided herein is recombinantly expressed. In some embodiments, the recombinant soy leghemoglobin provided herein further comprises a heterologous polypeptide, such as a histidine tag, TAP (tandem affinity purification) tag, TEV cleavage site, a FLAG tag, a GST tag, and/or an immunoglobulin domain. In some embodiments, such heterologous polypeptide enhances or facilitates purification and/or detection of the heme-containing polypeptide. In some embodiments, the recombinant polypeptide is expressed in a host cell (e.g., from an exogenous nucleic acid, such as an expression vector present in the host cell). In some embodiments, the host cells are bacteria cells, yeast cells, insect cells, or mammalian cells (e.g., a mammalian cell line). In some embodiments, the host cells are yeast cells. In some embodiments, the host cell is Pichia Pastoris. It will be understood that Pichia pastoris has been reclassified as Komagataella species, such as Komagataella phaffii, Komagataella pastoris, or Komagataella pseudopastoris, though the term “ Pichia pastoris ” is still in use and may refer to any appropriate Komagataella species. Representative recombinant heme- containing polypeptides using Pichia Pastoris host cells are disclosed in U.S. Patent Nos. 9,938,327; 10,273,492; 10,798,958, which are hereby incorporated by reference herein in their entireties. In some embodiments, the host cell is Escherichia coli.
[123] In certain aspects, provided herein is a growth medium for use in culturing hemoglobin-dependent bacteria (e.g., for use in abacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use), the growth medium comprising a soy leghemoglobin provided herein. In some embodiments, the growth medium comprises hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use). In certain embodiments, provided herein is a soy leghemoglobin for use as a substitute for conventional hemoglobin or a derivative thereof in a growth medium for hemoglobin-dependent bacteria.
[124] In certain aspects, provided herein is a method of culturing hemoglobin- dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use), the method comprising incubating the hemoglobin-dependent bacteria in a growth medium that comprises a soy leghemoglobin provided herein. In some aspects, provided herein is a method of culturing hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use), the method comprising (a) adding a soy leghemoglobin provided herein and hemoglobin- dependent bacteria to a growth medium; and (b) incubating the hemoglobin-dependent bacteria in the growth medium.
[125] In certain aspects, provided herein is a composition comprising a growth medium comprising a soy leghemoglobin provided herein and hemoglobin-dependent bacteria.
[126] In certain aspects, provided herein is a bioreactor comprising hemoglobin- dependent bacteria (e.g, for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) in a growth medium comprising a soy leghemoglobin provided herein. In some embodiments, provided herein is a method of culturing hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use), the method comprising comprises incubating the hemoglobin-dependent bacteria in a bioreactor provided herein.
[127] In some embodiments, the growth medium comprises at least 0.001 g/L, at least 0.005 g/L, at least 0.01 g/L, at least 0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, at least 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L, at least 0.1 g/L, at least 0.2 g/L, at least 0.3 g/L, at least 0.4 g/L, at least 0.5 g/L, at least 0.75 g/L, at least 1 g/L, at least 1.25 g/L, at least 1.5 g/L, at least 1.75 g/L, at least 2 g/L, at least 2.25 g/L, at least 2.5 g/L, at least 2.75 g/L, at least 3 g/L, at least 3.25 g/L, at least 3.5 g/L, at least 3.75 g/L, at least 4 g/L, or at least 4.25 g/L of a soy leghemoglobin provided herein. In some embodiments, the growth medium comprises at least 0.005 g/L and no more than 1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.02 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.05 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.2 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.5 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises yeast extract, soy peptone A2SC 19649, Soy peptone El 10 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, glucidex 21 D, and/or glucose. In some embodiments, the growth media comprises about 5 g/L glucose, about 10 g/L yeast extract 19512, about 10 g/L soy peptone A2 SC 19649, about 10 g/L soypeptone El 10 19885, about 2.5 g/L dipotassium phosphate K2HP04, and about 0.5 g/L L-cysteine-HCl. In some embodiments, the growth medium is at a pH of 5.5 to 7.5. In certain embodiments, the growth medium is at a pH of about 6.5. In some embodiments of the methods and compositions provided herein, the growth medium does not comprise hemoglobin or a derivative thereof. In certain embodiments, the growth medium does not comprise animal products. In certain embodiments, the growth medium does not comprise a heme-containing polypeptide sourced from an animal.
[128] In some embodiments of the methods and compositions provided herein, the hemoglobin-dependent bacteria ( e.g ., for use in abacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) can be any bacteria that require the presence of hemoglobin or a hemoglobin derivative for optimal growth (i.e. for optimal growth in the absence of a soy leghemoglobin provided herein). In some embodiments of the methods and compositions provided herein, the hemoglobin- dependent bacteria are bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella. In some embodiments, the hemoglobin- dependent bacteria are of the genus Prevotella. In some embodiments, the hemoglobin- dependent bacteria are of the species Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopes, Prevotella shahii, Prevotella zoogleof ormans, or Prevotella veroralis. In some embodiments, the hemoglobin- dependent bacteria are of the species Alistipes indistinctus, Alistipes shahii, Alistipes timonensis, Bacillus coagulans, Bacteroides acidifaciens, Bacteroides cellulosilyticus, Bacteroides eggerthii, Bacteroides intestinalis, Bacteroides uniformis, Collinsella aerofaciens, Cloacibacillus evryensis, Clostridium cadaveris, Clostridium cocleatum, Cutibacterium acnes, Eisenbergiella sp., Erysipelotrichaceae sp., Eubacterium hallii/Anaerobutyricum halii, Eubacterium infirmum, Megasphaera micronuciformis, Parabacteroides distasonis, Peptoniphilus lacrimalis, Rarimicrobium hominis, Shuttleworthia satelles, or Turicibacter sanguinis.
[129] In some embodiments of the methods and compositions provided herein, the hemoglobin-dependent bacteria ( e.g ., for use in bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) are a strain of the species Prevotella histicola. In some embodiments, the Prevotella histicola strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g, at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain B 50329. In certain embodiments, the Prevotella histicola strain is a strain that comprises at least at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329). In certain embodiments, the Prevotella histicola strain is a strain that comprises at least at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329). In certain embodiments, the Prevotella histicola strain is Prevotella Strain B 50329 (NRRL accession number B 50329).
[130] In some embodiments, the Prevotella histicola strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain C (ATCC Deposit Number PTA-126140, deposited on September 10, 2019). In certain embodiments, the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Prevotella Strain C (PTA-126140). In certain embodiments, the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Prevotella Strain C (PTA-126140). In certain embodiments, the Prevotella histicola strain is Prevotella Strain C (PTA-126140).
[131] In some embodiments, the hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) are a strain of Prevotella bacteria comprising one or more proteins listed in Table 1. In some embodiments, the hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) are from a strain of Prevotella substantially free of one or more of the proteins listed in Table 2.
[132] In some embodiments, the growth medium comprises at least 0.001 g/L, at least 0.005 g/L, at least 0.01 g/L, at least 0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, at least 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L, at least 0.1 g/L, at least 0.2 g/L, at least 0.3 g/L, at least 0.4 g/L, at least 0.5 g/L, at least 0.75 g/L, at least 1 g/L, at least 1.25 g/L, at least 1.5 g/L, at least 1.75 g/L, at least 2 g/L, at least 2.25 g/L, at least 2.5 g/L, at least 2.75 g/L, at least 3 g/L, at least 3.25 g/L, at least 3.5 g/L, at least 3.75 g/L, at least 4 g/L, or at least 4.25 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises at least 0.005 g/L and no more than 1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.05 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.02 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.2 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.5 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 1 g/L of a soy leghemoglobin provided herein. In some embodiments, the growth medium comprises yeast extract, soy peptone A2SC 19649, Soy peptone El 10 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, glucidex 21 D, and/or glucose. In some embodiments, the growth media comprises about 5 g/L glucose, about 10 g/L yeast extract 19512, about 10 g/L soy peptone A2 SC 19649, about 10 g/L soypeptone El 10 19885, about 2.5 g/L dipotassium phosphate K2HP04, and about 0.5 g/L L-cysteine-HCl. In some embodiments, the growth medium is at a pH of 5.5 to 7.5. In certain embodiments, the growth medium is at a pH of about 6.5. [133] In some embodiments of the methods and compositions provided herein, the growth medium does not comprise conventional hemoglobin (e.g., comprises soy leghemoglobin) (e.g., not comprising a hemoglobin sourced from an animal) or a derivative thereof. In certain embodiments, the growth medium does not comprise animal products. In certain embodiments, the growth medium does not comprise a heme-containing polypeptide sourced from an animal.
[134] In some embodiments of the methods and compositions provided herein, the hemoglobin-dependent bacteria (e.g., for use in abacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) grow at an increased rate in the growth medium comprising a soy leghemoglobin provided herein compared to the rate at which the hemoglobin-dependent bacteria grow in the same growth medium but without the soy leghemoglobin . In some embodiments, the rate at which the hemoglobin-dependent bacteria grow in the growth medium comprising a soy leghemoglobin provided herein is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least
160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least
220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least
280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least
340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, or at least
400% higher than the rate at which the hemoglobin-dependent bacteria grow in the same growth medium but without the soy leghemoglobin. In some embodiments, the growth rate is increased by 200% to 400%.
[135] In certain embodiments of the methods and compositions provided herein the hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) grow to a higher cell density in the growth medium comprising a soy leghemoglobin provided herein, compared to the cell density to which the hemoglobin-dependent bacteria grow in the same growth medium but without the soy leghemoglobin. In some embodiments, the hemoglobin-dependent bacteria grow to a cell density in the growth medium comprising a soy leghemoglobin provided herein that is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, or at least 400% higher than the cell density to which the hemoglobin-dependent bacteria grow in the same growth medium but without the soy leghemoglobin. In some embodiments, the bacterial cell density is 200% to 400% higher.
[136] In certain aspects, provided herein is a composition comprising hemoglobin- dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use) disclosed herein and a heme-containing polypeptide soy leghemoglobin disclosed herein.
BRIEF DESCRIPTION OF FIGURES
[137] FIG. 1 shows the growth curves for Strain A ( Prevotella histicola strain B 50329 (NRRL accession number B 50329)) with various sources of hemoglobin.
[138] FIG. 2 shows the growth curves for Strain A ( Prevotella histicola strain B 50329 (NRRL accession number B 50329)) with various sources and concentrations of hemoglobin.
[139] FIG. 3 shows the growth curves for Parahacteroides Strain A with various sources and concentrations of hemoglobin.
[140] FIG. 4 shows the growth curves for Bacteroides Strain B with various sources and concentrations of hemoglobin.
[141] FIG. 5 shows the growth curves for Alistipes Strain C with various sources and concentrations of hemoglobin.
[142] FIG. 6 shows an exemplary qPCR standard curve for the detection of soy leghemoglobin with TaqMan qPCR assay.
DETAILED DESCRIPTION
[143] In certain aspects, provided herein are bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) comprising bacteria (or components thereof, such as mEVs) and at least one component of soy leghemoglobin (e.g., a nucleic acid soy leghemoglobin component and/or a protein soy leghemoglobin component). In certain aspects, provided herein are methods of making and/or using such compositions. In some aspects, provided herein are methods of determining the presence and/or amount of at least one component of soy leghemoglobin in such a composition.
[144] In certain aspects, provided herein are methods and compositions that allow for the culturing of hemoglobin-dependent bacteria (e.g., for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use). Specifically, disclosed herein are a soy leghemoglobin that can be added to culture media to facilitate the growth of hemoglobin-dependent bacteria. These heme -containing polypeptides may be purified directly from various organisms, or a processed form thereof (e.g., flour, protein hydrolysate, meals, etc.). Alternatively, the soy leghemoglobinmay be recombinantly produced, e.g., recombinantly expressed in a host cell.
[145] Thus, in certain aspects, provided herein are methods and compositions for culturing hemoglobin-dependent bacteria in growth media that includes a soy leghemoglobin provided herein. In some aspects, provided herein are compositions (e.g., growth media) comprising a soy leghemoglobin provided herein that are useful for culturing hemoglobin-dependent bacteria, as well as methods of making and/or using such compositions.
Definitions
[146] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.
[147] As used herein, “anaerobic conditions” are conditions with reduced levels of oxygen compared to normal atmospheric conditions. For example, in some embodiments anaerobic conditions are conditions wherein the oxygen levels are partial pressure of oxygen (pCh) no more than 8%. In some instances, anaerobic conditions are conditions wherein the pO2 is no more than 2%. In some instances, anaerobic conditions are conditions wherein the pO2 is no more than 0.5%. In certain embodiments, anaerobic conditions may be achieved by purging a bioreactor and/or a culture flask with a gas other than oxygen such as, for example, nitrogen and/or carbon dioxide (CO2).
[148] As used herein, the term “bacterial composition” includes compositions comprising bacteria and/or components thereof, such as mEVs. In some embodiments, the bacterial composition can be a pharmaceutical composition, a pharmaceutical agent, and/or a solid dosage form. In some embodiments, the bacterial composition can be a medicinal product, a medical food, a food product, and/or a dietary supplement. As provided herein, disclosure related to pharmaceutical agents, solid dosage forms, and/or pharmaceutical compositions and methods of preparing and/or evaluating the same also relate to other types of bacterial compositions as well as of preparing and/or evaluating bacterial composition generally.
[149] As used herein, “derivatives” of hemoglobin include compounds that are derived from hemoglobin that can facilitate growth of hemoglobin-dependent bacteria. Examples of derivatives of hemoglobin include hemin and protoporphyrin.
[150] The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. The term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
[151] As used herein, “heme-containing polypeptide” refers to a polypeptide that covalently or noncovalently binds to a heme moiety.
[152] “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, T, et al, Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al, J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al (1988) SIAM J Applied Math 48: 1073). For example, the BEAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)).
[153] “Microbiome” broadly refers to the microbes residing on or in body site of a subject or patient. Microbes in a microbiome may include bacteria, viruses, eukaryotic microorganisms, and/or viruses. Individual microbes in a microbiome may be metabolically active, dormant, latent, or exist as spores, may exist planktonically or in biofdms, or may be present in the microbiome in sustainable or transient manner. The microbiome may be a commensal or healthy-state microbiome or a disease-state microbiome. The microbiome may be native to the subject or patient, or components of the microbiome may be modulated, introduced, or depleted due to changes in health state (e.g., precancerous or cancerous state) or treatment conditions (e.g., antibiotic treatment, exposure to different microbes). In some aspects, the microbiome occurs at a mucosal surface. In some aspects, the microbiome is a gut microbiome. In some aspects, the microbiome is a tumor microbiome.
[154] “Non-animal-derived polypeptide” refers to a polypeptide that is not purified from an animal source. A non-animal -derived polypeptide includes both polypeptides purified from non-animal sources and polypeptides that are recombinantly expressed. Thus, for example, the term “non-animal-derived polypeptide” would include a polypeptide that is recombinantly expressed from a sequence that encodes an animal polypeptide, e.g., corresponds to a polypeptide encoded by an animal gene.
[155] “Strain” refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species. The genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic signatures between different strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively. Hemoglobin-dependent Bacteria
[156] In some aspects, provided herein are methods and compositions for culturing hemoglobin-dependent bacteria. The hemoglobin-dependent bacteria may be for use in a bacterial composition (e.g., pharmaceutical composition, pharmaceutical agent, solid dosage form, medicinal product, medical food, food product, and/or dietary supplement) and/or for therapeutic use (e.g., in a subject, e.g., human). As used herein, “hemoglobin dependent bacteria” (also referred to herein as “hemoglobin-dependent bacteria”) refers to bacteria for which growth rate is slowed and/or maximum cell density is reduced when cultured in growth media lacking conventional hemoglobin (e.g., soy leghemoglobin), a hemoglobin derivative or a heme-containing polypeptide disclosed herein when compared to the same growth media containing conventional hemoglobin (e.g., soy leghemoglobinf), a hemoglobin derivative or a heme-containing polypeptide disclosed herein. In some embodiments, the hemoglobin-dependent bacteria are bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
[157] In some embodiments, the hemoglobin-dependent bacteria are of the genus Prevotella. In some embodiments, the hemoglobin-dependent bacteria are of the species Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola,
Prevotella melanogenica, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopes, Prevotella shahii, Prevotella zoogleof ormans, or Prevotella veroralis. [158] In some embodiments, the hemoglobin-dependent bacteria are of the species Alistipes indistinctus, Alistipes shahii, Alistipes timonensis, Bacillus coagulans, Bacteroides acidifaciens, Bacteroides cellulosilyticus, Bacteroides eggerthii, Bacteroides intestinalis, Bacteroides uniformis, Collinsella aerofaciens, Cloacibacillus evryensis, Clostridium cadaveris, Clostridium cocleatum, Cutibacterium acnes, Eisenbergiella sp., Erysipelotrichaceae sp., Eubacterium hallii/Anaerobutyricum halii, Eubacterium infirmum, Megasphaera micronuciformis, Parabacteroides distasonis, Peptoniphilus lacrimalis, Rarimicrobium hominis, Shuttleworthia satelles, or Turicibacter sanguinis.
[159] In some embodiments, the hemoglobin-dependent Prevotella strain is Prevotella Strain B 50329 (NRRL accession number B 50329). In some embodiments, the hemoglobin-dependent P evotella strain is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity ( e.g ., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain B 50329.
[160] In some embodiments, the Prevotella histicola strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to a nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Prevotella Strain C (ATCC Deposit Number PTA-126140, deposited on September 10, 2019). In certain embodiments, the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) to the genomic sequence of the Prevotella Strain C (PTA-126140). In certain embodiments, the Prevotella histicola strain is a strain that comprises at least 99% sequence identity (e.g., at least 99.1% sequence identity, at least 99.2% sequence identity, at least 99.3% sequence identity, at least 99.4% sequence identity, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9%, or 100% sequence identity) of thel6S sequence of the Prevotella Strain C (PTA-126140). In certain embodiments, the Prevotella histicola strain is Prevotella Strain C (PTA-126140).
[161] In some embodiments, the hemoglobin-dependent Prevotella strain is a strain of Prevotella bacteria comprising one or more (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more) proteins listed in Table 1 and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more) genes encoding proteins listed in Table 1. In some embodiments, the hemoglobin- dependent Prevotella strain comprises all of the proteins listed in Table 1 and/or all of the genes encoding the proteins listed in Table 1.
Table 1: Exemplary Prevotella proteins
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
[162] In some embodiments, the Prevotella bacteria is a strain of Prevotella bacteria free or substantially free of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) proteins listed in Table 2 and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) genes encoding proteins listed in Table 2. In some embodiments, Prevotella bacteria is free of all of the proteins listed in Table 2 and/or all of the genes encoding the proteins listed in Table 2.
[163] Table 2: Other Prevotella proteins
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
[164] In some embodiments, the hemoglobin-dependent Prevotella strain is a strain of Prevotella bacteria comprising one or more of the proteins listed in Table 1 and that is free or substantially free of one or more proteins listed in Table 2. In some embodiments, the hemoglobin-dependent Prevotella strain is a strain of Prevotella bacteria that comprises all of the proteins listed in Table 1 and/or all of the genes encoding the proteins listed in Table 1 and that is free of all of the proteins listed in Table 2 and/or all of the genes encoding the proteins listed in Table 2.
[165] Under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, the Prevotella Strain C. was deposited on September 10, 2019, with the American Type Culture Collection (ATCC) of 10801 University Boulevard, Manassas, Va. 20110-2209 USA and was assigned ATCC Accession Number PTA-126140.
[166] Applicant represents that the ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited plasmid, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicant acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
Components of Soy Leghemoglobin
[167] Soy leghemoglobin can be used in growth media to facilitate the in vitro culturing of bacteria.
[168] As demonstrated herein, culturing bacteria in growth media comprising soy leghemoglobin results in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) comprising said bacteria or agents therefrom (e.g., mEVs), and comprising soy leghemoglobin or components thereof. The present disclosure provides such compositions and use thereof, as well as methods to test said compositions to detect the presence of soy leghemoglobin or components thereof.
[169] Various components of soy leghemoglobin are present and can be detected in the bacterial compositions described herein, including soy leghemoglobin nucleic acids and/ soy leghemoglobin proteins.
[170] In some embodiments, the component of soy leghemoglobin is a soy leghemoglobin nucleic acid. As used herein, the soy leghemoglobin nucleic acid is intended to include DNA (e.g., genomic DNA, cDNA) and RNA (e.g, mRNA, tRNA, rRNA, coding RNA, non-coding RNA, small RNA, etc.). The nucleic acid molecule can be single- stranded or double-stranded. In certain embodiments, the soy leghemoglobin nucleic acid is soy leghemoglobin genomic DNA. The soy leghemoglobin nucleic acid can comprise a coding sequence (e.g., a sequence encoding a soy leghemoglobin protein). In some embodiments, the soy leghemoglobin nucleic acid comprises only a portion of a sequence encoding a soy leghemoglobin protein. In some embodiments, the soy leghemoglobin nucleic acid comprises a non-coding sequence.
[171] In certain embodiments, the nucleic acid is soy leghemoglobin DNA. In some embodiments, the soy leghemoglobin DNA comprises a sequence encoding a soy leghemoglobin protein. In some embodiments, the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin A (LB A). In some embodiments, the soy leghemoglobin DNA comprises a sequence encoding soy leghemoglobin C2 (LGB2).
[172] In certain embodiments, the bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)of the present disclosure comprise a soy leghemoglobin protein. In some embodiments, the soy leghemoglobin protein is soy leghemoglobin A (LB A). In some embodiments, the soy leghemoglobin protein is a sequence encoding soy leghemoglobin C2 (LGB2).
[173] Exemplary nucleic sequences of components of soy leghemoglobin are presented below in Table 3.
[174] As demonstrated herein, culturing bacteria in growth media comprising soy leghemoglobin results in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) comprising said bacteria or agents therefrom (e.g., mEVs), and comprising soy leghemoglobin. The present disclosure provides such compositions and use thereof, as well as methods to test said compositions to detect the presence of soy leghemoglobin.
[175] In certain aspects, the methods and compositions provided herein relate to methods of testing bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)to detect (e.g., assay for) the presence of a soy leghemoglobin (or fragments thereof) in the composition. Any suitable methods described herein or those known in the art can be used to detect a soy leghemoglobin. In certain embodiments, samples for detection can be readily prepared by dissolving the composition in an appropriate buffer/medium prior to testing.
[176] Provided herein are soy leghemoglobins that are able to be used in culture media to facilitate the growth of otherwise hemoglobin-dependent bacteria (e.g., for use in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) and/or for therapeutic use). Soy leghemoglobin is a polypeptide that is capable of binding covalently and/or noncovalently to a heme moiety. In some embodiments, the soy leghemoglobin comprises a heme moiety. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or
100% of the soy leghemoglobins used in the methods and/or compositions provided herein are bound by heme. In some embodiments, about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the soy leghemoglobins used in the methods and/or compositions provided herein are bound by heme.
[177] In some embodiments, the soy leghemoglobin is a globin and can include a globin fold, which comprises a series of seven to nine alpha helices. Globin type proteins can be of any class (e.g., class I, class II, or class III), and in some embodiments, can transport or store oxygen. For example, a soy leghemoglobin can be a non-symbiotic type of hemoglobin or a leghemoglobin. A soy leghemoglobin can be a monomer, i.e., a single polypeptide chain, or can be a dimer, a trimer, tetramer, and/or higher order oligomers.
[178] Non-limiting examples of non-animal hemoglobins can include an androglobin, a cytoglobin, a globin E, a globin X, a globin Y, a hemoglobin, a leghemoglobin, a flavohemoglobin, Hell's gate globin I, a myoglobin, an erythrocruorin, a beta hemoglobin, an alpha hemoglobin, a protoglobin, a cyanoglobin, a cytoglobin, a histoglobin, a neuroglobins, a chlorocruorin, a truncated hemoglobin (e.g., HbN or HbO), a truncated 2/2 globin, a hemoglobin 3 (e.g. , Glb3), a cytochrome, or a peroxidase.
[179] In some embodiments, the soy leghemoglobin may be purified from plants (e.g., legumes). In some embodiments, the soy leghemoglobin is that of an organism of the genus Glycine. In some embodiments, soy leghemoglobin is recombinantly expressed. [180] In certain embodiments, soy leghemoglobins can be from a legume such as Glycine max (soybean); sometimes referred to Glycine hispida. In some embodiments, the soy leghemoglobinis recombinantly expressed.
[181] In certain embodiments, soy leghemoglobins can be a non-symbiotic hemoglobin. The non-symbiotic hemoglobin can be from any plant. In some embodiments, a non-symbiotic hemoglobin can be from a plant selected from the group consisting of soybean, sprouted soybean, alfalfa, golden flax, black bean, black eyed pea, northern bean, tobacco, pea, garbanzo, moong bean, cowpeas, pinto beans, pod peas, quinoa, sesame, sunflower, wheat berries, spelt, barley, wild rice, and rice. In some embodiments, the soy leghemoglobinis recombinantly expressed.
[182] In certain embodiments, recombinantly expressed non-animal hemoglobin can be isolated from fungi such as Saccharomyces cerevisiae, Pichia pastoris,
Magnaporthe oryzae, Fusarium graminearum, or Fusarium oxysporum. In certain embodiments, the non-animal hemoglobin is recombinantly expressed in Pichia pastoris.
[183] In certain embodiments, recombinantly expressed non-animal hemoglobin can be isolated from one or more bacteria such as Escherichia coli, Bacillus suhtilis, Bacillus megaterium, Synechocistis sp., Bacillus suhtilis, Aquifex aeolicus, Methylacidiphilum infernorum (Hell’s Gate), or thermophilic bacteria (e.g, that grow at temperatures greater than 45°C) such as Thermophilus or Thermohifidafusca.
[184] In certain embodiments, recombinantly expressed non-animal hemoglobin can be isolated from algae such as Chlamydomonas eugametos.
[185] In certain embodiments, recombinantly expressed non-animal hemoglobin can be isolated from protozoans such as Paramecium caudatum or Tetrahymena pyriformis.
[186] The sequences and structure of numerous non-animal hemoglobins are known. See for example, Reedy, et a , Nucleic Acids Research, 2008, Vol. 36, Database issue D307-D313 and the Heme Protein Database available on the world wide web at http://hemeprotein.info/heme.php. See also WO2014/110539 and WO2014/110532.
[187] In some embodiments, the soy leghemoglobin is symbiotic.
[188] The present disclosure also contemplates recombinantly expressed heme- containing polypeptides, such as those found in plants, bacteria, cyanobacteria, fungus, algae, grain, legume, and/or protozoa.
[189] In certain embodiments, the soy leghemoglobin is a polypeptide listed in Table 3. In certain embodiments, the soy leghemoglobinis a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with an amino acid sequence listed in Table 3. In certain embodiments, soy leghemoglobin is a polypeptide encoded by a cDNA sequence listed in Table 3. In certain embodiments, the soy leghemoglobinis a polypeptide encoded by a cDNA sequence having at least 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with a cDNA sequence listed in Table 3. In some embodiments, the soy leghemoglobin is recombinantly expressed.
[190] In certain embodiments, the soy leghemoglobin is a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with
SEQ ID NO: 4 or GenBank: NP_001235248.2.
[191] In certain embodiments, the component of soy leghemoglobin is a nucleic acid that encodes the amino acid sequence sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with SEQ ID NO: 4 or GenBank: NP_001235248.2.
[192] In certain embodiments, the component of soy leghemoglobin is a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with SEQ ID NO: 5 or GenBank: NP_001235928.1.
[193] In certain embodiments, the component of soy leghemoglobin is a nucleic acid that encodes the amino acid sequence sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with SEQ ID NO: 5 or GenBank: NP_001235928.1.
[194] In certain embodiments, the soy leghemoglobin is a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
[195] In certain embodiments, the soy component of leghemoglobin is a nucleic acid that encodes the amino acid sequence sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
[196] In certain embodiments, the soy leghemoglobin is a polypeptide comprising an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN ).
[197] In certain embodiments, the component of soy leghemoglobin is a nucleic acid that encodes the amino acid sequence sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN ).
[198] In making the changes in the amino sequences of a polypeptide, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (<RTI 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[199] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. [200] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well-known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
Table 3: cDNA and Amino Acid Sequences of Exemplary Soy Heme-containing Polypeptides
SEQ ID NO: 4 Glycine max (soybean) leghemoglobin C2 amino acid sequence (GenBank: NP_001235248.2)
1 mgaftekqea lvsssfeafk anipqysvvf ytsilekapa akdlfsflsn gvdpsnpklt 61 ghaeklfglv rdsagqlkan gtvvadaalg sihaqkaitd pqdfvvkeal lktikeavgd 121 kwsdelssaw evaydelaaa ikkaf
SEQ ID NO: 5 Glycine max (Soybean) ( Glycine hispida) leghemoglobin A amino acid sequence (GenBank: NP_001235928.1)
MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDP
TNPKLTGHAEKLFALVRDSAGQLKASGTVVADAALGSVHAQKAVTDPQFVVV
KEALLKTIKAAVGDKWSDELSRAWEVAYDELAAAIKKA
SEQ ID NO: 114 Glycine max (soybean) leghemoglobin C2 amino acid sequence (UniProtKB - P02236 (LGB2 _ SOYBN))
1 mgaftekqea lvsssfcafk anipqysvvf ytsilekapa akdlfsflsn gvdpsnpklt 61 ghaeklfglv rdsagqlkan gtvvadaalg sihaqkaitd pqfwvkeal lktikeavgd 121 kwsdelssaw evaydelaaa ikkaf
SEQ ID NO: 115 Glycine max (Soybean) ( Glycine hispida) leghemoglobin A amino acid sequence (UniProtKB - P02238 (LGBA _ SOYBN ))
MVAFTEKQDALVSSSFEAFKANIPQYSVVFYTSILEKAPAAKDLFSFLANGVDP
TNPKFTGHAEKFFAFVRDSAGQFKASGTVVADAAFGSVHAQKAVTDPQFVVV
KEAFFKTIKAAVGDKWSDEFSRAWEVAYDEFAAAIKKA Detection of Components of Soy Leghemoglobin
[201] In certain aspects, the methods and compositions provided herein relate to methods of testing bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) to detect (e.g., assay for) the presence of a component of soy leghemoglobin in the compositionAny suitable methods described herein or those known in the art can be used to detect a component of soy leghemoglobin. In certain embodiments, samples for detection can be readily prepared by dissolving the composition form in an appropriate buffer/medium prior to testing.
[202] In some embodiments, the component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid. In some embodiments, the soy leghemoglobin nucleic acid is soy leghemoglobin DNA. In some embodiments, the soy leghemoglobin DNA comprises a sequence from soy leghemoglobin A (LB A). In some embodiments, the soy leghemoglobin DNA comprises a sequence from soy leghemoglobin C2 (LGB2). In certain embodiments, the soy leghemoglobin nucleic acid (e.g., DNA) is detected using a nucleic acid amplification assay, a sequencing assay, and/or a microarray assay. In preferred embodiments, the presence of a component of soy leghemoglobin (e.g., nucleic acid) is detected using a polymerase chain reaction (PCR) assay, such as quantitative polymerase chain reaction (qPCR) assay or digital PCR.
[203] In some embodiments, the component of soy leghemoglobin is a soy leghemoglobin protein. In some embodiments, the soy leghemoglobin protein is Soy Leghemoglobin A. In preferred embodiments, the soy leghemoglobin protein is detected by high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC). In some embodiments, the soy leghemoglobin protein is detected by mass spectrometry (e.g., tandem mass spectrometry, MALDI-TOF).
Nucleic Acid Detection
[204] In certain aspects, the provided herein are methods related to the detection of soy leghemoglobin nucleic acids.
[205] In some embodiments, the soy leghemoglobin nucleic acid may be isolated, however, it will be appreciated by those skilled in the art that many detection assays (e.g., PCR, e.g., qPCR or digital PCR) can be performed to detect the nucleic acid in a sample without isolating the nucleic acid. A nucleic acid can be isolated using standard molecular biology techniques and the sequence information in the public database records, e.g., NCBI, Uniprot, etc. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the present invention can be isolated using standard hybridization and cloning techniques ( e.g ., as described in Green and Sambrook, ed., Molecular Cloning: A Laboratory Manual, Fourth ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2014).
[206] In other embodiments, amplification-based assays can be used to determine the presence of amount of the soy leghemoglobin DNA. In such amplification-based assays, the nucleic acid sequences act as a template in an amplification reaction (e.g., Polymerase Chain Reaction (PCR)). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample (see e.g., Kralik and Ricchi (2017) Front. Microbiol. 8:108). Comparison to appropriate controls, e.g. amplification of a bacterial nucleic acid sequence, provides a measure of the copy number or amount of the soy leghemoglobin nucleic acid.
[207] Methods of “quantitative” amplification are well-known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis is described in Ginzonger, el al. (2000) Cancer Research 60:5405-5409. The known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR may also be used in the methods of the present invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and SYBR green.
[208] Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science 241:1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh, et al. (1989 ) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, digital PCR, and linker adapter PCR, etc.
[209] Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. etal. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
[210] For determining the presence or amount of soy leghemoglobin RNA by amplification methods, the RNA molecules can first be converted to cDNA by a reverse transcriptase (primer-specific cDNA synthesis), prior to performing the amplification methods. Various amplification and detection methods can be used. For example, it is within the scope of the present invention to reverse transcribe RNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, or reverse transcribe mRNA into cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall, etal., PCR Methods and Applications 4: 80-84 (1994). Real time PCR may also be used.
[211] Other known amplification methods which can be utilized herein include but are not limited to the so-called “NASBA” or “3SR” technique described in PNAS USA 87: 1874-1878 (1990) and also described in Nature 350 (No. 6313): 91-92 (1991); Q-beta amplification as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (as described in G. T. Walker et al., Clin. Chem. 42: 9-13 (1996) and European Patent Application No. 684315; target mediated amplification, as described by PCT Publication W09322461; PCR; ligase chain reaction (LCR) (see, e.g.. Wu and Wallace, Genomics 4, 560 (1989), Landegren etal, Science 241, 1077 (1988)); self-sustained sequence replication (SSR) (see, e.g., Guatelli etal., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)); and transcription amplification (see, e.g., Kwoh etal, Proc. Natl. Acad. Sci. USA 86, 1173 (1989)).
[212] In some embodiments, the presence of soy leghemoglobin nucleic acids can be detected in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) provided herein by sequencing nucleic acids present in the composition. In some embodiments, the sequencing assay used is a next generation sequencing (NGS) assay. Nucleic acid sequencing processes include, but are not limited to chain termination sequencing, sequencing by ligation, sequencing by synthesis, pyrosequencing, ion semiconductor sequencing, single-molecule real-time sequencing, and/or 454 sequencing. In some embodiments, the NGS Modality is any of the following: SwabSeq, 1 Amplicon, 384 well plate, 96 Nextera barcode set, UDI’s, NextSeq; SwabSeq - 1 Amplicon, 384 well plate, 384 Truseq UDI barcode set, using NextSeq; or SwabSeq - 1 Amplicon, 384 well plate, 4000 UDI Truseq barcode set, NovaSeq. SwabSeq - Multiplex, 384 well plate, CDI barcode set, NovaSeq.
[213] Many techniques are known in the state of the art for determining absolute and relative levels of a nucleic acid in a sample. In some embodiments, the presence and amount of soy leghemoglobin nucleic acid can be detected on a DNA array, chip or a microarray. Labeled nucleic acids of a test sample (those present in the composition) obtained from a subject may be hybridized to a solid surface comprising the soy leghemoglobin nucleic acid (e.g., DNA or RNA). Positive hybridization signal is obtained with the sample containing the soy leghemoglobin nucleic acid. Methods of preparing DNA arrays and their use are well-known in the art (see, e.g., U.S. Pat. Nos: 6,618,6796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S. 20030157485 and Schena et al. (1995) Science 20, 467-470; Gerhold et al. (1999) Trends In Biochem. Sci. 24, 168-173; and Lennon et al. (2000) Drug Discovery Today 5, 59-65, which are herein incorporated by reference in their entirety).
[214] Methods of detecting a soy leghemoglobin nucleic acid include, but are not limited to, hybridization-based assays. Hybridization-based assays include traditional “direct probe” methods, such as Southern blots or dot blots. The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g., membrane or glass) bound methods or array-based approaches.
[215] In some embodiments, detecting a soy leghemoglobin DNA in a sample involves a Southern Blot. In a Southern Blot, the genomic DNA or fragments thereof (typically separated on an electrophoretic gel) is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the soy leghemoglobin DNA with control probe signal from analysis of bacterial DNA provides an estimate of the amount of the soy leghemoglobin nucleic acid. To increase the specificity of the assay, a probe hybridizes the soy leghemoglobin DNA under stringent conditions. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%; preferably 85%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989). A preferred, non- limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C. The form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example, 32P and 35S. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of suitably labeled bases.
[216] Alternatively, a Northern blot may be utilized for evaluating the presence or amount of RNA in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)of the present disclosure. In a Northern blot, RNA is hybridized to a probe specific for the soy leghemoglobin RNA. Comparison of the intensity of the hybridization signal from the probe for the soy leghemoglobin RNA with control probe signal from analysis of bacterial RNA provides an estimate of the relative amount of the soy leghemoglobin RNA. A simpler version of the Southern blot and the Northern blot may be performed using a dot blot format, in which the sample comprising the soy leghemoglobin DNA is simply spotted (without electrophoretic separation) on a platform and the hybridization method is carried out.
Protein Detection
[217] In certain aspects, the methods and compositions provided herein relate to the detection of soy leghemoglobin proteins. The presence or amount of a soy leghemoglobin protein in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)s described herein can be detected by various methods known in the art. Exemplary methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluore scent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn. pp 217-262, 1991 which is incorporated by reference). Preferred are binder-ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.
[218] For example, ELISA and RIA procedures may be conducted such that a desired protein standard (e.g., a known soy leghemoglobin protein, the presence of which is being tested in a composition) is labeled (with a radioisotope such as 125I or 35S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample (e.g., the soy leghemoglobin protein present in the composition), brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, the soy leghemoglobin protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-biomarker proteinantibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable.
[219] The above techniques may be conducted essentially as a “one-step” or “two- step” assay. A “one-step” assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. A “two-step” assay involves washing before contacting, the mixture with labeled antibody. Other conventional methods may also be employed as suitable.
[220] Enzymatic and radiolabeling of soy leghemoglobin proteins and/or the antibodies may be effected by conventional means. Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.
[221] It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient. [222] It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support.
[223] Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.
[224] Other techniques may be used to detect a soy leghemoglobin protein according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti-soy leghemoglobin protein antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including 125I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
[225] Antibodies that may be used to detect a soy leghemoglobin protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the protein to be detected. An antibody may have a Kd of at most about 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, or 10-12 M. The phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant. An antibody may bind preferentially to the target soy leghemoglobin protein relative to other proteins, such as related proteins. Antibodies may be prepared according to methods known in the art.
[226] In some embodiments, agents that specifically bind to a soy leghemoglobin protein other than antibodies are used, such as peptides or small molecules. Peptides or small molecules that specifically bind to a biomarker protein can be identified by any means known in the art. For example, specific peptide binders of a target soy leghemoglobin protein can be screened for using peptide phage display libraries.
[227] A soy leghemoglobin protein or fragment thereof may also be detected using mass spectrometry and/or HPLC and/or UPLC as for detection of a small molecule as described below.
Small Molecule Detection
[228] In certain embodiments, the methods and compostitions provided herein relate to the detection of soy leghemoglobin small molecules.
[229] In certain embodiments a chromatography method is used to detect a soy leghemoglobin small molecule (or protein). Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), etc. In certain embodiments, the small molecule or protein detected using a chromatography method is a pigment
[230] Any one or combination of the methods decribed herein can be used to detect and quantify the amount of at least one component of soy leghemoglobin present in the bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) provided herein. In preferred embodiments, the chromatography is HPLC or UPLC. These methods provide sensitivity that allows separation and detection of a trace amount of at least one component of soy leghemoglobin present in the composition
[231] Small molecules, nucleic acids (see above for mass spectrometry-based sequencing), or proteins or fragments thereof of soy leghemoglobin can be detected and quantified using mass spectrometry with or without separation techniques. Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.
[232] A mass spectrum is a plot of the ion signal as a function of the mass-to- charge ratio. These spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical identity or structure of molecules and other chemical compounds.
[233] Various mass spectrometry-based methods can be utilized to detect the small molecules, nucleic acids, or proteins or fragments thereof of soy leghemoglobin including, but are not limited to, tandem mass spectrometry (MS/MS), MALDI-TOF (a combination of a matrix-assisted laser desorption/ionization source with a time-of-flight mass analyzer), inductively coupled plasma-mass spectrometry (ICP-MS), accelerator mass spectrometry (AMS), thermal ionization-mass spectrometry (TIMS), isotope ratio mass spectrometry (IRMS), and spark source mass spectrometry (SSMS).
[234] A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation. For example, one mass analyzer can isolate one peptide from many entering a mass spectrometer. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID). A third mass analyzer then sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer overtime, as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), blackbody infrared radiative dissociation (BIRD), electron-detachment dissociation (EDD) and surface-induced dissociation (SID). An important application using tandem mass spectrometry is in protein identification.
[235] Mass spectrometry-based detection of soy leghemoglobin small molecules, nucleic acids, or proteins or fragments thereof, can be enhanced by coupling it with chromatographic and/or other separation techniques. Separation may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip, liquid chromatography, gas chromatography). Electrophoresis is a method which can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip. Examples of gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof. A gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient. Examples of capillaries used for electrophoresis include capillaries that interface with an electrospray.
[236] Capillary electrophoresis (CE) is preferred for separating complex hydrophilic molecules and highly charged solutes. CE technology can also be implemented on microfluidic chips. Depending on the types of capillary and buffers used, CE can be further segmented into separation techniques such as capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (cITP) and capillary electrochromatography (CEC). An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and/or base and an organic such as an alcohol or acetonitrile.
[237] Capillary isotachophoresis (cITP) is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities. Capillary zone electrophoresis (CZE), also known as free-solution CE (FSCE), is based on differences in the electrophoretic mobility of the species, determined by the charge on the molecule, and the frictional resistance the molecule encounters during migration which is often directly proportional to the size of the molecule. Capillary isoelectric focusing (CIEF) allows weakly-ionizable amphoteric molecules, to be separated by electrophoresis in a pH gradient. CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.
Soy Leghemoglobin Detection
[238] In certain aspects, the methods and compositions provided herein relate to the detection of soy leghemoglobin (or fragments thereof). The presence or amount of a soy leghemoglobin in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein can be detected by various methods known in the art. Exemplary methods include, but are not limited to, immunodiffusion, Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluore scent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn pp 217-262, 1991 which is incorporated by reference). Preferred are binder-ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.
[239] In some embodiments, the soy leghemoglobin is detected by high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC). In some embodiments, the soy leghemoglobin is detected by mass spectrometry (e.g., tandem mass spectrometry, MALDI-TOF). In some embodiments, soy leghemoglobin is detected by enzyme-linked immunosorbent assay (ELISA).
[240] For example, ELISA and RIA procedures may be conducted such that a desired protein standard (e.g., a known soy leghemoglobin protein, the presence of which is being tested in a composition) is labeled (with a radioisotope such as 125I or 35S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample (e.g., the soy leghemoglobin protein present in the composition), brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, the soy leghemoglobin protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-biomarker proteinantibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable.
[241] The above techniques may be conducted essentially as a “one-step” or “two- step” assay. A “one-step” assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. A “two-step” assay involves washing before contacting, the mixture with labeled antibody. Other conventional methods may also be employed as suitable.
[242] Enzymatic and radiolabeling of soy leghemoglobin proteins and/or the antibodies may be detected by conventional means. Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be detected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.
[243] It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient.
[244] It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support.
[245] Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.
[246] Other techniques may be used to detect a soy leghemoglobin protein according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti- soy leghemoglobin protein antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including 125I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
[247] Antibodies that may be used to detect a soy leghemoglobin protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the biomarker protein to be detected. An antibody may have a Kd of at most about 10-6 M, 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, or 10-12M. The phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant. An antibody may bind preferentially to the target soy leghemoglobin protein relative to other proteins, such as related proteins. Antibodies may be prepared according to methods known in the art.
[248] In some embodiments, agents that specifically bind to a soy leghemoglobin protein other than antibodies are used, such as peptides or small molecules. Peptides or small molecules that specifically bind to a biomarker protein can be identified by any means known in the art. For example, specific peptide binders of a target soy leghemoglobin hemoglobin protein can be screened for using peptide phage display libraries.
[249] An soy leghemoglobin protein or fragments thereof may also be detected using mass spectrometry and/or HPLC and/or UPLC.
[250] In certain embodiments a chromatography method is used to detect a soy leghemoglobin (or fragments thereof). Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), etc.
[251] Any one or combination of the methods decribed herein can be used to detect and quantify the amount of soy leghemoglobin present in the bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) provided herein.
[252] In some embodiments, the chromatography is HPLC or UPLC. These methods provide sensitivity that allows separation and detection of a trace amount of soy leghemoglobin present in the composition.
[253] Small moleculesor proteins or fragments thereof of soy leghemoglobin can be detected and quantified using mass spectrometry with or without separation techniques. Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.
[254] A mass spectrum is a plot of the ion signal as a function of the mass-to- charge ratio. These spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical identity or structure of molecules and other chemical compounds.
[255] Various mass spectrometry-based methods can be utilized to detect the small molecules, or proteins or fragments thereof of soy leghemoglobin including, but are not limited to, tandem mass spectrometry (MS/MS), MALDI-TOF (a combination of a matrix- assisted laser desorption/ionization source with a time-of-flight mass analyzer), inductively coupled plasma-mass spectrometry (ICP-MS), accelerator mass spectrometry (AMS), thermal ionization-mass spectrometry (TIMS), isotope ratio mass spectrometry (IRMS), and spark source mass spectrometry (SSMS).
[256] A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation. For example, one mass analyzer can isolate one peptide from many entering a mass spectrometer. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID). A third mass analyzer then sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer overtime, as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), blackbody infrared radiative dissociation (BIRD), electron-detachment dissociation (EDD) and surface-induced dissociation (SID). An important application using tandem mass spectrometry is in protein identification.
[257] Mass spectrometry-based detection of soy leghemoglobin small molecules, or proteins or fragments thereof, can be enhanced by coupling it with chromatographic and/or other separation techniques. Separation may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip, liquid chromatography, gas chromatography). Electrophoresis is a method which can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip. Examples of gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof. A gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient. Examples of capillaries used for electrophoresis include capillaries that interface with an electrospray.
[258] Capillary electrophoresis (CE) is preferred for separating complex hydrophilic molecules and highly charged solutes. CE technology can also be implemented on microfluidic chips. Depending on the types of capillary and buffers used, CE can be further segmented into separation techniques such as capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (cITP) and capillary electrochromatography (CEC). An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and/or base and an organic such as an alcohol or acetonitrile.
[259] Capillary isotachophoresis (cITP) is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities. Capillary zone electrophoresis (CZE), also known as free-solution CE (FSCE), is based on differences in the electrophoretic mobility of the species, determined by the charge on the molecule, and the frictional resistance the molecule encounters during migration which is often directly proportional to the size of the molecule. Capillary isoelectric focusing (CIEF) allows weakly-ionizable amphoteric molecules, to be separated by electrophoresis in a pH gradient. CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.
[260] As described herein, isolated and purified soy leghemoglobins can be derived from non-animal sources such as plants, bacteria, cyanobacteria, fungus, algae, grain, legume, and/or protozoa. In some embodiments, the isolated and purified proteins can be obtained from genetically modified organisms such as genetically modified bacteria or yeast. In some embodiments, the isolated and purified polypeptides are chemically synthesized or obtained via in vitro synthesis. In some embodiments, the non-animal- derived polypeptide is recombinantly expressed.
[261] In some embodiments, a soy leghemoglobin can be recombinantly produced using polypeptide expression techniques (e.g., cloning and expressing an exogenous nucleic acid encoding a heme -containing polypeptide in host cells, such as bacterial cells (e.g., Escherichia coli), insect cells, fungal cells such as yeast cells (e.g., Pichia Pastoris), plant cells, or mammalian cells). The exogenous nucleic acid may be operably linked to a regulatory sequence (e.g., promoter). The exogenous nucleic acid may be in a vector, such as an expression vector. In some embodiments, the exogenous nucleic acid may be in a viral vector suitable for transmission of DNA into host cells (e.g., insect cells, mammalian cells). The recombinant polypeptides may be expressed and purified as an intracellular protein, a secreted protein, and/or an insoluble protein (e.g., contained in inclusion bodies), which can be solubilized using urea and slowly refolded in a native condition.
[262] In some embodiments, the recombinant proteins comprise a heterologous sequence (e.g., a histidine tag, TAP (tandem affinity purification) tag, TEV cleavage site, a FLAG tag, a GST tag, and/or an immunoglobulin domain) that may be useful in purification and/or detection of the polypeptide.
[263] In certain embodiments, soy leghemoglobin can be purified on the basis of their molecular weight, for example, by size exclusion chromatography, ultrafiltration through membranes, or density centrifugation. In some embodiments, the polypeptides can be separated based on their surface charge, for example, by isoelectric precipitation, anion exchange chromatography, or cation exchange chromatography. Polypeptides can also be separated on the basis of their solubility, for example, by ammonium sulfate precipitation, isoelectric precipitation, surfactants, detergents, or solvent extraction. Polypeptides can also be separated by their affinity to another molecule, using, for example, hydrophobic interaction chromatography, reactive dyes, or hydroxyapatite. Affinity chromatography can also use antibodies having specific binding affinity for the protein of interest, nickel NTA for His-tagged recombinant proteins, lectins to bind to sugar moieties on a glycoprotein, or other molecules which specifically bind the polypeptides of interest.
[264] In certain embodiments, soy leghemoglobin can be purified from e.g., whole roots or root nodules (e.g., soy roots or soy root nodules). Whole roots or root nodules can be harvested and lysed, for example in 20 mM potassium phosphate pH 7.4, 100 mM potassium chloride and 5 mM EDTA using a grinder-blender. During this process, leghemoglobin is released into the buffer. Root-nodule lysate containing leghemoglobin can be cleared from cell debris by filtration through 5 μm filter. In some embodiments, filtration is followed by centrifugation (7000 x g, 20 min). Clarified lysate containing leghemoglobin is then filtered through 200 nm filter and applied to an anion-exchange chromatography column (High Prep Q; High Prep DEAE, GE Healthcare) on a fast protein liquid chromatography machine (GE Healthcare). Leghemoglobin is collected in the flowthrough fractions and concentrated over 3 kDa filtration membrane to a desired concentration.
Purity (partial abundance) of the purified leghemoglobin is analyzed by SDS-PAGE gel: in the lysate, leghemoglobin can be present at 20-40 %, while after the anion-exchange purification, it can be present at 70-80 %. In other embodiments, soybean leghemoglobin flowthrough from the anion-exchange chromatography is applied onto size -exclusion chromatography (Sephacryl S-100 HR, GE Healthcare). Soybean leghemoglobin is eluted as two fractions corresponding to dimeric and monomeric species. Purity (partial abundance) of leghemoglobin is analyzed by SDS-PAGE and can be ~ 90-100 %.
[265] Proteins in legume root-nodule lysates can be transferred to 10 mM sodium carbonate pH 9.5, 50 mM sodium chloride buffer, filtered through 200 nm filter, and applied onto an anion-exchange chromatography column on a fast protein liquid chromatography instrument (GE Healthcare). Leghemoglobin can bind the anion exchange chromatography matrix and the bound leghemoglobin can be eluted using a sodium chloride gradient. Purity (partial abundance) of leghemoglobin can be analyzed by SDS- PAGE and can be ~ 60-80 %.
[266] Undesired small molecules from legume roots can be removed from purified leghemoglobin by passing the leghemoglobin solution over the anion-exchange resin. In some embodiments, the anion exchange resin is FFQ, DEAE, Amberlite IRA900, Dowex 22, or Dowex 1x4. Leghemoglobin purified either by ammonium sulfate fractionation (60 % wt/v and 90 % wt/v ammonium sulfate) or by anion-exchange chromatography is buffer exchanged into 20 mM potassium phosphate pH 7.4, 100 mM sodium chloride, and the solution is passed over one of the above mentioned anion-exchange resins. Flowthrough that contains leghemoglobin can be collected.
[267] In certain embodiments, standard polypeptide synthesis techniques (e.g. , liquid-phase polypeptide synthesis techniques or solid-phase polypeptide synthesis techniques) can be used to produce soy leghemoglobinssynthetically. In some embodiments, cell-free translation techniques can be used to produce soy leghemoglobinproteins synthetically.
[268] The present disclosure contemplates purification of monomeric soy leghemoglobin (e.g., leghemoglobin) or multimeric soy leghemoglobin (e.g., tetrameric hemoglobin). In some embodiments, each subunit of the tetrameric hemoglobin may be individually expressed and isolated from inclusion bodies in E. coli. The denatured b- subunits can be refolded and reconstituted with hemin in the presence of native reduced α- chains to produce functional, tetrameric hemoglobin (e.g., Fronticelli etal. (1991) J Protein Chem 10:495-501). Alternatively, the α and β subunits can be coexpressed to produce a large amount of intact, soluble tetrameric hemoglobin in E. Coli or P. pastoris (e.g., Hoffman etal. (1990) P roc Natl Acad Sci USA 87:8521-8525; Anwised et al. (2016) Protein J 35:256-268). In certain embodiments, α genes can be fused to create a di-α gene, which is inserted in an operon with a copy of the b-gene to express a tetramer that does not dissociate into α1β1 dimers under a physiological condition (e.g., Shen et al. (1993) Proc Natl Acad Sci USA 90:8108-8112).
[269] In some embodiments, the soy leghemoglobin is sterilized, e.g., prior to combining with other components of a growth media. Sterilization may be by Ultra High Temperature (UHT) processing, autoclaving or filtering. In some embodiments, the soy leghemoglobinis autoclaved. In some embodiments, soy leghemoglobin is filtered, e.g., the soy leghemoglobinis sterilized prior to use (e.g., prior to addition to growth media or with sterilization of the growth media) in methods to culture hemoglobin-dependent bacteria (e.g., for use in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) and/or for therapeutic use). The soy leghemoglobins provided herein can allow for large-scale manufacture and/or GMP grade manufacture of hemoglobin- dependent bacteria, e.g., for therapeutic use and/or in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements).
Growth Media
[270] In some embodiments, provided herein is growth media comprising a soy leghemoglobin disclosed herein. In certain embodiments, the growth media comprises an amount of a soy leghemoglobin disclosed herein sufficient to support growth of hemoglobin-dependent bacteria (e.g., for use in a bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) and/or for therapeutic use). In certain embodiments, the growth medium comprises at least 0.001 g/L, at least 0.005 g/L, at least 0.01 g/L, at least 0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, at least 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L, at least 0.1 g/L, at least 0.2 g/L, at least 0.3 g/L, at least 0.4 g/L, at least 0.5 g/L, at least 0.75 g/L, at least 1 g/L, at least 1.25 g/L, at least 1.5 g/L, at least 1.75 g/L, at least 2 g/L, at least 2.25 g/L, at least 2.5 g/L, at least 2.75 g/L, at least 3 g/L, at least 3.25 g/L, at least 3.5 g/L, at least 3.75 g/L, at least 4 g/L, or at least 4.25 g/L of a soy leghemoglobin. In some embodiments, the growth media comprises at least 0.005 g/L and no more than 1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.02 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.05 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.2 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.5 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 1 g/L of a soy leghemoglobin disclosed herein. In some embodiments, the growth medium comprises about 0.01 g/L to about 0.2 g/L of a soy leghemoglobin. In certain embodiments, the growth medium comprises at least 0.001 g/L, at least 0.005 g/L, at least 0.01 g/L, at least 0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, at least 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L, at least 0.1 g/L, at least 0.2 g/L, at least 0.3 g/L, at least 0.4 g/L, at least 0.5 g/L, at least 0.75 g/L, at least 1 g/L, at least 1.25 g/L, at least 1.5 g/L, at least 1.75 g/L, at least 2 g/L, at least 2.25 g/L, at least 2.5 g/L, at least 2.75 g/L, at least 3 g/L, at least 3.25 g/L, at least 3.5 g/L, at least 3.75 g/L, at least 4 g/L, or at least 4.25 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.02 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.05 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.2 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.5 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 1 g/L of a soy leghemoglobin. In some embodiments, the growth medium comprises about 0.01 g/L to about 0.2 g/L of a soy leghemoglobin. In some embodiments of the methods and compositions provided herein, the growth media does not comprise hemoglobin or a derivative thereof (or other soy leghemoglobin) that is sourced from an animal. In some embodiments, the growth media does not comprise animal products.
[271] In some embodiments the growth media may contain sugar, yeast extracts, plant based peptones, buffers, salts, trace elements, surfactants, anti-foaming agents, and/or vitamins.
[272] In some embodiments, the growth media comprise yeast extract, soy peptone A2SC 19649, Soy peptone El 10 19885, dipotassium phosphate, monopotassium phosphate, L-cysteine-HCl, ammonium chloride, glucidex 21 D, and/or glucose. [273] In some embodiments, the growth media comprises 5 g/L to 15g/L yeast extract 19512. In some embodiments, the growth media comprises 10 g/L yeast extract 19512.
[274] In some embodiments, the growth media comprises 10 g/L to 15 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 12.5 g/L soy peptone A2SC 19649. In some embodiments, the growth media comprises 10 g/L soy peptone A2SC 19649.
[275] In some embodiments, the growth media comprises 10 g/L to 15 g/L Soy peptone El 10 19885. In some embodiments, the growth media comprises 12.5 g/L Soy peptone El 10 19885. In some embodiments, the growth media comprises 10 g/L soy peptone El 10 19885.
[276] In some embodiments, the growth media comprises 1 g/L to 3 g/L dipotassium phosphate. In some embodiments, the growth media comprises 1.59 g/L dipotassium phosphate. In some embodiments, the growth media comprises 2.5 g/L dipotassium phosphate.
[277] In some embodiments, the growth media comprises 0 g/L to 1.5 g/L monopotassium phosphate. In some embodiments, the growth media comprises 0.91 g/L monopotassium phosphate. In some embodiments, the growth media does not comprise monopotassium phosphate.
[278] In some embodiments, the growth media comprises 0.1 g/L to 1.0 g/L L- cysteine-HCl. In some embodiments, the growth media comprises 0.5 g/L L-cysteine-HCl.
[279] In some embodiments, the growth media comprises 0 g/L to 1.0 g/L ammonium chloride. In some embodiments, the growth media comprises 0.5 g/L ammonium chloride. In some embodiments, the growth media does not comprise ammonium chloride.
[280] In some embodiments, the growth media comprises 0 g/L to 30 g/L glucidex 21 D. In some embodiments, the growth media comprises 25 g/L glucidex 21 D. In some embodiments, the growth media does not comprise glucidex 21 D.
[281] In some embodiments, the growth media comprises 5 g/L to 15g/L glucose. In some embodiments, the growth media comprises 10 g/L glucose. In some embodiments, the growth media comprises 5 g/L glucose.
[282] In certain embodiments, the growth media comprises a soy leghemoglobin provided herein, about 10 g/L yeast extract 19512, about 12.5 g/L soy peptone A2SC 19649, about 12.5 g/L soy peptone El 10 19885, about 1.59 g/L dipotassium phosphate, about 0.91 g/L monopotassium phosphate, about 0.5 g/L ammonium chloride, about 25 g/L glucidex 21 D, and/or about 10 g/L glucose. In some embodiments, the growth medium is the growth medium of Table 5.
[283] In certain embodiments, the growth media comprises a soy leghemoglobin provided herein, about 10 g/L yeast extract 19512, about 10 g/L soy peptone A2SC 19649, about 10 g/L soy peptone El 10 19885, about 2.5 g/L dipotassium phosphate, about 0.5 g/L L-cysteine-HCl, and/or about 5 g/L glucose. In some embodiments, the growth medium is the growth medium of Table 6.
[284] In certain embodiments, the growth media is at a pH of 5.5 to 7.5. In some embodiments, the growth media is at a pH of about 6.5.
[285] In some embodiments, prior to being added to the growth media, a soy leghemoglobin is prepared as a liquid mixture and sterilized by autoclaving or filtration. In some embodiments, the soy leghemoglobin is added to the growth media, which is then sterilized as described below.
In some embodiments, the media is sterilized. Sterilization may be by Ultra High Temperature (UHT) processing, autoclaving or filtering. The UHT processing is performed at very high temperature for short periods of time. The UHT range may be from 135-180°C. Lor example, the medium may be sterilized from between 10 to 30 seconds at 135°C.
Culturing Methods
[286] In certain aspects, provided herein are methods and/or compositions that facilitate the growth of hemoglobin-dependent bacteria (e.g., for use in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) and/or for therapeutic use). Such methods may comprise incubating the hemoglobin- dependent bacteria in a growth media provided herein. The methods may comprise maintaining the temperature and pH of the growth media as disclosed herein. The culturing may begin in a relatively small volume of growth media (e.g., 1 L) where bacteria are allowed to reach the log phase of growth. Such culture may be transferred to a larger volume of growth media (e.g. , 20 L) for further growth to reach a larger biomass. Depending on the need of the final amount of biomass, such transfer may be repeated more than once. The methods may comprise the incubation of the hemoglobin-dependent bacteria in bioreactors.
[287] In certain aspects, the hemoglobin-dependent bacteria are incubated at a temperature of 35°C to 39°C. In some embodiments, the hemoglobin-dependent bacteria are incubated at a temperature of about 37°C.
[288] In certain embodiments, the methods and/or compositions provided herein increase the growth rate of hemoglobin-dependent bacteria such that hemoglobin-dependent bacteria grow at an increased rate in the growth media comprising a soy leghemoglobin disclosed herein, compared to the rate at which the hemoglobin-dependent bacteria grow in the same growth media but without the soy leghemoglobin disclosed herein. In some embodiments, the rate at which the hemoglobin-dependent bacteria grow in the growth media comprising a soy leghemoglobin disclosed herein is higher than the rate at which the hemoglobin-dependent bacteria grow in the same growth media but without the soy leghemoglobin disclosed herein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least
160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least
220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least
280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least
340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, or at least
400%. In some embodiments, the growth rate is increased by about 200% to about 400%. The rate may be measured as the cell density (as measured by e.g., optical density at the wavelength of 600 nm (OD600)) reached within a given amount of time. In certain embodiments, such rate is measured and compared during the log phase (or exponential phase) of the bacterial growth, optionally wherein the log phase is early log phase.
[289] In certain embodiments, the methods and/or compositions provided herein increase the bacterial cell density such that the hemoglobin-dependent bacteria grow to a higher bacterial cell density in the growth media comprising a soy leghemoglobin disclosed herein, compared to the cell density to which the hemoglobin-dependent bacteria grow in the same growth media but without the soy leghemoglobin disclosed herein. In some embodiments, the hemoglobin-dependent bacteria grow to a cell density in the growth media comprising a soy leghemoglobin disclosed herein is higher than the cell density to which the hemoglobin-dependent bacteria grow in the same growth media but without the soy leghemoglobin disclosed herein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, or at least 400 %. In some embodiments, the bacterial cell density higher than about 200% to about 400%. The cell density may be measured (e.g. , by OD600 or by cell counting) at the stationary phase of bacterial growth, optionally wherein the stationary phase is early stationary phase. In some embodiments, the stationary phase is determined as the phase where the growth rate is retarded followed by an exponential phase of growth (e.g., from a growth curve). In other embodiments, the stationary phase is determined by the low glucose level in the growth media.
[290] In some embodiments, the methods provided herein comprise incubating the hemoglobin-dependent bacteria under anaerobic atmosphere. In certain aspects, provided herein are methods of culturing hemoglobin-dependent bacteria under anaerobic atmosphere comprising CO2. In some embodiments, the anaerobic atmosphere comprises greater than 1% CO2. In some embodiments, the anaerobic atmosphere comprises greater than 5% CO2. In some embodiments, the anaerobic atmosphere comprises at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% CO2. In some embodiments, the anaerobic atmosphere comprises at least 10% CO2. In some embodiments, the anaerobic atmosphere comprises at least 20% CO2. In some embodiments, the anaerobic atmosphere comprises from 10% to 40% CO2. In some embodiments, the anaerobic atmosphere comprises from 20% to 30% CO2. In some embodiments, the anaerobic atmosphere comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% CO2. In some embodiments, the anaerobic atmosphere comprises about 25% CO2.
[291] In certain aspects, the anaerobic atmosphere comprises N2. In some embodiments, the anaerobic atmosphere comprises less than 95% N2. In some embodiments, the anaerobic atmosphere comprises less than 90% N2. In some embodiments, the anaerobic atmosphere comprises less than 95%, less than 92%, less than 90%, less than 87%, less than 85%, less than 82%, less than 80%, less than 77% N2. In some embodiments, the anaerobic atmosphere comprises less than 85% N2. In some embodiments, the anaerobic atmosphere comprises less than 80% N2. In some embodiments, the anaerobic atmosphere comprises from 65% to 85% N2. In some embodiments, the anaerobic atmosphere comprises from 70% to 80% N2. In some embodiments, the anaerobic atmosphere comprises about 65%, about 66%, about 67%, about 28%, about 69%, about 70%, about 71%, about 72% about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% N2. In some embodiments, the anaerobic atmosphere comprises about 75% N2.
[292] In some embodiments, the anaerobic atmosphere consists essentially of CO2 and N2. In some embodiments, the anaerobic atmosphere comprises about 25% CO2 and about 75% N2. In some embodiments, the anaerobic atmosphere comprises about 20% CO2 and about 80% N2. In some embodiments, the anaerobic atmosphere comprises about 30% CO2 and about 70% N2.
[293] Thus, in some embodiments provided herein are methods of culturing hemoglobin-dependent bacteria under anaerobic conditions comprising a greater level of CO2 compared to conventional anaerobic culture conditions (e.g. , at a level of greater than 1% CO2, e.g., at a level of greater than 5% CO2, such as at a level of about 25% CO2). In certain embodiments, provided herein are bioreactors comprising hemoglobin-dependent bacteria being cultured under conditions comprising a greater level of CO2 compared to conventional anaerobic culture conditions (e.g., at a level of greater than 1% CO2, such as at a level of about 25% CO2). In some embodiments, the methods and compositions provided herein result in increased bacterial yield compared to conventional culture conditions.
[294] In certain aspects, provided herein are methods of culturing hemoglobin- dependent bacteria under anaerobic conditions comprising a lower level of N2 compared to conventional anaerobic culture conditions (e.g., at a level of less than 95% N2, e.g., at a level of less than 90% N2, such as at a level of about 75% N2). In certain embodiments, provided herein are bioreactors comprising hemoglobin-dependent bacteria being cultured under conditions comprising a lower level of N2 compared to conventional anaerobic culture conditions (e.g. , at a level of less than 95% N2 such as at a level of about 75% N2).
In some embodiments, the methods and compositions provided herein result in increased bacterial yield compared to conventional culture conditions.
[295] In certain aspects, provided herein are methods of culturing hemoglobin- dependent bacteria, the method comprises the steps of a) purging a bioreactor with an anaerobic gaseous mixture comprising greater than 1% CO2; and b) culturing the hemoglobin-dependent bacteria in the bioreactor purged in step a). In some embodiments, the anaerobic gaseous mixture comprises greater than 1% CO2. In some embodiments, the anaerobic gaseous mixture comprises at least about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40% CO2. In some embodiments, the anaerobic gaseous mixture comprises at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,
26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% CO2. In some embodiments, the anaerobic gaseous mixture comprises from 5% to 35% CO2, 10% to 40% CO2, 10% to 30% CO2, 15% to 30% CO2, 20% to 30% CO2, 22% to 28% CO2, or 24%, to 26% CO2. In some embodiments, the anaerobic gaseous mixture comprises greater than 5% CO2. In some embodiments, the anaerobic gaseous mixture comprises at least 10% CO2. In some embodiments, the anaerobic gaseous mixture comprises at least 20% CO2. In some embodiments, the anaerobic gaseous mixture comprises from 10% to 40% CO2. In some embodiments, the anaerobic gaseous mixture comprises from 20% to 30% CO2. In some embodiments, the anaerobic gaseous mixture comprises about 25% CO2.
[296] In certain aspects, provided herein are methods of culturing hemoglobin- dependent bacteria, the method comprises the steps of a) purging a bioreactor with an anaerobic gaseous mixture comprising less than 95% N2; and b) culturing the hemoglobin- dependent bacteria in the bioreactor purged in step a). In some embodiments, the anaerobic gaseous mixture comprises less than 95% N2. In some embodiments, the anaerobic gaseous mixture comprises less than 95%, less than 92%, less than 90%, less than 87%, less than 85%, less than 82%, less than 80%, less than 77% N2. In some embodiments, the anaerobic gaseous mixture comprises about 65%, about 66%, about 67%, about 28%, about 69%, about 70%, about 71%, about 72% about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85% N2. In some embodiments, the anaerobic gaseous mixture comprises less than 95% N2. In some embodiments, the anaerobic gaseous mixture comprises less than 90% N2. In some embodiments, the anaerobic gaseous mixture comprises from 65% to 85% N2. In some embodiments, the anaerobic gaseous mixture comprises from 70% to 80% N2CO2. In some embodiments, the anaerobic gaseous mixture comprises about 75% N2.
[297] In some embodiments, the anaerobic gaseous mixture consists essentially of CO2 and N2. In some embodiments, the anaerobic gaseous mixture comprises about 25% CO2 and about 75% N2. In some embodiments, the anaerobic atmosphere comprises about 20% CO2 and about 80% N2. In some embodiments, the anaerobic atmosphere comprises about 30% CO2 and about 70% N2.
[298] In some embodiments, the anaerobic gaseous mixture comprises CO2 and N2 in a ratio of about 1:99, about 2:98, about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about 9:91, about 10:90, 11:89, about 12:88, about 13:87, about 14:86, about 15:85, about 16:84, about 17:83, about 18:82, about 19:81, about 20:80, 21:79, about 22:78, about 23:77, about 24:76, about 25:75, about 26:74, about 27:73, about 28:72, about 29:71, about 30:70, 31:69, about 32:68, about 33:67, about 34:66, about 35:65, about 36:64, about 37:63, about 38:62, about 39:61, or about 40:50 CO2 to N2. In some embodiments, the mixed gas composition provides an atmosphere in the bioreactor comprising CO2 and N2 in a ratio of about 25:75.
[299] In some embodiments, an anaerobic gaseous mixture is continuously added to the bioreactor during culturing. In some embodiments, the continuously added anaerobic gaseous mixture is added at a rate of 0.01 to 0.1 vvm. In some embodiments the continuously added anaerobic gaseous mixture is added at a rate of 0.02vvm. In some embodiments, the continuously added anaerobic gaseous mixture comprises any one of gaseous mixtures described above.
[300] In some embodiments, the methods provided herein further comprises the step of inoculating a growth media with the hemoglobin-dependent bacteria, wherein the bacteria are cultured in the growth media according to the methods provided herein. In some embodiments, the volume of the inoculated hemoglobin-dependent bacteria is between 0.01% and 10% v/v of the growth media (e.g., about 0.1% v/v of the growth media, about 0.5% v/v of the growth media, about 1% v/v of the growth media, about 5% v/v of the growth media). In some embodiments, the volume of hemoglobin-dependent bacteria is about 1 mL.
[301] In some embodiments, inoculum can be prepared in flasks or in smaller bioreactors where growth is monitored. For example, the inoculum size may be between approximately 0.1% v/v and 5% v/v of the total bioreactor volume. In some embodiments, the inoculum is 0.1-3% v/v, 0.1-1% v/v, 0.1-0.5% v/v, or 0.5-1% v/v of the total bioreactor volume. In some embodiments, the inoculum is about 0.1% v/v, about 0.2% v/v, about 0.3% v/v, about 0.4%, v/v, about 0.5% v/v, about 0.6% v/v, about 0.7% v/v, about 0.8% v/v, about 0.9% v/v, about 1% v/v, about 1.5% v/v, about 2% v/v, about 2.5% v/v, about 3% v/v, about 4%, v/v, or about 5% v/v of the total bioreactor volume.
[302] In some embodiments, before the inoculation, the bioreactor is prepared with growth medium at desired pH and temperature. The initial pH of the culture medium may be different than the process set-point. pH stress may be detrimental at low cell concentration; the initial pH could be between pH 7.5 and the process set-point. For example, pH may be set between 4.5 and 8.0, preferably 6.5. During the fermentation, the pH can be controlled through the use of sodium hydroxide, potassium hydroxide, or ammonium hydroxide. The temperature may be controlled from 25°C to 45°C, for example at 37°C.
[303] In some embodiments, depending on strain and inoculum size, the bioreactor fermentation time can vary. For example, fermentation time can vary from 5 hours to 48 hours. In some embodiments, fermentation time may be from 5 hours to 24 hours, 8 hours to 24 hours, 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 24 hours, 10 hours to 18 hours, 10 hours to 16 hours, 10 hours to 14 hours, 10 hours to 12 hours, 12 hours to 24 hours, 12 hours to 18 hours, 12 hours to 16 hours, or 12 hours to 14 hours.
[304] In some embodiments, culturing the hemoglobin-dependent bacteria comprises agitating the culture at a RPM of 50 to 300. In some embodiments, the hemoglobin-dependent bacteria is agitated at a RPM of about 150. [305] For example, in some embodiments, a culturing method comprises culturing the hemoglobin-dependent bacteria for at least 5 hours (e.g., at least 10 hours). In some embodiments, the hemoglobin-dependent bacteria is cultured for 10-24 hours. In some embodiments, the hemoglobin-dependent bacteria is cultured for 14 to 16 hours. In some embodiments, the method further comprises the step of inoculating about 5% v/v of the cultured bacteria in a growth media. In some embodiments, the growth media is about 20L in volume. In some embodiments, the hemoglobin-dependent bacteria is cultured for 10-24 hours. In some embodiments, the hemoglobin-dependent bacteria is cultured for 12-14 hours. In some embodiments, the method further comprises the step of inoculating about 0.5 %v/v of the cultured bacteria in a growth medium. In some embodiments, the growth medium is about 3500L in volume. In some embodiments, the hemoglobin-dependent bacteria is cultured for 10-24 hours. In some embodiments, the hemoglobin-dependent bacteria is cultured for 12-14 hours. In some embodiments, the hemoglobin-dependent bacteria is cultured at least until a stationary phase is reached.
[306] In certain embodiments, the culturing method further comprises the step of harvesting the cultured bacteria. The harvest time may be based on either glucose level being below 2 g/L or when stationary phase is reached. In some embodiments, the method further comprises the step of centrifuging the cultured bacteria after harvesting (e.g., to produce a cell paste). In some embodiments, the method further comprises diluting the cell paste with a stabilizer solution to produce a cell slurry. In some embodiments, the method further comprises the step of lyophilizing the cell slurry to produce a powder. In some embodiments, the method further comprises irradiating the powder with gamma radiation.
[307] For example, in some embodiments, once fermentation complete, the culture is cooled (e.g, to 10°C) and centrifuged collecting the cell paste. A stabilizer may be added to the cell paste and mixed thoroughly. Harvesting may be performed by continuous centrifugation. Product may be resuspended with various excipients to a desired final concentration. Excipients can be added for cryo protection or for protection during lyophilization. Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti -oxidants. Prior to lyophilization, droplets of cell pellets may be mixed with excipients and submerged in liquid nitrogen.
[308] In certain embodiments, the cell slurry may be lyophilized. Lyophilization of material, including live bacteria, may begin with primary drying. During the primary drying phase, the ice is removed. Here, a vacuum is generated and an appropriate amount of heat is supplied to the material for the ice to sublime. During the secondary drying phase, product bound water molecules may be removed. Here, the temperature is raised higher than in the primary drying phase to break any physico-chemical interactions that have formed between the water molecules and the product material. The pressure may also be lowered further to enhance desorption during this stage. After the freeze-drying process is complete, the chamber may be filled with an inert gas, such as nitrogen. The product may be sealed within the freeze dryer under dry conditions, preventing exposure to atmospheric water and contaminants. The lyophilized material may be gamma irradiated (e.g., 17.5 kGy).
Bioreactors
[309] In certain aspects, provided herein are bioreactors comprising growth media provided herein (i.e., a growth media comprising a soy leghemoglobin disclosed herein and/or hemoglobin-dependent bacteria (e.g., for use in bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) and/or for therapeutic use) provided herein. In some embodiments, the hemoglobin-dependent bacteria are Prevotella bacteria (e.g., aPrevotella strain provided herein). In some embodiments, provided herein are methods of culturing bacteria in such bioreactors.
[310] In certain embodiments, the bioreactor is under the anaerobic conditions mentioned above. In certain aspects, provided herein are bioreactors comprising hemoglobin-dependent bacteria under an anaerobic atmosphere disclosed above. In certain aspects, provided herein are bioreactors of various sizes. In some embodiments, the bioreactors are at least 1L in volume, at least 5L in volume, at least 10L in volume, at least 15L in volume, at least 20L in volume, at least 30L in volume, at least 40L in volume, at least 50L in volume, at least 100L in volume, at least 200L in volume, at least 250L in volume, at least 500L in volume, at least 750L in volume, at least 1000L in volume, at least 1500L in volume, at least 2000L in volume, at least 2500L in volume, at least 3000L in volume, at least 3500L in volume, at least 4000L in volume, at least 5000L in volume, at least 7500L in volume, at least 10,000L in volume, at least 15,000L in volume, or at least 20,000L in volume. In some embodiments, the bioreactors are about 1L in volume, about 5L in volume, about 10L in volume, about 15L in volume, about 20L in volume, about 30L in volume, about 40L in volume, about 50L in volume, about 100L in volume, about 200L in volume, about 250L in volume, about 500L in volume, about 750L in volume, about 1000L in volume, about 1500L in volume, about 2000L in volume, about 2500L in volume, about 3000L in volume, about 3500L in volume, about 4000L in volume, about 5000L in volume, about 7500L in volume, about 10,000L in volume, about 15,000L in volume, or about 20,000L in volume.
Modified Bacteria and mEVs
[311] In some embodiments, the bacteria of the bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement)or from which the mEVs of the composition are obtained are modified to reduce toxicity or other adverse effects, to enhance delivery) (e.g., oral delivery) (e.g., by improving acid resistance, muco- adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the bacteria and/or mEVs (e.g., either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the bacteria and/or mEVs (such as smEVs and/or pmEVs) (e.g., through modified production of polysaccharides, pili, fimbriae, adhesins). In some embodiments, the engineered bacteria described herein are modified to improve bacteria and/or mEV (such as smEV and/or pmEV) manufacturing (e.g., higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times). For example, in some embodiments, the engineered bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may result in the overexpression and/or underexpression of one or more genes. The engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
[312] In some aspects, the bacteria and/or mEVs (such as smEVs and/or pmEVs) described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety. [313] In some embodiments, the therapeutic moiety is a cancer-specific moiety. In some embodiments, the cancer-specific moiety has binding specificity for a cancer cell (e.g., has binding specificity for a cancer-specific antigen). In some embodiments, the cancer-specific moiety comprises an antibody or antigen binding fragment thereof. In some embodiments, the cancer-specific moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the cancer-specific moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In some embodiments, the cancer-specific moiety is a bipartite fusion protein that has two parts: a first part that binds to and/or is linked to the bacterium and a second part that is capable of binding to a cancer cell (e.g., by having binding specificity for a cancer-specific antigen). In some embodiments, the first part is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the first part has binding specificity for the mEV (e.g., by having binding specificity for a bacterial antigen). In some embodiments, the first and/or second part comprises an antibody or antigen binding fragment thereof. In some embodiments, the first and/or second part comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the first and/or second part comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In certain embodiments, co-administration of the cancer- specific moiety with the pharmaceutical agent (either in combination or in separate administrations) increases the targeting of the pharmaceutical agent to the cancer cells.
[314] In some embodiments, the bacteria and/or mEVs described herein can be modified such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (e.g., a magnetic bead). In some embodiments, the magnetic and/or paramagnetic moiety is comprised by and/or directly linked to the bacteria. In some embodiments, the magnetic and/or paramagnetic moiety is linked to and/or a part of a bacteria- or an mEV-binding moiety that binds to the bacteria or mEV. In some embodiments, the bacteria- or mEV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the bacteria- or mEV-binding moiety has binding specificity for the bacteria or mEV (e.g., by having binding specificity for a bacterial antigen). In some embodiments, the bacteria- or mEV- binding moiety comprises an antibody or antigen binding fragment thereof. In some embodiments, the bacteria- or mEV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the bacteria- or mEV-binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In certain embodiments, co-administration of the magnetic and/or paramagnetic moiety with the bacteria or mEVs (either together or in separate administrations) can be used to increase the targeting of the mEVs (e.g., to cancer cells and/or a part of a subject where cancer cells are present.
Production of Processed Microbial Extracellular Vesicles (pmEVs)
[315] In certain aspects, the pmEVs described herein can be prepared using any method known in the art.
[316] In some embodiments, the pmEVs are prepared without a pmEV purification step. For example, in some embodiments, bacteria from which the pmEVs described herein are released are killed using a method that leaves the bacterial pmEVs intact, and the resulting bacterial components, including the pmEVs, are used in the methods and compositions described herein. In some embodiments, the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the bacteria are killed using UV irradiation.
[317] In some embodiments, the pmEVs described herein are purified from one or more other bacterial components. Methods for purifying pmEVs from bacteria (and optionally, other bacterial components) are known in the art. In some embodiments, pmEVs are prepared from bacterial cultures using methods described in Thein, et al. (./. Proteome Res. 9(12):6135-6147 (2010)) or Sandrini, et al. ( Bio-protocol 4(21): el287 (2014)), each of which is hereby incorporated by reference in its entirety. In some embodiments, the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g., at 10,000- 15,000 x g for 10- 15 min at room temperature or 4°C). In some embodiments, the supernatants are discarded and cell pellets are frozen at -80°C. In some embodiments, cell pellets are thawed on ice and resuspended in 100 mM Tris-HCl, pH 7.5 supplemented with
1 mg/mL DNase I. In some embodiments, cells are lysed using an Emulsiflex C-3 (Avestin, Inc.) under conditions recommended by the manufacturer. In some embodiments, debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min at 4°C. In some embodiments, supernatants are then centrifuged at 120,000 x g for 1 hour at 4°C. In some embodiments, pellets are resuspended in ice-cold 100 mM sodium carbonate, pH 11, incubated with agitation for 1 hr at 4°C, and then centrifuged at 120,000 x g for 1 hour at 4°C. In some embodiments, pellets are resuspended in 100 mM Tris-HCl, pH 7.5, re- centrifuged at 120,000 x g for 20 min at 4°C, and then resuspended in 0.1 M Tris-HCl, pH 7.5 or in PBS. In some embodiments, samples are stored at -20°C.
[318] In certain aspects, pmEVs are obtained by methods adapted from Sandrini et al, 2014. In some embodiments, bacterial cultures are centrifuged at 10,000-15,500 x g for 10-15 min at room temp or at 4°C. In some embodiments, cell pellets are frozen at -80°C and supernatants are discarded. In some embodiments, cell pellets are thawed on ice and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA supplemented with 0.1 mg/mL lysozyme. In some embodiments, samples are incubated with mixing at room temp or at 37°C for 30 min. In some embodiments, samples are re-frozen at -80°C and thawed again on ice. In some embodiments, DNase I is added to a final concentration of 1.6 mg/mL and MgC12 to a final concentration of 100 mM. In some embodiments, samples are sonicated using a QSonica Q500 sonicator with 7 cycles of 30 sec on and 30 sec off. In some embodiments, debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min. at 4°C. In some embodiments, supernatants are then centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in 10 mM Tris-HCl, pH 8.0, 2% Triton X-100 and incubated 30-60 min with mixing at room temperature. In some embodiments, samples are centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in PBS and stored at -20°C.
[319] In certain aspects, a method of forming (e.g., preparing) isolated bacterial pmEVs, described herein, comprises the steps of: (a) centrifuging a bacterial culture, thereby forming a first pellet and a first supernatant, wherein the first pellet comprises cells; (b) discarding the first supernatant; (c) resuspending the first pellet in a solution; (d) lysing the cells; (e) centrifuging the lysed cells, thereby forming a second pellet and a second supernatant; (f) discarding the second pellet and centrifuging the second supernatant, thereby forming a third pellet and a third supernatant; (g) discarding the third supernatant and resuspending the third pellet in a second solution, thereby forming the isolated bacterial pmEVs.
[320] In some embodiments, the method further comprises the steps of: (h) centrifuging the solution of step (g), thereby forming a fourth pellet and a fourth supernatant; (i) discarding the fourth supernatant and resuspending the fourth pellet in a third solution. In some embodiments, the method further comprises the steps of: (j) centrifuging the solution of step (i), thereby forming a fifth pellet and a fifth supernatant; and (k) discarding the fifth supernatant and resuspending the fifth pellet in a fourth solution. [321] In some embodiments, the centrifugation of step (a) is at 10,000 x g. In some embodiments the centrifugation of step (a) is for 10-15 minutes. In some embodiments, the centrifugation of step (a) is at 4 °C or room temperature. In some embodiments, step (b) further comprises freezing the first pellet at -80 °C . In some embodiments, the solution in step (c) is 100mM Tris-HCl, pH 7.5 supplemented with lmg/ml DNasel. In some embodiments, the solution in step (c) is 10mM Tris-HCl, pH 8.0, ImM EDTA, supplemented with 0.1 mg/ml lysozyme. In some embodiments, step (c) further comprises incubating for 30 minutes at 37 °C or room temperature. In some embodiments, step (c) further comprises freezing the first pellet at -80 °C . In some embodiments, step (c) further comprises adding DNase I to a final concentration of 1.6mg/ml. In some embodiments, step (c) further comprises adding MgCl2to a final concentration of lOOmM. In some embodiments, the cells are lysed in step (d) via homogenization. In some embodiments, the cells are lysed in step (d) via emulsiflex C3. In some embodiments, the cells are lysed in step (d) via sonication. In some embodiments, the cells are sonicated in 7 cycles, wherein each cycle comprises 30 seconds of sonication and 30 seconds without sonication. In some embodiments, the centrifugation of step (e) is at 10,000 x g. In some embodiments, the centrifugation of step (e) is for 15 minutes. In some embodiments, the centrifugation of step
(e) is at 4 °C or room temperature.
[322] In some embodiments, the centrifugation of step (f) is at 120,000 x g. In some embodiments, the centrifugation of step (f) is at 110,000 x g. In some embodiments, the centrifugation of step (f) is for 1 hour. In some embodiments, the centrifugation of step
(f) is for 15 minutes. In some embodiments, the centrifugation of step (f) is at 4 °C or room temperature. In some embodiments, the second solution in step (g) is 100 mM sodium carbonate, pH 11. In some embodiments, the second solution in step (g) is lOmM Tris-HCl pH 8.0, 2% triton X-100. In some embodiments, step (g) further comprises incubating the solution for 1 hour at 4 °C. In some embodiments, step (g) further comprises incubating the solution for 30-60 minutes at room temperature. In some embodiments, the centrifugation of step (h) is at 120,000 x g. In some embodiments, the centrifugation of step (h) is at
110,000 x g. In some embodiments, the centrifugation of step (h) is for 1 hour. In some embodiments, the centrifugation of step (h) is for 15 minutes. In some embodiments, the centrifugation of step (h) is at 4 °C or room temperature. In some embodiments, the third solution in step (i) is 100mM Tris-HCl, pH 7.5. In some embodiments, the third solution in step (i) is PBS. In some embodiments, the centrifugation of step (j) is at 120,000 x g. In some embodiments, the centrifugation of step (j) is for 20 minutes. In some embodiments, the centrifugation of step (j) is at 4 °C or room temperature. In some embodiments, the fourth solution in step (k) is 100mM Tris-HCl, pH 7.5 or PBS.
[323] pmEVs obtained by methods provided herein may be further purified by size based column chromatography, by affinity chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35- 60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C.
[324] In some embodiments, to confirm sterility and isolation of the pmEV preparations, pmEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated pmEVs may be DNase or proteinase K treated.
[325] In some embodiments, the sterility of the pmEV preparations can be confirmed by plating a portion of the pmEVs onto agar medium used for standard culture of the bacteria used in the generation of the pmEVs and incubating using standard conditions.
[326] In some embodiments select pmEVs are isolated and enriched by chromatography and binding surface moieties on pmEVs. In other embodiments, select pmEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
[327] The pmEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019). [328] In some embodiments, pmEVs are lyophilized.
[329] In some embodiments, pmEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).
[330] In some embodiments, pmEVs are UV irradiated.
[331] In some embodiments, pmEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
[332] In some embodiments, pmEVs are acid treated.
[333] In some embodiments, pmEVs are oxygen sparged (e.g., at 0.1 vvm for two hours).
[334] The phase of growth can affect the amount or properties of bacteria. In the methods of pmEV preparation provided herein, pmEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
Production of Secreted Microbial Extracellular Vesicles (smEVs)
[335] In certain aspects, the smEVs described herein can be prepared using any method known in the art.
[336] In some embodiments, the smEVs are prepared without an smEV purification step. For example, in some embodiments, bacteria described herein are killed using a method that leaves the smEVs intact and the resulting bacterial components, including the smEVs, are used in the methods and compositions described herein. In some embodiments, the bacteria are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the bacteria are killed using UV irradiation. In some embodiments, the bacteria are heat-killed.
[337] In some embodiments, the smEVs described herein are purified from one or more other bacterial components. Methods for purifying smEVs from bacteria are known in the art. In some embodiments, smEVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015) or Jeppesen, et al. Cell 177:428 (2019), each of which is hereby incorporated by reference in its entirety. In some embodiments, the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g. , at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C). In some embodiments, the culture supernatants are then passed through filters to exclude intact bacterial cells (e.g., a 0.22 pm filter). In some embodiments, the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS. In some embodiments, filtered supernatants are centrifuged to pellet bacterial smEVs (e.g., at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C). In some embodiments, the smEVs are further purified by resuspending the resulting smEV pellets (e.g., in PBS), and applying the resuspended smEVs to an Optiprep (iodixanol) gradient or gradient (e.g., a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (e.g., at 200,000 x g for 4-20 hours at 4°C). smEV bands can be collected, diluted with PBS, and centrifuged to pellet the smEVs (e.g. , at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C). The purified smEVs can be stored, for example, at -80°C or -20°C until use.
In some embodiments, the smEVs are further purified by treatment with DNase and/or proteinase K.
[338] For example, in some embodiments, cultures of bacteria can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria. Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells. Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. For example, for ammonium sulfate precipitation, 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4°C. Precipitations can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C. The resulting pellets contain bacteria smEVs and other debris. Using ultracentrifugation, filtered supernatants can be centrifuged at 100,000- 200,000 x g for 1-16 hours at 4°C. The pellet of this centrifugation contains bacteria smEVs and other debris such as large protein complexes. In some embodiments, using a filtration technique, such as through the use of an Amicon Ultra spin filter or by tangential flow filtration, supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
[339] Alternatively, smEVs can be obtained from bacteria cultures continuously during growth, or at selected time points during growth, for example, by connecting a bioreactor to an alternating tangential flow (ATF) system (e.g., XCell ATF from Repligen). The ATF system retains intact cells (>0.22 um) in the bioreactor, and allows smaller components (e.g., smEVs, free proteins) to pass through a filter for collection. For example, the system may be configured so that the <0.22 um filtrate is then passed through a second filter of 100 kDa, allowing species such as smEVs between 0.22 um and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor. Alternatively, the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture. smEVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
[340] smEVs obtained by methods provided herein may be further purified by size-based column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, e.g., 4-24 hours at 4°C.
[341] In some embodiments, to confirm sterility and isolation of the smEV preparations, smEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated smEVs may be DNase or proteinase K treated.
[342] In some embodiments, for preparation of smEVs used for in vivo injections, purified smEVs are processed as described previously (G. Norheim, et al. PLoS ONE.
10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing smEVs are resuspended to a final concentration of 50 μg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v). In some embodiments, for preparation of smEVs used for in vivo injections, smEVs in PBS are sterile-filtered to < 0.22 um.
[343] In certain embodiments, to make samples compatible with further testing (e.g., to remove sucrose prior to TEM imaging or in vitro assays), samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using fdtration (e.g., Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
[344] In some embodiments, the sterility of the smEV preparations can be confirmed by plating a portion of the smEVs onto agar medium used for standard culture of the bacteria used in the generation of the smEVs and incubating using standard conditions.
[345] In some embodiments, select smEVs are isolated and enriched by chromatography and binding surface moieties on smEVs. In other embodiments, select smEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
[346] The smEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019).
[347] In some embodiments, smEVs are lyophilized.
[348] In some embodiments, smEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).
[349] In some embodiments, smEVs are UV irradiated.
[350] In some embodiments, smEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
[351] In some embodiments, smEVs s are acid treated.
[352] In some embodiments, smEVs are oxygen sparged (e.g., at 0.1 vvm for two hours).
[353] The phase of growth can affect the amount or properties of bacteria and/or smEVs produced by bacteria. For example, in the methods of smEV preparation provided herein, smEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
[354] The growth environment (e.g., culture conditions) can affect the amount of smEVs produced by bacteria. For example, the yield of smEVs can be increased by an smEV inducer, as provided in Table 4. Table 4: Culture Techniques to Increase smEV Production
Figure imgf000113_0001
[355] In the methods of smEVs preparation provided herein, the method can optionally include exposing a culture of bacteria to an smEV inducer prior to isolating smEVs from the bacterial culture. The culture of bacteria can be exposed to an smEV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
Solid Dosage Forms
[356] In certain embodiments, provided herein are solid dosage forms (e.g., pharmaceutical compositions having a solid dosage form) comprising a pharmaceutical agent that contains bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component. In some embodiments, the pharmaceutical agent can optionally contain one or more additional components, such as a cryoprotectant. The pharmaceutical agent can be lyophilized (e.g., resulting in a powder). The pharmaceutical agent can be combined with one or more excipients (e.g., pharmaceutically acceptable excipients) in the solid dosage form.
[357] In certain aspects provided herein are solid dosage forms of pharmaceutical compositions. In certain embodiments, the solid dosage form comprises a pharmaceutical agent (e.g., bacteria and/or an agent (e.g., component) of bacterial origin, such as mEVs, a powder comprising bacteria and/or an agent (e.g., component) of bacterial origin, such as mEVs) and one or more disintegration agents. In certain embodiments, the total pharmaceutical agent mass is at least 5%, 10%, 15%, 20% or 25 % of the total mass of the pharmaceutical composition. In some embodiments the total pharmaceutical agent mass is no more than 45%, 40%, 35%, 30%, or 25% of the total mass of the pharmaceutical composition. In some embodiments, the total mass of the one or more disintegrating agents is at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total mass of the pharmaceutical composition. In some embodiments, the total mass of the one or more disintegrating agents is no more than 70%, 65%, 60%, or 55% of the total mass of the pharmaceutical composition. In some embodiments, the one or more disintegration agents comprise low-substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac- Di-Sol), and/or crospovidone (PVPP).
[358] In certain embodiments, the solid dosage forms provided herein comprise L- HPC. In some embodiments, the L-HPC is (or comprises L-HPC) of grade LH-B1. In certain embodiments, the total L-HPC mass is at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition. In certain embodiments, the total L-HPC mass is no more than 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition. In certain embodiments, the total L-HPC mass is about 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,
38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition.
[359] In certain embodiments, the solid dosage forms provided herein comprise Ac-Do-Sol. In some embodiments, the Ac -Di-Sol is (or comprises Ac -Di-Sol) of grade SD- 711. In certain embodiments, the total Ac -Di-Sol mass is at least 0.01%, 0.1%, 1%, 2%,
3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition. In certain embodiments, the total Ac-Di-Sol mass is no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition. In certain embodiments, the total Ac-Di-Sol mass is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition.
[360] In certain embodiments, the solid dosage forms provided herein comprise PVPP. In certain embodiments, the total PVPP mass is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition. In certain embodiments, the total PVPP mass is no more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition. In certain embodiments, the total PVPP mass is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition.
[361] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is at least 5% and no more than 35% of the total mass of the pharmaceutical composition, (ii) L-HPC (e.g., L- HPC of grade LH-B 1) having a total L-HPC mass that is at least 22% (e.g., at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,
38%, 39%, 40%, 41%, or 42%) and no more than 42% (e.g., no more than 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,
40%, 41%, or 42%) of the total mass of the pharmaceutical composition; (iii) Ac-Di-Sol (e.g., Ac-Di-Sol of grade SD-711) having a total Ac-Di-Sol mass that is at least 0.01% (e.g., at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16%) and no more than 16% (e.g., no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16%) of the total mass of the pharmaceutical composition; and (iv) PVPP having a total PVPP mass that is at least 5% (e.g., at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%) and no more than 25% (no more than 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%) of the total mass of the pharmaceutical composition. In certain embodiments, the total L-HPC mass plus the total Ac-Di-Sol mass plus the total PVPP mass is at least 35%, 40%, 45%, or 50% of the total mass of the pharmaceutical composition. In some embodiments, the solid dosage form comprises: a total L-HPC mass is about 32% of the total mass of the pharmaceutical composition; a total Ac-Di-Sol mass is about 6% of the total mass of the pharmaceutical composition; and a total PVPP mass is about 15% of the total mass of the pharmaceutical composition.
[362] In certain embodiments, the solid dosage forms provided herein further comprise mannitol. In certain embodiments, the mannitol is (or comprises) mannitol SD200. In certain embodiments, the total mannitol mass is at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% of the total mass of the pharmaceutical composition. In certain embodiments, the total mannitol mass is no more than 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,
34%, 35%, 36%, 37%, 38%, 39% or 40% of the total mass of the pharmaceutical composition. In certain embodiments, the total mannitol mass is about 10%, 11%, 12%, 13%, 14%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%,
20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5% or 40% of the total mass of the pharmaceutical composition.
[363] In certain embodiments, the solid dosage forms provided herein comprise magnesium stearate. In certain embodiments, the total magnesium stearate mass is at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11% of the total mass of the pharmaceutical composition. In certain embodiments, the total magnesium stearate mass is no more than 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11% of the total mass of the pharmaceutical composition. In certain embodiments, the total magnesium stearate mass is about 0.01%, 0.1%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, or 11% of the total mass of the pharmaceutical composition.
[364] In certain embodiments, the solid dosage forms provided herein comprise colloidal silica. In certain embodiments, the colloidal silica is (or comprises) Aerosil 200. In certain embodiments, the total colloidal silica mass is at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11% of the total mass of the pharmaceutical composition. In certain embodiments, the total colloidal silica mass is no more than 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11% of the total mass of the pharmaceutical composition. In certain embodiments, the total colloidal silica mass is about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 11% of the total mass of the pharmaceutical composition.
[365] In certain aspects provided herein are solid dosage forms of pharmaceutical compositions. In certain embodiments, the solid dosage form comprises a pharmaceutical agent, wherein the pharmaceutical agent comprises bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component and a diluent. In certain embodiments, the total pharmaceutical agent mass is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total mass of the pharmaceutical composition. In some embodiments, the total pharmaceutical agent mass is no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the total mass of the pharmaceutical composition.
[366] In some embodiments, the total mass of the diluent is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, or 95% of the total mass of the pharmaceutical composition. In some embodiments, the total mass of the diluent is no more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the total mass of the pharmaceutical composition. In some embodiments, the diluent comprises mannitol.
[367] In certain embodiments, the solid dosage form provided herein comprises a lubricant. In certain embodiments, the total lubricant mass is at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 0.5% to about 1.5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 1% of the total mass of the pharmaceutical composition. In some embodiments, the lubricant comprises magnesium stearate.
[368] In certain embodiments, the solid dosage forms provided herein comprise a glidant. In some embodiments, the glidant is colloidal silicon dioxide. In certain embodiments, the total glidant mass is at least 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,
0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is no more than 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.25% to about 0.75% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.5% of the total mass of the pharmaceutical composition.
[369] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 20% to about 50% of the total mass of the pharmaceutical composition; (ii) a diluent (e.g., mannitol) having a total mass that is about 50% to 80% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the pharmaceutical composition.
[370] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is at least 5% and no more than 95% of the total mass of the pharmaceutical composition; (ii) a diluent (e.g., mannitol) having a total mass that is at least 1% and no more than 95% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is at least 0.1% and no more than 5% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is at least 0.01% and no more than 2% of the total mass of the pharmaceutical composition.
[371] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 8% to about 92% of the total mass of the pharmaceutical composition; (ii) a diluent (e.g., mannitol) having a total mass that is about 5% to 90% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the pharmaceutical composition.
[372] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 30% to about 50% of the total mass of the pharmaceutical composition; (ii) a diluent (e.g., mannitol) having a total mass that is about 45% to 70% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the pharmaceutical composition.
[373] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 50% of the total mass of the pharmaceutical composition; (ii) a diluent (e.g., mannitol) having a total mass that is about 48.5% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the pharmaceutical composition. In certain embodiments, the solid dosage forms provided herein comprise: (i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 13.51 % of the total mass of the pharmaceutical composition; (ii) a diluent (e.g., mannitol) having a total mass that is about 84.99% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the pharmaceutical composition. [374] In certain embodiments, the solid dosage forms provided herein comprise:
(i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 90.22% of the total mass of the pharmaceutical composition; (ii) a diluent (e.g., mannitol) having a total mass that is about 8.28% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the pharmaceutical composition.
[375] Thus, in certain embodiments, provided herein are solid dosage forms comprising a pharmaceutical agent that contains bacteria. The bacteria can be live bacteria (e.g. , powder or biomass thereof); non-live (dead) bacteria (e.g. , powder or biomass thereof); non replicating bacteria (e.g., powder or biomass thereof); gamma irradiated bacteria (e.g., powder or biomass thereof); and/or lyophilized bacteria (e.g., powder or biomass thereof).
[376] In certain embodiments, provided herein are solid dosage forms comprising a pharmaceutical agent that contains mEVs. The mEVs can be from culture media (e.g., culture supernatant). The mEVs can be from live bacteria (e.g., powder or biomass thereof); the mEVs can be from non-live (dead) bacteria (e.g, powder or biomass thereof); the mEVs can be from non-replicating bacteria (e.g., powder or biomass thereof); the mEVs can be from gamma irradiated bacteria (e.g. , powder or biomass thereof); and/or the mEVs can be from lyophilized bacteria (e.g., powder or biomass thereof).
[377] In some embodiments, the pharmaceutical agent comprises mEVs substantially or entirely free of bacteria (e.g, whole bacteria), bacteria (e.g., live bacteria, dead (e.g., killed) , non-replicating bacteria, attenuated bacteria. In some embodiments, the pharmaceutical agents and/or pharmaceutical compositions comprise both mEVs and bacteria (e.g., whole bacteria) (e.g., live bacteria, killed bacteria, attenuated bacteria). In some embodiments, the pharmaceutical agents comprise bacteria and/or mEVs from one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the hemoglobin-dependent bacteria strains. In some embodiments, the pharmaceutical agents comprise bacteria and/or mEVs from one of the bacteria strains or species described herein, e.g., from bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttlew orthia, Turicibacter, or Veillonella.
[378] In some embodiments, the pharmaceutical agents comprise lyophilized bacteria and/or mEVs. In some embodiments, the pharmaceutical agent comprises gamma irradiated bacteria and/or mEVs. The mEVs (such as smEVs and/or pmEVs) can be gamma irradiated after the mEVs are isolated (e.g., prepared).
[379] In some embodiments, to quantify the numbers of mEVs (such as smEVs and/or pmEVs) and/or bacteria present in a sample, electron microscopy (e.g., EM of ultrathin frozen sections) can be used to visualize the mEVs (such as smEVs and/or pmEVs) and/or bacteria and count their relative numbers. Alternatively, nanoparticle tracking analysis (NTA), Coulter counting, or dynamic light scattering (DLS) or a combination of these techniques can be used. NTA and the Coulter counter count particles and show their sizes. DLS gives the size distribution of particles, but not the concentration. Bacteria frequently have diameters of 1-2 um (microns). The full range is 0.2-20 um. Combined results from Coulter counting and NTA can reveal the numbers of bacteria and/or mEVs (such as smEVs and/or pmEVs) in a given sample. Coulter counting reveals the numbers of particles with diameters of 0.7-10 um. For most bacterial and/or mEV (such as smEV and/or pmEV) samples, the Coulter counter alone can reveal the number of bacteria and/or mEVs (such as smEVs and/or pmEVs) in a sample. pmEVs are 20-600 nm in diameter. For NTA, a Nanosight instrument can be obtained from Malvern Pananlytical. For example, the NS300 can visualize and measure particles in suspension in the size range 10-2000nm. NTA allows for counting of the numbers of particles that are, for example, 50- 1000 nm in diameter. DLS reveals the distribution of particles of different diameters within an approximate range of 1 nm - 3 um.
[380] mEVs can be characterized by analytical methods known in the art (e.g. , Jeppesen, et al. Cell 177:428 (2019)).
[381] In some embodiments, the bacteria and/or mEVs may be quantified based on particle count. For example, total protein content of a bacteria and/or mEV preparation can be measured using NTA. NTA can be performed using Zetaview.
[382] In some embodiments, the bacteria and/or mEVs may be quantified based on the amount of protein, lipid, or carbohydrate. For example, total protein content of a bacteria and/or preparation can be measured using the Bradford assay or BCA. [383] In some embodiments, mEVs are isolated away from one or more other bacterial components of the source bacteria or bacterial culture. In some embodiments, bacteria are isolated away from one or more other bacterial components of the source bacterial culture. In some embodiments, the pharmaceutical agent further comprises other bacterial components.
[384] In certain embodiments, the mEV preparation obtained from the source bacteria may be fractionated into subpopulations based on the physical properties (e.g., sized, density, protein content, binding affinity) of the subpopulations. One or more of the mEV subpopulations can then be incorporated into the pharmaceutical agents of the invention.
[385] In certain aspects, provided herein are pharmaceutical compositions and/or solid dosage forms comprising pharmaceutical agents that comprise bacteria and/or mEVs (such as smEVs and/or pmEVs) useful for the treatment and/or prevention of disease (e.g. , a cancer, an autoimmune disease, an inflammatory disease, a metabolic disease, or a dysbiosis), as well as methods of making and/or identifying such bacteria and/or mEVs, and methods of using pharmaceutical agents and pharmaceutical compositions and/or solid dosage forms thereof (e.g. , for the treatment of a cancer, an autoimmune disease, an inflammatory disease, or a metabolic disease, either alone or in combination with other therapeutics). In some embodiments, the pharmaceutical agents comprise both mEVs (such as smEVs and/or pmEVs) and bacteria (e.g., whole bacteria) (e.g., live bacteria, dead (e.g., killed) bacteria, non-replicating bacteria, attenuated bacteria). In some embodiments, the pharmaceutical agents comprise bacteria in the absence of mEVs (such as smEVs and/or pmEVs). In some embodiments, the pharmaceutical agents comprise mEVs (such as smEVs and/or pmEVs) in the absence of bacteria. In some embodiments, the pharmaceutical agents comprise mEVs (such as smEVs and/or pmEVs) and/or bacteria from hemoglobin- dependent bacteria. In some embodiments, the pharmaceutical agents comprise bacteria and/or mEVs from one of the bacteria strains or species described herein, e.g., from bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides,
Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propioni bacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella. [386] In certain aspects, provided are pharmaceutical agents, and/or pharmaceutical compositions and/or solid dosage forms thereof, for administration to a subject (e.g., human subject). In some embodiments, the pharmaceutical agents are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format. In some embodiments, the pharmaceutical agent is combined with an adjuvant such as an immuno- adjuvant (e.g. , a STING agonist, a TLR agonist, or a NOD agonist).
[387] In some embodiments, the pharmaceutical composition and/or solid dosage form comprises at least one carbohydrate.
[388] In some embodiments, the pharmaceutical composition and/or solid dosage form comprises at least one lipid. In some embodiments, the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22: 1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0).
[389] In some embodiments, the pharmaceutical composition and/or solid dosage form comprises at least one mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
[390] In some embodiments, the pharmaceutical composition and/or solid dosage form comprises at least one vitamin. The at least one vitamin can be fat-soluble or water- soluble vitamins. Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin. [391] In some embodiments, the pharmaceutical composition and/or solid dosage form comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a glidant, a diluent, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
[392] Suitable excipients that can be included in the solid dosage form can be one or more pharmaceutically acceptable excipients known in the art. For example, see Rowe, Sheskey, and Quinn, eds., Handbook of Pharmaceutical Excipients, sixth ed.; 2009; Pharmaceutical Press and American Pharmacists Association.
[393] In some embodiments, the pharmaceutical agent can be prepared as a powder (e.g., for resuspension).
[394] In some embodiments, the pharmaceutical composition can be prepared as a powder (e.g., for resuspension).
[395] Solid dosage forms can similarly be prepared for other types of bacterial compositions, such as medicinal products, medical foods, food products, and/or dietary supplements.
Tablets and Minitablets
[396] The solid dosage form described herein can be, e.g. , a tablet or a minitablet. Further, a plurality of minitablets can be in (e.g. , loaded into) a capsule.
[397] In some embodiments, the solid dosage form comprises a tablet (> 4mm) (e.g., 5mm-17mm). For example, the tablet is a 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm or 18mm tablet. The size refers to the diameter of the tablet, as is known in the art. As used herein, the size of the tablet refers to the size of the tablet prior to application of an enteric coating.
[398] In some embodiments, the solid dosage form comprises a minitablet. The minitablet can be in the size range of lmm-4 mm range. E.g., the minitablet can be a 1mm minitablet, 1.5 mm minitablet, 2mm minitablet, 3mm minitablet, or 4mm minitablet. The size refers to the diameter of the minitablet, as is known in the art. As used herein, the size of the minitablet refers to the size of the minitablet prior to application of an enteric coating.
[399] The minitablets can be in a capsule. The capsule can be a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule. The capsule that contains the minitablets can comprise HPMC (hydroxyl propyl methyl cellulose) or gelatin. The minitablets can be inside a capsule: the number of minitablets inside a capsule will depend on the size of the capsule and the size of the minitablets. As an example, a size 0 capsule can contain 31-35 (an average of 33) minitablets that are 3 mm minitablets.
[400] In some embodiments, the solid dosage form (e.g., tablet or minitablet) is enterically coated (e.g., comprises an enteric coating; e.g., is coated with an enteric coating).
Capsules
[401] The solid dosage form described herein can be a capsule.
[402] The solid dosage forms can comprise capsules. In some embodiments, the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule. In some embodiments, the capsule comprises HPMC (hydroxyl propyl methyl cellulose) or gelatin. In some embodiments, the capsule comprises HPMC (hydroxyl propyl methyl cellulose). In some embodiments, the capsule is banded.
[403] In some embodiments, the solid dosage form (e.g., capsule) is enterically coated (e.g., comprises an enteric coating; e.g., is coated with an enteric coating).
Exemplary Coatings
[404] The solid dosage form (e.g. , tablet or minitablet or capsule) described herein can be enterically coated, e.g., with one enteric coating layer or with two layers of enteric coating, e.g., an inner enteric coating and an outer enteric coating. The inner enteric coating and outer enteric coating are not identical (e.g., the inner enteric coating and outer enteric coating do not contain the same components in the same amounts). The enteric coating can allow for release of the pharmaceutical agent, e.g. , in the small intestine, e.g. , upper small intestine, e.g., duodenum and/or jejunum.
[405] Release of the pharmaceutical agent in the small intestine, e.g. , in the upper small intestine, e.g. , in the duodenum, or in the jejunum, allows the pharmaceutical agent to target and affect cells (e.g., epithelial cells and/or immune cells) located at these specific locations, e.g., which can cause a local effect in the small intestine and/or cause a systemic effect (e.g., an effect outside of the gastrointestinal tract).
[406] EUDRAGIT is the brand name for a diverse range of polymethacrylate- based copolymers. It includes anionic, cationic, and neutral copolymers based on methacrylic acid and methacrylic/acrylic esters or their derivatives. [407] Examples of other materials that can be used in the enteric coating (e.g., the one enteric coating or the inner enteric coating and/or the outer enteric coating) include cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, shellac (esters of aleurtic acid), plastics, plant fibers, zein, AQUA-ZEIN® (an aqueous zein formulation containing no alcohol), amylose starch, starch derivatives, dextrins, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), methyl methacrylate- methacrylic acid copolymers, and/or sodium alginate.
[408] The enteric coating (e.g., the one enteric coating or the inner enteric coating and/or the outer enteric coating) can include a methacrylic acid ethyl acrylate (MAE) copolymer (1:1).
[409] The one enteric coating can include methacrylic acid ethyl acrylate (MAE) copolymer (1:1) (such as Kollicoat MAE 100P).
[410] The one enteric coating can include a Eudragit coplymer, e.g. , a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g. , Eudragit FS 30 D).
[411] Other examples of materials that can be used in the enteric coating (e.g. , the one enteric coating or the inner enteric coating and/or the outer enteric coating) include those described in, e.g., U.S. 6312728; U.S. 6623759; U.S. 4775536; U.S. 5047258; U.S. 5292522; U.S. 6555124; U.S. 6638534; U.S. 2006/0210631; U.S. 2008/200482; U.S. 2005/0271778; U.S. 2004/0028737; WO 2005/044240.
[412] See also, e.g., U.S. 9233074, which provides pH dependent, enteric polymers that can be used with the solid dosage forms provided herein, including methacrylic acid copolymers, polyvinylacetate phthalate, hydroxypropylmethyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate and cellulose acetate phthalate; suitable methacrylic acid copolymers include: poly (methacrylic acid, methyl methacrylate) 1:1 sold, for example, under the Eudragit LI 00 trade name; poly(methacrylic acid, ethyl acrylate) 1:1 sold, for example, under the Eudragit L100-55 trade name; partially- neutralized poly(methacrylic acid, ethyl acrylate) 1 : 1 sold, for example, under the Kollicoat MAE-100P trade name; and poly(methacrylic acid, methyl methacrylate) 1:2 sold, for example, under the Eudragit S100 trade name. [413] In certain aspects, the solid dosage form (e.g. , tablet or minitablet or capsule) described herein further comprises a sub-coating. In some embodiments, the solid dosage form comprises a sub-coating, e.g., in addition to the enteric coating, e.g., the sub-coating is beneath the enteric coating (e.g., between the solid dosage form and the enteric coating). In some embodiments, the sub-coating comprises Opadry QX, e.g., Opadry QX Blue.
Exemplary Doses
[414] The dose of the pharmaceutical agent (e.g. , for human subjects) is the dose per capsule or tablet or per total number of minitablets used in a capsule.
[415] In embodiments where dose is determined by total cell count, total cell count can be determined by Coulter counter.
[416] In some embodiments wherein the pharmaceutical agent comprises bacteria, the dose is total cell count of about 1 x 107 to about 1 x 1013 cells (e.g., wherein cell number is determined by total cell count, which is determined by Coulter counter) per capsule or tablet or per total number of minitablets used in a capsule.
[417] In some embodiments, wherein the pharmaceutical agent comprises bacteria, the dose is about 3 x 1010 or about 1.5 x 1011 cells (e.g., wherein cell number is determined by total cell count, which is determined by Coulter counter) per capsule or tablet or total per total number of minitablets used in a capsule. In some embodiments, wherein the pharmaceutical agent comprises bacteria, the dose is about 8 x 1010 or about 1.6 x 1011 , , or about 3.2 x 1011 cells (e.g., wherein cell number is determined by total cell count, which is determined by Coulter counter) per capsule or tablet or total per total number of minitablets used in a capsule.
[418] In some embodiments, wherein the pharmaceutical agent comprises mEVs, the dose of mEVs is about 1 x 105 to about 2 x 1012 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
[419] In some embodiments, wherein the pharmaceutical agent comprises mEVs, the dose of mEVs is about 2x106 to about 2x1016 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)), wherein the dose is per capsule or tablet or per total number of minitablets in a capsule.
[420] In some embodiments, the pharmaceutical agent dose can be a milligram (mg) dose determined by weight the pharmaceutical agent (e.g., a powder comprising bacteria and/or an agent of bacterial origin, such as mEVs). The dose of the pharmaceutical agent is per capsule or tablet or per total number of minitablets, e.g., in a capsule.
[421] For example, to administer a 1x dose of the pharmaceutical agent of about 400 mg, about 200 mg of the pharmaceutical agent is present per capsule and two capsules are administered, resulting in a dose of about 400 mg. The two capsules can be administered, for example, 1x or 2x daily.
[422] For example, for a minitablet: about 0.1 to about 3.5 mg (0.1, 0.35, 1.0, 3.5 mg) of the pharmaceutical agent can be contained per minitablet. The minitablets can be inside a capsule: the number of minitablets inside a capsule will depend on the size of the capsule and the size of the minitablets. For example, an average of 33 (range of 31-35)
3mm minitablets fit inside a size 0 capsule. As an example, 0.1- 3.5 mg of the pharmaceutical agent per minitablet, the dose range will be 3.3 mg- 115.5 mg (for 33 minitablets in size 0 capsule) per capsule (3.1 mg- 108.5 mg for 31 minitablets in size 0 capsule) (3.5 mg- 122.5 mg for 35 minitablets in size 0 capsule). Multiple capsules and/or larger capsule(s) can be administered to increase the administered dose and/or can be administered one or more times per day to increase the administered dose.
[423] In some embodiments, the dose can be about 3 mg to about 125 mg of the pharmaceutical agent, per capsule or tablet or per total number of minitablets, e.g., in a capsule.
[424] In some embodiments, the dose can be about 35 mg to about 1200 mg (e.g., about 35 mg, about 125 mg, about 350 mg, or about 1200 mg) of the pharmaceutical agent.
[425] In some embodiments, the dose of the pharmaceutical agent can be about 30 mg to about 3500 mg (about 25, about 50, about 75, about 100, about 150, about 250, about 300, about 350, about 400, about 500, about 600, about 750, about 1000, about 1250, about 1300, about 2000, about 2500, about 3000, or about 3500 mg).
[426] A human dose can be calculated appropriately based on allometric scaling of a dose administered to a model organism (e.g., mouse).
[427] In some embodiments, one or two tablets or capsules can be administered one or two times a day.
[428] The pharmaceutical agent contains the bacteria and/or an agent of bacterial origin, such as mEVs, or contains a powder comprising bacteria and/or an agent of bacterial origin, such as mEVs, and can also contain one or more additional components, such as a cryoprotectant etc. [429] In some embodiments, the mg (by weight) dose of the pharmaceutical agent is, e.g., about 1 mg to about 500 mg per capsule, or per tablet, or per total number of minitablets, e.g., used in a capsule.
[430] Other bacterial compositions (e.g., a pharmaceutical composition, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement) provided herein can also have the same dose as described herein for pharmacreutical agents.
Exemplary Methods of Using Bacterial Compositions
[431] The bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)described herein allow, e.g., for oral administration of bacteria (or components thereof, such as mEVs) contained therein.
[432] The solid dosage forms having the disclosed combinations and/or amounts of disintegration agents provide a decrease in disintegration times (e.g., 2-fold, 4-fold, 6- fold, 8-fold), which can further result in an increase in therapeutic efficacy and/or physiological effect as compared to the same solid dosage forms that do not have the disclosed combinations of the disintegration agents.
[433] The bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein can be used in the treatment and/or prevention of a cancer, inflammation, autoimmunity, a metabolic condition, or a dysbiosis.
[434] The bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements described herein can be used in the treatment and/or prevention of bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection).
[435] The bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements described herein can be used to decrease inflammatory cytokine expression (e.g., decreased IL-8, IL-6, IL-Ib, and/or TNFα expression levels).
[436] Methods of using a pharmaceutical composition and/or solid dosage form (e.g. , for oral administration) (e.g. , for pharmaceutical use) comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and soy leghemoglobin, and wherein the solid dosage form further comprises the disclosed disintegration agents are described herein.
[437] The methods and administered pharmaceutical composition and/or solid dosage forms described herein allow, e.g. , for oral administration of a pharmaceutical agent contained therein. The pharmaceutical composition and/or solid dosage form can be administered to a subject is a fed or fasting state. The pharmaceutical composition and/or solid dosage form can be administered, e.g., on an empty stomach (e.g., one hour before eating or two hours after eating). The pharmaceutical composition and/or solid dosage form can be administered one hour before eating. The pharmaceutical composition and/or solid dosage form can be administered two hours after eating.
[438] The methods and administered bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement) described herein allow, e.g., for oral administration of bacteria (or components thereof, such as mEVs) contained therein. The bacterial composition can be administered to a subject is a fed or fasting state. The bacterial composition can be administered, e.g., on an empty stomach (e.g., one hour before eating or two hours after eating). The bacterial composition can be administered one hour before eating. The bacterial composition can be administered two hours after eating.
[439] Bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) for use in the treatment and/or prevention of a cancer, inflammation, autoimmunity, a metabolic condition, or a dysbiosis are provided herein.
[440] Use of a bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement) for the preparation of a medicament for the treatment and/or prevention of a cancer, inflammation, autoimmunity, a metabolic condition, or a dysbiosis is provided herein.
[441] Use of a bacterial composition (e.g., a pharmaceutical composition, a pharmaceutical agent, a solid dosage form, a medicinal product, a medical food, a food product, and/or a dietary supplement) for the preparation of a medicament for the treatment and/or prevention of bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection) is provided herein.
[442] Use of bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) for the preparation of a medicament for decreasing inflammatory cytokine expression (e.g., decreased IL-8, IL-6, IL-Ib, and/or TNFα expression levels) is provided herein.
Method of Making Solid Dosage Forms
[443] In certain aspects, provided herein are methods of preparing a solid dosage form of a pharmaceutical composition, the method comprising (a) combining into a pharmaceutical composition (i) a pharmaceutical agent (e.g., comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein or a powder comprising bacteria and/or mEVs (such as smEVs and/or pmEVs and comprising at least one component of soy leghemoglobin), and (ii) at least one diluent, at least one lubricant, at least one glidant, and/or at least one (e.g., one, two, or three) disintegration agent. In some embodiments, the at least one diluent comprises mannitol. In some embodiments, the at least one lubricant comprises magnesium stearate. In some embodiments, the at least one glidant comprises colloidal silicon dioxide. In some embodiments, the at least one disintegration agent comprises low-substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac-Di-Sol), and/or crospovidone (PVPP).
[444] In certain embodiments, the total pharmaceutical agent mass is at least 5%, 10%, 15%, 20% or 25 % of the total mass of the pharmaceutical composition. In some embodiments the total pharmaceutical agent mass is no more than 45%, 40%, 35%, 30%, or 25% of the total mass of the pharmaceutical composition. In some embodiments, the total mass of the one or more disintegrating agents is at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total mass of the pharmaceutical composition. In some embodiments, the total mass of the one or more disintegrating agents is no more than 70%, 65%, 60%, or 55% of the total mass of the pharmaceutical composition.
[445] In some embodiments, the one or more disintegration agents comprise low- substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac-Di-Sol), and/or crospovidone (PVPP). In certain embodiments, the solid dosage forms provided herein comprise L-HPC. In some embodiments, the L-HPC is of grade LH-B1. In certain embodiments, the total L-HPC mass is at least 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition. In certain embodiments, the total L-HPC mass is no more than 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition. In certain embodiments, the total L-HPC mass is about 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, or 42% of the total mass of the pharmaceutical composition. In certain embodiments, the solid dosage forms provided herein comprise Ac -Do-Sol. In some embodiments, the Ac-Di-Sol is of grade SD-711. In certain embodiments, the total Ac -Di- Sol mass is at least 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition. In certain embodiments, the total Ac-Di-Sol mass is no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition. In certain embodiments, the total Ac-Di-Sol mass is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or 16% of the total mass of the pharmaceutical composition. In certain embodiments, the solid dosage forms provided herein comprise PVPP. In certain embodiments, the total PVPP mass is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition. In certain embodiments, the total PVPP mass is no more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition. In certain embodiments, the total PVPP mass is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the total mass of the pharmaceutical composition.
[446] In certain embodiments, the method further comprises compressing the pharmaceutical composition, thereby forming a tablet or a minitablet. In some embodiments, the method further comprises enterically coating the tablet or minitablet, thereby preparing the enterically coated tablet. In certain embodiments, the method further comprises loading the minitablets into a capsule.
[447] The methods of preparing a solid dosage form of a pharmaceutical composition can comprise blending, encapsulation, banding, and coating of capsules. [448] In certain aspects, provided herein are methods of preparing a solid dosage form of a pharmaceutical composition, the method comprising combining (e.g., blending) into a pharmaceutical composition a pharmaceutical agent (e.g., comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein or a powder comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein) and one or more additional components described herein. In certain aspects, provided herein are methods of preparing a solid dosage form of a pharmaceutical composition, the method comprising combining into a pharmaceutical composition a pharmaceutical agent (e.g., comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein or a powder comprising bacteria and/or mEVs (such as smEVs and/or pmEVs) and a soy leghemoglobin component disclosed herein) and a diluent. In certain embodiments, the total pharmaceutical agent mass is at least 20%, 25%, 30%, 35%, 40%, 45%, 50% or 55% of the total mass of the pharmaceutical composition. In some embodiments the total pharmaceutical agent mass is no more than 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% of the total mass of the pharmaceutical composition.
[449] In some embodiments, the total mass of the diluent is at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the total mass of the pharmaceutical composition. In some embodiments, the total mass of the diluent is no more than 80%, 75%, 70%, 65%, 60%, 55%, 50%, or 45% of the total mass of the pharmaceutical composition. In some embodiments, the diluent comprises mannitol.
[450] In certain embodiments, the method further comprises combining a lubricant. In certain embodiments, the total lubricant mass is at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is no more than 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 0.5% to about 1.5% of the total mass of the pharmaceutical composition. In certain embodiments, the total lubricant mass is about 1% of the total mass of the pharmaceutical composition. In some embodiments, the lubricant comprises magnesium stearate.
[451] In certain embodiments, the method further comprises combining a glidant. In some embodiments, the glidant is colloidal silicon dioxide. In certain embodiments, the total glidant mass is at least 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is no more than 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, or 2% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.25% to about 0.75% of the total mass of the pharmaceutical composition. In certain embodiments, the total glidant mass is about 0.5% of the total mass of the pharmaceutical composition.
[452] In certain embodiments, the method comprises combining: (i) a pharmaceutical agent having a total pharmaceutical agent mass that is at least 20% and no more than 55% of the total mass of the pharmaceutical composition, (ii) a diluent (e.g., mannitol) having a total mass that is at least 45% and no more than 80% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is at least 0.1% and no more than 5% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is at least 0.01% and no more than 2% of the total mass of the pharmaceutical composition.
[453] In certain embodiments, the method comprises combining: (i) a pharmaceutical agent having a total pharmaceutical agent mass that is about 20% to about 50% of the total mass of the pharmaceutical composition, (ii) a diluent (e.g., mannitol) having a total mass that is about 50% to 80% of the total mass of the pharmaceutical composition; (iii) a lubricant (e.g., magnesium stearate) having a total mass that is about 1% of the total mass of the pharmaceutical composition; and (iv) a glidant (e.g., colloidal silicon dioxide) having a total mass that is about 0.5% of the total mass of the pharmaceutical composition.
[454] In some embodiments, the method further comprises loading the pharmaceutical composition into a capsule (e.g., encapsulation).
[455] In some embodiments, the method further comprises banding the capsule after loading.
[456] In some embodiments, the method further comprises enterically coating the capsule. Additional Aspects of the Solid Dosage Forms
[457] The solid dosage forms, e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin, and wherein the solid dosage form further comprises the described disintegration agents, can provide a therapeutically effective amount of the pharmaceutical agent to a subject, e.g., a human.
[458] The solid dosage forms, e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin, and wherein the solid dosage form further comprises the described disintegration agents, can provide a non-natural amount of the therapeutically effective components (e.g., present in the pharmaceutical agent) to a subject, e.g. , a human.
[459] The solid dosage forms, e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin, and wherein the solid dosage form further comprises the described disintegration agents, can provide an unnatural quantity of the therapeutically effective components (e.g. , present in the pharmaceutical agent) to a subject, e.g. , a human.
[460] The solid dosage forms, e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin, and wherein the solid dosage form further comprises the described disintegration agents, can bring about one or more changes to a subject, e.g. , human, e.g. , to treat or prevent a disease or a health disorder.
[461] The solid dosage forms, e.g., as described herein, comprising a pharmaceutical agent (e.g., a therapeutically effective amount thereof), wherein the pharmaceutical agent comprises bacteria and/or microbial extracellular vesicles (mEVs) and comprises at least one component of soy leghemoglobin, and wherein the solid dosage form further comprises the described disintegration agents, has potential for significant utility, e.g. , to affect a subject, e.g. , a human, e.g. , to treat or prevent a disease or a health disorder. Administration
[462] In certain aspects, provided herein is a method of delivering bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)de scribed herein to a subject.
[463] The dosage regimen can be any of a variety of methods and amounts, and can be determined by one skilled in the art according to known clinical factors. As is known in the medical arts, dosages for any one patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the particular microorganism to be administered, duration and route of administration, the kind and stage of the disease, for example, tumor size, and other compounds such as drugs being administered concurrently or near-concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art. In the present methods, appropriate minimum dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate. The dose of a pharmaceutical agent (e.g., in a solid dosage form) described herein may be appropriately set or adjusted in accordance with the dosage form, the route of administration, the degree or stage of a target disease, and the like.
[464] In some embodiments, the dose administered to a subject is sufficient to prevent disease (e.g., autoimmune disease, inflammatory disease, metabolic disease, or cancer), delay its onset, or slow or stop its progression, or relieve one or more symptoms of the disease. One skilled in the art will recognize that dosage will depend upon a variety of factors including the strength of the particular agent (e.g., pharmaceutical agent) employed, as well as the age, species, condition, and body weight of the subject. The size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular pharmaceutical agent and the desired physiological effect.
[465] In accordance with the above, in therapeutic applications, the dosages of the pharmaceutical agents used in accordance with the invention vary depending on the active agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. For example, for cancer treatment, the dose should be sufficient to result in slowing, and preferably regressing, the growth of a tumor and most preferably causing complete regression of the cancer, or reduction in the size or number of metastases As another example, the dose should be sufficient to result in slowing of progression of the disease for which the subject is being treated, and preferably amelioration of one or more symptoms of the disease for which the subject is being treated.
[466] Separate administrations can include any number of two or more administrations, including two, three, four, five or six administrations. One skilled in the art can readily determine the number of administrations to perform or the desirability of performing one or more additional administrations according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein. Accordingly, the methods provided herein include methods of providing to the subject one or more administrations of a solid dosage form, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results.
[467] The time period between administrations can be any of a variety of time periods. The time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response. In one example, the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be no more than the time period for a subject to mount an immune response, such as no more than about one week, no more than about ten days, no more than about two weeks, or no more than about a month.
Immune Disorders
[468] In some embodiments, the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein relate to the treatment or prevention of a disease or disorder associated a pathological immune response, such as an autoimmune disease, an allergic reaction and/or an inflammatory disease. In some embodiments, the disease or disorder is an inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis). In some embodiments, the disease or disorder is psoriasis. In some embodiments, the disease or disorder is psoriatic arthritis. In some embodiments, the disease or disorder is atopic dermatitis. In some embodiments, the disease or disorder is asthma.
[469] The methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein can be used to treat any subject in need thereof. As used herein, a “subject in need thereof’ includes any subject that has a disease or disorder associated with a pathological immune response (e.g., an inflammatory bowel disease), as well as any subject with an increased likelihood of acquiring a such a disease or disorder.
[470] The bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein can be used, for example, as a pharmaceutical composition for preventing or treating (reducing, partially or completely, the adverse effects of) an autoimmune disease, such as chronic inflammatory bowel disease, systemic lupus erythematosus, psoriasis, muckle-wells syndrome, rheumatoid arthritis, multiple sclerosis, or Hashimoto's disease; an allergic disease, such as a food allergy, pollenosis, or asthma; an infectious disease, such as an infection with Clostridium difficile; an inflammatory disease such as a TNF -mediated inflammatory disease (e.g., an inflammatory disease of the gastrointestinal tract, such as pouchitis, a cardiovascular inflammatory condition, such as atherosclerosis, or an inflammatory lung disease, such as chronic obstructive pulmonary disease); a pharmaceutical agent and/or a pharmaceutical composition for suppressing rejection in organ transplantation or other situations in which tissue rejection might occur; a supplement, food, or beverage for improving immune functions; or a reagent for suppressing the proliferation or function of immune cells.
[471] In some embodiments, the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) provided herein are useful for the treatment of inflammation. In certain embodiments, the inflammation of any tissue and organs of the body, including musculoskeletal inflammation, vascular inflammation, neural inflammation, digestive system inflammation, ocular inflammation, inflammation of the reproductive system, and other inflammation, as discussed below.
[472] Immune disorders of the musculoskeletal system include, but are not limited, to those conditions affecting skeletal joints, including joints of the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle, and foot, and conditions affecting tissues connecting muscles to bones such as tendons. Examples of such immune disorders, which may be treated with the methods and compositions described herein include, but are not limited to, arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis, tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis, myositis, and osteitis (including, for example, Paget's disease, osteitis pubis, and osteitis fibrosa cystic).
[473] Ocular immune disorders refers to a immune disorder that affects any structure of the eye, including the eye lids. Examples of ocular immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, blepharitis, blepharochalasis, conjunctivitis, dacryoadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis.
[474] Examples of nervous system immune disorders which may be treated with the methods and solid dosage forms described herein include, but are not limited to, encephalitis, Guillain-Barre syndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis, myelitis and schizophrenia. Examples of inflammation of the vasculature or lymphatic system which may be treated with the methods and compositions described herein include, but are not limited to, arthrosclerosis, arthritis, phlebitis, vasculitis, and lymphangitis.
[475] Examples of digestive system immune disorders which may be treated with the methods and solid dosage forms described herein include, but are not limited to, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, and proctitis. Inflammatory bowel diseases include, for example, certain art-recognized forms of a group of related conditions. Several major forms of inflammatory bowel diseases are known, with Crohn's disease (regional bowel disease, e.g., inactive and active forms) and ulcerative colitis (e.g., inactive and active forms) the most common of these disorders. In addition, the inflammatory bowel disease encompasses irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis. Other less common forms of IBD include indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet’s disease, sarcoidosis, scleroderma, IBD-associated dysplasia, dysplasia associated masses or lesions, and primary sclerosing cholangitis.
[476] Examples of reproductive system immune disorders which may be treated with the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements)s described herein include, but are not limited to, cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis, and vulvodynia.
[477] The methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein may be used to treat autoimmune conditions having an inflammatory component. Such conditions include, but are not limited to, acute disseminated alopecia universalise, Behcet's disease, Chagas' disease, chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, diabetes mellitus type 1, giant cell arteritis, goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto's disease, Henoch- Schonlein purpura, Kawasaki's disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, Muckle-Wells syndrome, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmune haemolytic anemia, interstitial cystitis, Lyme disease, morphea, psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.
[478] The methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein may be used to treat T-cell mediated hypersensitivity diseases having an inflammatory component. Such conditions include, but are not limited to, contact hypersensitivity, contact dermatitis (including that due to poison ivy), uticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dustmite allergy) and gluten-sensitive enteropathy (Celiac disease).
[479] Other immune disorders which may be treated with the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel, skin allografts, skin homografts, and heart valve xengrafts, sewrum sickness, and graft vs host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, Sexary's syndrome, congenital adrenal hyperplasis, nonsuppurative thyroiditis, hypercalcemia associated with cancer, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensistivity reactions, allergic conjunctivitis, keratitis, herpes zoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonary tuberculosis chemotherapy, idiopathic thrombocytopenic purpura in adults, secondary thrombocytopenia in adults, acquired (autoimmune) haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia of childhood, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, solid organ transplant rejection, sepsis. Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosus, psoriasis, chronic obstructive pulmonary disease, and inflammation accompanying infectious conditions (e.g., sepsis).
Metabolic Disorders
[480] In some embodiments, the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein relate to the treatment or prevention of a metabolic disease or disorder a, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), Nonalcoholic Steatohepatitis (NASH) or a related disease. In some embodiments, the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema. In some embodiments, the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein relate to the treatment of Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH).
[481] The methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein can be used to treat any subject in need thereof. As used herein, a “subject in need thereof’ includes any subject that has a metabolic disease or disorder, as well as any subject with an increased likelihood of acquiring a such a disease or disorder.
[482] The bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein can be used, for example, for preventing or treating (reducing, partially or completely, the adverse effects of) a metabolic disease, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), Nonalcoholic Steatohepatitis (NASH), or a related disease. In some embodiments, the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema. Cancer
[483] In some embodiments, the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein relate to the treatment of cancer. In some embodiments, any cancer can be treated using the methods described herein. Examples of cancers that may treated by methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infdtrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma;
Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fimgoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
[484] In some embodiments, the cancer comprises a solid tumor.
[485] In some embodiments, the cancer comprises breast cancer (e.g., triple negative breast cancer).
[486] In some embodiments, the cancer comprises colorectal cancer (e.g., microsatellite stable (MSS) colorectal cancer).
[487] In some embodiments, the cancer comprises renal cell carcinoma. [488] In some embodiments, the cancer comprises lung cancer (e.g., non small cell lung cancer).
[489] In some embodiments, the cancer comprises bladder cancer.
[490] In some embodiments, the cancer comprises gastroesophageal cancer.
[491] In some embodiments, the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) provided herein relate to the treatment of a leukemia. The term "leukemia" includes broadly progressive, malignant diseases of the hematopoietic organs/systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Non-limiting examples of leukemia diseases include, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, and promyelocytic leukemia.
[492] In some embodiments, the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) provided herein relate to the treatment of a carcinoma. The term "carcinoma" refers to a malignant growth made up of epithelial cells tending to infdtrate the surrounding tissues, and/or resist physiological and non-physiological cell death signals and gives rise to metastases. Non-limiting exemplary types of carcinomas include, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, and carcinoma scroti.
[493] In some embodiments, the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) provided herein relate to the treatment of a sarcoma. The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance. Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing' s sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
[494] Additional exemplary neoplasias that can be treated using the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein include Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, plasmacytoma, colorectal cancer, rectal cancer, and adrenal cortical cancer.
[495] In some embodiments, the cancer treated is a melanoma. The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Non-limiting examples of melanomas are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
[496] Particular categories of tumors that can be treated using methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above. Particular types of tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma, hypemephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma including small cell, non-small and large cell lung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon carcinoma, rectal carcinoma, hematopoietic malignancies including all types of leukemia and lymphoma including: acute myelogenous leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin' s lymphoma, non-Hodgkin' s lymphoma, plasmacytoma, colorectal cancer, and rectal cancer.
[497] Cancers treated in certain embodiments also include precancerous lesions, e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucous fibrosis, actinic (solar) elastosis and cervical dysplasia.
[498] Cancers treated in some embodiments include non-cancerous or benign tumors, e.g., of endodermal, ectodermal or mesenchymal origin, including, but not limited to cholangioma, colonic polyp, adenoma, papilloma, cystadenoma, liver cell adenoma, hydatidiform mole, renal tubular adenoma, squamous cell papilloma, gastric polyp, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma. Other Diseases and Disorders
[499] In some embodiments, the methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein relate to the treatment of liver diseases. Such diseases include, but are not limited to, Alagille Syndrome, Alcohol-Related Liver Disease, Alpha- 1 Antitrypsin Deficiency, Autoimmune Hepatitis, Benign Liver Tumors, Biliary Atresia, Cirrhosis, Galactosemia, Gilbert Syndrome, Hemochromatosis, Hepatitis A, Hepatitis B, Hepatitis C, Hepatic Encephalopathy, Intrahepatic Cholestasis of Pregnancy (ICP), Lysosomal Acid Lipase Deficiency (LAL-D), Liver Cysts, Liver Cancer, Newborn Jaundice, Primary Biliary Cholangitis (PBC), Primary Sclerosing Cholangitis (PSC), Reye Syndrome, Type I Glycogen Storage Disease, and Wilson Disease.
[500] The methods and bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) described herein may be used to treat neurodegenerative and neurological diseases. In certain embodiments, the neurodegenerative and/or neurological disease is Parkinson’s disease, Alzheimer’s disease, prion disease, Huntington’s disease, motor neuron diseases (MND), spinocerebellar ataxia, spinal muscular atrophy, dystonia, idiopathicintracranial hypertension, epilepsy, nervous system disease, central nervous system disease, movement disorders, multiple sclerosis, encephalopathy, peripheral neuropathy or post-operative cognitive dysfunction.
Dysbiosis
[501] In recent years, it has become increasingly clear that the gut microbiome (also called the “gut microbiota”) can have a significant impact on an individual’s health through microbial activity and influence (local and/or distal) on immune and other cells of the host (Walker, W.A., Dysbiosis. The Microbiota in Gastrointestinal Pathophysiology. Chapter 25. 2017; Weiss and Thierry, Mechanisms and consequences of intestinal dysbiosis. Cellular and Molecular Life Sciences. (2017) 74(16):2959-2977. Zurich Open Repository and Archive, doi: https://doi.org/10.1007/s00018-017-2509-x)).
[502] A healthy host-gut microbiome homeostasis is sometimes referred to as a “eubiosis” or “normobiosis,” whereas a detrimental change in the host microbiome composition and/or its diversity can lead to an unhealthy imbalance in the microbiome, or a “dysbiosis” (Hooks and O’Malley. Dysbiosis and its discontents. American Society for Microbiology. Oct 2017. Vol. 8. Issue 5. mBio 8:e01492-17. https://doi.org/10.1128/mBio.01492-17). Dysbiosis, and associated local or distal host inflammatory or immune effects, may occur where microbiome homeostasis is lost or diminished, resulting in: increased susceptibility to pathogens; altered host bacterial metabolic activity; induction of host proinflammatory activity and/or reduction of host anti inflammatory activity. Such effects are mediated in part by interactions between host immune cells (e.g., T cells, dendritic cells, mast cells, NK cells, intestinal epithelial lymphocytes (IEC), macrophages and phagocytes) and cytokines, and other substances released by such cells and other host cells.
[503] A dysbiosis may occur within the gastrointestinal tract (a “gastrointestinal dysbiosis” or “gut dysbiosis”) or may occur outside the lumen of the gastrointestinal tract (a “distal dysbiosis”). Gastrointestinal dysbiosis is often associated with a reduction in integrity of the intestinal epithelial barrier, reduced tight junction integrity and increased intestinal permeability. Citi, S. Intestinal Barriers protect against disease, Science 359: 1098- 99 (2018); Srinivasan et al., TEER measurement techniques for in vitro barrier model systems. J. Lab. Autom. 20: 107-126 (2015). A gastrointestinal dysbiosis can have physiological and immune effects within and outside the gastrointestinal tract.
[504] The presence of a dysbiosis has been associated with a wide variety of diseases and conditions including: infection, cancer, autoimmune disorders (e.g., systemic lupus erythematosus (SLE)) or inflammatory disorders (e.g., functional gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease), neuroinflammatory diseases (e.g., multiple sclerosis), transplant disorders (e.g., graft-versus-host disease), fatty liver disease, type I diabetes, rheumatoid arthritis, Sjogren’s syndrome, celiac disease, cystic fibrosis, chronic obstructive pulmonary disorder (COPD), and other diseases and conditions associated with immune dysfunction. Lynch et al., The Human Microbiome in Health and Disease, N. Engl. J. Med .375:2369-79 (2016), Carding et al., Dysbiosis of the gut microbiota in disease. Microb. Ecol. Health Dis. (2015); 26: 10: 3402/mehd.v26.2619; Levy et al, Dysbiosis and the Immune System, Nature Reviews Immunology 17:219 (April 2017)
[505] Exemplary bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) disclosed herein can treat a dysbiosis and its effects by modifying the immune activity present at the site of dysbiosis. As described herein, such compositions can modify a dysbiosis via effects on host immune cells, resulting in, e.g., an increase in secretion of anti-inflammatory cytokines and/or a decrease in secretion of pro- inflammatory cytokines, reducing inflammation in the subject recipient or via changes in metabolite production.
[506] Exemplary bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) disclosed herein that are useful for treatment of disorders associated with a dysbiosis contain one or more types of immunomodulatory bacteria and/or mEVs (microbial extracellular vesicles) derived from such bacteria. Such compositions are capable of affecting the recipient host’s immune function, in the gastrointestinal tract, and/or a systemic effect at distal sites outside the subject’s gastrointestinal tract.
[507] Exemplary bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) disclosed herein that are useful for treatment of disorders associated with a dysbiosis contain a population of immunomodulatory bacteria of a single bacterial species (e.g., a single strain) and/or mEVs derived from such bacteria. Such compositions are capable of affecting the recipient host’s immune function, in the gastrointestinal tract, and /or a systemic effect at distal sites outside the subject’s gastrointestinal tract.
[508] In one embodiment, bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) containing an isolated population of immunomodulatory bacteria or mEVs derived from such bacteria are administered (e.g., orally) to a mammalian recipient in an amount effective to treat a dysbiosis and one or more of its effects in the recipient. The dysbiosis may be a gastrointestinal tract dysbiosis or a distal dysbiosis.
[509] In another embodiment, bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) of the instant invention can treat a gastrointestinal dysbiosis and one or more of its effects on host immune cells, resulting in an increase in secretion of anti-inflammatory cytokines and/or a decrease in secretion of pro-inflammatory cytokines, reducing inflammation in the subject recipient.
[510] In another embodiment, the bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) can treat a gastrointestinal dysbiosis and one or more of its effects by modulating the recipient immune response via cellular and cytokine modulation to reduce gut permeability by increasing the integrity of the intestinal epithelial barrier.
[511] In another embodiment, the bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) can treat a distal dysbiosis and one or more of its effects by modulating the recipient immune response at the site of dysbiosis via modulation of host immune cells.
[512] Other bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) are useful for treatment of disorders associated with a dysbiosis, which compositions contain one or more types of bacteria or mEVs capable of altering the relative proportions of host immune cell subpopulations, e.g., subpopulations of T cells, immune lymphoid cells, dendritic cells, NK cells and other immune cells, or the function thereof, in the recipient.
[513] Other exemplary bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) are useful for treatment of disorders associated with a dysbiosis, which compositions contain a population of immunomodulatory bacteria or mEVs of a single bacterial species e.g., a single strain) capable of altering the relative proportions of immune cell subpopulations, e.g., T cell subpopulations, immune lymphoid cells, NK cells and other immune cells, or the function thereof, in the recipient subject.
[514] In one embodiment, the invention provides methods of treating a gastrointestinal dysbiosis and one or more of its effects by orally administering to a subject in need thereof bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) which alters the microbiome population existing at the site of the dysbiosis. The bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) can contain one or more types of immunomodulatory bacteria or mEVs or a population of immunomodulatory bacteria or mEVs of a single bacterial species (e.g., a single strain).
[515] In one embodiment, the invention provides methods of treating a distal dysbiosis and one or more of its effects by orally administering to a subject in need thereof bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) which alters the subject’s immune response outside the gastrointestinal tract. The bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) can contain one or more types of immunomodulatory bacteria or mEVs or a population of immunomodulatory bacteria or mEVs of a single bacterial species (e.g., a single strain).
[516] In exemplary embodiments, bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) useful for treatment of disorders associated with a dysbiosis stimulate secretion of one or more anti-inflammatory cytokines by host immune cells. Anti-inflammatory cytokines include, but are not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGF , and combinations thereof. In other exemplary embodiments, bacterial compositions (e.g., pharmaceutical compositions, pharmaceutical agents, solid dosage forms, medicinal products, medical foods, food products, and/or dietary supplements) useful for treatment of disorders associated with a dysbiosis that decrease (e.g., inhibit) secretion of one or more pro-inflammatory cytokines by host immune cells. Pro-inflammatory cytokines include, but are not limited to, IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIPlα, MIRIb, TNFα, and combinations thereof. Other exemplary cytokines are known in the art and are described herein.
[517] In another aspect, the invention provides a method of treating or preventing a disorder associated with a dysbiosis in a subject in need thereof, comprising administering (e.g., orally administering) to the subject a composition in the form of a probiotic or medical food comprising bacteria or mEVs in an amount sufficient to alter the microbiome at a site of the dysbiosis, such that the disorder associated with the dysbiosis is treated. [518] In another embodiment, a composition of the instant invention in the form of a probiotic or medical food may be used to prevent or delay the onset of a dysbiosis in a subject at risk for developing a dysbiosis.
EXEMPLARY EMBONIMENTS
[519] Provided in exemplary embodiment 1 is a pharmaceutical composition comprising: a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin.
[520] Provided in exemplary embodiment 2 is a pharmaceutical composition of embodiment 1, wherein the at least one component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid.
[521] Provided in exemplary embodiment 3 is a pharmaceutical composition of embodiment 2, wherein the soy leghemoglobin nucleic acid is soy leghemoglobin DNA.
[522] Provided in exemplary embodiment 4 is a pharmaceutical composition of embodiment 3, wherein the soy leghemoglobin DNA comprises a sequence encoding soybean leghemoglobin A (LB A) or soybean leghemoglobin C2 (LGB2).
[523] Provided in exemplary embodiment 5 is a pharmaceutical composition of embodiment 1, wherein the at least one component of soy leghemoglobin comprises a soybean leghemoglobin protein.
[524] Provided in exemplary embodiment 6 is a pharmaceutical composition of embodiment 5, wherein the soy leghemoglobin protein is soy leghemoglobin A or soybean leghemoglobin C2 (LGB2).
[525] Provided in exemplary embodiment 7 is a pharmaceutical composition of any one of embodiments 1-6, wherein the at least one component of soy leghemoglobin is purified from soy roots or soy root nodules.
[526] Provided in exemplary embodiment 8 is a pharmaceutical composition of any one of embodiments 1-7, wherein the at least one component of soy leghemoglobin is recombinantly expressed.
[527] Provided in exemplary embodiment 9 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2. [528] Provided in exemplary embodiment 10 is a pharmaceutical composition of embodiment 9, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO:
4 or GenBank: NP_001235248.2.
[529] Provided in exemplary embodiment 11 is a pharmaceutical composition of embodiment 9 or 10, wherein the at least one component of soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
[530] Provided in exemplary embodiment 12 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
[531] Provided in exemplary embodiment 13 is a pharmaceutical composition of embodiment 12, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
[532] Provided in exemplary embodiment 14 is a pharmaceutical composition of embodiment 12 or 13, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
[533] Provided in exemplary embodiment 15 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
[534] Provided in exemplary embodiment 16 is a pharmaceutical composition of embodiment 15, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO:
5 or GenBank: NP_001235928.1.
[535] Provided in exemplary embodiment 17 is a pharmaceutical composition of embodiment 15 or 16, wherein the at least one component of soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
[536] Provided in exemplary embodiment 18 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
[537] Provided in exemplary embodiment 19 is a pharmaceutical composition of embodiment 18, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
[538] Provided in exemplary embodiment 20 is a pharmaceutical composition of embodiment 18 or 19, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence SEQ ID NO: 5 or GenBank: NP_001235928.1.
[539] Provided in exemplary embodiment 21 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
[540] Provided in exemplary embodiment 22 is a pharmaceutical composition of embodiment 21, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO:
114 or UniProtKB - P02236 (LGB2 _ SOYBN).
[541] Provided in exemplary embodiment 23 is a pharmaceutical composition of embodiment 21 or 22, wherein the at least one component of soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
[542] Provided in exemplary embodiment 24 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
[543] Provided in exemplary embodiment 25 is a pharmaceutical composition of embodiment 24, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN).
[544] Provided in exemplary embodiment 26 is a pharmaceutical composition of embodiment 24 or 25, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ _OYBN) . [545] Provided in exemplary embodiment 27 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGB A SOYBN) .
[546] Provided in exemplary embodiment 28 is a pharmaceutical composition of embodiment 27, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO:
115 or UniProtKB - P02238 (LGBA _ SOYBN ).
[547] Provided in exemplary embodiment 29 is a pharmaceutical composition of embodiment 27 or 28, wherein the at least one component of soy leghemoglobin comprises the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
[548] Provided in exemplary embodiment 30 is a pharmaceutical composition of any one of embodiments 1-8, wherein the at least one component of soy leghemoglobin comprises comprises a nucleic acid that encodes a sequence with at least 70% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
[549] Provided in exemplary embodiment 31 is a pharmaceutical composition of embodiment 30, wherein the at least one component of soy leghemoglobin comprises comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
[550] Provided in exemplary embodiment 32 is a pharmaceutical composition of embodiment 30 or 31, wherein the at least one component of soy leghemoglobin comprises comprises a nucleic acid that encodes the amino acid sequence SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN).
[551] Provided in exemplary embodiment 33 is a pharmaceutical composition of embodiment 8, wherein the recombinantly expressed polypeptide comprises a heterologous polypeptide.
[552] Provided in exemplary embodiment 34 is a pharmaceutical composition of embodiment 33, wherein the heterologous polypeptide comprises a histidine tag, TAP (tandem affinity purification) tag, TEV cleavage site, a FLAG tag, a GST tag, and/or an immunoglobulin domain.
[553] Provided in exemplary embodiment 35 is a pharmaceutical composition of any one of embodiments 8, 33, or 34, wherein the recombinant polypeptide is expressed and purified from an exogenous nucleic acid in a host cell. [554] Provided in exemplary embodiment 36 is a pharmaceutical composition of embodiment 35, wherein the exogenous nucleic acid is in a vector.
[555] Provided in exemplary embodiment 37 is a pharmaceutical composition of embodiment 36, wherein the vector is an expression vector.
[556] Provided in exemplary embodiment 38 is a pharmaceutical composition of any one of embodiments 35-37, wherein the host cell is a bacteria, yeast, insect, or mammalian cell line.
[557] Provided in exemplary embodiment 39 is a pharmaceutical composition of embodiment 38, wherein the host cell is Pichia Pastoris.
[558] Provided in exemplary embodiment 40 is a pharmaceutical composition of any one of embodiments 1-39, wherein the at least one component of soy leghemoglobin is a soy leghemoglobin recombinantly expressed in Pichia pastoris.
[559] Provided in exemplary embodiment 41 is a pharmaceutical composition of any one of embodiments 1-40, wherein the bacteria are hemoglobin-dependent bacteria.
[560] Provided in exemplary embodiment 42 is a pharmaceutical composition of any one of embodiments 1-41, wherein the bacteria are of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Foumierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
[561] Provided in exemplary embodiment 43 is a pharmaceutical composition of embodiment 42, wherein the bacteria are of the genus Foumierella.
[562] Provided in exemplary embodiment 44 is a pharmaceutical composition of embodiment 43, wherein the Foumierella are Foumierella Strain B (ATCC Deposit Number PTA- 126696).
[563] Provided in exemplary embodiment 45 is a pharmaceutical composition of embodiment 42, wherein the bacteria are of the genus Prevotella.
[564] Provided in exemplary embodiment 46 is a pharmaceutical composition of embodiment 45, wherein the bacteria are Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella ftisca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, or Prevotella veroralis.
[565] Provided in exemplary embodiment 47 is a pharmaceutical composition of embodiment 45, wherein the bacteria are of the species Prevotella histicola.
[566] Provided in exemplary embodiment 48 is a pharmaceutical composition of embodiment 45, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
[567] Provided in exemplary embodiment 49 is a pharmaceutical composition of embodiment 45, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain C (ATTC Deposit Number PTA-126140).
[568] Provided in exemplary embodiment 50 is a pharmaceutical composition of embodiment 45, wherein the Prevotella are Prevotella Strain B 50329 (NRRL accession number B 50329).
[569] Provided in exemplary embodiment 51 is a pharmaceutical composition of embodiment 45, wherein the Prevotella are Prevotella Strain C (ATTC Deposit Number PTA-126140).
[570] Provided in exemplary embodiment 52 is a pharmaceutical composition of embodiment 45, wherein the Prevotella bacteria (i) comprise one or more proteins listed in Table 1, and/or (ii) are substantially free of a protein listed in Table 2.
[571] Provided in exemplary embodiment 53 is a pharmaceutical composition of any one of embodiments 1 to 52, wherein the bacterial are live, attenuated, or dead.
[572] Provided in exemplary embodiment 54 is a pharmaceutical composition of any one of embodiments 1 to 53, wherein the bacteria are lyophilized bacteria. [573] Provided in exemplary embodiment 55 is a pharmaceutical composition of any one of embodiments 1-54, wherein the pharmaceutical agent comprises mEVs.
[574] Provided in exemplary embodiment 56 is a pharmaceutical composition of embodiment 55, wherein the mEVs are secreted mEVs (smEVs).
[575] Provided in exemplary embodiment 57 is a pharmaceutical composition of embodiment 55, wherein the mEVs are processed mEVs (pmEVs).
[576] Provided in exemplary embodiment 58 is a pharmaceutical of any one of embodiments 55 to 57, wherein the mEVs are from hemoglobin-dependent bacteria.
[577] Provided in exemplary embodiment 59 is a pharmaceutical composition of any one of embodiments 55 to 58, wherein the mEVs are from bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Foumierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
[578] Provided in exemplary embodiment 60 is a pharmaceutical composition of embodiment 59, wherein the mEVs are from bacteria of the genus Foumierella.
[579] Provided in exemplary embodiment 61 is a pharmaceutical composition of embodiment 60, wherein the Foumierella are Foumierella Strain B (ATCC Deposit Number PTA- 126696).
[580] Provided in exemplary embodiment 62 is a pharmaceutical composition of embodiment 59, wherein the mEVs are from bacteria of the genus Prevotella.
[581] Provided in exemplary embodiment 63 is a pharmaceutical composition of embodiment 62, wherein the bacteria are Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevote 11a pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, or Prevotella veroralis.
[582] Provided in exemplary embodiment 64 is a pharmaceutical composition of embodiment 62, wherein the mEVs are from bacteria of the species Pr votella histicola.
[583] Provided in exemplary embodiment 65 is a pharmaceutical composition of embodiment 62, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
[584] Provided in exemplary embodiment 66 is a pharmaceutical composition of embodiment 62, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain C (ATTC Deposit Number PTA-126140).
[585] Provided in exemplary embodiment 67 is a pharmaceutical composition of embodiment 62, wherein the Prevotella are Prevotella Strain B 50329 (NRRL accession number B 50329).
[586] Provided in exemplary embodiment 68 is a pharmaceutical composition of embodiment 62, wherein the Prevotella are Prevotella Strain C (ATTC Deposit Number PTA-126140).
[587] Provided in exemplary embodiment 69 is a pharmaceutical composition of embodiment 62, wherein the Prevotella bacteria (i) comprise one or more proteins listed in Table 1, and/or (ii) are substantially free of a protein listed in Table 2.
[588] Provided in exemplary embodiment 70 is a pharmaceutical composition of any one of embodiments 55 to 69, wherein the mEVs are lyophilized mEVs.
[589] Provided in exemplary embodiment 71 is a pharmaceutical composition of any one of embodiments 1 to 70, further comprising a cryoprotectant.
[590] Provided in exemplary embodiment 72 is a solid dosage form comprising: (i) a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin; and (ii) at least one diluent, at least one lubricant, at least one glidant, and/or at least one disintegration agent. [591] Provided in exemplary embodiment 73 is a solid dosage form of embodiment 72, wherein the at least one diluent has a total mass that is at least 1% and no more than 95% of the total mass of the solid dosage form.
[592] Provided in exemplary embodiment 74 is a solid dosage form of embodiment 72, wherein the at least one diluent comprises mannitol.
[593] Provided in exemplary embodiment 75 is a solid dosage form of any one of embodiments 72 to 74, wherein the at least one lubricant has a total mass that is at least
0.1% and no more than 5% of the total mass of the solid dosage form.
[594] Provided in exemplary embodiment 76 is a solid dosage form of any one of embodiments 72 to 75, wherein the at least one lubricant comprises magnesium stearate.
[595] Provided in exemplary embodiment 77 is a solid dosage form of any one of embodiments 72 to 76, wherein the at least one glidant has a total mass that is at least 0.01% and no more than 2% of the total mass of the solid dosage form.
[596] Provided in exemplary embodiment 78 is a solid dosage form of any one of embodiments 72 to 77, wherein the at least one glidant comprises colloidal silicon dioxide.
[597] Provided in exemplary embodiment 79 is a solid dosage form of any one of embodiments 72 to 78, wherein the at least one disintegration agent has a total mass that is at least 40% of the total mass of the solid dosage form.
[598] Provided in exemplary embodiment 80 is a solid dosage form of any one of embodiments 72 to 79, wherein the at least one disintegration agent comprises low- substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac-Di-Sol), and/or crospovidone (PVPP).
[599] Provided in exemplary embodiment 81 is a solid dosage form of any one of embodiments 72 to 80, wherein the the at least one disintegration agent comprises low- substituted hydroxypropyl cellulose (L-HPC), croscarmellose sodium (Ac-Di-Sol), and crospovidone (PVPP).
[600] Provided in exemplary embodiment 82 is a solid dosage form of embodiment 80 or 81, wherein the L-HPC has a total L-HPC mass that is at least 22% and no more than 42% of the total mass of the solid dosage form.
[601] Provided in exemplary embodiment 83 is a solid dosage form of any one of embodiments 80 to 82, wherein the L-HPC is L-HPC of grade LH-B1. [602] Provided in exemplary embodiment 84 is a solid dosage form of any one of embodiments 80 to 83, wherein the Ac-Di-Sol has a total Ac -Di-Sol mass that is at least 0.01% and no more than 16% of the total mass of the solid dosage form.
[603] Provided in exemplary embodiment 85 is a solid dosage form of any one of embodiments 80 to 84, wherein the Ac-Di-Sol is Ac-Di-Sol of grade SD-711.
[604] Provided in exemplary embodiment 86 is a solid dosage form of any one of embodiments 80 to 85, wherein the PVPP has a total PVPP mass that is at least 5% and no more than 25% of the total mass of the solid dosage form.
[605] Provided in exemplary embodiment 87 is a solid dosage form of any one of embodiments 80 to 86, wherein the total L-HPC mass plus the total Ac-Di-Sol mass plus the total PVPP mass is at least 40% of the total mass of the solid dosage form.
[606] Provided in exemplary embodiment 88 is a solid dosage form of any one of embodiments 75 to 82, wherein the total L-HPC mass is at least 22% and no more than 42% of the total mass of the solid dosage form; the total Ac-Di-Sol mass is at least 0.01% and no more than 16% of the total mass of the solid dosage form; and the total PVPP mass is at least 5% and no more than 25% of the total mass of the solid dosage form.
[607] Provided in exemplary embodiment 89 is a solid dosage form of any one of embodiments 75 to 83, wherein the total L-HPC mass is about 32% of the total mass of the solid dosage form; the total Ac-Di-Sol mass is about 6% of the total mass of the solid dosage form; and the total PVPP mass is about 15% of the total mass of the solid dosage form.
[608] Provided in exemplary embodiment 90 is a solid dosage form of any one of embodiments 67 to 84, wherein the pharmaceutical agent has a total pharmaceutical agent mass that is at least 5% and no more than 65% of the total mass of the solid dosage form.
[609] Provided in exemplary embodiment 91 is a solid dosage form of embodiment 85, wherein the pharmaceutical agent has a total pharmaceutical agent mass that is at least 5% and no more than 35% of the total mass of the solid dosage form.
[610] Provided in exemplary embodiment 92 is a solid dosage form of embodiment 85, wherein the total pharmaceutical agent mass is about 25% of the total mass of the solid dosage form.
[611] Provided in exemplary embodiment 93 is a solid dosage form of any one of embodiments 67 to 87, wherein the solid dosage form is a tablet. [612] Provided in exemplary embodiment 94 is a solid dosage form of embodiment 88, wherein tablet is a 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, or 18mm tablet.
[613] Provided in exemplary embodiment 95 is a solid dosage form of any one of embodiments 67 to 87, wherein the solid dosage form is a minitablet.
[614] Provided in exemplary embodiment 96 is a solid dosage form of embodiment 90, wherein the minitablet is a 1mm minitablet, 1.5 mm minitablet, 2mm minitablet, 3mm minitablet, or 4mm minitablet.
[615] Provided in exemplary embodiment 97 is a solid dosage form of embodiment 91 or 92, wherein a plurality of minitablets are contained in a capsule.
[616] Provided in exemplary embodiment 98 is a solid dosage form of any one of embodiments 67 to 92, further comprising an enteric coating.
[617] Provided in exemplary embodiment 99 is a solid dosage form of embodiment 93, wherein the enteric coating is a single enteric coating or more than one enteric coating.
[618] Provided in exemplary embodiment 100 is a solid dosage form of embodiment 93 or 94, wherein the enteric coating comprises an inner enteric coating and an outer enteric coating, and wherein the inner and outer enteric coatings are not identical.
[619] Provided in exemplary embodiment 101 is a solid dosage form of embodiment any one of embodiments 93 to 95, wherein the enteric coating comprises a methacrylic acid ethyl acrylate (MAE) copolymer (1:1).
[620] Provided in exemplary embodiment 102 is a solid dosage form of any one of embodiments 93 to 96, wherein the enteric coating comprises cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), a fatty acid, a wax, shellac (esters of aleurtic acid), a plastic, a plant fiber, zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch, a starch derivative, a dextrin, a methyl acrylate- methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), a methyl methacrylate-methacrylic acid copolymer, or sodium alginate. [621] Provided in exemplary embodiment 103 is a solid dosage form of any one of embodiments 93 to 97, wherein the enteric coating comprises an anionic polymeric material.
[622] Provided in exemplary embodiment 104 is a solid dosage form comprising: (i) a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin; and (ii) at least one diluent, at least one lubricant, and/or at least one glidant.
[623] Provided in exemplary embodiment 105 is a solid dosage form of embodiment 99, wherein the at least one diluent has a total mass that is at least 1% and no more than 95% of the total mass of the solid dosage form.
[624] Provided in exemplary embodiment 106 is a solid dosage form of embodiment 99 or 100, wherein the at least one diluent comprises mannitol.
[625] Provided in exemplary embodiment 107 is a solid dosage form of any one of embodiments 99 to 101, wherein the at least one lubricant has a total mass that is at least
0.1% and no more than 5% of the total mass of the solid dosage form.
[626] Provided in exemplary embodiment 108 is a solid dosage form of any one of embodiments 99 to 102, wherein the at least one lubricant comprises magnesium stearate.
[627] Provided in exemplary embodiment 109 is a solid dosage form of any one of embodiments 99 to 103, wherein the at least one glidant has a total mass that is at least 0.01% and no more than 2% of the total mass of the solid dosage form.
[628] Provided in exemplary embodiment 110 is a solid dosage form of any one of embodiments 99 to 104, wherein the at least one glidant comprises colloidal silicon dioxide.
[629] Provided in exemplary embodiment 111 is a solid dosage form of any one of embodiments 99 to 105, wherein the pharmaceutical agent has a total pharmaceutical agent mass that is at least 5% and no more than 95% of the total mass of the solid dosage form.
[630] Provided in exemplary embodiment 112 is a solid dosage form of embodiment 106, wherein the pharmaceutical agent has a total pharmaceutical agent mass that is at least 20% and no more than 50% of the total mass of the solid dosage form.
[631] Provided in exemplary embodiment 113 is a solid dosage form of embodiment 106, wherein the total pharmaceutical agent mass is about 30% to about 50% of the total mass of the solid dosage form. [632] Provided in exemplary embodiment 114 is a solid dosage form of any one of embodiments 99 to 108, wherein (i) the total pharmaceutical agent mass is at least 5% and no more than 95% of the total mass of the solid dosage form; (ii) the total diluent mass is at least 1% and no more than 95% of the total mass of the solid dosage form; (iii) the total lubricant mass is at least 0.1% and no more than 5% of the total mass of the solid dosage form; and (iv) the total glidant mass is at least 0.01% and no more than 2% of the total mass of the solid dosage form.
[633] Provided in exemplary embodiment 115 is a solid dosage form of any one of embodiments 99 to 108, wherein (i) the total pharmaceutical agent mass is about 20% to about 50% of the total mass of the solid dosage form; (ii) the total diluent mass is about 50% to 80% of the total mass of the solid dosage form; (iii) the total lubricant mass is about 1% of the total mass of the solid dosage form; and (iv) the total glidant mass is about 0.5% of the total mass of the solid dosage form.
[634] Provided in exemplary embodiment 116 is a solid dosage form of any one of embodiments 99 to 108, wherein (i) the total pharmaceutical agent mass is about 30% to about 50% of the total mass of the solid dosage form; (ii) the total diluent mass is about 45% to 70% of the total mass of the solid dosage form; (iii) the total lubricant mass is about 1% of the total mass of the solid dosage form; and (iv) the total glidant mass is about 0.5% of the total mass of the solid dosage form.
[635] Provided in exemplary embodiment 117 is a solid dosage form of any one of embodiments 99 to 108, wherein (i) the total pharmaceutical agent mass is about 50% of the total mass of the solid dosage form; (ii) the total diluent mass is about 48.5% of the total mass of the solid dosage form; (iii) the total lubricant mass is about 1% of the total mass of the solid dosage form; and (iv) the total glidant mass is about 0.5% of the total mass of the solid dosage form.
[636] Provided in exemplary embodiment 118 is a solid dosage form of any one of embodiments 104 to 113, wherein (i) the total pharmaceutical agent mass is about 13.51% of the total mass of the solid dosage form; (ii) the total diluent mass is about 84.99% of the total mass of the solid dosage form; (iii) the total lubricant mass is about 1% of the total mass of the solid dosage form; and (iv) the total glidant mass is about 0.5% of the total mass of the solid dosage form.
[637] Provided in exemplary embodiment 119 is a solid dosage form of any one of embodiments 104 to 113, wherein (i) the total pharmaceutical agent mass is about 90.22% of the total mass of the solid dosage form; (ii) the total diluent mass is about 8.28% of the total mass of the solid dosage form; (iii) the total lubricant mass is about 1% of the total mass of the solid dosage form; and (iv) the total glidant mass is about 0.5% of the total mass of the solid dosage form.
[638] Provided in exemplary embodiment 120 is a solid dosage form of any one of embodiments 104 to 119, wherein the solid dosage form is a capsule.
[639] Provided in exemplary embodiment 121 is a solid dosage form of embodiment 120, wherein the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule.
[640] Provided in exemplary embodiment 122 is a solid dosage form of embodiment 120 or 121, wherein the capsule is a size 0 capsule.
[641] Provided in exemplary embodiment 123 is a solid dosage form of any one of embodiments 104 to 122, further comprising an enteric coating.
[642] Provided in exemplary embodiment 124 is a solid dosage form of embodiment 123, wherein the solid dosage form is enteric coated to dissolve at pH 5.5.
[643] Provided in exemplary embodiment 125 is a solid dosage form of embodiment 123 or 124, wherein the enteric coating comprises a polymethacrylate-based copolymer.
[644] Provided in exemplary embodiment 126 is a solid dosage form of any one of embodiments 123 to 125, wherein the the enteric coating comprises poly(methacrylic acid- co-ethyl acrylate).
[645] Provided in exemplary embodiment 127 is a solid dosage form of any one of embodiments 123 to 126, wherein the enteric coating comprises amethacrylic acid ethyl acrylate (MAE) copolymer (1:1) (e.g., Kollicoat MAE 100P).
[646] Provided in exemplary embodiment 128 is a solid dosage form of any one of embodiments 123 to 127, wherein the enteric coating comprises a Eudragit copolymer, e.g., a Eudragit L (e.g., Eudragit L 100-55; Eudragit L 30 D-55), a Eudragit S, a Eudragit RL, a Eudragit RS, a Eudragit E, or a Eudragit FS (e.g., Eudragit FS 30 D).
[647] Provided in exemplary embodiment 129 is a solid dosage form of any one of embodiments 123 to 128, wherein the enteric coating comprises cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), hydroxypropyl methylcellulose phthalate (HPMCP), a fatty acid, a wax, shellac (esters of aleurtic acid), a plastic, a plant fiber, zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch, a starch derivative, a dextrin, a methyl acrylate- methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), a methyl methacrylate-methacrylic acid copolymer, or sodium alginate.
[648] Provided in exemplary embodiment 130 is a solid dosage form of any one of embodiments 123 to 129, wherein the enteric coating comprises an anionic polymeric material.
[649] Provided in exemplary embodiment 131 is a method of preventing or treating a disease of a subject comprising administering to the subject a pharmaceutical composition of any one of embodiments 1 to 71.
[650] Provided in exemplary embodiment 132 is a method of preventing or treating a disease in a subject comprising administering to the subject a solid dosage form of any one of embodiments 72 to 130.
[651] Provided in exemplary embodiment 133 is a use of a pharmaceutical composition of any one of embodiments 1 to 71 for the treatment or prevention of a disease of a subject.
[652] Provided in exemplary embodiment 134 is a use of a solid dosage form of any one of embodiments 72 to 130 for the treatment or prevention of a disease in a subject.
[653] Provided in exemplary embodiment 135 is a method or use of any one of embodiments 131 to 134, wherein the disease is a cancer, inflammation, autoimmunity, a metabolic condition, or a dysbiosis.
[654] Provided in exemplary embodiment 136 is a method or use of any one of embodiments 131 to 134, wherein the disease is bacterial septic shock, cytokine storm and/or viral infection (such as a coronavirus infection, an influenza infection, and/or a respiratory syncytial virus infection).
[655] Provided in exemplary embodiment 137 is a method or use of any one of embodiments 131 to 134, wherein the solid dosage form decreases inflammatory cytokine expression (e.g., decreased IL-8, IL-6, IL-Ib, and/or TNFα expression levels).
[656] Provided in exemplary embodiment 138 is a method of preparing a solid dosage form, the method comprising: (A) combining into a pharmaceutical composition: (i) a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin; and (ii) at least one diluent, at least one lubricant, at least one glidant, and/or at least one disintegration agent. (B) compressing the pharmaceutical composition into a solid dosage form.
[657] Provided in exemplary embodiment 139 is a method of embodiment 138, further comprising the step of enterically coating the solid dosage form to obtain an enterically coated solid dosage form.
[658] Provided in exemplary embodiment 140 is a method of embodiment 138 or 139, wherein the solid dosage form is a tablet.
[659] Provided in exemplary embodiment 141 is a method of embodiment 138 or 139, wherein the solid dosage form is a minitablet.
[660] Provided in exemplary embodiment 142 is a method of preparing a solid dosage form, the method comprising combining into a pharmaceutical composition: (i) a pharmaceutical agent, wherein the pharmaceutical agent comprises (a) bacteria and/or microbial extracellular vesicles (mEVs); and (b) at least one component of soy leghemoglobin; and (ii) at least one diluent, at least one lubricant, and/or at least one glidant.
[661] Provided in exemplary embodiment 143 is a method of embodiment 142, further comprising blending and/or loading the pharmaceutical composition into a capsule.
[662] Provided in exemplary embodiment 144 is a method of embodiment 143, further comprising banding the capsule.
[663] Provided in exemplary embodiment 145 is a method of embodiment 144, wherein the capsule is banded with an HPMC-based banding solution.
[664] Provided in exemplary embodiment 146 is a method of any one of embodiments 142 to 145, further comprising the step of enterically coating the solid dosage form to obtain an enterically coated solid dosage form.
[665] Provided in exemplary embodiment 147 is a method of any one of embodiments 142 to 146, wherein the solid dosage form is a capsule.
[666] Provided in exemplary embodiment 148 is a method of testing a pharmaceutical composition comprising bacteria and/or microbial extracellular vesicles (mEVs), the method comprising performing an assay to detect the presense of a component of a soy leghemoglobin in the pharmaceutical composition.
[667] Provided in exemplary embodiment 149 is a method of embodiment 148, wherein the component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid. [668] Provided in exemplary embodiment 150 is a method of embodiment 149, wherein the soyn leghemoglobin nucleic acid is soy leghemoglobin DNA.
[669] Provided in exemplary embodiment 151 is a method of embodiment 150, wherein the soybean leghemoglobin DNA comprises a sequence encoding soybean leghemoglobin A (LB A) or soy leghemoglobin C2 (LGB2).
[670] Provided in exemplary embodiment 152 is a method of embodiment 148, wherein the assay to detect the presence of a component of soybean leghemoglobin is a nucleic acid amplification assay, a sequencing assay, and/or a microarray assay.
[671] Provided in exemplary embodiment 153 is a method of embodiment 148 or 152, the assay to detect the presence of a component of soybean leghemoglobin is a polymerase chain reaction (PCR) assay.
[672] Provided in exemplary embodiment 154 is a method of embodiment 148, wherein the component of soy leghemoglobin is a soy leghemoglobin protein.
[673] Provided in exemplary embodiment 155 is a method of embodiment 154, wherein the soy leghemoglobin protein is soybean leghemoglobin A (LBA) or soy leghemoglobin C2 (LGB2) .
[674] Provided in exemplary embodiment 156 is a method of embodiment 154 or 155, wherein the soybean leghemoglobin protein is detected using an antibody specific for the soybean leghemoglobin protein, HPLC or UPLC.
[675] Provided in exemplary embodiment 157 is a method of any one of embodiments 154 to 156, wherein the pharmaceutical composition comprises bacteria.
[676] Provided in exemplary embodiment 158 is a method of embodiment 157, wherein the bacteria are hemoglobin-dependent bacteria.
[677] Provided in exemplary embodiment 159 is a method of embodiment 157 or 158, wherein the bacteria are of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Foumierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
[678] Provided in exemplary embodiment 160 is a method of embodiment 159, wherein the bacteria are of the genus Foumierella. [679] Provided in exemplary embodiment 161 is a method of embodiment 160, wherein the Fournierella are Fournierella Strain B (ATCC Deposit Number PTA- 126696).
[680] Provided in exemplary embodiment 162 is a method of embodiment 159, wherein the bacteria are of the genus Prevotella.
[681] Provided in exemplary embodiment 163 is a method of embodiment 162, wherein the bacteria are Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, or Prevotella veroralis.
[682] Provided in exemplary embodiment 164 is a method of embodiment 162, wherein the bacteria are of the species Prevotella histicola.
[683] Provided in exemplary embodiment 165 is a method of embodiment 162, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
[684] Provided in exemplary embodiment 166 is a method of embodiment 162, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain C (ATTC Deposit Number PTA- 126140).
[685] Provided in exemplary embodiment 167 is a method of embodiment 162, wherein the Prevotella are Prevotella Strain B 50329 (NRRL accession number B 50329).
[686] Provided in exemplary embodiment 168 is a method of embodiment 162, wherein the Prevotella are Prevotella Strain C (ATTC Deposit Number PTA- 126140). [687] Provided in exemplary embodiment 169 is a method of embodiment 162, wherein the Prevotella bacteria (i) comprise one or more proteins listed in Table 1, and/or (ii) are substantially free of a protein listed in Table 2.
[688] Provided in exemplary embodiment 170 is a method of any one of embodiments 157 to 169, wherein the bacterial are live, attenuated, or dead.
[689] Provided in exemplary embodiment 171 is a method of any one of embodiments 157 to 170, wherein the bacteria are lyophilized bacteria.
[690] Provided in exemplary embodiment 172 is a method of any one of embodiments 148 to 171, wherein the pharmaceutical composition comprises mEVs.
[691] Provided in exemplary embodiment 173 is a method of embodiment 172, wherein the mEVs are secreted mEVs (smEVs).
[692] Provided in exemplary embodiment 174 is a method of embodiment 172, wherein the mEVs are processed mEVs (pmEVs).
[693] Provided in exemplary embodiment 175 is a method of any one of embodiments 172 to 174, wherein the mEVs are from hemoglobin-dependent bacteria.
[694] Provided in exemplary embodiment 176 is a method of any one of embodiments 172 to 175, wherein the mEVs are from bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
[695] Provided in exemplary embodiment 177 is a method of embodiment 176, wherein the mEVs are from bacteria of the genus Fournierella.
[696] Provided in exemplary embodiment 178 is a method of embodiment 177, wherein the Fournierella are Fournierella Strain B (ATCC Deposit Number PTA- 126696).
[697] Provided in exemplary embodiment 179 is a method of embodiment 176, wherein the mEVs are from bacteria of the genus Prevotella.
[698] Provided in exemplary embodiment 180 is a method of embodiment 179, wherein the bacteria are Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopes, Prevotella shahii, Prevotella zoogleof ormans, or Prevotella veroralis.
[699] Provided in exemplary embodiment 181 is a method of embodiment 179, wherein the mEVs are from bacteria of the species Prevotella histicola.
[700] Provided in exemplary embodiment 182 is a method of embodiment 179, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
[701] Provided in exemplary embodiment 183 is a method of embodiment 179, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain C (ATTC Deposit Number PTA-126140).
[702] Provided in exemplary embodiment 184 is a method of embodiment 179, wherein the Prevotella are Prevotella Strain B 50329 (NRRL accession number B 50329).
[703] Provided in exemplary embodiment 185 is a method of embodiment 179, wherein the Prevotella are Prevotella Strain C (ATTC Deposit Number PTA-126140).
[704] Provided in exemplary embodiment 186 is a method of embodiment 179, wherein the Prevotella bacteria (i) comprise one or more proteins listed in Table 1, and/or (ii) are substantially free of a protein listed in Table 2.
EXAMPLES
Example 1: Exemplary Purification of Heme-containing Polypeptides
[705] All steps are carried out at 4°C or room temperature. Centrifugation steps are at 8000 x g for 20 mins, 4 °C or room temperature.
[706] The clarified tissue lysates or protein solutions prepared as described below are subjected to ammonium sulfate precipitation. Briefly, the clarified tissue lysates or protein solutions are buffered with 1M HEPES, pH 7.4 or 1 M Tris-HCl, pH 7.4, to a final concentration of 50 mM. Proteins are precipitated by addition of ammonium sulfate in at least two steps at 50% and 80% saturation. The lysate with ammonium sulfate is stirred gently for a minimum of 4 hours and a maximum of 16 hours at 4°C. The solution is centrifuged at 25,000 x g for 1 hour at 4 °C.
[707] Upon fractionation, all ammonium sulfate precipitate fractions of interest are stored at -20 °C until further use. Prior to their use in experiments, the precipitates are resuspended in 10 volumes of 50 mM potassium phosphate buffer, pH 7.4, containing 0.5 M NaCl. The suspensions are centrifuged and the supernatants are microfiltered through a 0.2 micron PES membrane. The filtrates are concentrated by ultrafiltration on a 3 kDa, 5 kDa, or 10 kDa molecular weight cutoff PES membrane on a Spectrum Labs KrosFlo hollow fiber tangential flow filtration system. The protein composition at individual fractionation step is monitored by SDS-PAGE and protein concentrations are measured by standard UV-Vis methods. Hemoglobin, myoglobin, or other heme-containing polypeptide is further separated by size exclusion chromatography (Sephacryl S-100 HR, GE Healthcare) based on the molecular size of the protein. The fraction is optionally further purified using an ion exchange chromatography (High Prep Q; Prep S; High Prep DEAE, GE Healthtcare). The fraction containing the heme -containing polypeptide is determined by SDS-PAGE and/or mass spectrometry.
Heme-Containing Polypeptide from Fish
[708] Carps are coarsely chopped and quickly frozen in liquid nitrogen. The frozen tissue is ground in liquid nitrogen with mortar and pestle until fine powder is formed. The powder is stored in -80 °C until further use.
[709] The ground carp powder or commercially available fish protein hydrolysates are suspended in 10 volumes of 50 mM potassium phosphate buffer, pH 8.0 and 0.4 M sodium chloride and stirred for 1 hour at 4°C. Soluble proteins are separated from the rest by centrifugation. The supernatant is subjected to ammonium sulfate precipitation described above. Heme-Containing Polypeptide from Non-Animal Source
[710] Commercially available legume flour is suspended in 10 volumes of 50 mM potassium phosphate buffer, pH 8.0 and 0.4 M sodium chloride. The lysate is clarified by centrifugation. The supernatant is used for ammonium sulfate precipitation described above.
[711] Pea total proteins: Dry green or yellow pea flour is used to extract total pea proteins. The flour is suspended in 10 volumes of 20 mM potassium phosphate buffer pH 8 and 100 mM sodium chloride and stirred for 1 hour. Soluble protein is separated from pea seed debris by centrifugation. The supernatant is collected and filtered through a 0.2 micron membrane and concentrated using a 10 KDa cutoff PES membrane.
[712] Lentil total proteins: Air-classified lentil flour is used to extract crude mixture of lentil proteins. Flour is suspended in 5 volumes of 20 mM potassium phosphate buffer pH 7.4 and 0.5 M sodium chloride and stirred for 1 hour. Soluble protein is separated from the unextracted protein and lentil seed debris by centrifugation (8000 g, 20 minutes ). The supernatant is collected and filtered through a 0.2 micron membrane and concentrated using a 10 KDa cutoff PES membrane.
[713] Chickpea / Garbanzo bean total proteins: Garbanzo bean flour is suspended in 5 volumes of 20 mM potassium phosphate buffer pH 7.4 and 0.5 M sodium chloride and stirred for 1 hour. Soluble protein is separated from the unextracted protein and chickpea seed debris by centrifugation (8000 g, 20 minutes ). The supernatant is collected and filtered through a 0.2 micron membrane and concentrated using a 10 KDa cutoff PES membrane.
[714] Soybean proteins: Soybean proteins are also extracted by suspending the defatted soy flour in 4-15 volumes (e.g., 5 volumes) of 20 mM sodium carbonate, pH 9 (or water, pH adjusted to 9 after addition of the flour) or 20 mM potassium phosphate buffer pH 7.4 and 100 mM sodium chloride. The slurry is stirred for one hour and centrifuged at 8000 x g for 20 minutes. The extracted proteins are ultrafiltered and then processed as above or alternatively, the supernatant is collected and filtered through a 0.2 micron membrane and concentrated using a 10 KDa cutoff PES membrane.
[715] Amaranth flour dehvdrins: Amaranth flour is suspended in 5 volumes of 0.5 M sodium chloride, pH 4.0 and stirred for 1 hour. Soluble protein is separated from the unextracted protein and debris by centrifugation (8000 x g, 20 minutes). The supernatant is collected and filtered through a 0.2 micron membrane and concentrated using a 3 KDa cutoff PES membrane. Further enrichment of dehydrins from this fraction is obtained by boiling the concentrated protein material, spinning at 8000 x g for 10 minutes, and collecting the supernatant.
[716] Pea globulins: Dry green pea flour is used to extract pea globulin proteins. The flour is suspended in 10 volumes of 50 mM potassium phosphate buffer pH 8 and 0.4 M sodium chloride and stirred for 1 hour. Soluble protein is separated from pea seed debris by centrifugation. The supernatant is subjected to ammonium sulfate fractionation in two steps at 50 % and 80 % saturation. The 80 % pellet containing globulins of interest is stored at -20 °C until further use. Protein is recovered from the pellet and prepared for use as described above.
[717] Soybean 7S and 11 S globulins: Globulins from soybean flour are isolated by first suspending lowfat/defatted soy flour in 4-15 volumes of 20 mM potassium phosphate pH 7.4. The slurry is centrifuged at 8000 x g for 20 mins or clarified by 5 micron filtration and the supernatant is collected. The crude protein extract contains both the 7S and 1 IS globulins. The solution is filtered using a 0.2 micron filter and concentrated using a 10 kDa molecular weight cutoff PES membrane on a Spectrum Labs KrosFlo hollow fiber tangential flow filtration system or by passing over the anion-exchange resin prior to use in experiments. The 1 IS globulins are separated from the 7S proteins by isoelectric precipitation. The pH of the crude protein extract is adjusted to 6.4 with dilute HC1, stirred for 30 min-1 hour and then centrifuged to collect the 1 IS precipitate and 7S proteins in the supernatant. The 1 IS fraction is resuspended with 10 mM potassium phosphate pH 7.4 and the protein fractions are micro-filtered and concentrated prior to use.
[718] Mung bean 8S globulins: Mung bean flour is used to extract 8S globulins by first suspending the flour in 4 volumes of 50 mM potassium phosphate buffer pH 7, 0.5M NaCl. After centrifugation, proteins in the supernatant are fractionated by addition of ammonium sulfate in 2 steps at 50 % and 90 % saturation respectively. The precipitate from the 90 % fraction contains the 8S globulins, which is saved at -20 °C until further use. 8S globulins are recovered from the pellet and prepared for use as described above.
[719] Mung bean globulins: Mung bean globins are also extracted by suspending the flour in 4 volumes of 20 mM sodium carbonate buffer, pH 9 (or water adjusted to pH 9 after addition of the mung bean flour). The slurry is centrifuged (or filtered) to remove solids, ultrafiltered, and then processed as described above. [720] Leghemoglobin. Soy root nodules are suspended and lysed in 20 mM potassium phosphate pH 7.4, 100 mM potassium chloride and 5 mM EDTA using a grinder-blender. During this process, leghemoglobin is released into the buffer. Root- nodule lysate containing leghemoglobin is cleared from cell debris by filtration through 5 micron filter. Filtration is followed by centrifugation (7000 x g, 20min). Clarified lysate containing leghemoglobin is then filtered through 0.2 micron filter and applied onto an anion-exchange chromatography column (High Prep Q; High Prep DEAE, GE Healthtcare) on a fast protein liquid chromatography instrument (GE Healthcare). Leghemoglobin is collected in the flowthrough fraction and concentrated over 3 kDa molecular weight cutoff PES membrane on a Spectrum Labs KrosFlo hollow fiber tangential flow filtration system to a desired concentration. Purity (partial abundance) of purified leghemoglobin is analyzed by SDS-PAGE gel. In lysate, leghemoglobin is typically present at 20-40 %, while after anion-exchange purification, it can be present at 70-80 %. The soybean leghemoglobin flowthrough from anion-exchange chromatography is optionally applied onto size- exclusion chromatography (Sephacryl S-100 HR, GE Healthcare). Soybean leghemoglobin elutes as two fractions corresponding to dimeric and monomeric species. Purity (partial abundance) of leghemoglobin is analyzed by SDS-PAGE and can be in the range of ~ 90- 100%. Analysis of UV-VIS spectra (250-700nm) can reveal spectral signature consistent with heme loaded leghemoglobin.
Example 2: Determining Proper Incorporation of the Heme Group in Purified Heme- Containing Polypeptides
[721] Recombinant heme-containing protein produced from any organism (e.g., E. Coli or P. pastoris) is analyzed for proper incorporation of the heme group using the pyridine hemochromogen method (Oinuma et al., (2003) J Biol Chem 278:29600-29608) using the molecular extinction coefficient of the α-peak at 557 nm of the hemochromogen of protoheme IX (e = 34.4 mM-1cm-1). Briefly, 200 μL of 0.5 M NaOH is added to 500 μL of purified protein and then 200 μL of pyridine and 5 μL of 0.1 M K3Fe(CN)6 are added to the mixture. Then, 10 μL of 0.5 M Na2S2O4 is added to heme -containing protein. The absorption spectrum of the sample is monitored from 340 to 700 nm using a Halo DB-20 UV-Vis spectrophotometer (Dynamica Ltd., VIC, AU). The heme content is reported as mol of heme per mol of globin, and the molar extinction coefficient of heme is calculated by substitution in the Beer-Lambert law. Furthermore, the iron content of the recombinant a-globin is measured with an atomic absorption flame emission spectrophotometer (PerkinElmer, Analyst 800, USA). The typical protein concentration used in UV-VIS experiments is 8-9 mM purified recombinant α-globin.
Example 3: Exemplary Manufacturing Process of Hemoglobin-dependent Bacteria
[722] A hemoglobin solution is also prepared by dissolving a piscine hemoglobin, an avian hemoglobin, a fungal hemoglobin, a plant hemoglobin, or a bacterial hemoglobin in 0.01 M NaOH. The solution is sterilized by autoclaving. A working concentration of 0.02 g/L or 0.2 g/L is used. A myoglobin solution, a leghemoglobin solution, or other heme- containing polypeptide solution (e.g., peroxidase, cytochrome c) is prepared similarly in phosphate buffer saline, pH 7.4, or 0.01 M NaOH. The solution is sterilized by autoclaving, and is added to the growth media at various working concentrations (e.g., 0.02 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L, or 0.5 g/L).
[723] An exemplary manufacturing process of hemoglobin-dependent bacteria, e.g., Prevotella histicola is presented herein. In this exemplary method, the hemoglobin- dependent bacteria are grown in growth media comprising the components listed in Table 5. The media is filter sterilized prior to use.
Table 5 : Exemplary Growth Media
Figure imgf000178_0001
Table 6: Another Exemplary Growth Media
Figure imgf000179_0001
[724] Briefly, a 1L bottle is inoculated with a lmL of a cell bank sample that had been stored at -80 °C. This inoculated culture is incubated in an anaerobic chamber at 37 °C, pH = 6.5 due to sensitivity of this strain to aerobic conditions. When the bottle reaches log growth phase (after approximately 14 to 16 hours of growth), the culture is used to inoculate a 20 L bioreactor at 5 % v/v. During log growth phase (after approximately 10 to 12 hours of growth), the culture is used to inoculate a 3500 L bioreactor at 0.5% v/v.
[725] Fermentation culture is continuously mixed with addition of a mixed gas at 0.02 VVM with a composition of 25 % CO2 and 75 % N2. pH is maintained at 6.5 with ammonium hydroxide and temperature is controlled at 37 °C. Harvest time is based on when the stationary phase is reached (after approximately 12 to 14 hours of growth).
[726] Once fermentation completes, the culture is cooled to 10 °C, centrifuged, and the resulting cell paste is collected. 10 % Stabilizer is added to the cell paste and mixed thoroughly (Stabilizer Concentration (in slurry): 1.5 % Sucrose, 1.5 % Dextran, 0.03 % Cysteine).
[727] For other growth conditions that can be used, see, e.g., WO 2019/051381, the disclosure of which is hereby incorporated by reference.
Table 7: Stabilizer Formulation
Figure imgf000179_0002
Growth Analysis
[728] Four replicates are performed for each growth analysis. 0.1 % inoculum from a frozen cell bank is used for each culture. Bacteria are grown in the media of Table 6 as described above. Kinetics of bacterial growth are measured by measuring the optical density (OD600) every 30 minutes on a plate reader for 48 hours while culturing in the anaerobic environment at 37 °C.
Example 4: Isolation and Purification of Sov Leghemoglobin
[729] A nucleic acid encoding Glycine max leghemoglobin C2 (Uniprot KB P02236) with an N-terminal His6 epitope tag and a TEV cleavage site is cloned into the pJexpress401 vector (DNA2.0), and transformed into E. coli BL21. Transformed cells are grown by fed-batch fermentation supplemented with kanamycin, 0.1 mM ferric chloride and 10 ug/ml 5 -aminolevulinic acid. Expression is induced by 0.3 mM isopropyl □-D-1- thiogalactopyranoside (IPTG) and cells are grown at 30 degrees °C for 24 hr. Cells are concentrated by centrifugation and resuspended in 20 mM potassium phosphate pH 7.8, 100 mM NaCl. Cells are lysed by high-pressure homogenization and clarified by centrifugation and microfiltration. Leghemoglobin is purified from the soluble lysate using zinc-charged IMAC sepharose fast flow resin (GE Healthcare). Bound leghemoglobin is eluted off the resin with 500 mM potassium phosphate monobasic, 100 mM NaCl. Purified leghemoglobin is neutralized and concentrated using ultrafiltration. Concentrated leghemoglobin is reduced with 20 mM Na dithionite. Na dithionite is removed by diafiltration. Leghemoglobin concentration is determined by soret peak absorbance and adjusted to 60-70 mg/ml. The final leghemoglobin product is frozen in liquid nitrogen, lyophilized, and stored at -20 degrees C. Purity (partial abundance) of leghemoglobin is analyzed by SDS-PAGE and determined to be about 80%. Analysis of UV-VIS spectra (250-700 nm) reveals spectral signature consistent with heme-loaded leghemoglobin.
[730] Glycine max leghemoglobin C2 and eight Pichia pastoris heme biosynthesis genes (listed in Table 8) are cloned into the Pichia pastoris expression vector pJA (BioGrammatics Inc.; Carlsbad, Calif.) under the control of the pAOXl methanol inducible promoter. Pichia pastoris strain Bgl 1 (BioGrammatics, Inc.) is transformed with linearized plasmids, and stable integrants are selected by antibiotic resistance. Table 8
Figure imgf000181_0001
[731] Transformed Pichia cells are grown by fed-batch fermentation and leghemoglobin expression is induced with methanol for 120 hours at 30 degrees °C. Cells are concentrated by centrifugation, resuspended in water, and lysed by high pressure homogenization. Solids are removed by treatment with Tramfloc 863 A, centrifugation, and 0.2 micron microfiltration (Koch Membrane Systems). The soluble lysate is concentrated and diafiltered with water using 3 kDa ultrafiltration (Spectrum Laboratories). The formulated lysate is partially purified using HPA25L anion exchange resin (Mitsubishi) to a final purity of about 40%. The partially purified leghemoglobin solution is re-formulated by concentration and water diafiltration using 3 kDa ultrafiltration (Spectrum Laboratories) and further purified using Q Fast Flow anion exchange resin (GE Lifesciences). The final leghemoglobin product is concentrated using 3 kD ultrafiltration and frozen at -20 degrees C. The final product is about 80% pure and contains 80 g/L leghemoglobin. See U.S. Patent No. 10,798,958. Example 5: Testing Sov Leghemoglobin as a Substitute for Animal-Sourced Hemoglobin in Growth Media
[732] A sample of Impossible™ meat acquired from a commercial grocer was used to generate solutions containing different concentrations of soy leghemoglobin as listed in Table 9.
Table 9: Soy leghemoglobin Solutions
Figure imgf000182_0001
[733] Solutions IM1 and IM2 were prepared by resuspending the Impossible™ meat in distilled water and adding 0.01 M NaOH to achieve a concentration similar to the concentration used for the animal-sourced hemoglobin or spirulina stock solutions. After resuspension, solids were removed from the solutions by centrifugation. Solutions IM3 and IM4 were prepared by resuspending the Impossible™ meat in either water or 0.01 M NaOH solution followed by heating until boiling. Solids were then removed by double filtration through a gauze filter. All solutions were autoclaved following solid removal and then added to growth media to test a range of 3 concentrations: 20 mg/L final concentration of soy leghemoglobin (this is the concentration currently used for animal-sourced hemoglobin); 100 mg/L final concentration of soy leghemoglobin; and 200 mg/L final concentration of soy leghemoglobin.
[734] As a positive control, a spirulina solution at 1 g/L concentration or an animal-sourced hemoglobin solution at 20 mg/L concentration was used. The SPY media (Table 9) with glucose without any other additives was used as a negative control.
[735] The following recipe of the growth media was used:
Table 10: SPY Media
Figure imgf000182_0002
[736] 5 g/L of glucose was used as a carbon source for all growth media.
[737] For all 4 IM solutions (IM1-IM4), growth of Strain A (Prevote lla histicola strain B 50329 (NRRL accession number B 50329)) was restored to levels similar to when cultured with animal-sourced hemoglobin or spirulina (FIG. 1).
[738] The higher concentrations of soy leghemoglobin did not improve the growth of Strain A (Prevotella histicola strain B 50329 (NRRL accession number B 50329)) beyond the positive controls and showed similar results as the lower concentration of soy leghemoglobin (FIG. 2), thereby demonstrating that the 20 mg/L concentration provides sufficient leghemoglobin to support the growth. Animal-sourced hemoglobin and spirulina solution provided consistent growth support for Strain A ( Prevotella histicola strain B 50329 (NRRL accession number B 50329)) (FIG. 2).
[739] Example 6: Testing Sov Leghemoglobin to Replace Animal Source Hemoglobin in Different Microbes
[740] To confirm that soy leghemoglobin will be sufficient to support growth of hemoglobin-dependent microbes, a sample of Impossible™ Meat was purchased and a soy leghemoglobin-containing solution was prepared using this sample.
[741] Solution prepared:
Table 11 : Sov leghemoglobin Solution
Figure imgf000183_0001
[742] Percentage of the solution was calculated based on estimation that the Impossible™ Meat contains 2% of soy leghemoglobin (the package states 2% or less). The actual concentration of soy leghemoglobin in the solution is expected to be equal or less than the calculated estimate.
[743] Solutions were prepared by resuspending the Impossible™ Meat in 0.0 IM NaOH solution, followed by heating until boiling and then clearing the Impossible™ Meat solids out by double filtering through a gauze filter. Final solution was autoclaved and then added to the growth media to test a range of 2 concentrations: 20mg/L of soy leghemoglobin (IM) final concentration; and 200mg/L of soy leghemoglobin final concentration.
[744] As positive control, animal (porcine) sourced hemoglobin solution at 20mg/L concentration was used. SPY base with glucose without any other additives was used as a negative control. [745] Growth media used for the experiment was SPY, according to the following recipe:
Table 12: SPY Media
Figure imgf000184_0001
[746] 5g/L of glucose was used as a carbon source for all media compositions. Complete media with glucose added is referred to as SPYG5 in FIGS. 3-5.
[747] The growth dynamics test was performed in a BioTek Epoch2 platereader under anaerobic conditions for 48 hours.
[748] As can be seen in the growth dynamics graph in FIG. 3, the negative control, SPYG5 media without any additives, was unable to support growth of a Parabacteroides strain. In both IM concentrations, the growth of a. Parabacteroides strain, Parabacteroides Strain A, was restored to the similar levels as animal source hemoglobin. The higher concentration of IM even exceeded the growth with animal sourced hemoglobin, therefore showing that soy leghemoglobin can be used to support full growth of this microbe.
[749] Similar to FIG. 3, in the growth dynamics graph in FIG. 4, the negative control, SPYG5 media without any additives, was unable to support growth of a Bacteroides strain. In both IM concentrations here, the growth of a Bacteroides strain, Bacteroides Strain B, was restored to the same level as with positive control - animal source hemoglobin. Soy leghemoglobin can be used to fully support growth of this microbe.
[750] In the growth dynamics graph in FIG. 5 for an Alistipes strain, Alistipes Strain C, the negative control, SPYG5 media without any additives, was unable to support growth of this microbe. In both IM concentrations, the growth of Alistipes Strain C was restored to the same level as with positive control - animal sourced hemoglobin. Example 7: PCR Detection of the Presence of Sov Leghemoglohin
[751] Soy hemoglobin is added to cultures of hemoglobin-dependent bacteria to support the growth of the bacteria, as described above. An aliquot of bacterial culture is resuspended in PBS, centrifuged to pellet the microbes, and filtered. The bacterial culture supernatant is collected, serial diluted, and then incubated with antibody-oligonucleotide conjugated probes. The antibodies are specific to soy hemoglobin. The supernatant is incubated with the conjugated probes to allow sufficient time for antibodies to bind to target epitope. Another inbucabtion step is performed to allow for ligation of proximal oligonucleotides. When the conjugated antibodies bind to an epitope and are in close proximity, the oligonucleotides can be ligated, serving as the template for PCR amplification and quantification. PCR is performed on the oligonucleotides to detect presence of soy hemoglobin.
Example 8: PCR Detection of the Presence of Sov Leghemoglobin in Impossible Meat
[752] The presence of soybean leghemoglobin A (gene name: LBA) was detected in Impossible® meat using TaqMan qPCR assay. Below is the experimental procedure for DNA purification from Impossible meat:
1. 1.6 grams of frozen Impossible® meat was homogenized in 4 mL of Gram+ Lysis Solution (Sigma Aldrich GenElute kit) containing lysozyme 45 mg/ml using a razor blade inside a 5cm Petri dish.
2. 3.6 mL of homogenized sample was recovered from the Petri dish.
3. Sample was incubated at 37°C for 30 minutes.
4. 40 μL of RNase I was added to the sample.
5. 40 μL of Proteinase K was added to the sample.
6. 500 μL of Lysis Solution C was added to the sample.
7. The sample was vortexed at full speed for 10 seconds and incubated at 56°C for 10 minutes.
8. The sample was centrifuged at 6000g for 1 minute to pellet insoluble material.
9. 1 mL of cleared supernatant was recovered and divided into two microcentrifuge tubes.
10. 300 uL of 100% ethanol was added to each tube.
11. The two tubes were vortexed at full speed for 10 seconds. 12. The sample was transferred from each tube to two spin columns.
13. The two spin columns were centrifuged at 12,000g for 1 minute.
14. The two spin columns were washed twice with wash buffer.
15. 200 μL of Elution Buffer was added to each spin column to elute the bound DNA.
16. The eluted DNA from each spin column was combined, and DNA concentration was determined using absorbance measurements at 260 nm and 280 nm.
17. 10-fold serial dilutions of eluted DNA were analyzed by qPCR using LB A TaqMan qPCR assay. qPCR standard curve was constructed using 10-fold serial dilutions of 543-bp DNA fragment containing LBA coding region (FIG. 6 ).
Table 13
Figure imgf000186_0001
[753] Summary:
1 16 ng/μL DNA concentration * 380 μL eluted DNA volume = 6 micrograms of DNA was recovered from 1.6 grams of Impossible meat
2 There were approximately 100 copies of soybean leghemoglobin A gene per nanogram of DNA.
3. Each gram of Impossible meat contains approximately 4 x 105 copies of soybean leghemoglobin A gene.
Incorporation by Reference
[754] All publications patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. Equivalents
[755] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS What is claimed is:
1. A pharmaceutical composition comprising: a pharmaceutical agent, wherein the pharmaceutical agent comprises
(a) bacteria and/or microbial extracellular vesicles (mEVs); and
(b) at least one component of soy leghemoglobin.
2. The pharmaceutical composition of claim 1, wherein the at least one component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid.
3. The pharmaceutical composition of claim 2, wherein the soy leghemoglobin nucleic acid is soy leghemoglobin DNA.
4. The pharmaceutical composition of claim 3, wherein the soy leghemoglobin DNA comprises a sequence encoding soybean leghemoglobin A (LBA) or soybean leghemoglobin C2 (LGB2).
5. The pharmaceutical composition of claim 1, wherein the at least one component of soy leghemoglobin comprises a soybean leghemoglobin protein.
6. The pharmaceutical composition of claim 5, wherein the soy leghemoglobin protein is soy leghemoglobin A or soybean leghemoglobin C2 (LGB2).
7. The pharmaceutical composition of any one of claims 1-6, wherein the at least one component of soy leghemoglobin is purified from soy roots or soy root nodules.
8. The pharmaceutical composition of any one of claims 1-7, wherein the at least one component of soy leghemoglobin is recombinantly expressed.
9. The pharmaceutical composition of any one of claims 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
10. The pharmaceutical composition of any one of claims 1-8, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 4 or GenBank: NP_001235248.2.
11. The pharmaceutical composition of any one of claims 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
12. The pharmaceutical composition of any one of claims 1-8, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 5 or GenBank: NP_001235928.1.
13. The pharmaceutical composition of any one of claims 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB -
P02236 (LGB2 _ SOYBN) .
14. The pharmaceutical composition of any one of claims 1-8, wherein the at least one component of soy leghemoglobin comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 114 or UniProtKB - P02236 (LGB2 _ SOYBN) .
15. The pharmaceutical composition of any one of claims 1-8, wherein the at least one component of soy leghemoglobin comprises an amino acid sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN ).
16. The pharmaceutical composition of any one of claims 1-8, wherein the at least one component of soy leghemoglobin comprises comprises a nucleic acid that encodes a sequence with at least 90% identity to the amino acid sequence of SEQ ID NO: 115 or UniProtKB - P02238 (LGBA _ SOYBN ).
17. The pharmaceutical composition of claim 8, wherein the recombinantly expressed polypeptide comprises a heterologous polypeptide.
18. The pharmaceutical composition of claim 17, wherein the heterologous polypeptide comprises a histidine tag, TAP (tandem affinity purification) tag, TEV cleavage site, a FLAG tag, a GST tag, and/or an immunoglobulin domain.
19. The pharmaceutical composition of any one of claims 1-18, wherein the bacteria are hemoglobin-dependent bacteria.
20. The pharmaceutical composition of any one of claims 1-19, wherein the bacteria are of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides,
Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
21. The pharmaceutical composition of claim 20, wherein the bacteria are of the genus Fournierella.
22. The pharmaceutical composition of claim 21, wherein the Fournierella are Fournierella Strain B (ATCC Deposit Number PTA- 126696).
23. The pharmaceutical composition of claim 20, wherein the bacteria are of the genus Prevotella.
24. The pharmaceutical composition of claim 23, wherein the bacteria are of the species Prevotella histicola.
25. The pharmaceutical composition of claim 24, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
26. The pharmaceutical composition of claim 24, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain C (ATTC Deposit Number PTA-126140).
27. The pharmaceutical composition of claim 24, wherein the Prevotella are Prevotella Strain B 50329 (NRRL accession number B 50329).
28. The pharmaceutical composition of claim 24, wherein the Prevotella are Prevotella Strain C (ATTC Deposit Number PTA-126140).
29. The pharmaceutical composition of any one of claims 1 to 28, wherein the bacteria are live, attenuated, or dead.
30. The pharmaceutical composition of any one of claims 1 to 28, wherein the bacteria are lyophilized bacteria.
31. The pharmaceutical composition of any one of claims 1-28, wherein the pharmaceutical agent comprises mEVs.
32. The pharmaceutical composition of claim 31, wherein the mEVs are secreted mEVs (smEVs).
33. The pharmaceutical composition of claim 31, wherein the mEVs are processed mEVs (pmEVs).
34. The pharmaceutical of any one of claims 31 to 33, wherein the mEVs are from hemoglobin-dependent bacteria.
35. The pharmaceutical composition of any one of claims 31 to 33, wherein the mEVs are from bacteria of the genus Actinomyces, Alistipes, Anaerobutyricum, Bacillus, Bacteroides, Cloacibacillus, Clostridium, Collinsella, Cutibacterium, Eisenbergiella, Erysipelotrichaceae, Eubacterium/Mogibacterium, Faecalibacterium, Fournierella, Fusobacterium, Megasphaera, Parabacteroides, Peptoniphilus, Peptostreptococcus, Porphyromonas, Prevotella, Propionibacterium, Rarimicrobium, Shuttleworthia, Turicibacter, or Veillonella.
36. The pharmaceutical composition of claim 35, wherein the mEVs are from bacteria of the genus Fournierella.
37. The pharmaceutical composition of claim 36, wherein the Fournier ella are Fournierella Strain B (ATCC Deposit Number PTA- 126696).
38. The pharmaceutical composition of claim 35, wherein the mEVs are from bacteria of the genus Prevotella.
39. The pharmaceutical composition of claim 38, wherein the mEVs are from bacteria of the species Prevotella histicola.
40. The pharmaceutical composition of claim 39, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
41. The pharmaceutical composition of claim 39, wherein the Prevotella comprise at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain C (ATTC Deposit Number PTA- 126140).
42. The pharmaceutical composition of claim 39, wherein the Prevotella are Prevotella Strain B 50329 (NRRL accession number B 50329).
43. The pharmaceutical composition of claim 39, wherein the Prevotella are Prevotella Strain C (ATTC Deposit Number PTA-126140).
44. The pharmaceutical composition of any one of claims 31 to 43, wherein the mEVs are lyophilized mEVs.
45. The pharmaceutical composition of any one of claims 1 to 44, further comprising a cryoprotectant.
46. A solid dosage form comprising:
(i) a pharmaceutical agent, wherein the pharmaceutical agent comprises
(a) bacteria and/or microbial extracellular vesicles (mEVs); and
(b) at least one component of soy leghemoglobin; and
(ii) at least one diluent, at least one lubricant, at least one glidant, and/or at least one disintegration agent.
47. A solid dosage form comprising:
(i) a pharmaceutical agent, wherein the pharmaceutical agent comprises
(a) bacteria and/or microbial extracellular vesicles (mEVs); and
(b) at least one component of soy leghemoglobin; and
(ii) at least one diluent, at least one lubricant, and/or at least one glidant.
48. A method of preventing or treating a disease of a subject comprising administering to the subject a pharmaceutical composition of any one of claims 1 to 47 or a solid dosage form of claim 46 or 47.
49. Use of a pharmaceutical composition of any one of claims 1 to 47 or a solid dosage form of claim 46 or 47 for the treatment or prevention of a disease of a subject.
50. A method of testing a pharmaceutical composition comprising bacteria and/or microbial extracellular vesicles (mEVs), the method comprising performing an assay to detect the presense of a component of a soy leghemoglobin in the pharmaceutical composition.
51. The method of claim 50, wherein the component of soy leghemoglobin comprises a soy leghemoglobin nucleic acid.
52. The method of claim 51, wherein the soyn leghemoglobin nucleic acid is soy leghemoglobin DNA.
53. The method of claim 52, wherein the soybean leghemoglobin DNA comprises a sequence encoding soybean leghemoglobin A (LB A) or soy leghemoglobin C2 (LGB2).
54. The method of claim 50, wherein the assay to detect the presence of a component of soybean leghemoglobin is a nucleic acid amplification assay, a sequencing assay, and/or a microarray assay.
55. The method of claim 50 or 54, the assay to detect the presence of a component of soybean leghemoglobin is a polymerase chain reaction (PCR) assay.
56. The method of claim 50, wherein the component of soy leghemoglobin is a soy leghemoglobin protein.
57. The method of claim 56, wherein the soy leghemoglobin protein is soybean leghemoglobin A (LBA) or soy leghemoglobin C2 (LGB2) .
58. The method of claim 56 or 57, wherein the soybean leghemoglobin protein is detected using an antibody specific for the soybean leghemoglobin protein, HPLC or UPLC.
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