WO2023146843A1 - Compositions de vésicules extracellulaires et méthodes d'utilisation - Google Patents

Compositions de vésicules extracellulaires et méthodes d'utilisation Download PDF

Info

Publication number
WO2023146843A1
WO2023146843A1 PCT/US2023/011420 US2023011420W WO2023146843A1 WO 2023146843 A1 WO2023146843 A1 WO 2023146843A1 US 2023011420 W US2023011420 W US 2023011420W WO 2023146843 A1 WO2023146843 A1 WO 2023146843A1
Authority
WO
WIPO (PCT)
Prior art keywords
evs
extracellular vesicles
inflammation
resolving
dried form
Prior art date
Application number
PCT/US2023/011420
Other languages
English (en)
Inventor
Adam CARTWRIGHT
Andrea Itano
Taylor A. Cormack
Original Assignee
Evelo Biosciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2022/017446 external-priority patent/WO2022182707A1/fr
Application filed by Evelo Biosciences, Inc. filed Critical Evelo Biosciences, Inc.
Publication of WO2023146843A1 publication Critical patent/WO2023146843A1/fr

Links

Classifications

    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2009—Inorganic compounds
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/485—Inorganic compounds
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • Inflammation can be a protective response to harmful stimuli, such as invading pathogens, damaged cells, toxic compounds, or cancerous cells.
  • harmful stimuli such as invading pathogens, damaged cells, toxic compounds, or cancerous cells.
  • excessive inflammatory responses, or inflammatory responses that fail to resolve can result in serious adverse effects, including tissue damage and even death.
  • EVs extracellular vesicles
  • EVs extracellular vesicles
  • the methods and compositions provided herein resolve an inflammatory response, e.g., as described herein.
  • inflammation-resolving EVs are identified by their ability to decrease inflammation in a delayed type hypersensitivity (DTH) model of inflammation, e.g., in a DTH model provided herein.
  • DTH delayed type hypersensitivity
  • inflammation-resolving EVs are obtained from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • inflammation-resolving EVs decrease inflammation in a delayed type hypersensitivity (DTH) model of inflammation, e.g., in a DTH model provided herein, and are obtained from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • DTH delayed type hypersensitivity
  • compositions comprising inflammation-resolving extracellular vesicles (EVs), such as EVs obtained from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria, have therapeutic effects.
  • EVs extracellular vesicles
  • EVs can be prepared as a biomass (e.g., isolated EVs can be resuspended in a buffer such as PBS).
  • inflammation- resolving extracellular vesicles such as EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria
  • EVs extracellular vesicles
  • EVs can be prepared as dried forms, such as powders and/or lyophilates.
  • inflammation-resolving extracellular vesicles such as EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria
  • solid dosage forms such as capsules, tablets, and/or mini-tablets.
  • a method of resolving inflammation in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the extracellular vesicles resolve peripheral inflammation.
  • the extracellular vesicles resolve inflammation via an antigen-independent mechanism that can reduce antigen-specific inflammation.
  • the inflammation-resolving extracellular vesicles comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • the subject e.g., a human subject
  • the subject e.g., a human subject
  • the subject e.g., a human subject
  • an immune disorder in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • the immune disorder comprises an autoimmune disease, an inflammatory disease, or an allergy.
  • the immune disorder comprises an inflammatory disease.
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a medicament for treating an immune disorder in a subject (e.g., a human subject).
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a method of treating an inflammatory disease in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the inflammatory disease comprises a Thl mediated inflammatory disease.
  • the inflammatory disease comprises a Th2 mediated inflammatory disease (such as asthma or atopic dermatitis).
  • the inflammatory disease comprises a Thl 7 mediated inflammatory disease (such as psoriasis).
  • the inflammation-resolving extracellular vesicles comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a medicament for treating an inflammatory disease in a subject (e.g., a human subject).
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a method of treating psoriasis in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a medicament for treating psoriasis in a subject (e.g., a human subject).
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a method of treating atopic dermatitis in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a medicament for treating atopic dermatitis in a subject (e.g., a human subject).
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • atopic dermatitis in a subject (e.g., a human subject).
  • a method of activating an anti-inflammatory cytokine response in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammationresolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the antiinflammatory cytokine response comprises interleukin- 10 (IL- 10) production.
  • the anti-inflammatory cytokine response comprises IL-27 production.
  • the anti-inflammatory cytokine response comprises IL- 10 and IL-27 production.
  • the inflammation-resolving extracellular vesicles comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a method of increasing anti-inflammatory cytokine secretion in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the anti-inflammatory cytokine is IL- 10.
  • the antiinflammatory cytokine is IL-27.
  • the anti-inflammatory cytokines are IL- 10 and IL-27.
  • the inflammation-resolving extracellular vesicles comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a method of instructing T cells to be less inflammatory in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammationresolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles, e.g., as described herein.
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • the T cells are instructed in mesenteric lymph nodes.
  • a method of inducing a functional change in CD4+ T cells in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammationresolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles, e.g., as described herein.
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • the CD4+ T cells are peripheral CD4+ T cells.
  • the CD4+ T cells are conventional CD4+ (CD4+ CD25-) T cells (Tcons).
  • the CD4+ T cells resolve inflammation, such as peripheral inflammation.
  • the CD4+ T cells resolve inflammation in an antigenindependent manner.
  • the inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles are orally administered (e.g., and travel to the small intestine)
  • dendritic cells interact with the inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles in the small intestine
  • the dendritic cells travel to the mesenteric lymph nodes, and T cells trafficking through the mesenteric lymph node encounter the dendritic cells.
  • a method of affecting T cells that traffic to mesenteric lymph nodes in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles, e.g., as described herein.
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a medicament for the preparation of a medicament for affecting T cells that traffic to mesenteric lymph nodes in a subject (e.g., a human subject).
  • a method of generating inflammationresolving CD4+ T cells in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammationresolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • the extracellular vesicles generate a population of CD4+ T cells that can resolve inflammation.
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a method of activating (e.g., stimulating) TLR2 in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • the inflammationresolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the extracellular vesicles for use in the activation (e.g., stimulation) of TLR2 in a subject (e.g., a human subject).
  • the EVs stimulate heterodimers formed by TLR2, TLR2/1 and TLR2/6, e.g., in a dose-response dependent manner, e.g., as described herein.
  • TLR2 activation is necessary but not sufficient for the generation of inflammation resolving CD4+ T cells.
  • TLR2 activation is necessary but not sufficient for the generation of inflammation resolving CD4+ T cells, but not their function in the periphery, e.g., as described herein.
  • a method of reducing inflammatory cytokine expression in a subject in need thereof, comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles, wherein a Type I interferon response is not reduced (e.g., not reduced to the same extent that the inflammatory cytokine expression is reduced), e.g., as determined by anti-viral TLR3 -mediated Type I interferon levels, e.g., as determined by IFNa and/or IFN0 levels, e.g., as described herein.
  • the inflammation-resolving extracellular vesicles e.g., a therapeutically effective dose
  • a Type I interferon response is not reduced (e.g., not reduced to the same extent that
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a Type I interferon response is not reduced.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a Type I interferon response is not reduced.
  • a method of reducing inflammatory cytokine expression comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles, wherein a Type II interferon response is not reduced (e.g., not reduced to the same extent that the inflammatory cytokine expression is reduced), e.g., as determined by Type II interferon levels, e.g., as determined by IFNy levels, e.g., as described herein.
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotell
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a Type II interferon response is not reduced.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a Type II interferon response is not reduced.
  • a method of reducing inflammatory cytokine expression comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles, wherein interferon-gamma production by T cells (e.g., CD4 T cells and/or CD8 T cells) is not reduced (e.g., not reduced to the same extent that the inflammatory cytokine expression is reduced), e.g., as described herein.
  • the inflammationresolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflin
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • T cells e.g., CD4 T cells and/or CD8 T cells
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • T cells e.g., CD4 T cells and/or CD8 T cells
  • a method of reducing inflammatory cytokine expression comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles, wherein generation of functional CD4+ Thl cells is not reduced (e.g., not reduced to the same extent that the inflammatory cytokine expression is reduced), e.g., as described herein.
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • generation of functional CD4+ Thl cells is not reduced.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • generation of functional CD4+ Thl cells is not reduced.
  • a method of reducing inflammatory cytokine expression comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles, wherein production of interferon-gamma from human memory CD8 T cells in response to a viral peptide pool (Cytomegalovirus, Epstein-Bar virus, and Influenza virus) is not reduced (e.g., not reduced to the same extent that the inflammatory cytokine expression is reduced), e.g., as described herein.
  • the inflammation- resolving extracellular vesicles (EVs) e.g., reducing IL-8, IL-6, IL-10, and/or TNFa expression levels
  • a dose e.g., a therapeutically effective dose
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a medicament for reducing inflammatory cytokine expression in a subject (e.g., a human subject), wherein production of interferon-gamma from human memory CD8 T cells in response to a viral peptide pool (Cytomegalovirus, Epstein-Bar virus, and Influenza virus) is not reduced.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • production of interferon-gamma from human memory CD8 T cells in response to a viral peptide pool is not reduced.
  • a method of inducing resolution of peripheral inflammation in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria.
  • the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • the dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the extracellular vesicles is administered in combination with an anti-TNF alpha antibody.
  • the dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • an TNF alpha antagonist e.g., TNF alpha antagonist or TNF alpha receptor antagonist
  • an TNF alpha antagonist e.g., ADALIMUMAB (Humira®), ETANERCEPT (Enbrel®), INFLIXIMAB (Remicade®; TA-650), CERTOLIZUMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB (Simpom®; CNTO 148), ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; MabThera®), ABATACEPT (Orencia®), TOCILIZUMAB (RoActemra /Actemra®).
  • the dose is orally administered.
  • the effect of the dose on peripheral inflammation is comparable to the effect of dexamethasone, tofacitinib, or an anti-TNF antibody, for example, in a DTK model, e.g., as described herein.
  • the duration of the effect of the dose on peripheral inflammation is comparable to the duration of the effect of dexamethasone, tofacitinib, or an anti-TNF antibody, for example, in a DTK model, e.g., as described herein.
  • the duration of the effect of the dose on peripheral inflammation exceeds the duration of the effect of dexamethasone, tofacitinib, or an anti-TNF antibody, for example, in a DTH model, e.g., as described herein.
  • the duration of the effect of the dose on peripheral inflammation exceeds the duration of the effect of dexamethasone or tofacitinib, for example, in a DTH model, e.g., as described herein.
  • more than one dose is administered to the subject and tachyphylaxis is not caused by more than one administration of the dose to the subject, e.g., as described herein.
  • the dose resolves inflammation that is not dependent on antigenic recognition, as described herein.
  • the dose resolves inflammation that is not dependent on antigen specificity, as described herein.
  • a method of inducing resolution of peripheral inflammation in an antigen-independent manner through CD4+ T cells in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • EVs extracellular vesicles
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the inflammation-resolving extracellular vesicles comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria. In some embodiments, the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • the dose is orally administered.
  • the resolution of peripheral inflammation does not involve an antigen-specific response to the EVs, e.g., as described herein.
  • the resolution of peripheral inflammation does not involve adaptive recognition of the EVs, e.g., as described herein.
  • the resolution of peripheral inflammation does not require B cells, e.g., as described herein.
  • the resolution of peripheral inflammation does not require CD8+ cells, e.g., as described herein.
  • a method of inducing functional changes to peripheral CD4+ T cells towards a pro-resolving utility in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the inflammation-resolving extracellular vesicles comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria. In some embodiments, the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a pro-resolving utility in a subject (e.g., a human subject).
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a pro-resolving utility in a subject (e.g., a human subject).
  • the dose is orally administered.
  • the inducing functional changes to peripheral CD4+ T cells promotes resolution of inflammation is antigen-independent, e.g., as described herein.
  • the EVs induce functional changes to peripheral CD4+ T cells towards a pro-resolving utility in a dose-response dependent manner, e.g., as described herein.
  • a method of generating CD4+ T cells that can resolve inflammation and do not secrete IL-10 in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • EVs extracellular vesicles
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the inflammation-resolving extracellular vesicles comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria. In some embodiments, the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g, a therapeutically effective dose
  • a composition e.g, a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • the dose is orally administered.
  • the CD4+ T cells induce IL-10 release (e.g., by another cell population), e.g., as described herein.
  • a method of inducing functional changes in conventional CD4+CD25- T cells to resolve peripheral inflammation in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of inflammation-resolving extracellular vesicles (EVs) and/or a composition (e.g., a solution, dried form and/or therapeutic composition) comprising the extracellular vesicles.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the inflammation-resolving extracellular vesicles comprise EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria. In some embodiments, the inflammation-resolving extracellular vesicles (EVs) comprise EVs from Prevotella histicola bacteria.
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • a subject e.g., a human subject
  • the dose is orally administered.
  • the inflammation-resolving extracellular vesicles comprise EVs from Prevotella histicola bacteria.
  • the inflammation-resolving extracellular vesicles are from a Prevotella histicola strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% 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 histicola Strain B (NRRL accession number B 50329).
  • the Prevotella histicola strain is the Prevotella histicola Strain B (NRRL accession number B 50329).
  • the inflammation-resolving extracellular vesicles comprise EVs from Veillonella parvula bacteria.
  • the inflammation-resolving extracellular vesicles are from a Veillonella parvula strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% 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 Veillonella parvula Strain A (ATCC Deposit Number PTA-125691).
  • 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 Veillonella parvula strain is Veillonella parvula Strain A (ATCC Deposit Number PTA-125691).
  • the inflammation-resolving extracellular vesicles comprise EVs from Harryflintia acetispora bacteria.
  • the inflammation-resolving extracellular vesicles are from a Harryflintia acetispora strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% 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 Harryflintia acetispora Strain A (ATCC Deposit Number PTA- 126694).
  • the Harryflintia acetispora strain is Harryflintia acetispora Strain A (ATCC Deposit Number PTA- 126694).
  • the subject e.g., a human subject
  • the immune disorder is arthrosclerosis, arthritis, phlebitis, vasculitis, and lymphangitis, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, proctitis, Crohn's disease, ulcerative colitis, irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis, eosinophilic enterocolitis, indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet’s disease, sarcoidosis, scleroderma, IBD- associated dysplasia, dysplasia, dysplasia, dysplasia, dysplasia, dysplasia, dysplasia, dys
  • the subject e.g., a human subject
  • the subject e.g., a human subject
  • the subject e.g., a human subject
  • the subject e.g., a human subject
  • the subject e.g., a human subject
  • the EVs are administered orally.
  • the inflammation-resolving extracellular vesicles (EVs) and/or a composition e.g., a solution, dried form and/or therapeutic composition
  • a composition e.g., a solution, dried form and/or therapeutic composition
  • the inflammation-resolving extracellular vesicles (EVs) and/or a composition comprising the extracellular vesicles induce IL- 10, IL-27, IL-6, IP-10, and/or TNLa secretion from in vitro cultured human PBMCs.
  • the inflammation-resolving extracellular vesicles (EVs) and/or a composition comprising the extracellular vesicles induce IL- 10, IL-27, IL-6, IP-10, and/or TNLa secretion from in vitro cultured human macrophages.
  • the inflammation-resolving extracellular vesicles (EVs) and/or a composition comprising the extracellular vesicles induce IL- 10, IL-27, IL-6, IP-10, and/or TNLa secretion from in vitro cultured human dendritic cells.
  • the inflammation-resolving extracellular vesicles (EVs) and/or a composition comprising the extracellular vesicles induce IL- 10, IL-6, and/or TNLa secretion from in vitro cultured human dendritic cells.
  • the inflammation-resolving extracellular vesicles (EVs)and/or a composition comprising the extracellular vesicles induce IL- 10, IL-27, IL-6, IP-10, and TNLa secretion from in vitro cultured U937 cells.
  • the dose is in the form of one or more capsules, optionally comprising an enteric-coating (e.g., enteric- coated capsules).
  • the dose is in the form of one or more tablets, optionally comprising an enteric-coating (e.g., enteric-coated tablets).
  • the dose is in the form of one or more mini-tablets.
  • the mini-tablets are enteric-coated mini-tablets.
  • the dose is in the form of a non-enteric coated capsule comprising one or more enteric-coated mini-tablets.
  • inflammation-resolving extracellular vesicles have therapeutic effects and are useful for the treatment and/or prevention of disease and/or health disorders.
  • Therapeutic compositions of biomass, solutions and/or dried forms containing inflammation-resolving extracellular vesicles (EVs) can be prepared.
  • dried forms having a moisture content below about 6% are better suited for downstream processing, In some embodiments, dried forms having a moisture content below about 6% have improved stability.
  • the solutions comprising the inflammation-resolving extracellular vesicles (EVs), such as EVs from Prevotella histicola, Harryflintia acetispora, or Veillonella parvula bacteria also comprise an excipient that contains a bulking agent, and optionally comprises one or more additional ingredients, such as a lyoprotectant.
  • EVs extracellular vesicles
  • the solutions comprising the inflammation-resolving extracellular vesicles also comprise an excipient that contains a lyoprotectant, and optionally comprises one or more additional ingredients, such as a bulking agent.
  • the dried forms comprising the inflammation-resolving extracellular vesicles also comprise an excipient that contains a bulking agent, and that optionally comprises one or more additional ingredients, such as a lyoprotectant.
  • the dried forms comprising the inflammation-resolving extracellular vesicles also comprise an excipient that contains a lyoprotectant, and optionally comprise one or more additional ingredients, such as a bulking agent.
  • Bulking agents and/or lyoprotectants are used when preparing extracellular vesicles (EVs) for drying, such as freeze drying and spray drying.
  • bulking agents including but not limited to sucrose, mannitol, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, and dextran (such as dextran 40k), make dried forms (such as powders and/or lyophilates) easier to handle after drying.
  • bulking agents improve the properties of a dried form.
  • lyoprotectants including but not limited to trehalose, sucrose, and lactose protect the EVs during drying, such as freeze-drying or spray drying.
  • the excipient functions to decrease drying cycle time.
  • the excipient functions to maintain therapeutic efficacy of the EVs. 1 [124]
  • the disclosure provides a dried form comprising inflammationresolving extracellular vesicles (EVs), wherein the dried form has a moisture content (e.g., as determined by the Karl Fischer method) of below about 6%.
  • the dried form provided herein has a moisture content (e.g., as determined by the Karl Fischer method) of below about 5%.
  • the dried form provided herein has a moisture content (e.g., as determined by the Karl Fischer method) of below about 4%.
  • the dried form provided herein has a moisture content (e.g., as determined by the Karl Fischer method) of between about 1% to about 4%.
  • the dried form provided herein has a moisture content (e.g., as determined by the Karl Fischer method) of between about 2% to about 4%.
  • the dried form provided herein has a moisture content (e.g., as determined by the Karl Fischer method) of between about 2% to about 3%.
  • the disclosure provides a lyophilate comprising inflammationresolving extracellular vesicles (EVs), wherein the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of below about 6%.
  • a moisture content e.g., as determined by the Karl Fischer method
  • the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of below about 5%.
  • the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of below about 4%.
  • the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of between about 1% to about 4%.
  • the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of between about 2% to about 4%.
  • the lyophilate has a moisture content (e.g., as determined by the Karl Fischer method) of between about 2% to about 3%.
  • the disclosure provides a powder comprising inflammation-resolving extracellular vesicles (EVs), wherein the powder has a moisture content (e.g., as determined by the Karl Fischer method) of below about 6%.
  • EVs extracellular vesicles
  • the powder has a moisture content (e.g., as determined by the Karl Fischer method) of below about 5%.
  • the powder has a moisture content (e.g., as determined by the Karl Fischer method) of below about 4%.
  • the powder has a moisture content (e.g., as determined by the Karl Fischer method) of between about 1% to about 4%.
  • the powder has a moisture content (e.g., as determined by the Karl Fischer method) of between about 2% to about 4%.
  • the powder has a moisture content (e.g., as determined by the Karl Fischer method) of between about 2% to about 3%.
  • the dried form comprises a powder.
  • the powder comprises a lyophilized powder.
  • the powder comprises a spray-dried powder.
  • the dried form comprises a lyophilate.
  • the lyophilate comprises a lyophilized powder.
  • the lyophilate comprises a lyophilized cake.
  • the disclosure provides a therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs), wherein the composition further comprises a pharmaceutically acceptable excipient.
  • EVs extracellular vesicles
  • the disclosure provides a solution comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a solution consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a solution comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a solution consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a solution comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • the disclosure provides a solution consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising the solution, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a dried form comprising inflammationresolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a dried form consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a dried form comprising inflammationresolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a dried form consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a dried form comprising inflammationresolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a dried form consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition comprising the dried form, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a powder comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • the disclosure provides a powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • the disclosure provides a powder comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a powder comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition comprising the powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a spray-dried powder comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a spray-dried powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a spray-dried powder comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a spray-dried powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a spray-dried powder comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • the disclosure provides a spray-dried powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition comprising the spray-dried powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilate comprising inflammationresolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilate consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • a lyophilate consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilate comprising inflammationresolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilate consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilate comprising inflammationresolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilate consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • a lyophilate consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising the lyophilate, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized powder comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • the disclosure provides a lyophilized powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilized powder comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilized powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and from an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilized powder comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilized powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and from an excipient that comprises a lyoprotectant.
  • a lyophilized powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and from an excipient that comprises a lyoprotectant.
  • the disclosure provides a therapeutic composition comprising the lyophilized powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized cake comprising inflammationresolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilized cake consisting essentially of inflammation-resolving extracellular vesicles (EVs) a and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilized cake comprising inflammationresolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • a lyophilized cake consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • the disclosure provides a lyophilized cake comprising inflammationresolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilized cake consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition consisting essentially of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • EVs extracellular vesicles
  • the disclosure provides a solution comprising inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • the disclosure provides a solution consisting essentially of inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • the disclosure provides a therapeutic composition comprising such solution, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a dried form comprising inflammationresolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • EVs extracellular vesicles
  • the disclosure provides a dried form consisting essentially of inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition comprising such dried form, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a powder comprising inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • EVs extracellular vesicles
  • the disclosure provides a powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition comprising such powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a spray-dried powder comprising inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • the disclosure provides a spray-dried powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • the disclosure provides a therapeutic composition comprising such spray-dried powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilate comprising inflammationresolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilate consisting essentially of inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition comprising such lyophilate, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized powder comprising inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • EVs extracellular vesicles
  • the disclosure provides a lyophilized powder consisting essentially of inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • EVs extracellular vesicles
  • the disclosure provides a therapeutic composition comprising such lyophilized powder, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a lyophilized cake comprising inflammationresolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • the disclosure provides a lyophilized cake consisting essentially of inflammation-resolving extracellular vesicles (EVs) and excipients of a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • the disclosure provides a therapeutic composition comprising such lyophilized cake, wherein the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient comprises a glidant, lubricant, and/or diluent.
  • the disclosure provides a method of treating a subject (for example, human) (for example, a subject in need of treatment), the method comprising:
  • inflammation-resolving extracellular vesicles (EVs) or a solution, dried form, or therapeutic composition provided herein is for use in treating a subject (for example, human) (for example, a subject in need of treatment).
  • the disclosure provides use of inflammation-resolving extracellular vesicles (EVs) or a solution, dried form, or therapeutic composition provided herein for the preparation of a medicament for treating a subject (for example, human) (for example, a subject in need of treatment).
  • a subject for example, human
  • a subject in need of treatment for example, a subject in need of treatment.
  • the inflammation-resolving extracellular vesicles (EVs) or solution, dried form, or therapeutic composition is orally administered (for example, is for oral administration).
  • the subject is in need of treatment (and/or prevention) of an immune disease.
  • the subject is in need of treatment (and/or prevention) of an autoimmune disease.
  • the subject is in need of treatment (and/or prevention) of an inflammatory disease.
  • the subject is in need of treatment (and/or prevention) of a metabolic disease.
  • the subject is in need of treatment (and/or prevention) of dysbiosis.
  • solution, dried form, therapeutic composition or use provided herein the solution, dried form, or therapeutic composition is administered in combination with an additional therapeutic agent.
  • the dried form is a powder.
  • the powder is a lyophilized powder.
  • the powder is a spray-dried powder.
  • the dried form is a lyophilate.
  • the lyophilate is a lyophilized powder.
  • the lyophilate is a lyophilized cake.
  • the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, Ficoll, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, or PVP-K30.
  • the bulking agent comprises mannitol.
  • the excipient comprises an additional ingredient.
  • the additional ingredient comprises trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B- cyclodextrin.
  • the excipient comprises mannitol and trehalose.
  • the excipient consists essentially of mannitol and trehalose.
  • the excipient comprises mannitol, trehalose, and sorbitol.
  • the excipient consists essentially of mannitol, trehalose, and sorbitol.
  • the excipient comprises trehalose.
  • the excipient consists essentially of trehalose.
  • the excipient is from a stock comprising one or more excipients, wherein the stock comprises a formula provided in provided in Table A, B, C, D, K, or P.
  • the dried form is a powder.
  • the powder is a lyophilized powder.
  • the powder is a spray-dried powder.
  • the dried form is a lyophilate.
  • the lyophilate is a lyophilized powder.
  • the lyophilate is a lyophilized cake.
  • the excipient solution comprises mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis).
  • the excipient solution comprises more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form comprises mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis).
  • the excipient of the solution or dried form comprises more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form comprises at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient of the solution or dried form comprises at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution consists essentially of mannitol and trehalose.
  • the excipient solution consists essentially of mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis).
  • the excipient solution consists essentially of mannitol and trehalose, wherein the excipient contains more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein neither the mannitol nor the trehalose is present in an amount of 5 mg/ml to 15 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the mannitol is not present in an amount of 5 mg/ml to 15 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the trehalose is not present in an amount of 5 mg/ml to 15 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein neither the mannitol nor the trehalose is present in an amount of 9 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the mannitol is not present in an amount of 9 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the trehalose is not present in an amount of 9 mg/ml.
  • the excipient comprises, or consists essentially of, mannitol and trehalose, and does not comprise methionine.
  • the dried form or therapeutic composition comprises, or consists essentially of, mannitol and trehalose, and the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts, for example, on a weight basis or a weight percent basis) in the dried form or therapeutic composition.
  • At least about 10% (by weight) of the solution or dried form is excipient stock.
  • the solution, dried form, or therapeutic composition provided herein about 30% to about 60% (by weight) of the solution or dried form is excipient stock.
  • the inflammation-resolving extracellular vesicles comprise at least about 1% of the total solids by weight of the dried form.
  • the inflammation-resolving extracellular vesicles (EVs) comprise about 1% to about 99% of the total solids by weight of the dried form.
  • the inflammation-resolving extracellular vesicles (EVs) comprise about 5% to about 90% of the total solids by weight of the dried form.
  • the inflammation-resolving extracellular vesicles (EVs) comprise about 1% to about 60% of the total solids by weight of the dried form.
  • the inflammation-resolving extracellular vesicles (EVs) comprise about 1% to about 20% of the total solids by weight of the powder or cake. In some embodiments of the dried form or therapeutic composition provided herein, the inflammation-resolving extracellular vesicles (EVs) comprise about 2% to about 10% of the total solids by weight of the dried form. In some embodiments of the dried form or therapeutic composition provided herein, the inflammation-resolving extracellular vesicles (EVs) comprise about 2% to about 6% of the total solids by weight of the dried form. In some embodiments of the dried form or therapeutic composition provided herein, the dried form comprises a moisture content below about 6% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content below about 5% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 0.5% to about 5% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 1% to about 5% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 1% to about 4% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 2% to about 5% (for example, as determined by Karl Fischer titration).
  • the dried form comprises a moisture content about 2% to about 4% (for example, as determined by Karl Fischer titration).
  • particle numeration is determined on a dried form by NTA. In some embodiments, particle numeration is determined on a dried form by NTA with use of a Zetaview camera.
  • particle numeration is determined on dried form resuspended in water, by NTA and with use of a Zetaview camera.
  • a solution, dried form, or therapeutic composition provided herein can contain EVs from one or more bacterial strain in addition to inflammation-resolving extracellular vesicles (EVs).
  • a solution, dried form, or therapeutic composition provided herein can contain EVs from one bacterial strain in addition to inflammation-resolving extracellular vesicles (EVs).
  • the bacterial strain used as a source of EVs may be selected based on the properties of the bacteria (e.g., growth characteristics, yield, ability to modulate an immune response in an assay or a subject).
  • a solution, dried form, or therapeutic composition provided herein can contain inflammation-resolving extracellular vesicles (EVs) from one or more strains of bacteria.
  • a solution, dried form, or therapeutic composition provided herein can contain inflammation-resolving extracellular vesicles (EVs) from one strain of bacteria.
  • a solution, dried form, or therapeutic composition provided herein can contain inflammation-resolving extracellular vesicles (EVs) from two strains of bacteria.
  • a solution, dried form, or therapeutic composition provided herein can contain inflammation-resolving extracellular vesicles (EVs) from three strains of bacteria.
  • the bacterial strain used as a source of inflammation-resolving EVs may be selected based on the properties of the bacteria (e.g., growth characteristics, yield, ability to modulate an immune response in an assay or a subject, ability to decrease inflammation in a DTH model).
  • inflammation-resolving extracellular vesicles (EVs) or a solution, dried form, or therapeutic composition provided herein comprising inflammationresolving extracellular vesicles (EVs) can be used for the treatment or prevention of a disease and/or a health disorder, e.g., in a subject (e.g., human).
  • a dried form (or a therapeutic composition thereof) provided herein comprising inflammation-resolving extracellular vesicles (EVs) can be prepared as a solid dose form, such as a tablet, a minitablet, a capsule, or a powder; or a combination of these forms (e.g., minitablets comprised in a capsule).
  • the solid dose form can comprise a coating (e.g., enteric coating).
  • the therapeutic composition comprises a solid dose form.
  • the therapeutic composition comprises a blend of freeze-dried powder of inflammation-resolving extracellular vesicles (EVs) and excipients (e.g., an encapsulated freeze- dried powder of the inflammation-resolving extracellular vesicles (EVs) provided herein and excipients).
  • the therapeutic composition comprises freeze-dried (e.g., lyophilized) powder of inflammation- resolving extracellular vesicles (EVs) in a capsule.
  • the capsule comprises gelatin or HPMC.
  • the capsule is enteric coated.
  • the excipients include one or more of mannitol, magnesium stearate and colloidal silicon dioxide. In some embodiments, the excipients include mannitol, magnesium stearate and colloidal silicon dioxide. In some embodiments, the therapeutic composition comprises freeze-dried (e.g., lyophilized) powder of inflammationresolving extracellular vesicles (EVs) in a tablet or mini -tablet. In some embodiments, the tablet or mini-tablet is enteric coated. In some embodiments, the excipients include one or more of silicified microcrystalline cellulose, crospovidone, magnesium stearate and colloidal silicon dioxide. In some embodiments, the excipients include silicified microcrystalline cellulose, crospovidone, magnesium stearate and colloidal silicon dioxide.
  • a dried form (or a therapeutic composition thereof) provided herein comprising inflammation-resolving extracellular vesicles (EVs) can be reconstituted.
  • a solution (or a therapeutic composition thereof) provided herein comprising inflammation-resolving extracellular vesicles (EVs) can be used as suspension, e.g., diluted to a suspension or used in undiluted form.
  • a therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) or a solution and/or dried form comprising inflammation-resolving extracellular vesicles (EVs) can be prepared as provided herein.
  • the therapeutic composition comprising a dried form can be formulated into a solid dose form, such as a tablet, a minitablet, a capsule, or a powder; or can be reconstituted in a suspension.
  • inflammation-resolving extracellular vesicles (EVs) or a solution, dried form, or therapeutic composition provided herein can comprise gamma irradiated inflammation-resolving extracellular vesicles (EVs).
  • the gamma irradiated inflammationresolving extracellular vesicles can be formulated into a therapeutic composition.
  • the gamma irradiated inflammation-resolving extracellular vesicles (EVs) can be formulated into a solid dose form, such as a tablet, a minitablet, a capsule, or a powder; or can be reconstituted in a suspension.
  • inflammation-resolving extracellular vesicles (EVs) or a solution, dried form, or therapeutic composition provided herein comprising inflammationresolving extracellular vesicles (EVs) can be orally administered.
  • inflammation-resolving extracellular vesicles (EVs) or a solution, dried form, or therapeutic composition provided herein comprising inflammationresolving extracellular vesicles (EVs) can be administered intranasally.
  • inflammation-resolving extracellular vesicles (EVs) or a solution, dried form, or therapeutic composition provided herein comprising inflammationresolving extracellular vesicles (EVs) can be administered by inhalation.
  • inflammation-resolving extracellular vesicles (EVs) or a solution, dried form, or therapeutic composition provided herein comprising inflammationresolving extracellular vesicles (EVs) can be administered intravenously.
  • inflammation-resolving extracellular vesicles (EVs) or a solution, dried form, or therapeutic composition provided herein comprising inflammationresolving extracellular vesicles (EVs) can be administered by injection.
  • compositions comprising inflammation-resolving extracellular vesicles (EVs) and/or solutions and/or dried forms comprising inflammation-resolving extracellular vesicles (EVs) useful for the treatment and/or prevention of a disease or a health disorder (e.g., adverse health disorders) (e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease), as well as methods of making and/or identifying such inflammation-resolving extracellular vesicles (EVs) and/or solutions and/or dried forms and therapeutic compositions, and methods of using such inflammation-resolving extracellular vesicles (EVs) and/or solutions and/or dried forms, and therapeutic compositions thereof (e.g., for the treatment of an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease, either alone or in combination
  • a disease or a health disorder
  • the therapeutic compositions can comprise both inflammationresolving extracellular vesicles (EVs) and whole bacteria, e.g., bacteria from which the EVs were obtained, such as live bacteria, killed bacteria, attenuated bacteria.
  • the therapeutic compositions comprise inflammation- resolving extracellular vesicles (EVs) in the absence of the bacteria from which they were obtained, such that over about 85%, over about 90%, or over about 95% (or over about 99%) of the bacteria-sourced content of the solutions and/or dried forms comprises inflammation-resolving extracellular vesicles (EVs).
  • the inflammation-resolving extracellular vesicles (EVs) can be isolated EVs, e.g., isolated by a method described herein.
  • the inflammation-resolving extracellular vesicles (EVs) or solution, dried form, or therapeutic composition comprises isolated inflammation-resolving extracellular vesicles (EVs) (e.g., from one or more strains of bacteria (e.g., a therapeutically effective amount thereof).
  • EVs extracellular vesicles
  • EVs inflammation-resolving extracellular vesicles
  • EVs inflammation-resolving extracellular vesicles
  • the inflammation-resolving extracellular vesicles (EVs) or solution, dried form, or therapeutic composition comprises isolated inflammation-resolving extracellular vesicles (EVs) (e.g., from one strain of bacteria (e.g., bacteria of interest) (e.g., a therapeutically effective amount thereof).
  • isolated inflammation-resolving extracellular vesicles (EVs) e.g., from one strain of bacteria (e.g., bacteria of interest) (e.g., a therapeutically effective amount thereof).
  • EVs extracellular vesicles
  • EVs isolated inflammation-resolving extracellular vesicles
  • the inflammation-resolving extracellular vesicles (EVs) or solution, dried form or therapeutic composition comprises inflammation-resolving extracellular vesicles (EVs) from one strain of bacteria.
  • the solution, dried form, or therapeutic composition comprises inflammation-resolving extracellular vesicles (EVs) from more than one strain of bacteria.
  • the inflammation-resolving extracellular vesicles are lyophilized.
  • the inflammation-resolving extracellular vesicles are gamma irradiated.
  • the inflammation-resolving extracellular vesicles are UV irradiated.
  • the inflammation-resolving extracellular vesicles are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
  • the inflammation-resolving extracellular vesicles (EVs) are acid treated.
  • the inflammation-resolving extracellular vesicles are oxygen sparged (e.g., at 0.1 wm for two hours).
  • LPS lipopoly
  • the inflammation-resolving extracellular vesicles are from engineered bacteria that are modified to enhance certain desirable properties.
  • the engineered bacteria are modified so that EVs produced therefrom will have reduced toxicity and adverse effects (e.g., by removing or deleting lipopolysaccharide (LPS)), enhanced oral delivery (e.g., by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, resistance to anti-microbial peptides and/or antibody neutralization), target desired cell types (e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages), improved bioavailability systemically or in an appropriate niche (e.g., mesenteric lymph nodes, Peyer’s patches, lamina intestinal, lymph nodes, and/or blood), enhanced immunomodulatory and/or therapeutic effect (e.g., either alone or in combination with another therapeutic agent), enhanced immune activation, and/or improved
  • LPS lipopolysaccharide
  • inflammation-resolving extracellular vesicles (EVs) and/or solutions and/or dried forms (or therapeutic compositions thereof) comprising inflammation-resolving extracellular vesicles (EVs) useful for the treatment and/or prevention of a disease or a health disorder (e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease), as well as methods of making and/or identifying such solutions and/or dried forms(or therapeutic compositions thereof), and methods of using such solutions and/or dried forms (e.g., for the treatment of an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease), either alone or in combination with one or more other therapeutics.
  • a disease or a health disorder e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease)
  • Therapeutic compositions containing inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can provide potency comparable to or greater than therapeutic compositions that contain the whole bacteria from which the EVs were obtained.
  • a therapeutic composition containing solutions and/or dried form can provide potency comparable to or greater than a comparable therapeutic composition that contains whole bacteria of the same bacterial strain from which the EVs were obtained.
  • Such EV- and/or solution- and/or dried form - containing therapeutic compositions can allow the administration of higher doses and elicit a comparable or greater (e.g., more effective) response than observed with a comparable therapeutic composition that contains whole bacteria of the same bacterial strain from which the EVs were obtained.
  • a therapeutic composition containing inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can contain less microbially-derived material (based on particle count or protein content), as compared to a therapeutic composition that contains the whole bacteria of the same bacterial strain from which the EVs were obtained, while providing an equivalent or greater therapeutic benefit to the subject receiving such therapeutic composition.
  • inflammation-resolving extracellular vesicles can be administered at doses e.g., of about IxlO 7 to about IxlO 15 particles, e.g., as measured by NTA.
  • the dose of EVs is about 1 x 10 5 to about 7 x 10 13 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • the dose of inflammation-resolving extracellular vesicles (EVs) is about I x lO 10 to about 7 x 10 13 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • NTA can be performed with Zetaview.
  • inflammation-resolving extracellular vesicles can be administered at doses e.g., of about 5 mg to about 900 mg total protein, e.g., as measured by Bradford assay.
  • inflammation-resolving extracellular vesicles can be administered at doses e.g., of about 5 mg to about 900 mg total protein, e.g., as measured by BCA assay.
  • provided herein are methods of treating a subject who has an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy) comprising administering to the subject a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form described herein.
  • an immune disorder e.g., an autoimmune disease, an inflammatory disease, an allergy
  • methods of treating a subject who has a metabolic disease comprising administering to the subject a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form described herein.
  • provided herein are methods of treating a subject who has a dysbiosis comprising administering to the subject a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form described herein.
  • methods of treating a subject who has a neurologic disease comprising administering to the subject a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form described herein.
  • the method further comprises administering to the subject an antibiotic.
  • the method further comprises the administration of an immune suppressant and/or an anti-inflammatory agent.
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution, dried form, and/or lyophilate can be for use in combination with one or more other immune effect modulators.
  • the method further comprises the administration of a metabolic disease therapeutic agent.
  • a therapeutic composition or inflammationresolving extracellular vesicles (EVs) and/or a solution and/or dried form for use in the treatment and/or prevention of a disease (e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease) or a health disorder, either alone or in combination with one or more other (e.g., additional) therapeutic agent.
  • a disease e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease) or a health disorder, either alone or in combination with one or more other (e.g., additional) therapeutic agent.
  • a therapeutic composition or inflammationresolving extracellular vesicles (EVs) and/or a solution and/or dried form for use in treating and/or preventing an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy) in a subject (e.g., human).
  • an immune disorder e.g., an autoimmune disease, an inflammatory disease, an allergy
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be used either alone or in combination with one or more other therapeutic agent for the treatment of the immune disorder.
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form for use in treating and/or preventing a dysbiosis in a subject (e.g., human).
  • the therapeutic composition or inflammationresolving extracellular vesicles (EVs) and/or a solution and/or dried form can be used either alone or in combination with therapeutic agent for the treatment of the dysbiosis.
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form for use in treating and/or preventing a metabolic disease in a subject (e.g., human).
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be used either alone or in combination with therapeutic agent for the treatment of the metabolic disease.
  • a therapeutic composition or inflammationresolving extracellular vesicles (EVs) and/or a solution and/or dried form for use in treating and/or preventing a dysbiosis in a subject (e.g., human).
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be used either alone or in combination with therapeutic agent for the treatment of the dysbiosis.
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form for use in treating and/or preventing a neurologic disease in a subject (e.g., human).
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be used either alone or in combination with one or more other therapeutic agent for treatment of the neurologic disorder.
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use in combination with an antibiotic.
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use in combination with another therapeutic bacterium and/or EVs obtained from one or more other bacterial strains (e.g., therapeutic bacterium).
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use in combination with one or more immune suppressant(s) and/or an anti-inflammatory agent(s).
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use in combination with one or more other metabolic disease therapeutic agents.
  • a therapeutic composition or inflammationresolving extracellular vesicles (EVs) and/or a solution and/or dried form for the preparation of a medicament for the treatment and/or prevention of a disease (e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease), either alone or in combination with another therapeutic agent.
  • a disease e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease
  • the use is in combination with another therapeutic bacterium and/or EVs obtained from one or more other bacterial strains (e.g., therapeutic bacterium).
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form for the preparation of a medicament for treating and/or preventing an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy) in a subject (e.g., human).
  • an immune disorder e.g., an autoimmune disease, an inflammatory disease, an allergy
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use either alone or in combination with another therapeutic agent for the immune disorder.
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form for the preparation of a medicament for treating and/or preventing a dysbiosis in a subject (e.g., human).
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use either alone or in combination with another therapeutic agent for the dysbiosis.
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form for the preparation of a medicament for treating and/or preventing a metabolic disease in a subject (e.g., human).
  • the therapeutic composition or inflammationresolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use either alone or in combination with another therapeutic agent for the metabolic disease.
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form for the preparation of a medicament for treating and/or preventing a dysbiosis in a subject (e.g., human).
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use either alone or in combination with another therapeutic agent for the dysbiosis.
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form for the preparation of a medicament for treating and or preventing a neurologic disease in a subject (e.g., human).
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use either alone or in combination with another therapeutic agent for the neurologic disorder.
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use in combination with an antibiotic.
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use in combination with another therapeutic bacterium and/or EVs obtained from one or more other bacterial strains (e.g., therapeutic bacterium).
  • the therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use in combination with one or more other immune suppressant(s) and/or an antiinflammatory agent(s).
  • the therapeutic composition or inflammationresolving extracellular vesicles (EVs) and/or a solution and/or dried form can be for use in combination with one or more other metabolic disease therapeutic agent(s).
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form, e.g., as described herein, comprising inflammation-resolving extracellular vesicles (EVs) can provide a therapeutically effective amount of inflammationresolving extracellular vesicles (EVs) to a subject, e.g., a human.
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form, e.g., as described herein, comprising inflammation-resolving extracellular vesicles (EVs) can provide a non- natural amount of the therapeutically effective components (e.g., present in the inflammation-resolving extracellular vesicles (EVs)) to a subject, e.g., a human.
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form, e.g., as described herein, comprising inflammation-resolving extracellular vesicles (EVs) can provide unnatural quantity of the therapeutically effective components (e.g., present in the EVs) to a subject, e.g., a human.
  • a therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form, e.g., as described herein, comprising inflammation-resolving extracellular vesicles (EVs) 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 therapeutic composition or inflammation-resolving extracellular vesicles (EVs) and/or a solution and/or dried form, e.g., as described herein, comprising inflammation-resolving extracellular vesicles (EVs) 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 stock comprising one or more excipients, wherein the stock comprises a bulking agent, wherein the stock is for use in combination with inflammation-resolving extracellular vesicles (EVs) (for example, a liquid preparation thereof) .
  • EVs extracellular vesicles
  • a stock comprising one or more excipients, wherein the stock comprises a bulking agent and a lyoprotectant, wherein the stock is for use in combination with inflammation-resolving extracellular vesicles (EVs) (for example, a liquid preparation thereof).
  • EVs extracellular vesicles
  • a stock comprising one or more excipients, wherein the stock comprises a lyoprotectant, wherein the stock is for use in combination with inflammation-resolving extracellular vesicles (EVs) (for example, a liquid preparation thereof).
  • the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, Ficoll, or PVP-K30.
  • the bulking agent comprises mannitol.
  • the excipient solution comprises an additional ingredient.
  • the additional ingredient comprises trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • the excipient solution comprises mannitol and trehalose.
  • the excipient solution consists essentially of mannitol and trehalose.
  • the excipient solution comprises mannitol, trehalose, and sorbitol.
  • the excipient solution consists essentially of mannitol, trehalose, and sorbitol.
  • the excipient solution comprises trehalose.
  • the excipient solution consists essentially of trehalose.
  • the excipient solution comprises mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis).
  • the excipient solution comprises more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises at least threefold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form comprises mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis).
  • the excipient of the solution or dried form comprises more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form comprises at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient of the solution or dried form comprises at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution consists essentially of mannitol and trehalose. In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the mannitol and the trehalose are not present in equal amounts (for example, the mannitol and the trehalose are present in unequal amounts; for example, on a weight basis or a weight percent basis). In some embodiments, the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution consists essentially of mannitol and trehalose, wherein the excipient solution contains at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains at least two-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis. In some embodiments, the excipient of the solution or dried form consists essentially of mannitol and trehalose, wherein the excipient of the solution or dried form contains at least three-fold more mannitol than trehalose, for example, on a weight basis or weight percent basis.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein neither the mannitol nor the trehalose is present in an amount of 5 mg/ml to 15 mg/ml. In some embodiments, the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the mannitol is not present in an amount of 5 mg/ml to 15 mg/ml. In some embodiments, the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the trehalose is not present in an amount of 5 mg/ml to 15 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein neither the mannitol nor the trehalose is present in an amount of 9 mg/ml. In some embodiments, the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the mannitol is not present in an amount of 9 mg/ml. In some embodiments, the excipient solution comprises, or consists essentially of, mannitol and trehalose, wherein the trehalose is not present in an amount of 9 mg/ml.
  • the excipient solution comprises, or consists essentially of, mannitol and trehalose, and does not comprise methionine.
  • a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P.
  • a stock comprising one or more excipients, wherein the stock comprises a formula provided in Table A, B, C, D, K, or P wherein the stock is for use in combination with extracellular vesicles (EVs) from inflammation-resolving extracellular vesicles (EVs) (for example, a liquid preparation thereof).
  • EVs extracellular vesicles
  • EVs extracellular vesicles
  • a liquid preparation comprises a cell culture supernatant, such as a bacterial cell culture supernatant, for example, as described herein.
  • the liquid preparation comprises a retentate, such as a concentrated retentate, for example, as described herein.
  • excipients are present in (for example, provided in) an excipient solution.
  • excipient solution include the stocks comprising one or more excipients provided in Tables A, B, C, D, K, or P.
  • the dried forms provided herein contain excipients from the excipient solution (such as a stock) once the moisture has been removed, such as by drying.
  • a liquid preparation that comprises inflammation- resolving extracellular vesicles (EVs) is combined with the stock of formula 7a (which comprises the excipients mannitol and trehalose) from Table A to prepare a solution.
  • the solution is dried to prepare a dried form.
  • the dried form comprises inflammationresolving extracellular vesicles (EVs), mannitol, and trehalose.
  • a “stock” refers to a solution comprising one or more excipients but no active ingredient (such as an extracellular vesicle ).
  • a stock is used to introduce one or more excipients into a preparation (such as a liquid preparation) comprising EVs.
  • the stock is a concentrated solution comprising a known amount of one or more excipients.
  • the stock is combined with a preparation (such as a liquid preparation) that comprises EVs to prepare a solution or dried form provided herein.
  • Fig. 1 is a graph showing the effects of orally-administered Prevotella EVs powder prepared in formula 7a in a delayed type hypersensitivity (DTH) model of inflammation. Inflammation is assessed as change in ear thickness (mm).
  • DTH delayed type hypersensitivity
  • Fig. 2 is a graph showing the effects of orally-administered Prevotella smEVs (EVs)s and intraperitoneally-administered Anti-TNFa antibody in a delayed type hypersensitivity (DTH) model of inflammation. Inflammation is assessed as change in ear thickness (mm).
  • DTH delayed type hypersensitivity
  • Fig. 3 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following dosing with vehicle (PBS), dexamethasone, o Prevotella smEVs (EVs) or Harryflintia smEVs either before (pre) or after (post) immunization with KLH-CFA.
  • PBS vehicle
  • dexamethasone o Prevotella smEVs
  • EVs Prevotella smEVs
  • Harryflintia smEVs either before (pre) or after (post) immunization with KLH-CFA.
  • Fig. 4 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following immunization with either IFA-PBS or CFA-PBS, dosing with vehicle (PBS), dexamethasone, or Prevotella smEVs (EVs), then immunization with KLH-CFA.
  • Fig. 5 is a graph showing changes in ear thickness 24 hours after KLH ear challenge in recipient mice following transfer of CD4+ T cells from KLH-CFA immunized donor mice dosed with vehicle ox Prevotella smEVs (EVs) ox Harryflintia smEVs.
  • EVs Prevotella smEVs
  • Fig. 6 is a graph showing changes in ear thickness 24 hours after KLH ear challenge in recipient mice following transfer of CD4+ T cells from isotype control- or anti-TLR2-treated KLH-CFA immunized mice dosed with vehicle or Prevotella smEVs (EVs) into KLH-CFA immunized recipient mice.
  • Fig. 7 is a graph showing changes in ear thickness 24 hours after KLH ear challenge in recipient mice following transfer of CD8+ T cells from KLH-CFA immunized donor mice dosed with vehicle ox Prevotella smEVs (EV) or Veillonella smEVs.
  • EV Prevotella smEVs
  • Veillonella smEVs Veillonella smEVs.
  • Figs. 8A and 8B are two panels.
  • Fig. 8A is a graph showing 24 hour DTH measurements after treatment with Prevotella smEVs (Prevotella EV) or dexamethasone. Inflammation is assessed as change in ear thickness (mm).
  • Fig. 8B is a graph showing spleen weight (mg) after treatment with Prevotella smEVs (Prevotella EV) or dexamethasone.
  • Figs. 9A-9D are four panels.
  • Fig. 9A is a graph showing 24 hour DTH measurements after treatment with Prevotella smEVs (Prevotella EV). Inflammation is assessed as change in ear thickness (mm).
  • Figs. 9A-9D are four panels.
  • FIGS 9B-9D are graphs showing levels of IFN-y (Fig. 9B), IFN-a (Fig. 9C), IFN-P (Fig. 9D) in spleen cells (pg/ml) after treatment with Prevotella smEVs (Prevotella EV).
  • Figs. 10A-10D are four panels.
  • Fig. 10A is a graph showing the proportion of TH1 T cells, which are defined by their expression of the transcription factor T-bet (% T-bet positive), of total CD4 T cells in mice treated with either vehicle ox Prevotella histicola smEVs (Prevotella EV).
  • Figs. 10B and IOC are graphs showing amounts (pg/ml) of IFN-y (Fig. 10B) and TNF (Fig. IOC) from CD4 T cells treated with either vehicle ox Prevotella histicola smEVs (Prevotella EV).
  • Fig. 10D is a graph showing levels (pg/ml) of TNF produced by PMA/ionomycin-treated spleen cells from vehicle- or Prevotella histicola smEV (Prevotella EV)-treated mice.
  • Figs. 11A-1 ID are four panels.
  • Fig. 11A is a graph showing IFNy response (pg/ml) by DCs to CEF after treatment with three doses of Prevotella histicola smEVs (Prevotella EV) compared to the DC + CD8 T cell + CEF peptide co-culture control.
  • Figs. 11B- 1 ID are graphs showing IL-12p70 (Fig. 1 IB), TNFoc (Fig. 11C), and IL-6 (Fig.
  • cytokine levels (pg/ml) produced primarily by DCs after treatment with three doses of Prevotella histicola smEVs (Prevotella EV), compared to the DC + CD 8 T cell + CEF peptide control.
  • Figs. 12A-12G are seven panels showing that orally-dosed Prevotella histicola smEVs (Prevotella EV) induce robust resolution of inflammation superior to standard of care anti-inflammatory drugs.
  • mice were either dosed for 4 days (dO-4) pre-KLH-CFA immunization (d5) or for 4 days (d9- 12) post-KLH-CFA immunization with vehicle, dexamethasone, or Prevotella histicola smEVs (Prevotella EV). Ears were then measured and challenged with a KLH intradermal injection (dl2) and measured again 24 hours later (dl3). Graphs show change in ear thickness relative to baseline 24 hours after challenge.
  • mice were immunized with CFA-KLH (dO) then dosed daily for 4 days (d5-8) with either vehicle, Prevotella histicola smEVs (Prevotella EV) (all PO), dexamethasone, tofacitinib, or dO, 3, and 6 with anti-TNFa (all IP). Ears were then measured and challenged with a KLH intradermal injection (d8) and measured again 24 hours later (d9). Graphs show change in ear thickness relative to baseline 24 hours after challenge.
  • D-F Mice were immunized with CFA-KLH (dO) then dosed daily for 4 days (d5 -8) with either vehicle, dexamethasone, tofacitinib, or Prevotella histicola smEVs (Prevotella EV) or days 0, 3, and 6 with anti-TNF.
  • mice were re- dosed daily with indicated treatments (dl 15-118), then right ears were challenged with a KLH intradermal injection (dl 18) and measured 24 hours later. Graphs show change in ear thickness relative to vehicle 24 hours after each challenge. Statistical analysis compares challenge to the first challenge for each treatment.
  • mice were then immunized with KLH-CFA (d9) and ears were then measured and challenged with a KLH intradermal injection (dl 3) and measured again 24 hours later (dl 4).
  • Graphs show change in ear thickness relative to baseline 24 hours after challenge. All data are representative of at least 2 independent experiments. Bars indicated median.
  • Figs. 13A-13E are five panels showing Prevotella histicola smEVs (Prevotella EV) require homing of peripheral lymphocytes to the mesenteric lymph node for efficacy.
  • A-D Mice were treated with isotype control (5 pg) or anti-CD62L/a.4p7 (2.5 pg each) on days 0, and 2.
  • mesenteric lymph nodes were isolated, homogenized, and immune cell populations were analyzed by flow cytometry.
  • Graphs show absolute cell numbers of A) total cells within the mLN and B-D) indicated immune cell populations populations.
  • mice were immunized with KLH-CFA (dO) and treated with isotype control (5 pg) or anti-CD62L/a.4p7 (2.5 pg each) (dl, 3, 5, 7). Mice were then dosed for 4 days with vehicle, dexamethasone, or Prevotella histicola smEVs (Prevotella EV) (d5-8). Ears were then measured and challenged with a KLH intradermal injection (dl 3) and measured again 24 hours later (dl4). Graphs show change in ear thickness relative to baseline 24 hours after challenge. All data are representative of at least 2 independent experiments. Bars indicated A-D) mean ⁇ SD or E) median. Statistical analysis was performed using A-D) unpaired two-tailed t-test or D) one-way ANOVA with Dunnett’s multiple comparisons. ****p ⁇ 0.0001.
  • Figs. 14A-14E are five panels showing Prevotella histicola smEVs (Prevotella EV) induce a population of pro-resolving CD4+ T cells that can adoptively transfer efficacy.
  • ACT DTH adoptive cell transfer delayed type hypersensitivity
  • Donor mice were immunized with KLH-CFA (dO) and dosed for 4 days (d5-8) with vehicle or Prevotella histicola smEVs (Prevotella EV) and recipient mice were later immunized with KLH-CFA (d5).
  • spleens and lymph nodes were harvested, and CD4+ T cells were isolated from homogenized tissues. Isolated cells were then transferred by intraperitoneal injection into recipient mice (d9). After 4 days, the ears of recipient mice were then measured and challenged with a KLH intradermal injection (dl 2) and measured again 24 hours later (dl 3).
  • CD4+ T cells were isolated from lymphoid tissue of ACT DTH mice and transferred into KLH- immunized recipient mice. Four days after transfer, mice in both DTH and ACT DTH groups were challenged with KLH and ear swelling was measured 24 hours later.
  • Figs. 15A-15H are eight panels showing Prevotella histicola smEVs (Prevotella EV) require TLR2 signaling to induce pro-resolving CD4+ T cells that do not produce but rather induce the release of IL- 10 to resolve inflammation.
  • mice were then dosed for 4 days with vehicle, dexamethasone, or Prevotella histicola smEVs (Prevotella EV) (d5-8). Ears were then measured and challenged with a KLH intradermal injection (d8) and measured again 24 hours later (d9). Graphs show change in ear thickness relative to baseline 24 hours after challenge.
  • C-D) Donor mice were immunized with KLH-CFA (dO) and dosed for 4 days (d5-8) with vehicle ox Prevotella histicola smEVs (Prevotella EV) and recipient mice were later immunized with KLH-CFA (d5).
  • mice in E) donor groups or F) recipient groups were treated with isotype control or anti-TLR2 antibodies (Donor: dO, 3, 6; Recipient: d5, 8, 11). After dosing (d9), spleens and lymph nodes were harvested, and cells were isolated from homogenized tissues. Isolated cells were then transferred by intraperitoneal injection into recipient mice (d9). After 4 days, the ears of recipient mice were then measured and challenged with a KLH intradermal injection (dl2) and measured again 24 hours later (dl3).
  • Graphs show change in ear thickness relative to baseline 24 hours after challenge in mice receiving 1E7 CD4+ T cells from donor mice dosed with either vehicle or Prevotella histicola smEVs (Prevotella EV).
  • Mice were immunized with KLH-CFA (dO) and treated with isotype control or anti-ILlOR (dO, 3, 6). Mice were then dosed for 4 days with vehicle, dexamethasone, ox Prevotella histicola smEVs (Prevotella EV) (d5-8). Ears were then measured and challenged with a KLH intradermal injection (dl 3) and measured again 24 hours later (dl4).
  • Graphs show change in ear thickness relative to baseline 24 hours after challenge.
  • F IL10 WT or IL10 KO mice were immunized with CFA-KLH (dO) then dosed for 4 days (d5 -8) with either vehicle, dexamethasone, or Prevotella histicola smEVs (Prevotella EV). Ears were then measured and challenged with a KLH intradermal injection (d8) and measured again 24 hours later (d9). Graphs show change in ear thickness relative to baseline 24 hours after challenge.
  • IL 10 KO donor mice were immunized with KLH-CFA (dO) and dosed for 4 days (d5-8) with Prevotella histicola smEVs (Prevotella EV) and recipient mice were later immunized with KLH-CFA (d5). After dosing (d9), spleens and lymph nodes were harvested, and cells were isolated from homogenized tissues. Isolated cells were then transferred by intraperitoneal injection into IL10 WT recipient mice (d9). After 4 days, the ears of recipient mice were then measured and challenged with a KLH intradermal injection (dl 2) and measured again 24 hours later (dl 3).
  • Graphs show change in ear thickness relative to baseline 24 hours after challenge in mice receiving 1E7 CD4+ T cells from donor mice dosed with either vehicle or Prevotella histicola smEVs (Prevotella EV) as indicated.
  • H IL10 WT donor mice were immunized with KLH-CFA (dO) and dosed for 4 days (d5-8) with vehicle ox Prevotella histicola smEVs (Prevotella EV) and recipient mice were later immunized with KLH-CFA (d5). After dosing (d9), spleens and lymph nodes were harvested, and cells were isolated from homogenized tissues.
  • Isolated cells were then transferred by intraperitoneal injection into IL10 KO recipient mice (d9). After 4 days, the ears of recipient mice were then measured and challenged with a KLH intradermal injection (dl 2) and measured again 24 hours later (dl 3). Graphs show change in ear thickness relative to baseline 24 hours after challenge in mice receiving 1E7 CD4+ T cells from donor mice dosed with either vehicle ox Prevotella histicola smEVs (Prevotella EV) as indicated. All data are representative of at least 2 independent experiments. Bars indicated mean ⁇ SD median.
  • Fig. 16 is a graph showing Prevotella histicola smEVs (Prevotella EV) induce a proresolving function in conventional CD4+ T cells and enhance the function of regulatory CD4+ T cells.
  • Donor mice were immunized with KLH-CFA (dO) and dosed for 4 days (d5-8) with Prevotella histicola smEVs (Prevotella EV) and recipient mice were later immunized with KLH- CFA (d5). After dosing (d9), spleens and lymph nodes were harvested, and cells were isolated from homogenized tissues (CD4 negative selection followed by CD25 positive selection).
  • Indicated numbers of isolated cells were then transferred by intraperitoneal injection into recipient mice (d9). After 4 days, the ears of recipient mice were then measured and challenged with a KLH intradermal injection (dl 2) and measured again 24 hours later (dl 3).
  • the disclosure provides inflammation-resolving extracellular vesicles (EVs), and solutions, dried forms and therapeutic compositions that contain inflammation-resolving extracellular vesicles (EVs), and methods for using the same.
  • the disclosure provides solutions and dried forms that contain inflammation-resolving extracellular vesicles (EVs), and methods for using the same.
  • the disclosure also provides therapeutic compositions that contain the solutions and/or dried forms.
  • EVs are secreted (for example, produced) by bacterial cells in culture. Such secreted extracellular vesicles may be referred to as secreted microbial extracellular vesicles (smEVs).
  • smEVs secreted microbial extracellular vesicles
  • EVs are prepared (for example, artificially prepared) by processing bacterial cells, for example, by methods that disrupt the bacterial membrane, such as sonication. Such artificially prepared may be referred to as processed microbial extracellular vesicles (pmEVs).
  • the resulting dried form is a lyophilate.
  • the dried form is a lyophilate.
  • a lyophilate is a lyophilized powder or a lyophilized cake.
  • the lyophilized cake is milled to produce a lyophilized powder.
  • the solutions and dried forms that contain inflammationresolving extracellular vesicles also comprise one or more excipients, such as a bulking agent, and/or a lyoprotectant.
  • bulking agents and lyoprotectants are used when preparing extracellular vesicles (EVs) for freeze drying.
  • bulking agents including but not limited to sucrose, mannitol, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, and dextran (such as dextran 40k), are added (for example, as a stock containing the same) to a liquid preparation of EVs (for example, obtained by isolating EVs from a bacterial culture) to prepare a dried form such as a lyophilate, making it easier to handle (and optionally, further formulate, for example, into a therapeutic composition) after drying.
  • PEG polyethylene glycol
  • dextran such as dextran 40k
  • lyoprotectants including but not limited to trehalose, sucrose, and lactose, are added (for example, as a stock containing the same) to a liquid preparation of EVs (for example, obtained by isolating EVs from a bacterial culture) to protect the EVs while lyophilizing or spray drying.
  • a bulking agent and/or lyoprotectant is included from an excipient stock that is added to EVs (for example, purified and/or concentrated EVs) to produce a solution, and/or to produce a dried form upon subsequent drying, for example, of the solution.
  • a dried form such as a lyophilate contains between about 5% and about 100% EV solids by weight. In some embodiments, prior to drying (such as by lyophilization), the total solids, including EVs and excipients, are between about 2% and about 20% by weight.
  • the resulting lyophilate (for example, lyophilized cake) has a uniform appearance, and is a white to off-white.
  • the resulting lyophilate (for example, lyophilized cake) obtained after freeze-drying is a white to off-white, fine and smooth granular powder (for example, after milling (for example, grinding) the lyophilized cake).
  • Particles in a lyophilate (after a solution containing EVs is lyophilized) contain Prevotella histicola EVs, and may also include other components from the culture media, such as cell debris, LPS, and/or proteins.
  • the lyophilate obtained after freeze-drying with the excipients and/or conditions provided herein is a white to off-white, fine and smooth granular lyophilate powder.
  • the lyophilates containing inflammation-resolving extracellular vesicles (EVs) described herein are prepared to have a moisture content (for example, as determined by the Karl Fischer method) of below about 10% (for example, below about 9%, below about 8%, below about 7%, below about 6%, below about 5% or below about 4%, for example, about 1% to about 4%, about 1.5% to about 4%, about 2% to about 4%, about 2% to about 3%) upon completion of freeze drying.
  • a moisture content for example, as determined by the Karl Fischer method
  • the lyophilate are better suited for downstream processing, for example, for use in a therapeutic composition. In some embodiments, by preparing lyophilates to have a moisture content below about 6%, the lyophilate has improved stability, e.g., upon storage.
  • adjuvant or “Adjuvant therapy” broadly refers to an agent that affects an immunological or physiological response in a patient or subject (e.g., human).
  • an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumor, help absorb an antigen presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines.
  • an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent.
  • an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.
  • administering broadly refers to a route of administration of a composition (e.g., a therapeutic composition) to a subject.
  • routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection.
  • Administration by injection includes intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration.
  • a therapeutic composition described herein can be administered in any form by any effective route, including but not limited to oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), implanted, intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial.
  • transdermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.
  • a therapeutic composition described herein is administered orally, rectally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.
  • a therapeutic composition described herein is administered orally or intravenously.
  • a therapeutic composition described herein is administered orally.
  • the term “antibody” may refer to both an intact antibody and an antigen binding fragment thereof.
  • Intact antibodies are glycoproteins that include at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the term “antibody” includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies and antigen-binding antibody fragments.
  • antigen binding fragment and “antigen-binding portion” of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to bind to an antigen.
  • binding fragments encompassed within the term "antigen-binding fragment” of an antibody include Fab, Fab', F(ab')2, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody.
  • These antibody fragments can be obtained using conventional recombinant and/or enzymatic techniques and can be screened for antigen binding in the same manner as intact antibodies.
  • a “carbohydrate” refers to a sugar or polymer of sugars.
  • saccharide polysaccharide
  • carbohydrate oligosaccharide
  • Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule.
  • Carbohydrates generally have the molecular formula CnFbnOn.
  • a carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide.
  • the most basic carbohydrate is a monosaccharide, such as glucose, galactose, mannose, ribose, arabinose, xylose, and fructose.
  • Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose.
  • Carbohydrates may contain modified saccharide units such as 2’ -deoxyribose wherein a hydroxyl group is removed, 2 ’-fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2’ -fluororibose, deoxyribose, and hexose).
  • Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.
  • Cellular augmentation broadly refers to the influx of cells or expansion of cells in an environment that are not substantially present in the environment prior to administration of a composition and not present in the composition itself.
  • Cells that augment the environment include immune cells, stromal cells, bacterial and fungal cells.
  • ‘Clade” refers to the OTUs or members of a phylogenetic tree that are downstream of a statistically valid node in a phylogenetic tree.
  • the clade comprises a set of terminal leaves in the phylogenetic tree that is a distinct monophyletic evolutionary unit and that share some extent of sequence similarity.
  • a “combination” can refer to EVs from one source strain with another agent, e.g., another EV (e.g., from another strain), with bacteria (e.g., of the same or different strain that the EV was obtained from), or with another therapeutic agent.
  • the combination can be in physical co-existence, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the EVs and other agent.
  • the term “consists essentially of’ means limited to the recited elements and/or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.
  • a “dried form” that contains extracellular vesicles (EVs) refers to the product resulting from drying a solution that contains EVs.
  • the drying is performed, for example, by freeze drying (lyophilization) or spray drying.
  • the dried form is a powder.
  • a powder refers to a type of dried form and includes a lyophilized powder and a spray-dried powder, obtained by a method such as spray drying. See also WO 2022/132738.
  • Dysbiosis refers to a state of the microbiota or microbiome of the gut or other body area, including, e.g., mucosal or skin surfaces (or any other microbiome niche) in which the normal diversity and/or function of the host gut microbiome ecological networks ( ’’microbiome”) are disrupted.
  • a state of dysbiosis may result in a diseased state, or it may be unhealthy under only certain conditions or only if present for a prolonged period.
  • Dysbiosis may be due to a variety of factors, including, environmental factors, infectious agents , host genotype, host diet and/or stress.
  • a dysbiosis may result in: a change (e.g., increase or decrease) in the prevalence of one or more bacteria types (e.g., anaerobic), species and/or strains, change (e.g., increase or decrease) in diversity of the host microbiome population composition; a change (e.g., increase or reduction) of one or more populations of symbiont organisms resulting in a reduction or loss of one or more beneficial effects; overgrowth of one or more populations of pathogens (e.g., pathogenic bacteria); and/or the presence of, and/or overgrowth of, symbiotic organisms that cause disease only when certain conditions are present.
  • the term “decrease” or “deplete” means a qualitative or quantitative difference between a reference and a value that is less than the reference, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the reference, or such that the value is 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 of the reference, or such that the value is undetectable after a treatment when compared to a reference representative of a pre-treatment state.
  • Properties that may be decreased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size (e.g., in an animal tumor model)).
  • the term “effective dose” is the amount of the therapeutic composition that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, with the least toxicity to the subject.
  • engineered bacteria are any bacteria that have been genetically altered from their natural state by human activities, and the progeny of any such bacteria.
  • Engineered bacteria include, for example, the products of targeted genetic modification, the products of random mutagenesis screens and the products of directed evolution.
  • epitope means a protein determinant capable of specific binding to an antibody or T cell receptor.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which an antibody is capable of binding.
  • EVs Extracellular vesicles
  • smEVs vesicles derived from bacteria
  • EVs are comprised of bacterial lipids and/or bacterial proteins and/or bacterial nucleic acids and/or bacterial carbohydrate moieties, and are isolated from culture supernatant.
  • the natural production of these vesicles can be artificially enhanced (for example, increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (for example, by media or temperature alterations).
  • EV compositions may be modified to reduce, increase, add, or remove bacterial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (for example, lymph node), absorption (for example, gastrointestinal), and/or yield (for example, thereby altering the efficacy).
  • purified EV composition or “EV composition” refers to a preparation of EVs that have been separated from at least one associated substance found in a source material (for example, separated from at least one other bacterial component) or any material associated with the EVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components.
  • Extracellular vesicles may also be obtained from mammalian cells and from can be obtained from microbes such as archaea, fungi, microscopic algae, protozoans, and parasites. Extracellular vesicles from any of these sources can be prepared into a solution and/or dried form as described herein.
  • Extracellular vesicles may be artificially-produced vesicles prepared from bacteria, such as pmEVs, for example, obtained by chemically disrupting (for example, by lysozyme and/or lysostaphin) and/or physically disrupting (for example, by mechanical force) bacterial cells and separating the bacterial membrane components from the intracellular components through centrifugation and/or ultracentrifugation, or other methods, can also be prepared into a solution and/or dried form as described herein.
  • bacteria such as pmEVs
  • lysozyme and/or lysostaphin obtained by chemically disrupting (for example, by lysozyme and/or lysostaphin) and/or physically disrupting (for example, by mechanical force) bacterial cells and separating the bacterial membrane components from the intracellular components through centrifugation and/or ultracentrifugation, or other methods, can also be prepared into a solution and/or dried form as described herein.
  • 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.
  • “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 etal. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., etal., 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 etal.
  • the term “immune disorder” refers to any disease, disorder or disease symptom caused by an activity of the immune system, including autoimmune diseases, inflammatory diseases and allergies.
  • Immune disorders include, but are not limited to, autoimmune diseases (e.g., psoriasis, atopic dermatitis, lupus, scleroderma, hemolytic anemia, vasculitis, type one diabetes, Grave’s disease, rheumatoid arthritis, multiple sclerosis, Goodpasture’s syndrome, pernicious anemia and/or myopathy), inflammatory diseases (e.g., acne vulgaris, asthma, celiac disease, chronic prostatitis, glomerulonephritis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis and/or interstitial cystitis), and/or an allergies (e.g., food allergies, drug allergies and/or
  • autoimmune diseases
  • Immunotherapy is treatment that uses a subject’s immune system to treat disease (e.g., immune disease, inflammatory disease, metabolic disease, cancer) and includes, for example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.
  • disease e.g., immune disease, inflammatory disease, metabolic disease, cancer
  • checkpoint inhibitors e.g., cancer vaccines, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.
  • the term “increase” means a qualitative or quantitative difference between a reference and a value that is more than the reference, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 10- fold, 100-fold, 10 A 3 fold, 10 A 4 fold, 10 A 5 fold, 10 A 6 fold, and/or 10 A 7 fold of the reference, e.g., where the difference is between a reference representative of a pre-treatment state and a value that is representative of a post-treatment state.
  • Properties that may be increased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size (e.g., in an animal tumor model).
  • ‘Innate immune agonists” or “immuno-adjuvants” are small molecules, proteins, or other agents that specifically target innate immune receptors including Toll-Like Receptors (TLR), NOD receptors, RLRs, C-type lectin receptors, SHNG-cGAS Pathway components, inflammasome complexes.
  • TLR Toll-Like Receptors
  • NOD receptors NOD receptors
  • RLRs C-type lectin receptors
  • SHNG-cGAS Pathway components inflammasome complexes.
  • LPS is a TLR-4 agonist that is bacterially derived or synthesized and aluminum can be used as an immune stimulating adjuvant
  • immuno-adjuvants are a specific class of broader adjuvant or adjuvant therapy.
  • SUNG agonists include, but are not limited to, 2'3'- cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GMP, 2'2'-cGAMP, and 2'3'-cGAM(PS)2 (Rp/Sp) (Rp, Sp-isomers of the bis-phosphorothioate analog of 2'3'- cGAMP).
  • TLR agonists include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 and TLR11.
  • NOD agonists include, but are not limited to, N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyldipeptide (MDP)), gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), and desmuramylpeptides (DMP).
  • MDP N-acetylmuramyl-L-alanyl-D-isoglutamine
  • iE-DAP gamma-D-glutamyl-meso-diaminopimelic acid
  • DMP desmuramylpeptides
  • ITS is a piece of non-functional RNA located between structural ribosomal RNAs (rRNA) on a common precursor transcript often used for identification of eukaryotic species in particular fungi.
  • rRNA structural ribosomal RNAs
  • the rRNA of fungi that forms the core of the ribosome is transcribed as a signal gene and consists of the 8S, 5.8S and 28S regions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28S regions, respectively.
  • isolated or “enriched” encompasses a microbe, an EV (such as a bacterial EV) or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man.
  • Isolated bacteria or EVs may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
  • isolated bacteria or EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure, e.g., substantially free of other components.
  • a “lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans).
  • Metal refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or bacterial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or bacterial metabolic reaction.
  • 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 biofilms, 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 or dysbiotic 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 or treatment conditions (e.g., antibiotic treatment, exposure to different microbes).
  • the microbiome occurs at a mucosal surface.
  • the microbiome is a gut microbiome.
  • a “microbiome profile” or a “microbiome signature” of a tissue or sample refers to an at least partial characterization of the bacterial makeup of a microbiome.
  • a microbiome profile indicates whether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bacterial strains are present or absent in a microbiome.
  • ‘Modified” in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form.
  • Bacterial modification can result from engineering bacteria. Examples of bacterial modifications include genetic modification, gene expression modification, phenotype modification, formulation modification, chemical modification, and dose or concentration. Examples of improved properties are described throughout this specification and include, e.g., attenuation, auxotrophy, homing, or antigenicity.
  • Phenotype modification might include, by way of example, bacteria growth in media that modify the phenotype of a bacterium such that it increases or decreases virulence.
  • “Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, e.g., the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species.
  • the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence.
  • the entire genomes of two entities are sequenced and compared.
  • select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared.
  • OTUs that share > 97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU. See e.g., Claesson MJ, Wang Q, O’Sullivan O, Greene-Diniz R, Cole JR, Ross RP, and O’Toole PW. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.
  • MLSTs For complete genomes, MLSTs, specific genes, other than 16S, or sets of genes OTUs that share > 95% average nucleotide identity are considered the same OTU. See e.g., Achtman M, and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequently defined by comparing sequences between organisms.
  • OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof.
  • Operational Taxonomic Units (OTUs) with taxonomic assignments made to, e.g., genus, species, and phylogenetic clade are provided herein.
  • a gene is “overexpressed” in a bacteria if it is expressed at a higher level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions.
  • a gene is “underexpressed” in a bacteria if it is expressed at a lower level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions.
  • polynucleotide and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Polynucleotides may have any three-dimensional structure, and may perform any function.
  • loci locus
  • locus defined from linkage analysis, exons, introns
  • messenger RNA messenger RNA
  • miRNA micro RNA
  • siRNA silencing RNA
  • transfer RNA transfer RNA
  • ribosomal RNA ribozymes
  • cDNA recomb
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.
  • a substance is “pure” if it is substantially free of other components.
  • the terms “purify,” “purifying” and “purified” refer to an EV (such as an EV from bacteria) preparation or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production.
  • An EV preparation or compositions may be considered purified if it is isolated at or after production, such as from one or more other bacterial components, and a purified microbe or bacterial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “purified.”
  • purified EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • EV compositions (or preparations) are, e.g., purified from residual habitat products.
  • the term “purified EV composition” or “EV composition” refers to a preparation that includes EVs from bacteria that have been separated from at least one associated substance found in a source material (e.g., separated from at least one other bacterial component) or any material associated with the EVs in any process used to produce the preparation. It also refers to a composition that has been significantly enriched or concentrated. In some embodiments, the EVs are concentrated by 2 fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 1000- fold, 10,000-fold or more than 10,000-fold.
  • ‘Residual habitat products” refers to material derived from the habitat for microbiota within or on a subject.
  • fermentation cultures of microbes can contain contaminants, e.g., other microbe strains or forms (e.g., bacteria, virus, mycoplasm, and/or fungus).
  • microbes live in feces in the gastrointestinal tract, on the skin itself, in saliva, mucus of the respiratory tract, or secretions of the genitourinary tract (i.e., biological matter associated with the microbial community).
  • Substantially free of residual habitat products means that the microbial composition no longer contains the biological matter associated with the microbial environment on or in the culture or human or animal subject and is 100% free, 99% free, 98% free, 97% free, 96% free, or 95% free of any contaminating biological matter associated with the microbial community.
  • Residual habitat products can include abiotic materials (including undigested food) or it can include unwanted microorganisms.
  • Substantially free of residual habitat products may also mean that the microbial composition contains no detectable cells from a culture contaminant or a human or animal and that only microbial cells are detectable.
  • substantially free of residual habitat products may also mean that the microbial composition contains no detectable viral (including bacteria, viruses (e.g., phage)), fungal, mycoplasmal contaminants.
  • it means that fewer than lxl0' 2 %, lxl0' 3 %, lxl0' 4 %, lxl0' 5 %, lxl0' 6 %, lxl0' 7 %, lxl0' 8 % of the viable cells in the microbial composition are human or animal, as compared to microbial cells. There are multiple ways to accomplish this degree of purity, none of which are limiting.
  • contamination may be reduced by isolating desired constituents through multiple steps of streaking to single colonies on solid media until replicate (such as, but not limited to, two) streaks from serial single colonies have shown only a single colony morphology.
  • reduction of contamination can be accomplished by multiple rounds of serial dilutions to single desired cells (e.g., a dilution of 10' 8 or 1 O' 9 ), such as through multiple 10-fold serial dilutions. This can further be confirmed by showing that multiple isolated colonies have similar cell shapes and Gram staining behavior.
  • telomere binding refers to the ability of an antibody to bind to a predetermined antigen or the ability of a polypeptide to bind to its predetermined binding partner.
  • an antibody or polypeptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 1 O' 7 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by KD) that is at least 10- fold less, at least 100-fold less or at least 1000-fold less than its affinity for binding to a nonspecific and unrelated antigen/binding partner (e.g., BSA, casein).
  • specific binding applies more broadly to a two component system where one component is a protein, lipid, or carbohydrate or combination thereof and engages with the second component which is a protein, lipid, carbohydrate or combination thereof in a specific way.
  • 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 nonnative plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof.
  • strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome.
  • strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.
  • subject refers to any mammal.
  • a subject or a patient described as “in need thereof’ refers to one in need of a treatment (or prevention) for a disease.
  • Mammals i.e., mammalian animals
  • mammals include humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents).
  • the subject may be a human.
  • the subject may be a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
  • the subject may be healthy, or may be suffering from a disease or disorder at any developmental stage. In some embodiments, the subject is a human.
  • a therapeutic agent refers to an agent for therapeutic use.
  • a therapeutic agent is a composition comprising EVs (“an EV composition”) that can be used to treat and/or prevent a disease and/or condition.
  • the therapeutic agent is a pharmaceutical agent.
  • a medicinal product, medical food, a food product, or a dietary supplement comprises a therapeutic agent.
  • the therapeutic agent is in a solution, and in other embodiments, a dried form. The dried form embodiments may be produced, for example, by lyophilization or spray drying.
  • the dried form of the therapeutic agent is a lyophilized cake or powder.
  • the dried form of the therapeutic agent is a spray-dried powder.
  • the term “therapeutic composition” or “pharmaceutical composition” refers to a composition that comprises a therapeutically effective amount of a therapeutic agent (for example an EV composition described herein).
  • the therapeutic composition is (or is present in) a medicinal product, medical food, a food product, or a dietary supplement.
  • the term “treating” a disease in a subject or “treating” a subject having or suspected of having a disease refers to administering to the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening.
  • “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.
  • the term “preventing” a disease in a subject refers to administering to the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that onset of at least one symptom of the disease is delayed or prevented.
  • EVs solutions and/or dried forms, and therapeutic compositions, that comprise inflammation-resolving extracellular vesicles
  • solutions and/or dried forms, and therapeutic compositions, that comprise inflammationresolving extracellular vesicles (EVs) are provided herein.
  • bacteria from which inflammation-resolving extracellular vesicles (EVs) are obtained are lyophilized.
  • bacteria from which inflammation-resolving extracellular vesicles (EVs) are obtained are gamma irradiated (e.g., at 17.5 or 25 kGy).
  • bacteria from which inflammation-resolving extracellular vesicles (EVs) are obtained are UV irradiated.
  • bacteria from which inflammation-resolving extracellular vesicles (EVs) are obtained are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
  • bacteria from which inflammation-resolving extracellular vesicles (EVs) are obtained are acid treated.
  • bacteria from which inflammation-resolving extracellular vesicles (EVs) are obtained are oxygen sparged (e.g., at 0.1 wm for two hours).
  • the inflammation-resolving EVs are lyophilized.
  • the inflammation-resolving EVs are spray dried.
  • the inflammation-resolving EVs are gamma irradiated (e.g., at
  • the inflammation-resolving EVs are UV irradiated.
  • the inflammation-resolving EVs are heat inactivated (e.g., at
  • the inflammation-resolving EVs are acid treated.
  • the inflammation-resolving EVs are oxygen sparged (e.g., at 0.1 wm for two hours).
  • the phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria.
  • EVs 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 inflammation-resolving EVs are from one strain of bacteria, e.g., a strain provided herein.
  • the inflammation-resolving EVs are from one strain of bacteria (e.g., a strain provided herein) or from more than one strain.
  • the inflammation-resolving extracellular vesicles are from a Prevotella histicola strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella histicola Strain B (NRRL accession number B 50329).
  • the Prevotella histicola strain is the Prevotella histicola Strain B (NRRL accession number B 50329).
  • the inflammation-resolving extracellular vesicles are from a Veillonella parvula strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of Veillonella parvula Strain A (ATCC Deposit Number PTA-125691).
  • the Veillonella parvula strain is Veillonella parvula Strain A (ATCC Deposit Number PTA-125691). See WO 2019/157003.
  • the inflammation-resolving extracellular vesicles are from a Harryflintia acetispora strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of Harryflintia acetispora Strain A (ATCC Deposit Number PTA- 126694).
  • the Harryflintia acetispora strain is Harryflintia acetispora Strain A (ATCC Deposit Number PTA- 126694). See
  • the bacteria from which the inflammation-resolving EVs are obtained are modified (e.g., engineered) to reduce toxicity or other adverse effects, to enhance delivery) (e.g., oral delivery) of the EVs (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 EVs (e.g., either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the EVs (e.g., through modified production of polysaccharides, pili, fimbriae, adhesins).
  • target desired cell types e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages
  • the engineered bacteria described herein are modified to improve EV 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 inflammation-resolving extracellular vesicles (EVs) described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.
  • the inflammation-resolving extracellular vesicles (EVs) described herein are engineered such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (e.g., a magnetic bead).
  • 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 an EV-binding moiety that that binds to the EV.
  • the EV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
  • the EV-binding moiety has binding specificity for the EV (e.g., by having binding specificity for a bacterial antigen).
  • the EV-binding moiety comprises an antibody or antigen binding fragment thereof.
  • the EV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
  • the EVs (such as secreted EVs (smEVs) from bacteria described herein) are 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 (for example, 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.
  • 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.
  • the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (for example, at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C).
  • the culture supernatants are then passed through filters to exclude intact bacterial cells (for example, 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 (for example, 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 (for example, in PBS), and applying the resuspended smEVs to an OptiPrepTM (iodixanol) gradient or gradient (for example, a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (for example, at 200,000 x g for 4-20 hours at 4°C).
  • OptiPrepTM iodixanol
  • gradient or gradient for example, a 30-60% discontinuous gradient, a 0-45% discontinuous gradient
  • smEV bands can be collected, diluted with PBS, and centrifuged to pellet the smEVs (for example, 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.
  • 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 bacterial 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 (for example, XCell ATF from Repligen).
  • ATF alternating tangential flow
  • the ATF system retains intact cells (> 0.22 pm) in the bioreactor, and allows smaller components (for example, smEVs, free proteins) to pass through a filter for collection.
  • the system may be configured so that the ⁇ 0.22 pm filtrate is then passed through a second filter of 100 kDa, allowing species such as smEVs between 0.22 pm 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, for example, 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 pm filter to exclude intact cells. To further increase purity, isolated smEVs may be DNase or proteinase K treated.
  • smEVs used for in vivo injections purified EVs 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 pg/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).
  • EVs in PBS are sterile- filtered to ⁇ 0.22 pm.
  • samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (for example, Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
  • filtration for example, Amicon Ultra columns
  • dialysis for example, 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.
  • smEVs are analyzed, for example, as described in Jeppesen et al. Cell 177:428 (2019).
  • smEVs are lyophilized.
  • smEVs are spray dried.
  • smEVs are gamma irradiated (for example, at 17.5 or 25 kGy).
  • smEVs are UV irradiated.
  • smEVs are heat inactivated (for example, at 50°C for two hours or at 90° C for two hours).
  • smEVs are acid treated.
  • smEVs are oxygen sparged (for example, at 0.1 wm 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, for example, 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 (for example, culture conditions) can affect the amount of EVs produced by bacteria.
  • the yield of smEVs can be increased by an smEV inducer, as provided in Table 1.
  • the methods 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.
  • the EVs (such as processed EVs (pmEVs) described herein) are prepared (for example, artificially 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 (for example, 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.
  • pmEVs are prepared from bacterial cultures using methods described in Thein etal. (J. 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 (for example, 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 1 mg/mL DNase I.
  • 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 hour 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.
  • 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 MgCh 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
  • 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 (for example, 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.
  • 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 .
  • the solution in step (c) is 100 mM Tris-HCl, pH 7.5 supplemented with 1 mg/ml DNasel. In some embodiments, the solution in step (c) is 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, supplemented with 0.1 mg/ml lysozyme.
  • 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 1.6 mg/ml. In some embodiments, step (c) further comprises adding MgCh to a final concentration of 100 mM.
  • 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.
  • the cells are sonicated in 7 cycles, wherein each cycle comprises 30 seconds of sonication and 30 seconds without sonication.
  • 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 (f) is for 15 minutes. In some embodiments, the centrifugation of step (f) is at 4°C or room temperature.
  • the second solution in step (g) is 100 mM sodium carbonate, pH 11. In some embodiments, the second solution in step (g) is 10 mM Tris-HCl pH 8.0, 2% triton X-100.
  • 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 100 mM Tris-HCl, pH 7.5.
  • the third solution in step (i) is PBS.
  • 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 100 mM 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 pm 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 are analyzed, for example, as described in Jeppesen et al. Cell 177:428 (2019).
  • pmEVs are lyophilized.
  • pmEVs are spray dried.
  • pmEVs are gamma irradiated (for example, at 17.5 or 25 kGy).
  • pmEVs are UV irradiated.
  • pmEVs are heat inactivated (for example, at 50°C for two hours or at 90° C for two hours).
  • pmEVs are acid treated.
  • pmEVs are oxygen sparged (for example, at 0.1 wm for two hours).
  • pmEVs can be isolated , for example, 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. Solutions and Dried Forms
  • a solution includes inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • a solution includes inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • a solution includes inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant. See also WO 2022/132738.
  • the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, Ficoll, and/or PVP-K30.
  • the excipient optionally includes an additional component such as trehalose, mannitol, sucrose, sorbitol, maltodextrin, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl- B-cyclodextrin.
  • a solution contains a liquid preparation of EVs and an excipient that comprises a bulking agent, for example, an excipient from a stock of a formula provided in one of Tables A, B, C, D, K, or P.
  • a solution includes a liquid preparation containing inflammation-resolving extracellular vesicles (EVs) (for example, obtained by isolating the EVs from a bacterial culture (such as the supernatant) or a retentate) and an excipient that comprises a bulking agent, for example, a liquid preparation containing inflammation-resolving extracellular vesicles (EVs) is combined with an excipient stock that comprises a bulking agent, for example, an excipient stock of a formula provided in one of Tables A, B, C, D, K, or P, to prepare the solution.
  • EVs inflammation-resolving extracellular vesicles
  • a “dried form” that contains inflammation-resolving extracellular vesicles (EVs) refers to the product resulting from drying a solution that contains inflammation-resolving extracellular vesicles (EVs).
  • the drying is performed by freeze drying (lyophilization) or spray drying.
  • the dried form is a powder.
  • a powder refers to a type of dried form and includes a lyophilized powder, but includes powders, such as spray-dried powders, obtained by methods such as spray drying.
  • the resulting product is a lyophilate.
  • the dried form is a lyophilate.
  • a lyophilate refers to a type of dried form and includes a lyophilized powder and lyophilized cake.
  • the lyophilized cake is milled (for example, ground) to produce a lyophilized powder. Milling refers to mechanical size reduction of solids. Grinding is a type of milling, for example, that can be performed on dried forms. See, for example, Seibert et al., “MILLING OPERATIONS IN THE PHARMACEUTICAL INDUSTRY” in Chemical Engineering in the Pharmaceutical Industry: R&D to Manufacturing. Edited by David J. am Ende (2011).
  • the disclosure also provides dried forms, in some embodiments, such as lyophilates, that comprise inflammation-resolving extracellular vesicles (EVs) (for example, inflammationresolving extracellular vesicles (EVs) and/or a combination of EVs described herein), and an excipient.
  • a dried form can include inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • a dried form can include inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • a dried form can include inflammationresolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • inflammation-resolving extracellular vesicles (EVs) are combined with an excipient that comprises a bulking agent and/or lyoprotectant, for example, to prepare a solution.
  • the solution is dried.
  • the resulting dried form (for example, lyophilate) contains inflammation-resolving extracellular vesicles (EVs) and a component(s) of the excipient, for example, bulking agent and/or lyoprotectant (for example, in dried form).
  • the disclosure also provides dried forms of inflammation-resolving extracellular vesicles (EVs) and an excipient.
  • the dried form is a lyophilate, for example, such as a lyophilized cake or lyophilized powder.
  • the dried form is a powder, for example, such as a spray-dried powder or lyophilized powder.
  • the bulking agent comprises mannitol, sucrose, maltodextrin, dextran, Ficoll, and/or PVP-K30.
  • the excipient includes an additional component such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • a dried form contains inflammation-resolving extracellular vesicles (EVs) and an excipient, for example, that comprises a bulking agent, for example, an excipient from a stock of a formula provided in one of Tables A, B, C, D, K, or P.
  • the dried form has a moisture content below about 6% (or below about 5%) (for example, as determined by Karl Fischer titration). In some embodiments, the dried form has about 10% to about 80% (by weight) of an excipient, for example, an excipient that comprises a bulking agent. In some embodiments, the dried form has about 10% to about 80% (by weight) of an excipient, for example, an excipient from a stock of a formula provided in one of Tables A, B, C, D, K, or P. In some embodiments, the inflammation-resolving extracellular vesicles (EVs) comprise about 1% to about 99% of the total solids by weight of the dried form.
  • EVs extracellular vesicles
  • the solutions and/or dried form comprise inflammation-resolving extracellular vesicles (EVs) substantially or entirely free of whole bacteria (for example, live bacteria, killed bacteria, and/or attenuated bacteria).
  • the solutions and/or dried form comprise both inflammation-resolving extracellular vesicles (EVs) and whole bacteria (for example, live bacteria, killed bacteria, and/or attenuated bacteria).
  • the solutions and/or dried form comprise gamma irradiated inflammation-resolving extracellular vesicles (EVs).
  • the inflammation-resolving extracellular vesicles (EVs) are gamma irradiated after the EVs are isolated (for example, prepared).
  • NTA nanoparticle tracking analysis
  • Coulter counting Coulter counting
  • DLS dynamic light scattering
  • Combined results from Coulter counting and NTA can reveal the numbers of bacteria and/or EVs from bacteria in a given sample.
  • Coulter counting reveals the numbers of particles with diameters of 0.7-10 pm.
  • the Coulter counter alone can reveal the number of bacteria and/or EVs in a sample.
  • NTA a Nanosight instrument can be obtained from Malvern Pananlytical.
  • the NS300 can visualize and measure particles in suspension in the size range 10-2000 nm.
  • 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 pm.
  • inflammation-resolving extracellular vesicles are characterized by analytical methods known in the art (for example, Jeppesen, et al. Cell 177:428 (2019)).
  • the inflammation-resolving extracellular vesicles are quantified based on particle count.
  • particle count of an EV preparation can be measured using NTA.
  • particle count of an EV preparation can be measured using NT A with Zetaview.
  • the inflammation-resolving extracellular vesicles are quantified based on the amount of protein, lipid, or carbohydrate.
  • total protein content of an EV preparation is measured using the Bradford assay or BCA.
  • the inflammation-resolving extracellular vesicles are isolated away from one or more other bacterial components of the source bacteria.
  • the solution and/or dried form further comprises other bacterial components.
  • the inflammation-resolving extracellular vesicles (EVs) liquid preparation obtained from the source bacteria may be fractionated into subpopulations based on the physical properties (for example, size, density, protein content, and/or binding affinity) of the subpopulations.
  • One or more of the EV subpopulations (for example, as a liquid preparation) can then be incorporated into the solutions and/or dried forms (such as powders and/or lyophilates) of the invention.
  • solutions and/or dried forms (and therapeutic compositions thereof) comprising inflammation-resolving extracellular vesicles (EVs) from bacteria useful for the treatment and/or prevention of disease (for example, an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease), as well as methods of making and/or identifying such EVs, and methods of using such solutions and/or dried forms (and therapeutic compositions thereof) (for example, for the treatment of an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease, either alone or in combination with other therapeutics).
  • an immune disorder e.g., an autoimmune disease, an inflammatory disease, an allergy
  • a dysbiosis e.g., a inflammatory disease, an allergy
  • a metabolic disease for example, for the treatment of an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbios
  • the therapeutic compositions comprise both inflammationresolving extracellular vesicles (EVs), and whole bacteria (for example, from which the EVs were obtained) (for example, live bacteria, killed bacteria, and/or attenuated bacteria).
  • EVs extracellular vesicles
  • whole bacteria for example, from which the EVs were obtained
  • live bacteria for example, live bacteria, killed bacteria, and/or attenuated bacteria
  • the solutions and/or dried forms comprise EVs from bacteria of one or more (e.g.,
  • the solutions and/or dried forms comprise EVs from bacteria of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) bacteria strains or species.
  • the solutions and/or dried forms comprise EVs from bacteria of one taxonomic group (e.g., class, order, family, genus, species or strain).
  • the solutions and/or dried forms comprise EVs from bacteria of one bacteria strain or species.
  • the therapeutic compositions comprise inflammation-resolving extracellular vesicles (EVs) in the absence of bacteria (for example, at least about 85%, at least about 90%, at least about 95%, or at least about 99% free of bacteria).
  • the therapeutic compositions comprise EVs from bacteria of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) taxonomic groups (e.g., class, order, family, genus, species or strain).
  • the therapeutic compositions comprise EVs from bacteria of one or more (e.g., 1,
  • the therapeutic compositions comprise EVs from bacteria of one taxonomic group (e.g., class, order, family, genus, species or strain). In some embodiments, the therapeutic compositions comprise EVs from bacteria of one bacteria strain or species.
  • the solution and/or dried form is added to or incorporated into a food product (for example, a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a probiotic, a food or beverage for patients, or an animal feed.
  • a food product for example, a food or beverage
  • a food or beverage for infants such as a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group
  • a functional food such as a beverage, a food or beverage for specified health use, a dietary supplement, a probiotic, a food or beverage for patients, or an animal feed.
  • the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquid
  • the solution and/or dried form is added to a food product or food supplement for animals, including humans.
  • the animals, other than humans, are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like.
  • Specific examples of the animals include pigs, cattle, horses, sheep, goats, chickens, ducks, ostriches, turkeys, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.
  • a solution and/or dried form provided herein is formulated into a therapeutic composition.
  • therapeutic compositions comprising a solution and/or dried form described herein.
  • the therapeutic composition comprises a solution and/or dried form provided herein and a pharmaceutically acceptable carrier.
  • the therapeutic composition comprises a pharmaceutically acceptable excipient, such as a glidant, lubricant, and/or diluent.
  • compositions comprising inflammation-resolving extracellular vesicles (EVs) useful for the treatment and/or prevention of inflammation (for example, Thl, Th2, or Thl7 inflammation), as well as methods of making and/or identifying such EVs, and methods of using such therapeutic compositions (for example, for the treatment of inflammation (for example, Thl, Th2, or Thl 7 inflammation) either alone or in combination with other therapeutics).
  • EVs extracellular vesicles
  • compositions comprising inflammation-resolving extracellular vesicles (EVs) useful for the treatment and/or prevention of disease (for example, an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease), as well as methods of making and/or identifying such EVs, and methods of using such therapeutic compositions (for example, for the treatment of an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease, either alone or in combination with other therapeutics).
  • the therapeutic compositions comprise both EVs and whole bacteria (for example, live bacteria, killed bacteria, attenuated bacteria).
  • the therapeutic compositions comprise EVs in the absence of bacteria (for example, at least about 85%, at least about 90%, at least about 95%, or at least about 99% free of bacteria).
  • the therapeutic compositions comprise EVs and/or bacteria from one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) taxonomic groups (e.g., class, order, family, genus, species or strain).
  • the therapeutic compositions comprise EVs and/or bacteria from one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) bacteria strains or species.
  • the therapeutic compositions comprise EVs and/or bacteria from one taxonomic group (e.g., class, order, family, genus, species or strain). In some embodiments, the therapeutic compositions comprise EVs and/or bacteria from one bacteria strain or species. In some embodiments, the therapeutic compositions comprise EVs and/or bacteria from one of the bacteria from a taxonomic group. In some embodiments, the therapeutic compositions comprise EVs and/or bacteria from one of the bacteria strains or species.
  • taxonomic group e.g., class, order, family, genus, species or strain.
  • the therapeutic compositions comprise EVs and/or bacteria from one bacteria strain or species.
  • therapeutic compositions for administration to a subject (e.g., human subject).
  • the therapeutic compositions 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 therapeutic composition is combined with an adjuvant such as an immuno-adjuvant (e.g., a STING agonist, a TLR agonist, or a NOD agonist).
  • an adjuvant such as an immuno-adjuvant (e.g., a STING agonist, a TLR agonist, or a NOD agonist).
  • the therapeutic composition comprises at least one carbohydrate.
  • the therapeutic composition 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) (EP A), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), dococosanoic acid (22:0
  • the therapeutic composition comprises at least one supplemental mineral or mineral source.
  • supplemental mineral or mineral source examples 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 therapeutic composition comprises at least one supplemental 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 Bl 2, 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 therapeutic composition comprises an excipient.
  • suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
  • the excipient is a buffering agent.
  • suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • the excipient comprises a preservative.
  • suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
  • the therapeutic composition comprises a binder as an excipient.
  • suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
  • the therapeutic composition comprises a lubricant as an excipient.
  • suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.
  • the therapeutic composition comprises a dispersion enhancer as an excipient.
  • Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.
  • the therapeutic composition comprises a disintegrant as an excipient.
  • the disintegrant is a non-effervescent disintegrant.
  • suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth.
  • the disintegrant is an effervescent disintegrant.
  • suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
  • the therapeutic composition is a food product (e.g., a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • a food product e.g., a food or beverage
  • a food or beverage such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like.
  • beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages
  • the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies.
  • the therapeutic composition is a food product for animals, including humans.
  • the animals, other than humans, are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like.
  • Specific examples of the animals include pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.
  • a therapeutic composition comprising a dried form is formulated as a solid dosage form, (also referred to as “solid dose form”) for example, for oral administration.
  • the solid dosage form comprises one or more excipients, for example, pharmaceutically acceptable excipients, in addition to the dried form.
  • the dried form in the solid dosage form contains isolated inflammation-resolving extracellular vesicles (EVs).
  • EVs inflammation-resolving extracellular vesicles
  • the solid dosage form comprises a tablet, a minitablet, a capsule, or a dried form (such as a powder and/or lyophilate); or a combination of these forms (for example, minitablets comprised in a capsule).
  • the solid dosage form described herein can be, e.g., a capsule.
  • 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 capsule.
  • 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 size of the capsule refers to the size of the tablet prior to application of an enteric coating.
  • the capsule is banded after loading (and prior to enterically coating the capsule).
  • the capsule is banded with an HPMC-based banding solution.
  • the solid dosage form comprises a tablet (> 4 mm) (e.g., 5 mm- 17 mm).
  • the tablet is a 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, or 18 mm 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 1 mm-4 mm range.
  • the minitablet can be a 1 mm minitablet, 1.5 mm minitablet, 2 mm minitablet, 3 mm minitablet, or 4 mm 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 capsule is banded after loading.
  • the capsule is banded with an HPMC-based banding solution.
  • a therapeutic composition comprising a solution and/or dried form (e.g., that comprises EVs and a bulking agent) can be formulated as a suspension (e.g., a dried form can be reconstituted; a solution can be diluted), e.g., for oral administration or for injection.
  • a solution and/or dried form e.g., that comprises EVs and a bulking agent
  • a suspension e.g., a dried form can be reconstituted; a solution can be diluted
  • a solution can be diluted
  • EVs can be in a buffer, e.g., a pharmaceutically acceptable buffer, e.g., saline or PBS.
  • the suspension can comprise one or more excipients, e.g., pharmaceutically acceptable excipients.
  • the suspension can comprise, e.g., sucrose or glucose.
  • the EVs in the solution or dried form e.g., that comprises EVs and a bulking agent
  • the EVs in the suspension can be gamma irradiated.
  • a solid dosage form (e.g., capsule, tablet or minitablet) 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 allows for release of the therapeutic agent (such as inflammation-resolving extracellular vesicles (EVs), dried forms, and/or solid dosage forms thereof), e.g., in the small intestine.
  • the therapeutic agent such as inflammation-resolving extracellular vesicles (EVs), dried forms, and/or solid dosage forms thereof
  • the therapeutic agent in the small intestine allows the therapeutic 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 gastrointestinal tract and/or cause a systemic effect (e.g., an effect outside of the gastrointestinal tract).
  • cells e.g., epithelial cells and/or immune cells located at these specific locations, e.g., which can cause a local effect in the gastrointestinal tract and/or cause a systemic effect (e.g., an effect outside of the gastrointestinal tract).
  • 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 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).
  • enteric coating examples 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 LI 00-55 trade name; partially-neutralized poly(methacrylic acid, ethyl acrylate) 1 : 1 sold, for example, under the Kollicoat MAE- 1 OOP trade name; and poly(methacrylic acid, methyl methacrylate) 1:2 sold, for example, under the Eu
  • the solid dose form (e.g., a capsule) can comprise a single layer coating, e.g., a nonenteric coating, such as HPMC (hydroxyl propyl methyl cellulose) or gelatin.
  • a nonenteric coating such as HPMC (hydroxyl propyl methyl cellulose) or gelatin.
  • the disclosure also provides methods of preparing solutions of inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • the bulking agent comprises mannitol, sucrose, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, dextran, Ficoll, or PVP-K30.
  • the excipient comprises a lyoprotectant.
  • the excipient optionally includes an additional component such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl- B-cyclodextrin.
  • an additional component such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl- B-cyclodextrin.
  • a liquid preparation of inflammationresolving extracellular vesicles (EVs)and an excipient that comprises a bulking agent are combined to prepare a solution.
  • a liquid preparation of inflammation-resolving extracellular vesicles (for example, obtained by isolating EVs from a bacterial culture (such as a supernatant or a retentate) and an excipient that comprises a bulking agent, for example, an excipient stock of a formula provided in one of Tables A, B, C, D, K, or P, are combined to prepare a solution.
  • a bacterial culture such as a supernatant or a retentate
  • an excipient that comprises a bulking agent for example, an excipient stock of a formula provided in one of Tables A, B, C, D, K, or P
  • a liquid preparation containing inflammation-resolving extracellular vesicles (for example, obtained by isolating EVs from a bacterial culture (such as a supernatant or a retentate) and an excipient that comprises a bulking agent are combined, for example, a liquid preparation containing inflammation-resolving extracellular vesicles (EVs) (for example, obtained by isolating EVs from a bacterial culture (such as a supernatant or a retentate) or a retentate) are combined with an excipient that comprises a bulking agent, for example, such as mannitol or an excipient of an excipient stock of a formula provided in one of Tables A, B, C, D, K, or P, to prepare the solution.
  • a bulking agent for example, such as mannitol or an excipient of an excipient stock of a formula provided in one of Tables A, B, C, D, K, or P, to
  • the disclosure also provides methods of preparing dried forms of inflammationresolving extracellular vesicles (EVs).
  • the method is used to prepare a lyophilate such as a lyophilized powder and/or a lyophilized cake.
  • the method is used to prepare a powder such as a lyophilized powder and/or a spray-dried powder.
  • the excipient comprises a bulking agent.
  • the bulking agent comprises mannitol, sucrose, polyethylene glycol (PEG, such as PEG 6000), cyclodextrin, maltodextrin, dextran, Ficoll, or PVP-K30.
  • the excipient comprises a lyoprotectant.
  • the excipient optionally includes an additional component such as trehalose, mannitol, sucrose, sorbitol, dextran, poloxamer 188, maltodextrin, PVP-K30, Ficoll, citrate, arginine, and/or hydroxypropyl-B-cyclodextrin.
  • a liquid preparation containing inflammation-resolving extracellular vesicles (for example, obtained by isolating EVs from a bacterial culture(such as a supernatant or a retentate) is be combined with an excipient that comprises a bulking agent, such as mannitol or an excipient of an excipient stock of a formula provided in one of Tables A, B, C, D, K, or P; and dried (for example, by lyophilization or spray drying) to thereby prepare a dried form.
  • a bulking agent such as mannitol or an excipient of an excipient stock of a formula provided in one of Tables A, B, C, D, K, or P
  • the dried form has a moisture content below about 6%, below about 5%, below about 4%, between about 0.5% to about 5%, between about 1% to about 5%, between about 1% to about 4%, between about 1.5% to about 4%, between about 2% and about 4%, or between about 2% to about 3%, (for example, as determined by Karl Fischer titration).
  • the dried form has about 10% to about 80% (by weight) of an excipient, for example, an excipient that comprises a bulking agent.
  • the dried form has about 10% to about 80% (by weight) of an excipient, for example, an excipient from a stock of a formula provided in one of Tables A, B, C, D, K, or P.
  • the inflammation-resolving extracellular vesicles (EVs) comprise about 1% to about 99% of the total solids by weight of the dried form.
  • the dried form is a lyophilate.
  • the lyophilate is a lyophilized powder or a lyophilized cake.
  • the dried form is a powder.
  • the powder is a lyophilized powder or a spray-dried powder.
  • the disclosure also provides methods of preparing therapeutic compositions.
  • the method includes combining a solution or dried form described herein with a pharmaceutically acceptable excipient, such as a glidant, lubricant, and/or diluent, thereby preparing a therapeutic composition.
  • a pharmaceutically acceptable excipient such as a glidant, lubricant, and/or diluent
  • the disclosure also provides methods of preparing therapeutic compositions, such as solid dosage forms, that contain a dried form described herein.
  • the solid dosage form is a capsule, tablet, or minitablet.
  • the disclosure also provides methods of making a solid dosage form (for example, for oral administration) (for example, for pharmaceutical use) that comprises a dried form.
  • the dried form comprises inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent.
  • the dried form comprises inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a lyoprotectant.
  • the dried form comprises inflammation-resolving extracellular vesicles (EVs) and an excipient that comprises a bulking agent and a lyoprotectant.
  • the dried form also contains one or more additional components.
  • the dried form is combined with one or more pharmaceutically acceptable excipients.
  • the solid dosage form is enterically coated, for example, with a coating described herein.
  • a dried form described herein is reconstituted in a liquid (for example, a buffer, juice, or water) to prepare a therapeutic composition.
  • a liquid for example, a buffer, juice, or water
  • a solution is resuspended (for example, diluted) in a liquid (for example, a buffer, juice, or water) to prepare a therapeutic composition.
  • a liquid for example, a buffer, juice, or water
  • a therapeutic composition comprising a dried form described herein is reconstituted in a liquid (for example, a buffer, juice, or water) to prepare a suspension.
  • a liquid for example, a buffer, juice, or water
  • a therapeutic composition comprising a solution is resuspended (for example, diluted) in a liquid (for example, a buffer, juice, or water) to prepare a suspension.
  • a liquid for example, a buffer, juice, or water
  • Dried forms (such as powders and/or lyophilates) (e.g., of inflammation-resolving extracellular vesicles (EVs)) can be gamma-irradiated at 17.5 kGy radiation unit at ambient temperature.
  • EVs extracellular vesicles
  • Frozen biomasses e.g., of inflammation-resolving extracellular vesicles (EVs)
  • EVs extracellular vesicles
  • the methods provided herein include the administration to a subject of a therapeutic composition described herein either alone or in combination with an additional therapeutic agent.
  • the additional therapeutic agent is an immunosuppressant, an anti-inflammatory agent, and/or a steroid.
  • the therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) is administered to the subject before the additional therapeutic agent is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
  • the therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) is administered to the subject after the additional therapeutic agent is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after or at least 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 or 30 days after).
  • the therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) and the additional therapeutic agent are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other).
  • an antibiotic is administered to the subject before the therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • an antibiotic is administered to the subject after therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 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 or 30 days after).
  • the therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) and the antibiotic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other).
  • the methods provided herein include the administration of a therapeutic composition described herein in combination with one or more additional therapeutic agents. In some embodiments, the methods disclosed herein include the administration of two therapeutic agents.
  • the therapeutic agent is an antibiotic.
  • Antibiotics broadly refers to compounds capable of inhibiting or preventing a bacterial infection. Antibiotics can be classified in a number of ways, including their use for specific infections, their mechanism of action, their bioavailability, or their spectrum of target microbe (e.g., Gram-negative vs. Grampositive bacteria, aerobic vs. anaerobic bacteria, etc.) and these may be used to kill specific bacteria in specific areas of the host (“niches”) (Leekha, et al 2011. General Principles of Antimicrobial Therapy. Mayo Clin Proc. 86(2): 156-167).
  • antibiotics can be used to selectively target bacteria of a specific niche.
  • antibiotics are administered after the therapeutic composition comprising EVs from Prevotella histicola bacteria. In some embodiments, antibiotics are administered before therapeutic composition comprising EVs from Prevotella histicola bacteria.
  • antibiotics can be selected based on their bactericidal or bacteriostatic properties.
  • Bactericidal antibiotics include mechanisms of action that disrupt the cell wall (e.g., 0-lactams), the cell membrane (e.g., daptomycin), or bacterial DNA (e.g., fluoroquinolones).
  • Bacteriostatic agents inhibit bacterial replication and include sulfonamides, tetracyclines, and macrolides, and act by inhibiting protein synthesis.
  • some drugs can be bactericidal in certain organisms and bacteriostatic in others, knowing the target organism allows one skilled in the art to select an antibiotic with the appropriate properties.
  • Antibiotics include, but are not limited to aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, and anti-mycobacterial compounds, and combinations thereof.
  • Aminoglycosides include, but are not limited to Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, and Spectinomycin. Aminoglycosides are effective, e.g., against Gram- negative bacteria, such as Escherichia coli, Klebsiella, Pseudomonas aeruginosa, and Francisella tularensis, and against certain aerobic bacteria but less effective against obligate/facultative anaerobes. Aminoglycosides are believed to bind to the bacterial 3 OS or 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Ansamycins include, but are not limited to, Geldanamycin, Herbimycin, Rifamycin, and Streptovaricin.
  • Geldanamycin and Herbimycin are believed to inhibit or alter the function of Heat Shock Protein 90.
  • Carbacephems include, but are not limited to, Loracarbef. Carbacephems are believed to inhibit bacterial cell wall synthesis.
  • Carbapenems include, but are not limited to, Ertapenem, Doripenem, Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal for both Gram-positive and Gram-negative bacteria as broad-spectrum antibiotics. Carbapenems are believed to inhibit bacterial cell wall synthesis.
  • Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin, Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole.
  • Cephalosporins are effective, e.g., against Gram-negative bacteria and against Gram-positive bacteria, including Pseudomonas, certain Cephalosporins are effective against methicillin- resistant Staphylococcus aureus (MRSA). Cephalosporins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin, and Telavancin. Glycopeptides are effective, e.g., against aerobic and anaerobic Gram-positive bacteria including MRSA and Clostridium difficile. Glycopeptides are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Lincosamides include, but are not limited to, Clindamycin and Lincomycin. Lincosamides are effective, e.g., against anaerobic bacteria, as well as Staphylococcus and Streptococcus. Lincosamides are believed to bind to the bacterial 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Lipopeptides include, but are not limited to, Daptomycin. Lipopeptides are effective, e.g., against Gram-positive bacteria. Lipopeptides are believed to bind to the bacterial membrane and cause rapid depolarization.
  • Macrolides include, but are not limited to, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective, e.g., against Streptococcus and Mycoplasma. Macrolides are believed to bind to the bacterial or 50S ribosomal subunit, thereby inhibiting bacterial protein synthesis.
  • Monobactams include, but are not limited to, Aztreonam. Monobactams are effective, e.g., against Gram-negative bacteria. Monobactams are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Nitrofurans include, but are not limited to, Furazolidone and Nitrofurantoin.
  • Oxazolidonones include, but are not limited to, Linezolid, Posizolid, Radezolid, and Torezolid. Oxazolidonones are believed to be protein synthesis inhibitors.
  • Penicillins include, but are not limited to, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cioxacillin, Dicloxacillin, Flucioxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin and Ticarcillin.
  • Penicillins are effective, e.g., against Gram-positive bacteria, facultative anaerobes, e.g., Streptococcus, Borrelia, and Treponema. Penicillins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Penicillin combinations include, but are not limited to, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, and Ticarcillin/clavulanate.
  • Polypeptide antibiotics include, but are not limited to, Bacitracin, Colistin, and Polymyxin B and E.
  • Polypeptide Antibiotics are effective, e.g., against Gram-negative bacteria. Certain polypeptide antibiotics are believed to inhibit isoprenyl pyrophosphate involved in synthesis of the peptidoglycan layer of bacterial cell walls, while others destabilize the bacterial outer membrane by displacing bacterial counter-ions.
  • Quinolones and Fluoroquinolone include, but are not limited to, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin.
  • Quinolones/Fluoroquinolone are effective, e.g., against Streptococcus and Neisseria.
  • Quinolones/Fluoroquinolone are believed to inhibit the bacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNA replication and transcription.
  • Sulfonamides include, but are not limited to, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co-trimoxazole), and Sulfonamidochrysoidine.
  • Sulfonamides are believed to inhibit folate synthesis by competitive inhibition of dihydropteroate synthetase, thereby inhibiting nucleic acid synthesis.
  • Tetracyclines include, but are not limited to, Demeclocy cline, Doxycycline, Minocycline, Oxytetracycline, and Tetracycline. Tetracyclines are effective, e.g., against Gramnegative bacteria. Tetracyclines are believed to bind to the bacterial 30S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Anti-mycobacterial compounds include, but are not limited to, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, and Streptomycin.
  • Suitable antibiotics also include arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprim amoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin, azithromycin, bacitracin, buforin II, carbomycin, cecropin Pl, clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate, gramicidin, imipenem, indolicidin, josamycin, magainan II, metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacin B-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, oleand
  • the additional therapeutic agent is an immunosuppressive agent, a DMARD, a pain-control drug, a steroid, a non-steroidal anti-inflammatory drug (NSAID), or a cytokine antagonist, and combinations thereof.
  • a DMARD a pain-control drug
  • a steroid a steroid
  • NSAID non-steroidal anti-inflammatory drug
  • cytokine antagonist a cytokine antagonist
  • Representative agents include, but are not limited to, cyclosporine, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprophen, cholin magnesium salicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib, acetominophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefanamic acid, meclofenamic acid
  • the additional therapeutic agent is an immunosuppressive agent.
  • immunosuppressive agents include, but are not limited to, corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis, TLR antagonists, inflammasome inhibitors, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines (e.g., vaccines used for vaccination where the amount of an allergen is gradually increased), cytokine inhibitors, such as anti-IL-6 antibodies, TNF
  • a method of delivering a therapeutic composition described herein e.g., a therapeutic composition inflammation- resolving extracellular vesicles (EVs)
  • the therapeutic composition is administered in conjunction with the administration of an additional therapeutic agent.
  • the therapeutic composition comprises inflammation-resolving extracellular vesicles (EVs) co-formulated with the additional therapeutic agent.
  • the therapeutic composition comprising inflammation-resolving extracellular vesicles (EVs) is co-administered with the additional therapeutic agent.
  • the additional therapeutic agent is administered to the subject before administration of the therapeutic composition that comprises inflammation-resolving extracellular vesicles (EVs) (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before).
  • EVs extracellular vesicles
  • the additional therapeutic agent is administered to the subject after administration of the therapeutic composition that comprises inflammation-resolving extracellular vesicles (EVs) (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes after, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after).
  • the same mode of delivery is used to deliver both the therapeutic composition that comprises inflammation-resolving extracellular vesicles (EVs) and the additional therapeutic agent.
  • different modes of delivery are used to administer the therapeutic composition that comprises inflammationresolving extracellular vesicles (EVs) and the additional therapeutic agent.
  • the therapeutic composition that comprises inflammation-resolving extracellular vesicles (EVs) is administered orally while the additional therapeutic agent is administered via injection (e.g., an intravenous and/or intramuscular injection).
  • the therapeutic composition described herein is administered once a day. In some embodiments, the therapeutic composition described herein is administered twice a day. In some embodiments, the therapeutic composition described herein is formulated for a daily dose. In some embodiments, the therapeutic composition described herein is formulated for twice a day dose, wherein each dose is half of the daily dose.
  • 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, 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 therapeutic composition that comprises inflammation-resolving extracellular vesicles (EVs) 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 general effective dose of the agents may range between 0.01 mg/kg body weight/day and 1000 mg/kg body weight/day, between 0.1 mg/kg body weight/day and 1000 mg/kg body weight/day, 0.5 mg/kg body weight/day and 500 mg/kg body weight/day, 1 mg/kg body weight/day and 100 mg/kg body weight/day, or between 5 mg/kg body weight/day and 50 mg/kg body weight/day.
  • the effective dose may be 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 mg/kg body weight/day or more, but the dose is not limited thereto.
  • the dose administered to a subject is sufficient to prevent disease (e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease), delay its onset, or slow or stop its progression, or relieve one or more symptoms of the disease.
  • disease e.g., an immune disorder (e.g., an autoimmune disease, an inflammatory disease, an allergy), a dysbiosis, or a metabolic disease)
  • dosage will depend upon a variety of factors including the strength of the particular agent (e.g., therapeutic 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 therapeutic agent and the desired physiological effect.
  • Suitable doses and dosage regimens can be determined by conventional range- finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
  • An effective dosage and treatment protocol can be determined by routine and conventional means, starting e.g., with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Animal studies are commonly used to determine the maximal tolerable dose ("MTD”) of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.
  • MTD maximal tolerable dose
  • the dosages of the therapeutic 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 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 therapeutic composition, 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 less than the time period for a subject to mount an immune response, such as less than about one week, less than about ten days, less than about two weeks, or less than about a month.
  • the delivery of an additional therapeutic agent in combination with the therapeutic composition described herein reduces the adverse effects and/or improves the efficacy of the additional therapeutic agent.
  • the effective dose of an additional therapeutic agent described herein is the amount of the additional therapeutic agent that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, with the least toxicity to the subject.
  • the effective dosage level can be identified using the methods described herein and will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions or agents administered, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.
  • an effective dose of an additional therapeutic agent will be the amount of the additional therapeutic agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the toxicity of an additional therapeutic agent is the level of adverse effects experienced by the subject during and following treatment.
  • Adverse events associated with additional therapy toxicity can include, but are not limited to, abdominal pain, acid indigestion, acid reflux, allergic reactions, alopecia, anaphylaxis, anemia, anxiety, lack of appetite, arthralgias, asthenia, ataxia, azotemia, loss of balance, bone pain, bleeding, blood clots, low blood pressure, elevated blood pressure, difficulty breathing, bronchitis, bruising, low white blood cell count, low red blood cell count, low platelet count, cardiotoxicity, cystitis, hemorrhagic cystitis, arrhythmias, heart valve disease, cardiomyopathy, coronary artery disease, cataracts, central neurotoxicity, cognitive impairment, confusion, conjunctivitis, constipation, coughing, cramping, cystitis, deep vein thrombosis, dehydration, depression, diarrhea, dizziness, dry mouth, dry skin, dyspepsi
  • the methods and therapeutic compositions 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.
  • 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.
  • 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 therapeutic compositions described herein can be used, for example, as a therapeutic (such as 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 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 rea
  • the methods provided herein 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
  • the inflammation comprises a Thl mediated inflammation.
  • the inflammation comprises a Th2 mediated inflammation (such as with asthma or atopic dermatitis).
  • the inflammation comprises a Thl 7 mediated inflammation (such as with psoriasis).
  • 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 aw, 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
  • the methods provided herein are useful for the treatment of psoriasis.
  • the methods provided herein are useful for the treatment of psoriatic arthritis.
  • the methods provided herein are useful for the treatment of atopic dermatitis.
  • 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 compositions 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 that may be treated with the methods and therapeutic compositions 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 therapeutic compositions described herein include, but are not limited to, cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo- ovarian abscess, urethritis, vaginitis, vulvitis, and vulvodynia.
  • the methods and therapeutic compositions 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, opsoclo
  • 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 dust mite allergy) and gluten-sensitive enteropathy (Celiac disease).
  • immune disorders which may be treated with the methods and therapeutic compositions include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, ulceris, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleuritis, pneumonitis, prostatitis, pyelonephritis, and stomatitis, 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 xenografts, serum sickness, and graft v
  • transplant rejection
  • 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 include 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 therapeutic compositions 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, hypertriglyceridemia, 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, hypertriglyceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • the methods and therapeutic compositions described herein relate to the treatment of NAFLD and NASH.
  • 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 therapeutic compositions 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, hypertriglyceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, NAFLD, 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 therapeutic compositions described herein 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, 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, newborn jaundice, primary biliary cholangitis (PBC), Primary Sclerosing Cholangitis (PSC), Reye syndrome, type I glycogen storage disease, and Wilson’s disease.
  • ICP intrahepatic cholestasis of pregnancy
  • LAL-D lysosomal acid lipase deficiency
  • the methods and therapeutic compositions described herein 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, idiopathic intracranial hypertension, epilepsy, nervous system disease, central nervous system disease, movement disorders, multiple sclerosis, encephalopathy, peripheral neuropathy or post-operative cognitive dysfunction.
  • 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.org/10.1007/s00018-017-2509-x)).
  • 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. 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.
  • the presence of a dysbiosis can be 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
  • exemplary therapeutic compositions disclosed herein 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.
  • compositions disclosed herein that are useful for treatment of disorders associated with a dysbiosis contain inflammation-resolving extracellular vesicles (EVs). 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.
  • EVs extracellular vesicles
  • compositions disclosed herein that are useful for treatment of disorders associated with a dysbiosis contain a population of inflammation-resolving extracellular vesicles (EVs). 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.
  • EVs extracellular vesicles
  • compositions containing an isolated population of inflammation-resolving extracellular vesicles 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.
  • compositions 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.
  • the therapeutic 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 therapeutic compositions 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.
  • compositions are useful for treatment of disorders associated with a dysbiosis, which compositions contain one or more types of bacteria and/or EVs 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.
  • 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 are useful for treatment of disorders associated with a dysbiosis, which compositions contain a population of inflammation-resolving extracellular vesicles (EVs) 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.
  • EVs extracellular vesicles
  • 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 a therapeutic composition which alters the microbiome population existing at the site of the dysbiosis.
  • the therapeutic composition can contain Prevotella histicola EVs.
  • 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 a therapeutic composition which alters the subject’s immune response outside the gastrointestinal tract.
  • the therapeutic composition can contain one or more types of EVs from immunomodulatory bacteria or a population of inflammation-resolving extracellular vesicles (EVs).
  • therapeutic 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, TGFP, and combinations thereof.
  • therapeutic 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, IFNy, IL-12p70, IL-la, IL-6, IL-8, MCP1, MIPla, MIPip, TNFa, 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 therapeutic composition in the form of a probiotic or medical food comprising inflammation-resolving extracellular vesicles (EVs) in an amount sufficient to alter the microbiome at a site of the dysbiosis, such that the disorder associated with the dysbiosis is treated.
  • administering e.g., orally administering
  • a therapeutic composition in the form of a probiotic or medical food comprising inflammation-resolving extracellular vesicles (EVs) in an amount sufficient to alter the microbiome at a site of the dysbiosis, such that the disorder associated with the dysbiosis is treated.
  • EVs extracellular vesicles
  • a therapeutic 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.
  • engineered bacteria for the production of the inflammation-resolving extracellular vesicles (EVs) described herein.
  • the engineered bacteria are modified to enhance certain desirable properties.
  • the engineered bacteria are modified to enhance the immunomodulatory and/or therapeutic effect of the EVs (e.g., either alone or in combination with another therapeutic agent), to reduce toxicity and/or to improve bacterial and/or EV manufacturing (e.g., higher oxygen tolerance, improved freeze- thaw tolerance, shorter generation times).
  • 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, CRISPR/Cas9, or any combination thereof.
  • the bacterium is modified by directed evolution.
  • the directed evolution comprises exposure of the bacterium to an environmental condition and selection of bacterium with improved survival and/or growth under the environmental condition.
  • the method comprises a screen of mutagenized bacteria using an assay that identifies enhanced bacterium.
  • the method further comprises mutagenizing the bacteria (e.g., by exposure to chemical mutagens and/or UV radiation) or exposing them to a therapeutic agent (e.g., antibiotic) followed by an assay to detect bacteria having the desired phenotype (e.g., an in vivo assay, an ex vivo assay, or an in vitro assay).
  • a therapeutic agent e.g., antibiotic
  • smEVs extracellular vesicles used in the studies were isolated from a strain of Prevotella histicola (Prevotella Strain B) (NRRL accession number B 50329).
  • Table B Stocks comprising excipients including polymers for stabilizing extracellular vesicles during lyophilization. The numerical values given are on a weight percent basis in the solution.
  • Table C Stocks comprising excipients including polymers for stabilizing extracellular vesicles during lyophilization. The numerical values given are on a weight percent basis in the solution.
  • Table D Stocks comprising excipients including polymers for stabilizing extracellular vesicles during lyophilization. The numerical values given are on a weight percent basis in the solution.
  • % Stabilizer refers to the percentage of the stock solution formula that was added to a liquid preparation of EVs on a weight basis.
  • % Moisture was determined by Karl Fischer titration. Zave was determined by dynamic light scattering (DLS). For particles per mass, particle numbers were determined by Z-view or NTA instrument; mass (mg) were decided by analytical balance.
  • the lyophilization cycle is optimized for each excipient formulation. Differences in the critical temperature and collapse temperature of the mixtures mean the shelf temperature during lyophilization is adjusted accordingly.
  • the optimization process involves 3 steps: initial screening, primary drying optimization, and secondary drying optimization.
  • the final cycle is confirmed to be sufficient to dry the material below 5% residual moisture.
  • the excipient formula chosen for optimization was excipient formula 7.
  • Table I Final lyophilization cycle optimized for extracellular vesicles stabilized with 47% (by volume) of excipient formula 7.
  • Example 2 Purification and preparation of extracellular vesicles (EVs) from bacteria
  • Extracellular vesicles are purified and prepared from bacterial cultures using methods known to those skilled in the art (S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011)).
  • bacterial cultures are centrifuged at 10,000-15,500 x g for 10-40 min at 4°C or room temperature to pellet bacteria.
  • Culture supernatants are then filtered to include material ⁇ 0.22 pm (for example, via a 0.22 pm or 0.45 pm filter) and to exclude intact bacterial cells.
  • Filtered supernatants are concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. Briefly, for ammonium sulfate precipitation, 1.5-3 M ammonium sulfate is added to filtered supernatant slowly, while stirring at 4°C.
  • Precipitations are incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C.
  • the pellets contain EVs and other debris.
  • using ultracentrifugation filtered supernatants are centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C.
  • the pellet of this centrifugation contains EVs and other debris.
  • using a filtration technique using an Amicon Ultra spin filter or by tangential flow filtration, supernatants are filtered so as to retain species of molecular weight > 50, 100, 300, or 500 kDa.
  • EVs are obtained from bacterial cultures continuously during growth, or at selected time points during growth, by connecting a bioreactor to an alternating tangential flow (ATF) system (e.g., XCell ATF from Repligen) according to manufacturer’s instructions.
  • ATF alternating tangential flow
  • the ATF system retains intact cells (> 0.22 pm) in the bioreactor, and allows smaller components (e.g., EVs, free proteins) to pass through a filter for collection.
  • the system may be configured so that the ⁇ 0.22 pm filtrate is then passed through a second filter of 100 kDa, allowing species such as EVs between 0.22 pm 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. EVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
  • EVs obtained by methods described above may be further purified 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 45% OptiPrepTM in PBS. If filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 45% OptiPrepTM. Samples are applied to a 0-45% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Alternatively, high resolution density gradient fractionation could be used to separate EVs based on density.
  • EVs 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 pm filter to exclude intact cells. To further increase purity, isolated EVs may be DNase or proteinase K treated.
  • samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (e.g., Amicon Ultra columns), dialysis, or ultracentrifugation (following 15-fold or greater dilution in PBS, 200,000 x g, 1-3 hours, 4°C) and resuspension in PBS.
  • filtration e.g., Amicon Ultra columns
  • dialysis e.g., dialysis
  • ultracentrifugation followeding 15-fold or greater dilution in PBS, 200,000 x g, 1-3 hours, 4°C
  • EVs may be heated, irradiated, and/or lyophilized prior to administration (as described herein).
  • Example 3 Manipulating bacteria through stress to produce various amounts of EVs and/or to vary content of EVs
  • Bacteria may be subjected to single stressors or stressors in combination. The effects of different stressors on different bacteria is determined empirically by varying the stress condition and determining the IC50 value (the conditions required to inhibit cell growth by 50%).
  • EV purification, quantification, and characterization occurs. EV production is quantified (1) in complex samples of bacteria and EVs by nanoparticle tracking analysis (NT A) or transmission electron microscopy (TEM); or (2) following EV purification by NTA, lipid quantification, or protein quantification. EV content is assessed following purification by methods described above.
  • NT A nanoparticle tracking analysis
  • TEM transmission electron microscopy
  • Bacteria are cultivated under standard growth conditions with the addition of sublethal concentrations of antibiotics. This may include 0.1-1 pg/mL chloramphenicol, or 0.1-0.3 pg/mL gentamicin, or similar concentrations of other antibiotics (e.g., ampicillin, polymyxin B). Host antimicrobial products such as lysozyme, defensins, and Reg proteins may be used in place of antibiotics. Bacterially-produced antimicrobial peptides, including bacteriocins and microcins may also be used.
  • Bacteria are cultivated under standard growth conditions, but at higher or lower temperatures than are typical for their growth. Alternatively, bacteria are grown under standard conditions, and then subjected to cold shock or heat shock by incubation for a short period of time at low or high temperatures respectively. For example, bacteria grown at 37°C are incubated for 1 hour at 4°C-18°C for cold shock or 42°C-50°C for heat shock.
  • bacteria are cultivated under conditions where one or more nutrients are limited. Bacteria may be subjected to nutritional stress throughout growth or shifted from a rich medium to a poor medium.
  • Some examples of media components that are limited are carbon, nitrogen, iron, and sulfur.
  • An example medium is M9 minimal medium (Sigma-Aldrich), which contains low glucose as the sole carbon source.
  • M9 minimal medium Sigma-Aldrich
  • iron availability is varied by altering the concentration of hemin in media and/or by varying the type of porphyrin or other iron carrier present in the media, as cells grown in low hemin conditions were found to produce greater numbers of EVs (S. Stubbs et al. Letters in Applied Microbiology. 29:31-36 (1999).
  • Media components are also manipulated by the addition of chelators such as EDTA and deferoxamine.
  • Bacteria are grown to saturation and incubated past the saturation point for various periods of time.
  • conditioned media is used to mimic saturating environments during exponential growth.
  • Conditioned media is prepared by removing intact cells from saturated cultures by centrifugation and filtration, and conditioned media may be further treated to concentrate or remove specific components.
  • Bacteria are cultivated in or exposed for brief periods to medium containing NaCl, bile salts, or other salts.
  • UV stress is achieved by cultivating bacteria under a UV lamp or by exposing bacteria to UV using an instrument such as a Stratalinker (Agilent). UV may be administered throughout the entire cultivation period, in short bursts, or for a single defined period following growth.
  • Stratalinker Agilent
  • Bacteria are cultivated in the presence of sublethal concentrations of hydrogen peroxide (250-1,000 pM) to induce stress in the form of reactive oxygen species. Anaerobic bacteria are cultivated in or exposed to concentrations of oxygen that are toxic to them.
  • Bacteria are cultivated in or exposed to detergent, such as sodium dodecyl sulfate (SDS) or deoxycholate. pH stress
  • EVs may be characterized by any one of various methods including, but not limited to, NanoSight characterization, SDS-PAGE gel electrophoresis, Western blot, ELISA, liquid chromatography-mass spectrometry and mass spectrometry, dynamic light scattering, lipid levels, total protein, lipid to protein ratios, nucleic acid analysis and/or zeta potential.
  • Nanoparticle tracking analysis is used to characterize the size distribution of purified EVs. Purified EV preparations are run on a NanoSight machine (Malvern Instruments) to assess EV size and concentration.
  • samples are run on a gel, for example a Bolt Bis-Tris Plus 4-12% gel (Thermo-Fisher Scientific), using standard techniques. Samples are boiled in lx SDS sample buffer for 10 minutes, cooled to 4°C, and then centrifuged at 16,000 x g for 1 min. Samples are then run on a SDS-PAGE gel and stained using one of several standard techniques (e.g., Silver staining, Coomassie Blue, Gel Code Blue) for visualization of bands.
  • a gel for example a Bolt Bis-Tris Plus 4-12% gel (Thermo-Fisher Scientific)
  • Samples are boiled in lx SDS sample buffer for 10 minutes, cooled to 4°C, and then centrifuged at 16,000 x g for 1 min. Samples are then run on a SDS-PAGE gel and stained using one of several standard techniques (e.g., Silver staining, Coomassie Blue, Gel Code Blue) for visualization of bands.
  • EV proteins are separated by SDS-PAGE as described above and subjected to Western blot analysis (Cvjetkovic et al., Sci. Rep. 6, 36338 (2016)) and are quantified via ELISA.
  • EV proteins present in EVs are identified and quantified by Mass Spectrometry techniques.
  • EV proteins may be prepared for LC-MS/MS using standard techniques including protein reduction using dithiothreitol solution (DTT) and protein digestion using enzymes such as LysC and trypsin as described in Erickson et al. (2017) Molecular Cell, 65(2): 361-70.
  • DTT dithiothreitol solution
  • peptides are prepared as described by Liu et al. (2010) J. Bacteriology, 192(11): 2852-60), Kieselbach and Oscarsson 2017 (Data Brief. 2017 Feb; 10: 426-431.), Vildhede et al, 2018 (Drug Metabolism and Disposition February 8, 2018).
  • peptide preparations are run directly on liquid chromatography and mass spectrometry devices for protein identification within a single sample.
  • peptide digests from different samples are labeled with isobaric tags using the iTRAQ Reagent- 8plex Multiplex Kit (Applied Biosystems, Foster City, CA) or TMT lOplex and 1 Iplex Label Reagents (Thermo Fischer Scientific, San Jose, CA, USA).
  • Each peptide digest is labeled with a different isobaric tag and then the labeled digests are combined into one sample mixture.
  • the combined peptide mixture is analyzed by LC-MS/MS for both identification and quantification.
  • a database search is performed using the LC-MS/MS data to identify the labeled peptides and the corresponding proteins.
  • the fragmentation of the attached tag generates a low molecular mass reporter ion that is used to obtain a relative quantitation of the peptides and proteins present in each EV.
  • LC-MS system includes a 4000 QTRAP triple quadrupole mass spectrometer (AB SCIEX) combined with 1100 Series pump (Agilent) and an HTS PAL autosampler (Leap Technologies).
  • the column is eluted by flowing a 5% mobile phase [10 mM ammonium formate, 0.1% formic acid in water] for 1 minute at a rate of 250 pL/minute followed by a linear gradient over 10 minutes to a solution of 40% mobile phase [acetonitrile with 0.1% formic acid].
  • the ion spray voltage is set to 4.5 kV and the source temperature is 450°C.
  • DLS measurements including the distribution of particles of different sizes in different EV preparations are taken using instruments such as the DynaPro NanoStar (Wyatt Technology) and the Zetasizer Nano ZS (Malvern Instruments).
  • Lipid levels are quantified using FM4-64 (Life Technologies), by methods similar to those described by A. J. McBroom et al. J Bacterial 188:5385-5392. and A. Frias, et al. Microb Ecol. 59:476-486 (2010). Samples are incubated with FM4-64 (3.3 pg/mL in PBS for 10 minutes at 37°C in the dark). After excitation at 515 nm, emission at 635 nm is measured using a Spectramax M5 plate reader (Molecular Devices).
  • Absolute concentrations are determined by comparison of unknown samples to standards (such as palmitoyloleoylphosphatidylglycerol (POPG) vesicles) of known concentrations.
  • POPG palmitoyloleoylphosphatidylglycerol
  • Lipidomics can be used to identify the lipids present in the EVs.
  • Protein levels are quantified by standard assays such as the Bradford and BCA assays.
  • the Bradford assays are run using Quick Start Bradford lx Dye Reagent (Bio-Rad), according to manufacturer’s protocols.
  • BCA assays are run using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Absolute concentrations are determined by comparison to a standard curve generated from BSA of known concentrations.
  • protein concentration can be calculated using the Beer-Lambert equation using the sample absorbance at 280 nm (A280) as measured on a Nanodrop spectrophotometer (Thermo-Fisher Scientific).
  • proteomics may be used to identify proteins in the sample.
  • Lipid protein ratios are generated by dividing lipid concentrations by protein concentrations. These provide a measure of the purity of vesicles as compared to free protein in each preparation.
  • Nucleic acids are extracted from EVs and quantified using a Qubit fluorimeter. Size distribution is assessed using a BioAnalyzer and the material is sequenced. Zeta Potential
  • the zeta potential of different preparations are measured using instruments such as the Zetasizer ZS (Malvern Instruments).
  • Enriched media is used to grow and prepare the bacteria for in vitro and in vivo use and, ultimately, for EV preparations.
  • media may contain sugar, yeast extracts, plantbased peptones, buffers, salts, trace elements, surfactants, anti-foaming agents, and vitamins.
  • Composition of complex components such as yeast extracts and peptones may be undefined or partially defined (including approximate concentrations of amino acids, sugars etc.).
  • Microbial metabolism may be dependent on the availability of resources such as carbon and nitrogen. Various sugars or other carbon sources may be tested.
  • media may be prepared and the selected bacterium grown as shown by Saarela etal., J. Applied Microbiology. 2005. 99: 1330-1339, which is hereby incorporated by reference. Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of the selected bacterium produced without milk-based ingredients.
  • the media is sterilized. Sterilization may be accomplished by Ultra High Temperature (UHT) processing.
  • UHT Ultra High Temperature
  • the 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.
  • Inoculum can be prepared in flasks or in smaller bioreactors and growth is monitored.
  • the inoculum size may be between approximately 0.5 and 3% of the total bioreactor volume.
  • bioreactor volume can be at least 2 L, 10 L, 80 L, 100 L, 250 L, 1000 L, 2500 L, 5000 L, 10,000 L.
  • the bioreactor Before the inoculation, the bioreactor is prepared with medium at desired pH, temperature, and oxygen concentration.
  • the initial pH of the culture medium may be different that the process set-point. pH stress may be detrimental at low cell centration; 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.
  • 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. Anaerobic conditions are created by reducing the level of oxygen in the culture broth from around 8 mg/L to 0 mg/L.
  • nitrogen or gas mixtures may be used in order to establish anaerobic conditions.
  • no gases are used and anaerobic conditions are established by cells consuming remaining oxygen from the medium.
  • the bioreactor fermentation time can vary. For example, fermentation time can vary from approximately 5 hours to 48 hours.
  • Reviving bacteria from a frozen state may require special considerations.
  • Production medium may stress cells after a thaw; a specific thaw medium may be required to consistently start a seed train from thawed material.
  • the kinetics of transfer or passage of seed material to fresh medium may be influenced by the current state of the bacteria (ex. exponential growth, stationary growth, unstressed, stressed).
  • Inoculation of the production fermenter(s) can impact growth kinetics and cellular activity.
  • the initial state of the bioreactor system must be optimized to facilitate successful and consistent production.
  • the fraction of seed culture to total medium (e.g., a percentage) has a dramatic impact on growth kinetics.
  • the range may be 1-5% of the fermenter’s working volume.
  • the initial pH of the culture medium may be different from the process set-point. pH stress may be detrimental at low cell concentration; the initial pH may be between pH 7.5 and the process set-point. Agitation and gas flow into the system during inoculation may be different from the process set-points. Physical and chemical stresses due to both conditions may be detrimental at low cell concentration.
  • Process conditions and control settings may influence the kinetics of microbial growth and cellular activity. Shifts in process conditions may change membrane composition, production of metabolites, growth rate, cellular stress, etc.
  • Optimal temperature range for growth may vary with strain. The range may be 20-40°C.
  • Optimal pH for cell growth and performance of downstream activity may vary with strain. The range may be pH 5-8. Gasses dissolved in the medium may be used by cells for metabolism. Adjusting concentrations of O2, CO2, and N2 throughout the process may be required. Availability of nutrients may shift cellular growth. Bacteria may have alternate kinetics when excess nutrients are available.
  • Bacteria may be preconditioned shortly before harvest to better prepare them for the physical and chemical stresses involved in separation and downstream processing. A change in temperature (often reducing to 20-5°C) may reduce cellular metabolism, slowing growth (and/or death) and physiological change when removed from the fermenter. Effectiveness of centrifugal concentration may be influenced by culture pH. Raising pH by 1 -2 points can improve effectiveness of concentration but can also be detrimental to cells. Bacteria may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization during downstream.
  • Separation methods and technology may impact how efficiently bacteria are separated from the culture medium.
  • Solids may be removed using centrifugation techniques. Effectiveness of centrifugal concentration can be influenced by culture pH or by the use of flocculating agents. Raising pH by 1 -2 points may improve effectiveness of concentration but can also be detrimental to cells.
  • Bacteria may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization during downstream. Additionally, Bacteria may also be separated via filtration. Filtration is superior to centrifugation techniques for purification if the cells require excessive g- minutes to successfully centrifuge. Excipients can be added before after separation.
  • 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 mixed with excipients are submerged in liquid nitrogen.
  • Harvesting can 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 antioxidants.
  • droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.
  • Lyophilization of material includes a freezing, primary drying, and secondary drying phase. Lyophilization begins with freezing.
  • the product material may or may not be mixed with a lyoprotectant or stabilizer prior to the freezing stage.
  • a product may be frozen prior to the loading of the lyophilizer, or under controlled conditions on the shelf of the lyophilizer.
  • the primary drying phase ice is removed via sublimation. Here, a vacuum is generated and an appropriate amount of heat is supplied to the material. The ice will sublime while keeping the product temperature below freezing, and below the material’s critical temperature (T c ).
  • the temperature of the shelf on which the material is loaded and the chamber vacuum can be manipulated to achieve the desired product temperature.
  • the temperature is generally 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 chamber may be filled with an inert gas, such as nitrogen.
  • the product may be sealed within the freeze dryer under dry conditions, in a glass vial or other similar container, preventing exposure to atmospheric water and contaminates.
  • smEVs Downstream processing of smEVs began immediately following harvest of the bioreactor. Centrifugation at 20,000 x g was used to remove the cells from the broth. The resulting supernatant was clarified using 0.22 pm filter. The EVs were concentrated and washed using tangential flow filtration (TFF) with flat sheet cassettes ultrafiltration (UF) membranes with 100 kDa molecular weight cutoff (MWCO). Diafiltration (DF) was used to washout small molecules and small proteins using 5 volumes of phosphate buffer solution (PBS). The retentate from TFF was spun down in an ultracentrifuge at 200,000 x g for 1 hour to form a pellet rich in EVs called a high-speed pellet (HSP).
  • TFF tangential flow filtration
  • UF ultrafiltration
  • MWCO molecular weight cutoff
  • the pellet was resuspended with minimal PBS and a gradient was prepared with OptiPrepTM density gradient medium and ultracentrifuged at 200,000 x g for 16 hours. Of the resulting fractions, 2 middle bands contained EVs. The fractions were washed with 15-fold PBS and the EVs spun down at 200,000 x g for 1 hour to create the fractionated HSP or fHSP. It was subsequently resuspended with minimal PBS, pooled, and analyzed for particles per mL and protein content. Dosing was prepared from the particle/mL count to achieve desired concentration. The EVs were characterized using a NanoSight NS300 by Malvern Panalytical in scatter mode using the 532 nm laser.
  • the equipment used in EV isolation includes a Sorvall RC-5C centrifuge with SLA- 3000 rotor; an Optima XE-90 Ultracentrifuge by Beckman-Coulter 45Ti rotor; a Sorvall wX+ Ultra Series Centrifuge by Thermo Scientific; and a Fiberlite F37L-8xl00 rotor.
  • Bacteria must be pelleted and filtered away from supernatant in order to recover EVs and not bacteria.
  • Pellet bacterial culture is generated by using a Sorvall RC-5C centrifuge with the SLA- 3000 rotor and centrifuge culture for a minimum of 15 min at a minimum of 7,000 rpm. And then decanting the supernatant into new and sterile container.
  • the supernatant is filtered through a 0.2 pm filter.
  • a 0.45 pm capsule filter is attached ahead of the 0.2 pm vacuum filter.
  • the filtered supernatant is stored at 4°C.
  • the filtered supernatant can then be concentrated using TFF.
  • Density gradients are used for EV purification. During ultracentrifugation, particles in the sample will move, and separate, within the graded density medium based on their ‘buoyant’ densities. In this way EVs are separated from other particles, such as sugars, lipids, or other proteins, in the sample.
  • the 45% gradient mixture is pipetted up and down to mix.
  • the sample is then pipetted into a labeled clean and sterile ultracentrifuge tube.
  • a 10 ml serological pipette is used to slowly add 13 ml of 35% gradient mixture.
  • 13ml of the 25% gradient mixture is added, followed by 13 ml of the 15% mixture and finally 6 ml of sterile lx PBS.
  • the ultracentrifuge tubes are balanced with sterile lx PBS.
  • the gradients are carefully placed in a rotor and the ultracentrifuge is set for 200,000 x g and 4°C. The gradients are centrifuged for a minimum of 16 hours.
  • a clean pipette is used to remove fraction(s) of interest, which are added to 15ml conical tube. These ‘purified’ EV samples are kept at 4°C.
  • lOx volume of PBS are added to purified EVs.
  • the ultracentrifuge is set for 200,000 x g and 4°C. Centrifuge and spun for 1 hour. The tubes are carefully removed from ultracentrifuge and the supernatant decanted.
  • the purified EVs are washed until all sample has been pelleted, lx PBS is added to the purified pellets, which are placed in a container. The container is placed on a shaker set at speed 70 at 4°C overnight or longer.
  • the ‘purified’ EV pellets are resuspended with additional sterile IxPBS.
  • the resuspended purified EV samples are stored at 4°C or at -80°C .
  • Example 8 Prevotella smEVs Lyophilate: DTH Efficacy
  • mice Female 5 week old C57BL/6 mice were purchased from Taconic Biosciences and acclimated at a vivarium for one week. Mice were primed with an emulsion of KLH and CFA (1 : 1) by subcutaneous immunization on day 0. Mice were orally gavaged daily with Prevotella histicola smEVs or dosed intraperitoneally with dexamethasone (positive control) at 1 mg/kg from days 6-8.
  • mice were anaesthetized with isoflurane, left ears were measured for baseline measurements with Fowler calipers and the mice were challenged intradermally with KLH in saline (10 pl) in the left ear and ear thickness measurements were taken at 24 hours.
  • smEVs Prevotella extracellular vesicles
  • the smEVs were lyophilized in excipient formula 7a.
  • the 24 hour ear measurement results are shown in Fig. 1. EVs made from Prevotella histicola and lyophilized in the excipient of formula 7a were tested in a dose range study with four doses (2E09, 2E07, 2E05, 2E03) for three days of dosing.
  • EVs from Prevotella histicola, Veillonella parvula, and Harryflintia acetispora bacteria are dried, such as by freeze drying or spray drying using one of the stocks provided in Table K.
  • Table K Stocks comprising excipients by relative concentration (%w:w).
  • Table L General conservative lyophilization cycle for EVs.
  • Example 10 Spray-Dried Powders of Prevotella histicola smEVs
  • Table P Stocks comprising excipients by relative concentration (%w:w)
  • Vian mannitol; Malt: maltodextrin; Tre: trehalose.
  • Spray drying was also performed using a stock that consisted of PEG6000-Mannitol- Trehalose (60:20:20). However, less dried product was recovered relative to other methods and was not analyzed further.
  • Prevotella histicola smEVs were spray dried or lyophilized in stock of formulation 7a (F7A) at two concentrations: 25X and 500X, with an inlet temperature of 130°C.
  • Example 11 Prevotella histicola smEVs and Anti-TNFa Antibody: DTH Efficacy
  • mice Female 5 week old C57BL/6 mice were purchased from Taconic Biosciences and acclimated at a vivarium for one week. Mice were primed with an emulsion of KLH and CFA (1: 1) by subcutaneous immunization on day 0. Mice were injected intraperitoneally on day 0, 3 and 6 with 3 mg/kg of anti-TNF alpha (Anti-TNFa) antibody (Clone: XT3.11 purchased from BioXCell) or equivalent isotype control (IgGl also purchased from BioXCell).
  • Anti-TNFa anti-TNF alpha
  • mice were orally gavaged daily with Prevotella histicola extracellular vesicles (smEVs) or dosed intraperitoneally with dexamethasone at 1 mg/kg from days 5-8. After dosing on day 8, mice were anaesthetized with isoflurane, left ears were measured for baseline measurements with Fowler calipers and the mice were challenged intradermally with KLH in saline (10 pl) in the left ear. Ear thickness measurements were taken at 24 hours.
  • smEVs Prevotella histicola extracellular vesicles
  • smEVs made from Prevotella histicola were tested alone or in combination with 3 mg/kg anti-TNF alpha antibody or equivalent isotype control (IgGl). Three mg/kg anti-TNF alpha antibody were also tested alone or with vehicle control.
  • the combination of Prevotella histicola smEVs with anti-TNF alpha was more efficacious than both Prevotella histicola smEVs alone and anti-TNF alpha antibody alone.
  • Example 12 Oral Dosing of Prevotella histicola smEVs or Harryflintia smEVs Does Not Inhibit the Immune System Under Non-Inflammatory Conditions
  • mice Two groups of mice were used in this study: ‘pre- immunization’ mice which were dosed with smEVs or controls prior to immunization, and ‘post- immunization’ mice, which were dosed with smEVs or controls after immunization. Starting on day -5, ‘pre-immunization’ mice were dosed daily for 4 days with PBS vehicle PO, dexamethasone (Img/kg, IP (intraperitoneally)), ox Prevotella smEVs or Harryflintia smEVs PO (orally).
  • mice On day 0, all mice were immunized by subcutaneous injection with KLH emulsified with Complete Freund’s Adjuvant (CFA). ‘Post-immunization’ mice were dosed daily starting on day 5 after immunization with sucrose vehicle, Prevotella smEVs or Harryflintia smEVs PO, or dexamethasone (1 mg/kg, IP) for 4 days. On day 8, baseline ear thickness was measured using calipers, then mice were challenged by intradermal ear injection with KLH. After 24 hours, the change in ear thickness was evaluated and compared to baseline measurements. Prevotella smEVs and Harryflintia smEVs were used at 2E9 parti cles/dose.
  • CFA Complete Freund’s Adjuvant
  • RESULTS AND CONCLUSION The results are shown in Fig. 3. Treatment with dexamethasone reduced inflammation in a subsequent DTK response. Dosing with Prevotella smEVs ox Harryflintia smEVs prior to immunization did not reduce inflammation that was subsequently induced by the intradermal ear challenge. Dosing with Prevotella smEVs or Harryflintia smEVs after immunization resulted in reduction of ear inflammation after the challenge. These data show that smEVs dosed in the absence of inflammation do not inhibit the immune response, but instead, resolve an ongoing inflammatory response.
  • Example 13 Antigen-Specific Response During Induction of Inflammation are Not Required for Resolution of Inflammation in a Subsequent KLH DTH Response by Prevotella smEVs
  • CFA Complete Freund’s adjuvant
  • IF A Incomplete Freud’s adjuvant
  • mice were immunized by subcutaneous injection with PBS emulsified with Complete or Incomplete Freund’s Adjuvant (CFA or IF A, respectively).
  • PBS vehicle PO per os
  • Prevotella smEVs PO mice were immunized with KLH emulsified with Complete Freund’s Adjuvant but no smEVs were administered after KLH- CFA immunization.
  • mice were challenged by intradermal ear injection with KLH. After 24 hours, the change in ear thickness was evaluated and compared to baseline measurements. Prevotella smEVs were used at 2E9 parti cles/dose.
  • RESULTS AND CONCLUSION The results are shown in Fig. 4. smEVs that were dosed only during induction of inflammation using CFA or IF A, prior to KLH sensitization, were able to inhibit the KLH DTH response after ear challenge. These data show that the induction of generalized inflammation in the absence of the relevant antigen used in the DTH (KLH) is sufficient for efficacy in the KLH DTH, and therefore the mechanism by which Prevotella smEVs induce their anti-inflammatory effect is antigen-independent. .
  • Example 14 Adoptive Transfer of CD4+ T cells from KLH-CFA Immunized Mice Dosed with Prevotella smEVs or Harryflintia smEVs Confers Immune Resolution in Recipient KLH-CFA Immunized Mice
  • brachial, axillary, and inguinal lymph nodes and spleens were harvested from donor mice and CD4+ T cells were enriched by negative selection on magnetic beads. Enriched cells were then counted, washed with PBS (300 x g, 10 mins, 4°C), and resuspended at 5x10 7 cells/mL in PBS. After enrichment, 5x10 6 CD4+ T cells were transferred into ‘recipient mice’ by IP injection. On day 12, baseline ear thickness was measured using calipers, then recipient mice were challenged by intradermal ear injection with KLH. After 24 hours, the change in ear thickness was evaluated and compared to baseline measurements. Prevotella smEVs and Harryflintia smEVs were used at 2E9 particles/dose.
  • TLR2 Signaling is Essential for the Generation of CD4+ T Cells That Can be Adoptively Transferred to Mediate Immune Resolution in a Recipient KLH DTH Model
  • TLR Toll-like Receptors
  • brachial, axillary, and inguinal lymph nodes and spleens were harvested from donor mice and CD4+ T cells were enriched by negative selection on magnetic beads. Enriched cells were then counted, washed with PBS (300 x g, 10 mins, 4°C), and resuspended at 5xl0 7 cells/mL in PBS. After enrichment, 5xlO 6 CD4+ T cells were transferred into ‘recipient mice’ by IP injection. On day 12, baseline ear thickness was measured using calipers, then recipient mice were challenged by intradermal ear injection with KLH. After 24 hours, the change in ear thickness was evaluated and compared to baseline measurements. Prevotella smEVs were used at 2E9 particles/dose.
  • CD4+ T cells isolated from mice treated with PBS vehicle and an anti-TLR2 blocking antibody did not inhibit the DTH response to KLH compared to CD4+ T cells from mice treated with PBS alone, or PBS with an isotype control antibody.
  • CD4+ T cells from mice treated with Prevotella smEVs and an isotype control antibody were efficacious in the model and able to inhibit ear inflammation after the intradermal KLH challenge, as previously shown.
  • CD4+ T cells transferred from mice that were dosed with Prevotella smEVs in the presence of anti-TLR2 antibody were not efficacious in recipient mice, and ear swelling was comparable to that of PBS-treated CD4+ T cells.
  • TLR2 signaling was essential for the Prevotella EV-induced generation of immune resolving CD4+ T cells as antibody-mediated blockade of this receptor in donor mice abrogated the efficacy of CD4+ T cells transferred from Prevotella EV-dosed mice.
  • Example 16 Adoptive Transfer of CD8+ T Cells from KLH-CFA Immunized Mice Dosed With Prevotella smEVs or Veillonella smEVs Does Not Confer Immune Resolution in Recipient KLH-CFA Immunized Mice
  • brachial, axillary, and inguinal lymph nodes and spleens were harvested from donor mice and CD8+ T cells were enriched by negative selection on magnetic beads. Enriched cells were then counted, washed with PBS (300 x g, 10 mins, 4°C), and resuspended at 5x10 7 cells/mL in PBS. After enrichment, 5x10 6 CD8+ T cells were transferred into ‘recipient mice’ by IP injection. On day 12, baseline ear thickness was measured using calipers, then recipient mice were challenged by intradermal ear injection with KLH. After 24 hours, the change in ear thickness was evaluated and compared to baseline measurements. Prevotella smEVs and Veillonella smEVs were used at 2x10 9 parti cles/dose.
  • RESULTS AND CONCLUSION The results are shown in Fig. 7. Unlike with CD4+ T cells, the adoptive cell transfer of CD8+ T cells from KLH-CFA inflamed Prevotella smEV- and Veillonella smEV-dosed donor mice into KLH-CFA inflamed recipient mice did not confer immune resolution of a KLH DTH model in the recipient mice. These data suggest that oral dosing of Prevotella smEVs and Veillonella smEVs induce a population of immune-resolving CD4+ T cells, but not CD8+ T cells, that can inhibit the DTH response in recipient mice.
  • Example 17 Preparation of a Solid Dosage Form Comprising Prevotella histicola smEVs
  • the Prevotella histicola smEVs in Table S are from strain Prevotella histicola Strain B 50329 (NRRL accession number B 50329).
  • Drug substance was prepared by lyophilization using excipient formula 7a.
  • HS DS high strength drug substance.
  • LS DS low strength drug substance.
  • LS DS was prepared by diluting HS DS lOx (using lyophilization excipients) before lyophilization.
  • Example 18 Preparation of a Solid Dosage Form Comprising Prevotella histicola smEVs
  • the Prevotella histicola smEVs in Table T are from strain Prevotella histicola Strain B 50329 (NRRL accession number B 50329).
  • Drug substance was prepared by lyophilization using excipient formula 7a.
  • HS DS high strength drug substance.
  • LS DS low strength drug substance.
  • LS DS was prepared by diluting HS DS lOx (using lyophilization excipients) before lyophilization.
  • the Prevotella histicola smEVs used to prepare drug substance (DS) are from strain Prevotella histicola Strain B 50329 (NRRL accession number B 50329).
  • Drug substance was prepared by lyophilization using excipient formula 7a (80% mannitol; 20% trehalose).
  • HS DS high strength drug substance.
  • LS DS low strength drug substance.
  • LS DS was prepared by diluting HS DS lOx (using lyophilization excipients) before lyophilization.
  • HS DS and LS DS were assessed in in vitro assays performed in human peripheral blood mononuclear cells (PBMCs), macrophages and dendritic cells.
  • PBMCs peripheral blood mononuclear cells
  • Cells were obtained from five donors; the five donors were run as biological replicates and data were expressed as average response.
  • Serial dilutions were performed: IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 , IxlO 7 , IxlO 8 , IxlO 9 , and 1x10 10 particles of both HS DS and LS DS were evaluated for their effects on cytokine secretion from the PBMCs, macrophages and dendritic cells. Similar trends were seen across all three cell populations.
  • HS DS and LS DS were able to induce IL- 10, IL-27, IL-6, IP- 10, and TNFa from all three cell populations. Dendritic cells produced low levels of IL-27 and IP- 10 in response to both DS. [716] HS DS and LS DS were assessed in in vitro assays performed in U937.
  • Antiviral responses are activated rapidly after viral infection in order to control and prevent dissemination of the virus.
  • Virus infection results in two general types of immune response. The first is a rapid-onset innate immune response against the virus, which involves the synthesis of Type 1 interferons and the stimulation of Natural Killer (NK) cells. If the infection proceeds beyond the first few rounds of viral replication, the innate immune response will trigger the adaptive immune response.
  • the adaptive immune response itself has two components, the humoral response (the synthesis of virus-specific antibodies by B lymphocytes) and the cell- mediated response (the synthesis of specific CD8+ cytotoxic T lymphocytes that kill infected cells).
  • T-cell levels do not tend to fade as quickly as antibodies after an infection or vaccination. And because T cells can recognize many more sites along the spike protein than can antibodies, they are better able to recognize mutated variants. Thus, having an intact T cell response is likely important for both mounting a successful anti-viral response as well as maintaining immune memory of viruses previously encountered.
  • Prevotella histicola EVs are obtained from Prevotella Strain B. Prevotella histicola EVs are formulated as a lyophilized powder. Preclinically, Prevotella histicola EVs are administered orally and are not systemically bioavailable. Prevotella histicola EVs exert their anti-inflammatory effects on peripheral tissue through engagement of cells of the intestine, including intestinal epithelial cells and immune cells in the lamina intestinal.
  • mice were immunized by subcutaneous injection with Keyhole Limpet Hemocyanin (KLH) emulsified with Complete Freund’s Adjuvant.
  • KLH Keyhole Limpet Hemocyanin
  • mice were dosed for 15 days with vehicle (phosphate buffered saline) and Prevotella histicola smEVs by oral gavage, or dexamethasone by intraperitoneal injection.
  • mice were challenged by intradermal ear injection with KLH. The DTH response was evaluated 24 hours post-challenge.
  • Interferons are cytokines that are secreted by host cells in response to virus infection and are the body's first line of antiviral defense. By inducing the expression of hundreds of IFN-stimulated genes, several of which have antiviral functions, IFNs block virus replication at many levels. There are two main types of IFN, type I and type II. Type I or 'viral' IFNs include IFN-a, IFN-0, IFN-co and IFN-T; type II IFN is IFN-y.
  • IFN-a and IFN-0 which are the best-characterized type I IFNs
  • IFN-y is produced only by certain cells of the immune system, including natural killer (NK) cells, CD4+ T helper 1 (TH1) cells and CD8+ cytotoxic T cells.
  • Drugs with broad immuno- suppressant activity such as corticosteroids, can inhibit the production of IFNs, which can lead to delayed viral clearance and adverse outcomes in various viral pneumonias. Therefore, a therapeutic approach that ameliorates inflammation without blocking the anti-viral IFN immune response would have an optimal safety profile.
  • spleen cells were isolated and then restimulated in vitro with PMA and ionomycin or polyinosinic-poly cytidylic acid (poly I:C).
  • PMA activates protein kinase C
  • ionomycin is a calcium ionophore
  • stimulation with these compounds bypasses the T cell membrane receptor complex and will lead to activation of several intracellular signaling pathways, resulting in T cell activation and production of a variety of cytokines.
  • Poly I:C is a molecule that mimics viral double-stranded RNA, and a potent ligand for Toll-like receptor 3, which induces interferon-alpha and interferon-beta from immune cells.
  • CD4 T cells help B cells to produce antibodies and help CD8+ T cells to kill virus-infected cells.
  • One of the dominant cytokines produced by T cells is interferon gamma, a key player in controlling viral infection.
  • One report has described a higher proportion of IFN-y- producing T helper 1 (THl)-like cells in patients with moderate disease than in patients with severe disease.
  • CD4+ T cells specific for the SARS-CoV-2 spike protein have been identified in acute infection and have a TH1 cell cytokine profile.
  • TH1 T cells In order to determine whether treatment with Prevotella histicola EVs reduces the numbers of TH1 T cells or inhibits their production of cytokines such as IFN-y and TNF, CD4 T cells were isolated from spleens and lymph nodes after a DTH response, and TH1 T cell numbers were determined by flow cytometry.
  • TH1 T cells which are defined by their expression of the transcription factor T-bet, were of a similar proportion (roughly 2%) of total CD4 T cells in mice treated with either vehicle ox Prevotella histicola EVs (Fig. 10A). Although PMA/ionomycin-stimulated CD4 T cells from mice treated with either vehicle or Prevotella histicola EVs produced the same amount of IFN- y (Fig. 10B), those from mice treated with Prevotella histicola EVs did produce about 25% less TNF than those from vehicle-treated mice (Fig. 10C).
  • DCs Human dendritic cells exposed to Prevotella histicola EVs enhance the production of viral defense cytokines in a mixed DC:CD8 T cell co-culture:
  • CD8+ T cells contribute to protective immune responses against SARS-CoV-2 in patients with CO VID-19.
  • CD8+ T cells play an essential role in controlling viral infection by killing virus-infected cells and producing effector cytokines such as IFN- y.
  • DCs that present viral antigens to CD8 T cells also produce cytokines such as IL- 12, TNF, and IL-6, which then induce the differentiation and activation of IFN- y- producing CD8 T cells.
  • the CEF peptide pool is composed of peptides from Cytomegalovirus, Epstein Bar virus, and Influenza virus, pathogens to which the majority of the human population has been exposed. Controls used were DCs only, DCs + T Cells only, and DCs + T cells + CEF peptide. After 24 hours of stimulation with CEF peptide, DC-CD8+ T cell supernatants were collected, and cytokines produced by both DCs and CD8 T cells were measured.
  • Prevotella histicola EVs do not broadly impair either innate or adaptive immune responses.
  • Prevotella histicola EVs are orally delivered and gut restricted, and its systemic effects are exerted through local interactions with cells of the gastro-intestinal tract.
  • Anti-viral responses such as CD4 and CD8 T cell production of IFN- y, innate anti-viral production of IFN-oc and IFN- , and the generation of effector T cell populations are all preserved after treatment with Prevotella histicola EVs.
  • Example 21 Extracellular vesicles isolated from Prevotella histicola (Prevotella smEVs) induce a population of inflammation resolving CD4+ T cells
  • Prevotella histicola EVs were isolated from broth culture supernatants using tangential flow filtration and gradient purification into PBS for oral dosing.
  • Prevotella histicola EV particle size and counts were estimated using Nanoparticle Tracking Analysis (NTA) and prepared to the relative particle concentration for each study.
  • NTA Nanoparticle Tracking Analysis
  • KLH keyhole limpet hemocyanin
  • Inflammation is often controlled clinically by administration of systemic steroidal immune-suppressive drugs such as dexamethasone.
  • systemic steroidal immune-suppressive drugs such as dexamethasone.
  • dexamethasone To compare suppression of the immune system between a glucocorticoid steroid, dexamethasone, and Prevotella histicola EVs, we dosed mice either pre- or post-KLH immunization with vehicle, dexamethasone, or Prevotella histicola EVs. Unlike dexamethasone, Prevotella histicola EVs did not exert any prophylactic suppression of immune responses, only displaying efficacy when administered following immunization (Fig. 12b), demonstrating that Prevotella histicola EVs control - or resolve - an aberrant inflammatory response rather than suppressing the immune system.
  • Prevotella histicola EVs were also compared to additional standard of care systemically administered anti-inflammatory drugs: dexamethasone, tofacitinib, and anti-TNF.
  • Prevotella histicola EVs performed comparably to all other drugs, despite being orally dosed (Fig. 12c).
  • mice were therefore immunized with PBS emulsified with CFA and subsequently dosed with vehicle, dexamethasone or Prevotella histicola EVs. Forty-eight hours after dosing, mice were then immunized with KLH emulsified in CFA , challenged 5 days later, and ear swelling was determined after 24 hours. We found that, firstly, Prevotella histicola EVs could reduce an antigen-specific, subsequent DTH response once dosed proceeding an antigen-independent immunization.
  • Prevotella histicola EVs remain gut- restricted and passes through the intestinal system within 24 hours. Orally dosed Prevotella histicola EVs offered comparable efficacy to systematically administered anti-inflammatory drugs.
  • Prevotella histicola EVs remain gut restricted and do not disseminate systemically, we used biodistribution imaging of murine organs following oral or intravenous dosing with fluorescently labeled Prevotella histicola EVs.
  • Oral Prevotella histicola EVs remained gut restricted and was passed within 24 hours, whereas intravenous injection led to accumulation of Prevotella histicola EVs in the liver, kidneys, and lungs. Therefore, Prevotella histicola EVs remain gut restricted whilst exhibiting resolution of inflammation within the periphery.
  • Prevotella histicola EVs require lymphocyte homing to the mesenteric lymph node to resolve peripheral inflammation. Restriction to the gut suggests that there exists a link between the intestine and the peripheral immune system to mediate the effects of Prevotella histicola EVs.
  • the major hub of communication between the gut and the immune system is the mesenteric lymph node (mLN), where peripheral lymphocytes home to and initiate appropriate responses under the control of tissue-migrating myeloid cells 2 .
  • mLN mesenteric lymph node
  • peripheral lymphocytes home to and initiate appropriate responses under the control of tissue-migrating myeloid cells 2 .
  • mice were immunized, treated with anti-CD62L and anti-o.407 integrin, then dosed with vehicle or Prevotella histicola EVs.
  • Prevotella histicola EVs could not reduce ear inflammation (Fig. 13e), suggesting that immune cell interactions within the mLN relay signals induced by Prevotella histicola EVs in the intestine to circulating lymphocytes.
  • Prevotella histicola EVs induce a resolution of peripheral inflammation in an antigenindependent manner through CD4+ T cells.
  • the long-lived efficacy and requirement of lymphocyte homing of Prevotella histicola EVs suggests a mechanism of action via modification of peripheral immune cells, specifically lymphocytes, within the mLN.
  • the adaptive immune system comprises professional antigen-presenting cells and lymphocytes to drive antigenspecific responses in either pro- or anti-inflammatory manners. It was unclear whether Prevotella histicola EVs were inducing a regulatory function in KLH-specific lymphocytes. Our data did not rule out an antigen-specific response to the EVs themselves.
  • 0T-II/Rag2 /_ mice only produce CD4+ T cells that recognize the MHC class Il-restricted ovalbumin peptide ISQAVHAAHAEINEAGR and lack CD8+ T cells and B cells and thus are incapable of recognizing and responding to Prevotella histicola EVs in an adaptive manner.
  • Prevotella histicola EVs were efficacious in the 0T-II/Rag2 / " system (Fig. 14a), showing that adaptive recognition of EVs was not required for efficacy.
  • the absence of CD8+ T cells and B cells demonstrates that neither cell type is required, indicating that the major cell type involved in inflammation resolution is CD4+ T cells.
  • Prevotella histicola EVs induce functional changes to peripheral CD4+ T cells towards a pro-resolving utility.
  • CD4+ T cells are a diverse subset of lymphocytes that have recently been shown to be extremely plastic with regards to both Th skewing 16-18 and pro- and antiinflammatory function 19 .
  • Our data suggested that, despite antigen-independence, Prevotella histicola EVs induced functional changes in peripheral CD4+ T cells.
  • ACT adoptive cell transfer
  • TLR2 Stimulation of TLR2 is necessary but not sufficient for Prevotella histicola EV- mediated resolution of peripheral inflammation.
  • the small intestinal lamina basement membrane comprises endothelial and immune cells that express several evolutionarily conserved Toll-like Receptors (TLR), recognizing a vast array of molecular patterns present on bacteria and viruses 20 .
  • TLR2 21 One of the most abundant TLRs expressed in the small intestine is TLR2 21 .
  • TLR2 21 One of the most abundant TLRs expressed in the small intestine.
  • TLR2 21 Using a HEK TLR reporter cell line, we showed that Prevotella histicola EVs could stimulate heterodimers formed by TLR2, TLR2/1 and TLR2/6 (Fig. 15a) in a dose-response dependent manner.
  • Prevotella histicola EVs generate CD4+ T cells that can resolve inflammation but do not secrete IL- 10, rather induce its release.
  • Our data have demonstrated a stepwise progression of Prevotella histicola EV-mediated modification of peripheral CD4+ T cells, requiring TLR2 signaling within the gut and gut-associated lymphoid tissue (GALT), the interaction of gut- resident/associated immune cells with CD4+ T cells in the mLN, and the dissemination of modified CD4+ T cells that can reduce inflammation.
  • GALT gut and gut-associated lymphoid tissue
  • IL- 10 is a key mediator of immune regulation, so we therefore asked whether this was involved in the control of peripheral inflammation.
  • Antibody blockade of the IL- 10 receptor (IL- 10R), which recognizes IL- 10 and initiates signaling cascades and reduces cellular transcriptional output within immune cells to inhibit their pro-inflammatory effector functions 22 , inhibited the efficacy of Prevotella histicola EVs (Fig. 15e), showing that resolution of inflammation was mediated by signaling through the IL-10R.
  • modified CD4+ T cells are not the source of IL-10, but possibly stimulate the release of IL- 10 in other cells.
  • IL-10 WT CD4+ T cells were transferred from Prevotella histicola EV-treated donors into IL- 10 KO recipients, these cells were unable to reduce ear swelling (Fig. 15h).
  • IL- 10 is not required for the generation of pro-resolving CD4+ T cells and that these cells are not the source of IL- 10, rather suggesting induction of release from other cells.
  • Prevotella histicola EVs induce functional changes in conventional CD4+CD25- T cells to resolve peripheral inflammation.
  • peripheral CD4+ T cells are directly involved in Prevotella histicola EV-mediated inflammation resolution.
  • immune regulation is attributed to regulatory T cells (Treg) that primarily control immune responses through the release of IL- 10 23,24 .
  • Treg regulatory T cells
  • adoptive transfer of IL-10-deficient CD4+ T cells can control peripheral inflammation, suggesting that inflammation resolution may be controlled by induction of a novel function in conventional (i.e., FoxP3-) CD4+ T cells.
  • Regulatory CD4+ T cells can be largely defined by high expression of CD25, and this can be used to positively enrich Tregs from CD4+ T cell populations.
  • CD25 Using magnetic enrichment, we isolated total CD4+ T cells and from this mixed population we enriched CD4+CD25+ Tregs to adoptively transfer in our ACT DTH model, also transferring CD4+CD25- conventional T cells (Tcon) as internal controls.
  • Tcon conventional T cells
  • the transfer of purified Tregs from vehicle treated mice conferred some efficacy.
  • the transfer of total CD4+ T cells, CD4+CD25- Tcons, and CD4+CD25+ Tregs from Prevotella histicola EV-treated mice all conferred significant resolution of inflammation.
  • Prevotella histicola EVs engage TLR2 in the gut to induce a population of pro-resolving CD4+ T cells within the mLN that traffic to the periphery and reduce inflammation through induction of IL- 10. Importantly, Prevotella histicola EVs enable reduction of peripheral inflammation whilst remaining gut restricted.
  • T helper 17 cells require T cell receptor ligation in the context of Tolllike receptor-activated monocytes. Proc National Acad Sci 104, 17034-17039 (2007).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Communicable Diseases (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des vésicules extracellulaires (VE) anti-inflammatoires, des solutions et des formes séchées (et des compositions thérapeutiques de celles-ci) de vésicules extracellulaires (VE) anti-inflammatoires qui peuvent être utiles en tant qu'agents thérapeutiques, et des méthodes d'utilisation de celles-ci.
PCT/US2023/011420 2022-01-25 2023-01-24 Compositions de vésicules extracellulaires et méthodes d'utilisation WO2023146843A1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US202263302862P 2022-01-25 2022-01-25
US63/302,862 2022-01-25
PCT/US2022/017446 WO2022182707A1 (fr) 2021-02-26 2022-02-23 Compositions et procédés pour réduire l'expression de cytokine
USPCT/US2022/017446 2022-02-23
US202263334407P 2022-04-25 2022-04-25
US63/334,407 2022-04-25
US202263381047P 2022-10-26 2022-10-26
US63/381,047 2022-10-26

Publications (1)

Publication Number Publication Date
WO2023146843A1 true WO2023146843A1 (fr) 2023-08-03

Family

ID=85381405

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/011420 WO2023146843A1 (fr) 2022-01-25 2023-01-24 Compositions de vésicules extracellulaires et méthodes d'utilisation

Country Status (1)

Country Link
WO (1) WO2023146843A1 (fr)

Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4775536A (en) 1986-02-24 1988-10-04 Bristol-Myers Company Enteric coated tablet and process for making
US5047258A (en) 1989-07-14 1991-09-10 Sterling Drug Inc. Aqueous spray-coating process
US5292522A (en) 1989-06-20 1994-03-08 Rohm Gmbh Aqueous film coating agent for solid medicaments
US6312728B1 (en) 1998-07-07 2001-11-06 Cascade Development, Inc. Sustained release pharmaceutical preparation
US6555124B1 (en) 1996-08-01 2003-04-29 Basf Aktiengesellschaft Use of (meth)acrylic acid copolymers to increase the permeability of mucous membranes
US6623759B2 (en) 1996-06-28 2003-09-23 Astrazeneca Ab Stable drug form for oral administration with benzimidazole derivatives as active ingredient and process for the preparation thereof
US6638534B1 (en) 1998-07-28 2003-10-28 Tanabe Seiyaku Co., Ltd. Preparation capable of releasing drug at target site in intestine
US20040028737A1 (en) 2002-08-12 2004-02-12 Kopran Research Laboratories Limited Enteric coated stable oral pharmaceutical composition of acid unstable drug and process for preparing the same
WO2005044240A2 (fr) 2003-10-31 2005-05-19 Dexcel, Ltd. Formulation stable contenant du lansoprazole
US20050271778A1 (en) 2002-12-20 2005-12-08 Roehm Gmbh & Co., Kg Method for producing coated pharmaceuticals and food supplements with concentration gradients in the coating thereof
US20060210631A1 (en) 2005-03-21 2006-09-21 Patel Ashish A Multi-particulate, modified-release composition
US20080200482A1 (en) 2005-07-12 2008-08-21 Evonik Roehm Gmbh Use of a Partially Neutralized, Anionic (Meth)Acrylate Copolymer as a Coating for the Production of a Medicament Releasing Active Substance at Reduced Ph Values
WO2011053653A2 (fr) * 2009-10-30 2011-05-05 Mayo Foundation For Medical Education And Research Préparations à base de prevotella histicola et traitement d'états auto-immuns
US9233074B2 (en) 2013-03-01 2016-01-12 Bpsi Holdings, Llc Delayed release film coatings containing calcium silicate and substrates coated therewith
WO2019051381A1 (fr) * 2017-09-08 2019-03-14 Evelo Biosciences, Inc. Vésicules extracellulaires provenant de prevotella
WO2019157003A1 (fr) 2018-02-06 2019-08-15 Evelo Biosciences, Inc. Compositions et méthodes pour traiter le cancer et des troubles immunitaires au moyen de bacteries veillonelles
WO2020252149A1 (fr) * 2019-06-11 2020-12-17 Evelo Biosciences, Inc. Vésicules extracellulaires microbiennes traitées
WO2020257248A1 (fr) * 2019-06-17 2020-12-24 Mayo Foundation For Medical Education And Research Préparations de prevotella et traitement de la bronchopneumopathie chronique obstructive (bpco) et d'autres affections pulmonaires
WO2020257390A1 (fr) * 2019-06-21 2020-12-24 Evelo Biosciences, Inc. Compositions et procédés de traitement d'une affection médiée par th2 à l'aide de prevotella
WO2021174041A1 (fr) * 2020-02-26 2021-09-02 Evelo Biosciences, Inc. Compositions et méthodes pour réduire l'expression des cytokines
WO2021252860A2 (fr) 2020-06-11 2021-12-16 Evelo Biosciences, Inc. Compositions et méthodes de traitement de maladies et de troubles à l'aide d'harryflintia acetispora
WO2022132738A1 (fr) 2020-12-14 2022-06-23 Evelo Biosciences, Inc. Préparations de vésicules extracellulaires

Patent Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4775536A (en) 1986-02-24 1988-10-04 Bristol-Myers Company Enteric coated tablet and process for making
US5292522A (en) 1989-06-20 1994-03-08 Rohm Gmbh Aqueous film coating agent for solid medicaments
US5047258A (en) 1989-07-14 1991-09-10 Sterling Drug Inc. Aqueous spray-coating process
US6623759B2 (en) 1996-06-28 2003-09-23 Astrazeneca Ab Stable drug form for oral administration with benzimidazole derivatives as active ingredient and process for the preparation thereof
US6555124B1 (en) 1996-08-01 2003-04-29 Basf Aktiengesellschaft Use of (meth)acrylic acid copolymers to increase the permeability of mucous membranes
US6312728B1 (en) 1998-07-07 2001-11-06 Cascade Development, Inc. Sustained release pharmaceutical preparation
US6638534B1 (en) 1998-07-28 2003-10-28 Tanabe Seiyaku Co., Ltd. Preparation capable of releasing drug at target site in intestine
US20040028737A1 (en) 2002-08-12 2004-02-12 Kopran Research Laboratories Limited Enteric coated stable oral pharmaceutical composition of acid unstable drug and process for preparing the same
US20050271778A1 (en) 2002-12-20 2005-12-08 Roehm Gmbh & Co., Kg Method for producing coated pharmaceuticals and food supplements with concentration gradients in the coating thereof
WO2005044240A2 (fr) 2003-10-31 2005-05-19 Dexcel, Ltd. Formulation stable contenant du lansoprazole
US20060210631A1 (en) 2005-03-21 2006-09-21 Patel Ashish A Multi-particulate, modified-release composition
US20080200482A1 (en) 2005-07-12 2008-08-21 Evonik Roehm Gmbh Use of a Partially Neutralized, Anionic (Meth)Acrylate Copolymer as a Coating for the Production of a Medicament Releasing Active Substance at Reduced Ph Values
WO2011053653A2 (fr) * 2009-10-30 2011-05-05 Mayo Foundation For Medical Education And Research Préparations à base de prevotella histicola et traitement d'états auto-immuns
US9233074B2 (en) 2013-03-01 2016-01-12 Bpsi Holdings, Llc Delayed release film coatings containing calcium silicate and substrates coated therewith
WO2019051381A1 (fr) * 2017-09-08 2019-03-14 Evelo Biosciences, Inc. Vésicules extracellulaires provenant de prevotella
WO2019157003A1 (fr) 2018-02-06 2019-08-15 Evelo Biosciences, Inc. Compositions et méthodes pour traiter le cancer et des troubles immunitaires au moyen de bacteries veillonelles
WO2020252149A1 (fr) * 2019-06-11 2020-12-17 Evelo Biosciences, Inc. Vésicules extracellulaires microbiennes traitées
WO2020257248A1 (fr) * 2019-06-17 2020-12-24 Mayo Foundation For Medical Education And Research Préparations de prevotella et traitement de la bronchopneumopathie chronique obstructive (bpco) et d'autres affections pulmonaires
WO2020257390A1 (fr) * 2019-06-21 2020-12-24 Evelo Biosciences, Inc. Compositions et procédés de traitement d'une affection médiée par th2 à l'aide de prevotella
WO2021174041A1 (fr) * 2020-02-26 2021-09-02 Evelo Biosciences, Inc. Compositions et méthodes pour réduire l'expression des cytokines
WO2021252860A2 (fr) 2020-06-11 2021-12-16 Evelo Biosciences, Inc. Compositions et méthodes de traitement de maladies et de troubles à l'aide d'harryflintia acetispora
WO2022132738A1 (fr) 2020-12-14 2022-06-23 Evelo Biosciences, Inc. Préparations de vésicules extracellulaires

Non-Patent Citations (72)

* Cited by examiner, † Cited by third party
Title
"Guide to Huge Computers", 1994, ACADEMIC PRESS
A. FRIAS ET AL., MICROB ECOL, vol. 59, 2010, pages 476 - 486
ACHTMAN MWAGNER M.: "Microbial diversity and the genetic nature of microbial species", NAT. REV. MICROBIOL., vol. 6, 2008, pages 431 - 440, XP037115178, DOI: 10.1038/nrmicro1872
BALAKRISHNAN, B. ET AL.: "Prevotella histicola Protects From Arthritis by Expansion of Allobaculum and Augmenting Butyrate Production in Humanized Mice", FRONT IMMUNOL, vol. 12, 2021, pages 609644
BERTELSEN, A.ELBORN, S. J.SCHOCK, B. C.: "Toll like Receptor signalling by Prevotella histicola activates alternative NF-oB signalling in Cystic Fibrosis bronchial epithelial cells compared to P. aeruginosa", PLOS ONE, vol. 15, 2020, pages 0235803
BLASTPBLASTNFASTA ATSCHUL, S. F. ET AL., J MOLEC BIOL, vol. 215, 1990, pages 403
BOSE, S.AGGARWAL, S.SINGH, D. V.ACHARYA, N.: "Extracellular vesicles: An emerging platform in gram-positive bacteria", MICROB CELL, vol. 7, 2020, pages 312 - 322
BRIAUD, P.CARROLL, R. K.: "Extracellular Vesicle Biogenesis and Functions in Gram-Positive Bacteria", INFECT IMMUN, vol. 88, 2020
BRINKMAN, C. C.PESKE, J. D.ENGELHARD, V. H.: "Peripheral Tissue Homing Receptor Control of Naive, Effector, and Memory CD8 T Cell Localization in Lymphoid and Non-Lymphoid Tissues", FRONT IMMUNOL, vol. 4, 2013, pages 241
CARDING ET AL.: "Dysbiosis of the gut microbiota in disease", MICROB. ECOL. HEALTH DIS., vol. 26, no. 10, 2015
CARILLO ET AL., SIAM J APPLIED MATH, vol. 48, 1988, pages 1073
CHAUDHRY, A. ET AL.: "Interleukin-10 Signaling in Regulatory T Cells Is Required for Suppression of Thl7 Cell-Mediated Inflammation", IMMUNITY, vol. 34, 2011, pages 566 - 578, XP028407954, DOI: 10.1016/j.immuni.2011.03.018
CITI, S.: "Intestinal Barriers protect against disease", SCIENCE, vol. 359, 2018, pages 1098 - 99
CLAESSON MJWANG QO'SULLIVAN OGREENE-DINIZ RCOLE JRROSS RPO'TOOLE PW.: "Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions", NUCLEIC ACIDS RES, vol. 3 8, 2010, pages 200
COX, C. A. ET AL.: "Both Th1 and Th17 Are Immunopathogenic but Differ in Other Key Biological Activities", J IMMUNOL, vol. 180, 2008, pages 7414 - 7422, XP055050390, DOI: 10.4049/jimmunol.180.11.7414
CVJETKOVIC ET AL., SCI. REP., vol. 6, 2016, pages 36338
DETTMER ET AL., MASS SPECTROM REV, vol. 26, no. 1, 2007, pages 51 - 78
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, no. I, 1984, pages 387
ERICKSON ET AL., MOLECULAR CELL, vol. 65, no. 2, 2017, pages 361 - 70
ESTERHAZY, D. ET AL.: "Compartmentalized gut lymph node drainage dictates adaptive immune responses", NATURE, vol. 569, 2019, pages 126 - 130, XP036771228, DOI: 10.1038/s41586-019-1125-3
EVANS, H. G.SUDDASON, T.JACKSON, I.TAAMS, L. S.LORD, G. M.: "Optimal induction of T helper 17 cells in humans requires T cell receptor ligation in the context of Toll-like receptor-activated monocytes", PROC NATIONAL ACAD SCI, vol. 104, 2007, pages 17034 - 17039, XP055399018, DOI: 10.1073/pnas.0708426104
G. NORHEIM ET AL., PL S ONE, vol. 10, no. 9, 2015, pages 0134353
G. NORHEIM ET AL., PLOS ONE, vol. 10, no. 9, 2015, pages 0134353
GORFU, G.RIVERA-NIEVES, J.LEY, K.: "Role of β7 Integrins in Intestinal Lymphocyte Homing and Retention", CURRMOLMED, vol. 9, 2009, pages 836 - 850
HONG, S.-W. ET AL.: "Immune tolerance of food is mediated by layers of CD4+ T cell dysfunction", NATURE, vol. 1-7, 2022
HOOKSO'MALLEY: "Dysbiosis and its discontents", vol. 8, October 2017, AMERICAN SOCIETY FOR MICROBIOLOGY
I. MACDONALDM. KUEHN., J BACTERIAL, vol. 188, no. 13, pages 5385 - 5392
JEPPE MADURA LARSEN: "The immune response to Prevotella bacteria in chronic inflammatory disease", IMMUNOLOGY, WILEY-BLACKWELL PUBLISHING LTD, GB, vol. 151, no. 4, 20 June 2017 (2017-06-20), pages 363 - 374, XP071277122, ISSN: 0019-2805, DOI: 10.1111/IMM.12760 *
JEPPESEN ET AL., CELL, vol. 177, 2019, pages 428
JOHNSON, J. L.JONES, M. B.COBB, B. A.: "Polysaccharide A from the Capsule of Bacteroides fragilis Induces Clonal CD4+ T Cell Expansion*", J BIOI CHEM, vol. 290, 2015, pages 5007 - 5014, XP055617980, DOI: 10.1074/jbc.M114.621771
JOHNSON, J. L.JONES, M. B.COBB, B. A.: "Polysaccharide-experienced effector T cells induce IL-10 in FoxP3+ regulatory T cells to prevent pulmonary inflammation", GLYCOBIOLOGY, vol. 28, 2017, pages 50 - 58
JONES, M. B. ET AL.: "CD45Rb-low effector T cells require IL-4 to induce IL-10 in FoxP3 Tregs and to protect mice from inflammation", PLOS ONE, vol. 14, 2019, pages 0216893
JUN, J. C. ET AL.: "T cell-intrinsic TLR2 stimulation promotes IL-10 expression and suppressive activity by CD45RbHi T cells", PLOS ONE, vol. 12, 2017, pages 0180688
KAWASAKI, T.KAWAI, T.: "Toll-Like Receptor Signaling Pathways", FRONT IMMUNOL, vol. 5, 2014, pages 461
KIESELBACHOSCARSSON, DATA BRIEF, vol. 10, February 2017 (2017-02-01), pages 426 - 431
KONSTANTINIDIS KTRAMETTE ATIEDJE JM: "The bacterial species definition in the genomic era", PHILOS TRANS R SOC LOND B BIOL SCI, vol. 361, 2006, pages 1929 - 1940
LEE, Y. K. ET AL.: "Late Developmental Plasticity in the T Helper 17 Lineage", IMMUNITY, vol. 30, 2009, pages 92 - 107, XP055190410, DOI: 10.1016/j.immuni.2008.11.005
LEEKHA ET AL.: "General Principles of Antimicrobial Therapy", MAYO CLIN PROC, vol. 86, no. 2, 2011, pages 156 - 167, XP055372896, DOI: 10.4065/mcp.2010.0639
LEVY ET AL.: "Dysbiosis and the Immune System", NATURE REVIEWS IMMUNOLOGY, vol. 17, April 2017 (2017-04-01), pages 219, XP055953368, DOI: 10.1038/nri.2017.7
LIU ET AL., J. BACTERIOLOGY, vol. 192, no. 11, 2010, pages 2852 - 60
LIU, H. ET AL.: "Bacterial extracellular vesicles as bioactive nanocarriers for drug delivery: Advances and perspectives", BIOACT MATER, vol. 14, 2021, pages 169 - 181
LUCKHEERAM, R. V.ZHOU, R.VERMA, A. D.XIA, B.: "CD4+T Cells: Differentiation and Functions", CLIN DEV IMMUNOL 2012, 2012, pages 925135
LYNCH ET AL.: "The Human Microbiome in Health and Disease", N. ENGL. J. MED, vol. 375, 2016, pages 2369 - 79
MARIETTA, E. V. ET AL.: "Suppression of Inflammatory Arthritis by Human Gut-Derived Prevotella histicola in Humanized Mice", ARTHRITIS RHEUMATOL, vol. 68, 2016, pages 2878 - 2888, XP055665204, DOI: 10.1002/art.39785
MATTEOLI, G. ET AL.: "Gut CD103+ dendritic cells express indoleamine 2,3-dioxygenase which influences T regulatory/T effector cell balance and oral tolerance induction", GUT, vol. 59, 2010, pages 595
MOWAT, A. M.AGACE, W. W.: "Regional specialization within the intestinal immune system", NAT REV IMMUNOL, vol. 14, 2014, pages 667 - 685
MURRAY, P. J.: "The primary mechanism of the IL-10-regulated antiinflammatory response is to selectively inhibit transcription", PROC NATIONAL ACAD SCI, vol. 102, 2005, pages 8686 - 8691
O'GARRA, A.VIEIRA, P. L.VIEIRA, P.GOLDFELD, A. E.: "IL-10-producing and naturally occurring CD4+ Tregs: limiting collateral damage", J CLIN INVEST, vol. 114, 2004, pages 1372 - 1378, XP055402442
OTSUBO, K. ET AL.: "Identification of FOXP3 -negative regulatory T-like (CD4+CD25+CD1271ow) cells in patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome", CLIN IMMUNOL, vol. 141, 2011, pages 111 - 120, XP028298105, DOI: 10.1016/j.clim.2011.06.006
PEARSON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 2444
PRICE, A. E. ET AL.: "A Map of Toll-like Receptor Expression in the Intestinal Epithelium Reveals Distinct Spatial, Cell Type-Specific, and Temporal Patterns", IMMUNITY, vol. 49, 2018, pages 560 - 575
ROBERTS ET AL., CURR PROTOC MOL BIOL, vol. 30, 2012, pages 1 - 24
ROCAMORA-REVERTE, L.MELZER, F. L.WURZNER, R.WEINBERGER, B.: "The Complex Role of Regulatory T Cells in Immunity and Aging", FRONT IMMUNOL, vol. 11, 2021, pages 616949
S. BIN PARK ET AL., PLOS ONE, vol. 6, no. 3, 2011, pages 17629
S. STUBBS ET AL., LETTERS IN APPLIED MICROBIOLOGY, vol. 29, 1999, pages 31 - 36
SAARELA ET AL., J. APPLIED MICROBIOLOGY., vol. 99, 2005, pages 1330 - 1339
SANDRINI ET AL., BIO-PROTOCOL, vol. 4, no. 21, 2014, pages 1287
SCHOCH ET AL.: "Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi", PNAS, vol. 109, 2012, pages 6241 - 6246, XP055194048, DOI: 10.1073/pnas.1117018109
SEIBERT ET AL.: "Chemical Engineering in the Pharmaceutical Industry: R&D to Manufacturing", 2011, article "MILLING OPERATIONS IN THE PHARMACEUTICAL INDUSTRY"
SHAHI, S. K. ET AL.: "Prevotella histicola, A Human Gut Commensal, Is as Potent as COPAXONE® in an Animal Model of Multiple Sclerosis", FRONT IMMUNOL, vol. 10, 2019, pages 462, XP055741469, DOI: 10.3389/fimmu.2019.00462
SHOUVAL, D. S. ET AL.: "Chapter Five Interleukin 10 Receptor Signaling Master Regulator of Intestinal Mucosal Homeostasis in Mice and Humans", ADV IMMUNOL, vol. 122, 2014, pages 177 - 210
SHREINER, A. B.KAO, J. Y.YOUNG, V. B.: "The gut microbiome in health and in disease", CURR OPIN GASTROEN, vol. 31, 2015, pages 69 - 75, XP055692938, DOI: 10.1097/MOG.0000000000000139
SRINIVASAN ET AL.: "TEER measurement techniques for in vitro barrier model systems", J. LAB. AUTOM., vol. 20, 2015, pages 107 - 126, XP055560328, DOI: 10.1177/2211068214561025
THEIN ET AL., J. PROTEOME RES., vol. 9, no. 12, 2010, pages 6135 - 6147
VIJAY-KUMAR, M.CHASSAING, B.KUMAR, M.BAKER, M.SINGH, V.: "Mammalian gut immunity", BIOMED J, vol. 37, 2014, pages 246
VILDHEDE ET AL., DRUG METABOLISM AND DISPOSITION, 8 February 2018 (2018-02-08)
VISEKRUNA, A. ET AL.: "Intestinal development and homeostasis require activation and apoptosis of diet-reactive T cells", J CLIN INVEST, vol. 129, 2019, pages 1972 - 1983
WAGNER, N. ET AL.: "L-selectin and β7 integrin synergistically mediate lymphocyte migration to mesenteric lymph nodes", EUR J IMMUNOL, vol. 28, 1998, pages 3832 - 3839, XP071220130, DOI: 10.1002/(SICI)1521-4141(199811)28:11<3832::AID-IMMU3832>3.0.CO;2-J
WEISSTHIERRY: "Mechanisms and consequences of intestinal dysbiosis", CELLULAR AND MOLECULAR LIFE SCIENCES, vol. 74, no. 16, 2017, pages 2959 - 2977, XP036272294, DOI: 10.1007/s00018-017-2509-x
WELTY, N. E. ET AL.: "Intestinal lamina propria dendritic cells maintain T cell homeostasis but do not affect commensalism", J EXP MED, vol. 210, 2013, pages 2011 - 2024
YU, S.GAO, N.: "Compartmentalizing intestinal epithelial cell toll-like receptors for immune surveillance", CELL MOL LIFE SCI, vol. 72, 2015, pages 3343 - 3353, XP035528149, DOI: 10.1007/s00018-015-1931-1
ZURICH, OPEN REPOSITORY AND ARCHIVE

Similar Documents

Publication Publication Date Title
TW202114718A (zh) 經加工的微生物胞外囊泡
US20230256032A1 (en) Prevotella histicola strain c as an oral therapy for inflammatory diseases
KR20230145050A (ko) 세포외 소포체 제제
TW202302125A (zh) 固體劑型
EP4284400A1 (fr) Préparations de vésicules extracellulaires de prevotella
WO2023114296A2 (fr) Préparations de vésicules extracellulaires
US20230277603A1 (en) Veillonella parvula strain as an oral therapy for neuroinflammatory diseases
TW202227111A (zh) 具有改善的崩散譜之固體劑型
WO2023146843A1 (fr) Compositions de vésicules extracellulaires et méthodes d&#39;utilisation
CN115768448A (zh) 使用马西里福涅拉氏菌治疗疾病和障碍的组合物和方法
US20240148797A1 (en) Compositions and methods for reducing cytokine expression
CN117136065A (zh) 普雷沃菌属细胞外囊泡制剂
WO2023200837A1 (fr) Compositions et méthodes de traitement d&#39;une inflammation à l&#39;aide de prevotella histicola
WO2022098961A1 (fr) Induction d&#39;effets immunitaires à l&#39;aide de bactéries veillonella parvula
WO2023239728A1 (fr) Compositions et méthodes de traitement d&#39;une inflammation à l&#39;aide de vésicules extracellulaires de prevotella histicola
TW202227110A (zh) 用棲組織普雷沃菌調節免疫反應之組成物及方法
WO2023114300A1 (fr) Préparations de vésicules extracellulaires de bactéries fournierella massiliensis
WO2023114295A1 (fr) Préparations de vésicules extracellulaires de veillonella parvula

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23707202

Country of ref document: EP

Kind code of ref document: A1