WO2022098961A1 - Inducing immune effects using veillonella parvula bacteria - Google Patents

Inducing immune effects using veillonella parvula bacteria Download PDF

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Publication number
WO2022098961A1
WO2022098961A1 PCT/US2021/058195 US2021058195W WO2022098961A1 WO 2022098961 A1 WO2022098961 A1 WO 2022098961A1 US 2021058195 W US2021058195 W US 2021058195W WO 2022098961 A1 WO2022098961 A1 WO 2022098961A1
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Prior art keywords
veillonella parvula
days
strain
total cells
bacteria
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PCT/US2021/058195
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French (fr)
Inventor
Kristie BARTH
Mark BODMER
Adam CARTWRIGHT
Taylor A. CORMACK
Andrea Itano
Holly PONICHTERA
Kritika RAMANI
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Evelo Biosciences, Inc.
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Publication of WO2022098961A1 publication Critical patent/WO2022098961A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • 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
    • A61K9/4891—Coated capsules; Multilayered drug free capsule shells

Definitions

  • bacterial compositions comprising Veillonella parvulci useful for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell and/or a decrease in expression of a pro-inflammatory cytokine by an immune cell) and methods of using such bacterial compositions (e.g., for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell)).
  • an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell and/or a decrease in expression of a pro-inflammatory cytokine by an immune cell
  • an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell
  • a method of inducing an immune effect in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a strain of a Veillonella parvula for the preparation of a medicament for inducing an immune effect in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for use in inducing an immune effect in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvulci strain for the preparation of a medicament for inducing an immune effect in a subject (e.g., a human subject).
  • the bacterial compositions comprise whole Veillonella parvula bacteria.
  • the Veillonella parvula bacteria are gamma irradiated.
  • the immune effect comprises an increase in expression of an anti-inflammatory cytokine by an immune cell.
  • the anti-inflammatory cytokine is IL- 10 and/or IL-27. In some embodiments, the antiinflammatory cytokine is IL- 10.
  • the immune effect comprises a decrease in expression of a pro-inflammatory cytokine by an immune cell.
  • the pro-inflammatory cytokine is IL-1[3, IL-6, TNF-a, IL-5, IL-4, IL-13, IL-17, and/or IL-8.
  • the pro-inflammatory cytokine is IL-5, IL-4, IL-13, and/or IL-17.
  • the immune effect comprises a decrease in mRNA transcript levels of a pro-inflammatory cytokine by an immune cell.
  • the mRNA transcript levels are //.-///. IL-6, TNF-a, IL-5, IL-4, IL-13, IL- 17 A, and/or IL-8 transcript levels.
  • the mRNA transcript levels are IL-5, IL-4, IL-13, and/or IL- 17 A transcript levels.
  • mRNA transcript levels are III 7f and/or Defb3 transcript levels.
  • the immune cells in which the immune effect are induced comprise a peripheral blood mononuclear cell (PBMC), a dendritic cell, and/or a macrophage.
  • PBMC peripheral blood mononuclear cell
  • dendritic cell a dendritic cell
  • macrophage a macrophage
  • a method of resolving an inflammatory response in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a bacterial composition comprising bacteria of a Veillonella parvulci strain for use in resolving an inflammatory response in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for resolving an inflammatory response in a subject (e.g., a human subject).
  • the inflammatory response comprises aThl and/or Th2 response.
  • compositions comprising Veillonella parvula useful for inducing an immune response in a human subject
  • the method comprising administering to the subject a population of T cells (e.g., CD4+ T cells) obtained from a different human subject, wherein the different human subject was administered Veillonella parvula.
  • T cells e.g., CD4+ T cells
  • a method of generating inflammation-resolving CD4+ T cells in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition ]) comprising a strain of Veillonella parvula.
  • a dose e.g., a therapeutically effective dose
  • a Veillonella parvula strain and/or a bacterial composition e.g., a pharmaceutical composition
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a subject e.g., a human subject
  • bacterial compositions comprising Veillonella parvula useful for affecting T cells to mediate an effect on inflammation (e.g., resolve inflammation) in a subject (e.g., a human subject), e.g., as described herein.
  • a method of affecting T cells to mediate an effect on inflammation e.g., resolve inflammation
  • a subject e.g., a human subject
  • administering e.g., orally administering
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a strain of a Veillonella parvula for the preparation of a medicament for affecting T cells to mediate an effect on inflammation (e.g., resolve inflammation) in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for use in affecting T cells to mediate an effect on inflammation (e.g., resolve inflammation) in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for affecting T cells to mediate an effect on inflammation (e.g., resolve inflammation) in 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 a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of Veillonella parvula.
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a medicament for instructing T cells to be less inflammatory in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for instructing T cells to be less inflammatory in a subject (e.g., a human subject).
  • the T cells are instructed in mesenteric lymph nodes.
  • the Veillonella parvulci strain (a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition)) is orally administered (e.g., and travels to the small intestine), dendritic cells interact with the Veillonella parvula strain 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 dose e.g., a therapeutically effective dose
  • a Veillonella parvula strain and/or a bacterial composition e.g., a pharmaceutical composition
  • 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 a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of Veillonella parvula.
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a subject e.g., a human subject
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical 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 bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament or affecting T cells that traffic to mesenteric lymph nodes in a subject (e.g., a human subject).
  • the T cells are affected in mesenteric lymph nodes.
  • bacterial compositions comprising Veillonella parvula useful for affecting B cells to mediate an effect on inflammation (e.g., resolve inflammation) in a subject (e.g., a human subject), e.g., as described herein.
  • a method of affecting B cells to mediate an effect on inflammation e.g., to resolve inflammation
  • a subject e.g., a human subject
  • administering e.g., orally administering
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • bacteria of a Veillonella parvula strain for use in affecting B cells to mediate an effect on inflammation (e.g., to resolve inflammation) in a subject (e.g., a human subject).
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a subject e.g., a human subject
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for affecting B cells to mediate an effect on inflammation (e.g., to resolve inflammation) in a subject (e.g., a human subject).
  • PBMC peripheral blood mononuclear cell
  • dendritic cell a dendritic cell
  • macrophage e.g., in a subject (e.g., a human subject).
  • a method of enhancing IL- 10 production in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a bacterial composition comprising bacteria of a Veillonella parvulci strain for use in enhancing IL- 10 production in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for enhancing IL-10 production in a subject (e.g., a human subject).
  • a method of activating TLR2 in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a method of activating TLR2 in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a strain of a Veillonella parvula for the preparation of a medicament for activating TLR2 in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for use in activating TLR2 in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for activating TLR2 in a subject (e.g., a human subject).
  • the TLR2 is activated in intestinal epithelial cells (IEC) in the subject. In some embodiments, the TLR2 is activated in immune cells in the lamina intestinal of the subject. In some embodiments, the activation of TLR2 results in increased expression of IL-10 by the subject.
  • IEC intestinal epithelial cells
  • a method of activating TLR1/2 and/or TLR2/6 heterodimers in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a strain of a Veillonella parvula for the preparation of a medicament for activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for use in activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject).
  • TLR2 activating TLR2 in a subject (e.g., a human subject).
  • TLR1/2 and/or TLR2/6 heterodimers are provided herein.
  • bacterial compositions comprising Veillonella parvula useful for elevating expression (e.g., in the small intestine) of a gene provided in Example 5.
  • a method of elevating expression (e.g., in the small intestine) of a gene provided in Example 5 in a subject comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a dose e.g., a therapeutically effective dose
  • a bacterial composition e.g., a pharmaceutical composition
  • a strain of a Veillonella parvula for the preparation of a medicament for elevating expression (e.g., in the small intestine) of a gene provided in Example 5 in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvulci strain for use in elevating expression (e.g., in the small intestine) of a gene provided in Example 5 in a subject (e.g., a human subject).
  • a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for elevating expression (e.g., in the small intestine) of a gene provided in Example 5 in a subject (e.g., a human subject).
  • the cell types found enriched amongst genes showing elevated expression are immune epithelial cells.
  • the cell types found enriched amongst genes showing elevated expression are immune cells (e.g., B cells; T cells; and/or myeloid cells).
  • the gene showing elevated expression is Spinkl, Tm4sf5, and/or Aocl.
  • the gene showing elevated expression is a gene provided in Table 2.
  • the gene showing elevated expression is associated with lymphocyte migration, gut-homing, and adhesion (e.g., Ccl25, CcrlO, Ccl22, Ccl24, Itgb7, Itgal, and/or Itgani).
  • the gene showing elevated expression is associated with T cell lineage maturation and activation (e.g., Cd69, Icos, Il2rg, Cd3d, Trbcl, Trbc2, Trac, Lat, Zap70, Lek, and/or Cd2).
  • the gene showing elevated expression is associated with B cell lineage maturation and activation (e.g., CD19, CD79a, CD79b, CD69, Ighd, Fcrll, Blk, Ikzf3, Tnfrsfl 3c, Jchain, Iglcl, and/or Iglc2) .
  • the gene showing elevated expression is associated with immune modulation (e.g., Foxp3, Lag3, Traf3ip3, Slamf6, 1133, Cd5, Adamdecl, and/or Nrli3).
  • the gene showing elevated expression is associated with intestinal epithelial cell homeostasis (barrier, metabolic, absorption) (e.g., Gpx2, Gstm3, Aqp8, Guca2b, Adipoq, Dgatl, Dgat2, Slc23al, and/or Slc51b).
  • the gene showing elevated expression is associated with host-protective pathways (e.g., Zgl6, Def 5a, Reg3a, Retnlb, Reg3g, Cfd, Lypd8, and/or Casp6).
  • the Veillonella parvula is Veillonella parvula strain A (ATCC Deposit Number PTA-125691).
  • the Veillonella parvula strain is a strain comprising at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Veillonella parvulci strain A.
  • 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 bacterial composition comprises one strain of bacteria, wherein the one strain of bacteria is a strain comprising at least 99.9% sequence identity to the nucleotide sequence of the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691). In some embodiments, the bacterial composition comprises one strain of bacteria, wherein the one strain of bacteria is the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition is administered in combination with an additional therapeutic.
  • the additional therapeutic is an IL-6 antagonist.
  • the additional therapeutic is an anti-IL-6 antagonist antibody.
  • the bacterial composition comprises at least about 3 x 10 10 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 3 x 10 10 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 4.5 x 10 10 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 1.5 x 10 n total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 7.5 x 10 n total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 1.5 x 10 12 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691). [74] In some embodiments, the bacterial composition comprises about 3 x 10 10 to about 1.5 x 10 12 total cells of Veillonella parvula, e.g., of Veillonella parvulci strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 4.5 x 10 10 to about 1.5 x 10 12 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 3 x 10 10 to about 1.5 x 10 11 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 4.5 x 10 10 to about 1.5 x 10 11 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 1.5 x 10 n to about 1.5 x 10 12 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 1.5 x 10 n to about 7.5 x 10 11 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 7.5 x 10 n to about 1.5 x 10 12 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition (e.g. , pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises about 1 x 10 10 total cells, about 2 x 10 10 total cells, about 3 x 10 10 total cells, about 4 x 10 10 total cells, about 4.5 x 10 10 total cells, about 5 x 10 10 total cells, about 6 x 10 10 total cells, about 7 x 10 10 total cells, about 8 x 10 10 total cells, about 9 x 10 10 total cells, about 1 x 10 11 total cells, about 1.5 x 10 11 total cells, about 2 x 10 11 total cells, about 3 x 10 11 total cells, about 4 x 10 11 total cells, about 5 x 10 11 total cells, about 6 x 10 11 total cells, about 7 x 10 11 total cells, about 7.5 x 10 11 total cells, about 8 x 10 11 total cells, about 9 x 10 11 total cells, about 1 x 10 12 total cells, about 1.5 x 10 12 total cells
  • the bacterial composition (e.g., composition of the total dose administered, e.g., once or twice daily) comprises at least 1 x 10 10 total cells (e.g., at least 1 x 10 10 total cells, at least 2 x 10 10 total cells, at least 3 x IO 10 total cells, at least 4 x IO 10 total cells, at least 4.5 x IO 10 total cells, at least 5 x IO 10 total cells, at least 6 x IO 10 total cells, at least 7 x IO 10 total cells, at least 8 x IO 10 total cells, at least 9 x IO 10 total cells, at least 1 x 10 11 total cells, at least 1.5 x 10 11 total cells, at least 2 x 10 11 total cells, at least 3 x 10 11 total cells, at least 4 x 10 11 total cells, at least 5 x 10 11 total cells, at least 6 x 10 11 total cells, at least 7 x 10 11 total cells, at least 7.5 x 10 11 total cells, at least 8 x 10
  • the bacterial composition comprises about 3 x 10 10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 4.5 x 10 10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 7.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 3 x 10 10 to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria.
  • the bacterial composition comprises about 4.5 x 10 10 to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 3 x 10 10 to about 1.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 4.5 x 10 10 to about 1.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10 n to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10 n to about 7.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 7.5 x 10 11 to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the Veillonella parvula bacteria are gamma irradiated.
  • solid dosage forms comprising the Veillonella parvula bacteria.
  • the solid dosage form comprises an enteric coating.
  • the solid dosage form is a capsule, e.g., an enteric coated capsule.
  • each capsule comprises about 3 x 10 10 total cells of the Veillonella parvula bacteria.
  • each capsule comprises about 4.5 x IO 10 total cells of the Veillonella parvula bacteria.
  • each capsule comprises about 1.5 x 10 11 total cells of the Veillonella parvula bacteria.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 capsules are administered, e.g., once or twice daily to a subject.
  • 1 capsule e.g., comprising about 3 x IO 10 total cells
  • 2 capsules e.g., each comprising about 3 x IO 10 total cells
  • 4 capsules e.g., each comprising about 3 x IO 10 total cells
  • 10 capsules are administered, e.g., once or twice daily to a subject.
  • 1 capsule e.g., comprising about 4.5 x IO 10 total cells
  • 2 capsules e.g., each comprising about 4.5 x IO 10 total cells
  • 4 capsules e.g., each comprising about 4.5 x IO 10 total cells
  • 10 capsules are administered, e.g., once or twice daily to a subject.
  • 1 capsule e.g., comprising about 1.5 x 10 n total cells
  • 2 capsules e.g., each comprising about 1.5 x 10 11 total cells
  • 5 capsules e.g., each comprising about 1.5 x 10 11 total cells
  • 10 capsules are administered, e.g., once or twice daily to a subject.
  • the Veillonella parvula bacteria in the capsule are lyophilized (e.g., in a powder). In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises sucrose and/or dextran. In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises sucrose, dextran, and/or L- cysteine HC1.
  • the capsule comprises excipients and the excipients include mannitol, colloidal silicon dioxide, magnesium stearate, hydroxypropyl methylcellulose, methacrylic acid ethyl acrylate copolymer, triethyl citrate, and/or talc.
  • the Veillonella parvula bacteria of the capsule are gamma irradiated.
  • the solid dosage form comprises a capsule.
  • the capsule is an enteric coated capsule.
  • the capsule comprises about 3 x IO 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules).
  • the capsule comprises about 4.5 x IO 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules).
  • the capsule comprises about 1.5 x 10 11 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules).
  • the Veillonella parvula bacteria in the capsule are lyophilized (e.g., in a powder). In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises sucrose and/or dextran. In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises sucrose, dextran, and/or L-cysteine HC1.
  • the capsule comprises excipients and the excipients include mannitol, colloidal silicon dioxide, magnesium stearate, hydroxypropyl methylcellulose, methacrylic acid ethyl acrylate copolymer, triethyl citrate, and/or talc.
  • the Veillonella parvula bacteria of the capsule are gamma irradiated.
  • the solid dosage form comprises a tablet.
  • the tablet is an enteric coated tablet.
  • the enteric coated tablet is from 5mm to 18mm in diameter. In some embodiments, the enteric coated tablet is 5.5mm in diameter. In some embodiments, the enteric coated tablet is 18mm in diameter.
  • the tablet comprises about 3 x IO 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets). In some embodiments, the tablet comprises about 4.5 x 10 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets).
  • the tablet comprises about 1.5 x 10 11 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets).
  • the Veillonella parvula bacteria in the tablet are lyophilized.
  • the Veillonella parvula bacteria of the tablet are gamma irradiated.
  • provided herein are solid dosage forms comprising the Veillonella parvulci bacteria.
  • the solid dosage form is a tablet, e.g., an enteric coated tablet.
  • the enteric coating comprises a polymethacrylate-based copolymer.
  • the enteric coating comprises a methacrylic acid ethyl acrylate (MAE) copolymer (1: 1). In some embodiments, the enteric coating comprises methacrylic acid ethyl acrylate (MAE) copolymer (1: 1) (such as Kollicoat MAE 100P).
  • MAE methacrylic acid ethyl acrylate
  • each tablet comprises about 3 x IO 10 total cells, 4.5 x 10 10 total cells or about 1.5 x 10 11 total cells of the Veillonella parvula bacteria.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tablets are administered, e.g., once or twice daily to a subject.
  • 1 tablet e.g., comprising about 3 x 10 10 total cells
  • 2 tablets e.g., each comprising about 3 x 10 10 total cells
  • 5 tablets are administered, e.g., once or twice daily to a subject.
  • 10 tablets e.g., each comprising about 3 x 10 10 total cells
  • 1 tablet e.g., comprising about 4.5 x 10 10 total cells
  • 2 tablets e.g., each comprising about 4.5 x 10 10 total cells
  • 5 tablets are administered, e.g., once or twice daily to a subject.
  • 10 tablets are administered, e.g., once or twice daily to a subject.
  • each tablet comprises about 1.5 x 10 11 total cells of the Veillonella parvula bacteria.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tablets are administered, e.g., once or twice daily to a subject.
  • 1 tablet e.g., comprising about 1.5 x 10 11 total cells
  • 2 tablets e.g., each comprising about 1.5 x 10 n total cells
  • 5 tablets e.g., each comprising about 1.5 x 10 11 total cells
  • 10 tablets e.g., each comprising about 1.5 x 10 11 total cells
  • the Veillonella parvula bacteria in the tablet are lyophilized (e.g., in a powder).
  • the Veillonella parvula bacteria in the tablet are lyophilized in a powder, and the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the Veillonella parvula bacteria in the tablet are lyophilized in a powder, and the powder further comprises sucrose and/or dextran. In some embodiments, the Veillonella parvula bacteria in the tablet are lyophilized in a powder, and the powder further comprises sucrose, dextran, and/or L-cysteine HC1. In some embodiments, the Veillonella parvula bacteria of the tablet are gamma irradiated.
  • the solid dosage form comprises a mini-tablet.
  • the mini-tablet is enteric coated.
  • the minitablet is from 1 mm to 4 mm in diameter.
  • the mini -tablet e.g., enteric coated mini-tablet
  • the solid dosage form comprises mini -tablets that comprise about 3 x 10 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a plurality of mini -tablets).
  • the solid dosage form comprises mini-tablets that comprise about 4.5 x IO 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a plurality of mini -tablets). In some embodiments, the solid dosage form comprises mini-tablets that comprise about 1.5 x 10 11 total cells of the Veillonella parvula bacteria (e.g., total dose of a plurality of minitablets). In some embodiments, the Veillonella parvula bacteria in the mini-tablets are lyophilized. In some embodiments, the Veillonella parvula bacteria of the mini-tablet are gamma irradiated.
  • the mini -tablets are contained in a capsule.
  • the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule.
  • the capsule comprises a non- enteric coating (e.g., gelatin) (e.g., is coated with a non-enteric coating).
  • the capsule comprises a non-enteric coating.
  • the capsule comprises gelatin.
  • the capsule comprises HPMC.
  • the mini-tablets e.g., enteric coated mini -tablets
  • the minitablets that comprise about 3 x 10 10 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC.
  • the minitablets e.g., enteric coated mini-tablets
  • the minitablets that comprise about 4.5 x 10 10 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC.
  • the mini-tablets e.g., enteric coated mini-tablets
  • the mini-tablets that comprise about 1.5 x 10 11 total cells of the Veillonella parvulci bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC.
  • the Veillonella parvula bacteria of the mini-tablet are gamma irradiated.
  • the bacterial composition comprising Veillonella parvula bacteria is prepared as a powder (e.g., for resuspension or for use in a solid dose form (such as a capsule)) or as a solid dose form, such as a tablet, a mini-tablet, a capsule, a pill, or a powder; or a combination of these forms (e.g., mini -tablets comprised in a capsule).
  • the powder can comprise lyophilized bacteria.
  • the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide.
  • the powder further comprises sucrose and/or dextran.
  • the powder further comprises sucrose, dextran, and/or L-cysteine HC1.
  • the Veillonella parvula bacteria are gamma irradiated.
  • the bacterial composition is administered orally. In some embodiments, the administration to the subject once daily. In some embodiments, the bacterial composition is administered in 2 or more doses (e.g., 3 or more, 4 or more or 5 or more doses).
  • the administration to the subject of the two or more doses are separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.
  • the bacterial composition is administered once daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
  • the bacterial composition is administered once daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks.
  • the bacterial composition is administered once daily for 8 weeks. In some embodiments, the bacterial composition is administered once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for at least 8 weeks.
  • the bacterial composition (e.g., pharmaceutical composition) comprises lyophilized Veillonella parvulci bacteria.
  • the lyophilized Veillonella parvula bacteria is formulated into a solid dose form, such as a tablet, a mini-tablet, a capsule, a pill, or a powder.
  • the lyophilized Veillonella parvula bacteria is contained in a capsule.
  • the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide.
  • the powder further comprises sucrose and/or dextran.
  • the powder further comprises sucrose, dextran, and/or L-cysteine HC1.
  • the powder is resuspended in a solution.
  • the lyophilized Veillonella parvula bacteria are resuspended in a solution.
  • the bacterial composition is formulated as a capsule or a tablet.
  • the bacterial formulation e.g., composition
  • the capsule is an enteric coated capsule.
  • the enteric coating allows the bacterial composition to be released in the upper small intestine, e.g., duodenum.
  • the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal (e.g., 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).
  • a non-human mammal e.g., 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 e.g., human subject
  • the immunoinflammatory disorder is arthrosclerosis, arthritis (e.g., psoriatic 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,
  • the subject has an immune disorder.
  • the immune disorder is atopic dermatitis, psoriasis, or asthma.
  • the atopic dermatitis is moderate atopic dermatitis.
  • the psoriasis is moderate psoriasis.
  • the asthma is moderate asthma.
  • the disclosure provides a bacterial composition described herein (e.g., in an amount described herein) for use in inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell).
  • the disclosure provides use of a bacterial composition described herein (e.g., in an amount described herein) for the preparation of a medicament for the induction of an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell).
  • an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell.
  • Figures 1A-1C show that Veillonella parvulci strain A induces IL-10 production from human immune cells.
  • Figure 1A shows IL-10 production in PBMC.
  • Figure IB shows IL-10 production in Dendritic cells.
  • Figure 1C shows IL-10 production in macrophages.
  • FIG. 1 shows stimulation of the production of TNF by pro-inflammatory macrophages.
  • Average TNF pg/ml
  • Veillonella parvula strain A- G.I. in an assay with pro-inflammatory macrophages after 24 hours of cell-microbe co-culture.
  • Error bars represent standard deviation (S.D.) of the mean.
  • Figures 3A-3B show effect of Veillonella parvula strain A on cytokine expression in a human dendritic cell: T cell in vitro co-culture system.
  • Cytokine is reported as pg/mL at 24 hours after addition of the CEF-peptide pool. Error bars on control treatments represent SD of duplicate wells.
  • FIG. 4 shows mouse KLH delayed-type hypersensitivity model. Shown are the 24-hour ear thickness change measurements. Error bars represent standard deviation. Statistical significance is measured from one-way ANOVA against vehicle. **** denotes pO.OOOL
  • Figure 5 shows reduction in ear inflammation after challenge on days 15 and 29.
  • Figure 6 shows mouse FITC model of cutaneous hypersensitivity. Results compiled for 4 individual experiments. Error bars represent standard deviation. Statistical significance is measured from one-way ANOVA against vehicle. * denotes p ⁇ 0.05 *** denotes p ⁇ 0.001, **** denotes p ⁇ 0.0001.
  • Figure 7 shows Veillonella parvula strain A- G.I. (gamma-irradiated) reduces MC903 atopic dermatitis-like skin inflammation with efficacy similar to systemic dexamethasone and tofacitinib. Ear inflammation measured daily. Total area under the curve (AUC) of the ear swelling measurements for the time course from days 1-14 were calculated. Error bars represent standard error. Statistical significance is measured from one-way ANOVA against vehicle. * * * * denotes p ⁇ 0.0001.
  • Figure 8 shows treatment with Veillonella parvula strain A- G.I. results in decrease in protein and mRNA levels in the ear. IL-4 protein and mRNA measurement in ears of treated mice. Error bars represent standard deviation. Statistical significance is measured from one-way ANOVA against vehicle. * denotes p ⁇ 0.05, ** denotes p ⁇ 0.01.
  • Figure 9 shows Veillonella parvula strain A- G.I. treatment reduces the production of TH2 and TH1 cytokines in the lymph node draining the site of inflammation. Ex vivo restimulation of cervical lymph nodes (cLN) and spleen using PMA and ionomycin. Error bars represent standard deviation. Statistical significance is measured from one-way ANOVA against vehicle. * denotes p ⁇ 0.05, *** denotes p ⁇ 0.001, **** denotes p ⁇ 0.0001.
  • FIG. 10 shows Veillonella parvula strain A- G.I. treatment reduces both ear inflammation and tissue expression of IL-17A. Compiled data from 5 individual studies. Ear inflammation measured on Day 8. Error bars represent standard error. Statistical significance is measured from one-way ANOVA against vehicle. **** denotes pO.OOOI.
  • Figure 11 shows disease scores in EAE model. Clinical weakness scores over 42 days of daily dosing with Veillonella parvula strain A- G.I. or vehicle. Error bars represent standard error.
  • Figure 12 shows the area under the curve (AUC) for total study and acute phase. Area under the curve for total study and acute phase. Error bars represent standard deviation (S.D.) **** denotes p ⁇ 0.0001 by unpaired t-test.
  • Figures 13A-13G shows that Veillonella parvula Strain A induces cytokine production in vitro and resolves inflammation in vivo.
  • Figure 13A shows IL-10 cytokine levels determined by performing a Meso Scale Discovery (MSD) assay of supernatants derived from human macrophages stimulated with different anaerobic bacterial strains for 24 hours and flushed with 1% oxygen. Data shown represent collective data from 6 independent human donors.
  • Figure 13B shows CXCL10/IP-10 levels determined by performing a MSD assay of supernatants derived from stimulated with different anaerobic bacterial strains for 24h and flushed with 1% oxygen. Data shown represent collective data from 6 independent human donors.
  • Figure 13C shows TNFa levels determined by performing a MSD assay of supernatants derived from stimulated with different anaerobic bacterial strains for 24h and flushed with 1% oxygen
  • Figure 13D shows human PBMCs, dendritic cells and macrophages stimulated with Veillonella parvula Strain A bacterial strains for 24 hours and flushed with 1% O2. Supernatants were collected to test for cytokine levels by MSD. Data shown represent collective data from 6 independent human donors DTH response to KLH. C57BL/6 mice were immunized with KLH and CFA on day 0 s.c. and challenged in the ear i.d. 9 days later with KLH.
  • Figure 13F shows dose dependent effects of Veillonella parvula Strain A (TCC 2. 16E+12).
  • Figure 13G shows that Veillonella parvula Strain A acts through IL-10R pathway to reduce ear inflammation.
  • Figures 14A-14B show that Veillonella parvula Strain A is gut restricted and transits through GI tract within 24 hours.
  • Figure 14A is a graph showing the reduction of signal indicating the presence of Veillonella parvula Strain A in the GI tract over time.
  • Figure 14B comprises graphs of the signal detected indicating the presence of Veillonella parvula Strain A in various tissues.
  • Figures 15A-15B show that Veillonella parvula Strain A alleviates skin inflammation in imiquimod-induced psoriasis.
  • Figure 15 A shows ear inflammation over the course of 7 days and area under the curve.
  • FIGS 16A-16E show that Veillonella parvulci Strain A displays efficacy when treating neuroinflammation in a model for relapsing remitting MS. EAE was induced in SJL mice by immunization with PLP 139-151 in CFA on day 0, hour 0 and PTX was administered on day 0, hour 2.
  • Figure 16A shows cumulative EAE scores of mice after for 41 days of oral prophylactic dosing with vehicle, Veillonella parvula Strain A (TCC- 8.46E+10), and fingolimod (1 mg/kg). Clinical scores were assessed daily forthe duration of the experiment.
  • FIG. 16B shows inflammation levels of the brain of spinal cords of mice treated with Veillonella parvula Strain A, vehicle or fingolimod.
  • Figure 16C shows the cumulative EAE scores of mice after day 10 to day 41 of oral therapeutic dosing with vehicle, Veillonella parvula Strain A (TCC- 8.46E+10), and fingolimod (1 mg/kg). Clinical scores were assessed daily forthe duration of the experiment. ****p ⁇ 0.00005 by Unpaired t-test with Welch’s correction was used to calculate p-value in cumulative AUC forthe EAE score.
  • Figure 16D is graph showing the inflammatory loci observed in the spinal cord cells.
  • Figures 17A-17B show that Veillonella parvula Strain A resolves inflammation in a Th2 cell driven atopic dermatitis model.
  • Figure 17A shows the change in ear thickness in FITC- driven atopic dermatitis (AD).
  • Figures 18A-18B show that Veillonella parvula Strain A resolves inflammation in a delayed type hypersensitivity T cell driven disease model in vivo.
  • FIGS 19A-19D show that Veillonella parvulci Strain is TLR2 dependent and TLR4 independent.
  • Figure 19A shows that Veillonella parvula Strain A stimulates both human TLR1/2 and TLR2/6 heterodimers, with greater potency observed for TLR2/6 heterodimer.
  • KLH-DTH was induced as previously described. Mice were orally dosed with vehicle or V. parvula Strain A (TCC- 2. 16E+12) from day 5 through 8. Ear inflammation was measured on day 9.
  • FIG 19B mice were treated with anti-TLR2 blocking antibody on days 2, 4, and 6 as indicated and change in ear thickness was measured.
  • Figure 21 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following dosing pre- or -post-immunization.
  • Figure 22 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following dosing with vehicle (PBS) or Veillonella Strain A-G. I after immunization with PBS-CFA or PBS-IFA prior to immunization with KLH-CFA and a KLH ear challenge.
  • Figure 23 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following adoptive transfer of CD4+ T cells from vehicle or Veillonella Strain A-G.I. treated mice into untreated KLH-CFA-immunized recipients.
  • Figure 24 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following treatment with vehicle or Veillonella Strain A-G.I. and depletion of B cells during the indicated time points.
  • Figure 25 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following treatment with vehicle or Veillonella Strain A-G.I. in combination with anti -IL-6.
  • Veillonella parvula strain A- G.I. is a pharmaceutical preparation of a single strain of Veillonella parvula, originally isolated from a fresh ileostomy sample of an IBD patient in remission, which has been gamma-irradiated.
  • Veillonella parvula strain A- G.I. can be manufactured by anaerobic fermentation, followed by lyophilization, gamma-irradiation, and encapsulation in a capsule with an enteric coating which protects the drug substance from low pH in the stomach. Because it is non-viable, it does not colonize the gut and has no detectable systemic exposure following oral dosing. In non-clinical studies its therapeutic effects are dose-dependent.
  • Veillonella parvula strain A- G.I falls into a new pharmacological class termed non -live bacterial pharmaceutical products. This is a unique positioning as an oral medicine which modulates the small intestinal axis (SINTAX) for systemic pharmacological effects, with no systemic exposure. Since the lumen of the gut is topologically on the outside of the body, this is effectively a topical drug with systemic activity. This is made possible by the discovery of the systemic control network that emanates from the gut, and the small intestine in particular.
  • SINTAX small intestinal axis
  • Adjuvant or “Adjuvant therapy” broadly refers to an agent that affects an immunological or physiological response in a patient or subject.
  • an adjuvant might increase the presence of an antigen over time or 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 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.
  • the bacterial compositions 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 intra
  • the bacterial compositions described herein are administered orally, rectally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously. In some preferred embodiments, the bacterial compositions described herein are 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), singlechain antibodies and antigen-binding antibody fragments.
  • antigen binding fragment and “antigen-binding portion” of an antibody, as used herein, refers 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.
  • 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.
  • “Operational taxonomic units,” “OTU” (or plural, “OTUs”) 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.
  • MMT multilocus sequence tags
  • OTUs that share 1197% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU (see e.g. Claesson M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ros R P, and O'Toole P W. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions.
  • OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU.
  • 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. Such characterization employs, e.g., WGS data or a whole genome sequence.
  • a “combination” of two or more monoclonal microbial strains includes the physical co-existence of the two monoclonal microbial strains, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the monoclonal microbial strains.
  • the term “decrease” or “deplete” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to 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)).
  • a “decrease” refers to a decrease as compared to amount produced (e.g., mRNA and/or protein) in the absence of the bacterial composition).
  • 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.
  • engineered bacteria are any bacteria that have been genetically altered from their natural state by human intervention 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.
  • genomic is used broadly to refer to any nucleic acid associated with a biological function.
  • the term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
  • “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J.
  • 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., 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 environmental allergies).
  • autoimmune diseases e.g., Lupus, Scleroderma, hemolytic anemia
  • Immunotherapy is treatment that uses a subject’s immune system to treat disease (e.g., immune disease) and includes, for example, checkpoint inhibitors, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.
  • disease e.g., immune disease
  • the term “increase” means a change, 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 greater after treatment when compared to a pre-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).
  • An “increase” refers to an increase as compared to amount produced (e.g., mRNA and/or protein) in the absence of the bacterial composition).
  • ‘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, STING-cGAS Pathway components, inflammasome complexes.
  • TLR Toll-Like Receptors
  • NOD receptors NOD receptors
  • RLRs C-type lectin receptors
  • STING-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.
  • STING 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, TLR1O and TLRI 1.
  • 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
  • isolated or “enriched” encompasses a microbe, bacteria 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 microbes 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 microbes 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.
  • purify refers to a microbe 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.
  • a microbe or a microbial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial 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 “isolated.”
  • purified microbes or microbial population 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.
  • the one or more microbial types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type.
  • Microbial compositions and the microbial components thereof are generally purified from residual habitat products.
  • Metal refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or microbial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or microbial metabolic reaction.
  • ‘Microbe” refers to any natural or engineered organism characterized as a bacterium, fungus, microscopic alga, protozoan, and the stages of development or life cycle stages (e.g., vegetative, spore (including sporulation, dormancy, and germination), latent, biofdm) associated with the organism.
  • 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 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.
  • 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 in a sample.
  • the microbiome profile indicates the relative or absolute amount of each bacterial strain detected in the sample.
  • ‘Modified” in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form.
  • bacterial modifications include genetic modification, gene expression, phenotype modification, formulation, 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 that increase or decrease virulence.
  • 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.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), micro RNA (miRNA), silencing RNA (siRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • 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.
  • U nucleotides are interchangeable with T nucleotides.
  • “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.
  • OTUs 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 Uond B Biol Sci 361: 1929-1940. OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU.
  • 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 substance is “pure” if it is substantially free of other components.
  • the terms “purify,” “purifying” and “purified” refer to a microbe 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.
  • a microbe 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 microbial 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 microbes 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.
  • Bacterial compositions and the microbial components thereof are, e.g., purified from residual habitat products.
  • ‘Residual habitat products” refers to material derived from the habitat for microbiota within or on a subject. For example, 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 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 human or animal and that only microbial cells are detectable. In one embodiment, substantially free of residual habitat products may also mean that the microbial composition contains no detectable viral (including microbial viruses (e.g., phage)), fungal, mycoplasmal contaminants.
  • microbial viruses e.g., phage
  • 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 10-9), 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.
  • Other methods for confirming adequate purity include genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, enzymatic and metabolic analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired constituents from contaminants.
  • specific 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 KF 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 non-specific 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.
  • subject refers to any animal.
  • a subject or a patient described as “in need thereof’ refers to one in need of a treatment 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 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 is a human subject.
  • ‘Strain” refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species.
  • the genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof.
  • regulatory region e.g., a promoter, a terminator, a riboswitch, a ribosome binding site
  • 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 .
  • treating refers to subjecting 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.
  • 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.
  • a “type” of bacteria may be distinguished from other bacteria by: genus, species, sub-species, strain or by any other taxonomic categorization, whether based on morphology, physiology, genotype, protein expression or other characteristics known in the art.
  • bacterial compositions comprising Veillonella parvulci useful for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell) and methods of using such bacterial compositions (e.g., for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell)), e.g., in a subject, e.g., in a human subject.
  • an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell
  • methods of using such bacterial compositions e.g., for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in
  • the bacterial compositions comprise whole Veillonella parvula bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In some embodiments, the Veillonella parvula bacteria are gamma irradiated. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises only one strain of bacteria, e.g., Veillonella parvula.
  • ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122.
  • the deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited plasmid, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicant acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
  • the Veillonella parvula is Veillonella parvula strain A (ATCC Deposit Number PTA-125691) (also referred to as “Veillonella parvula strain A”).
  • the Veillonella parvula strain is a strain comprising at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • sequence identity e.g., at least 99.5% sequence identity, at least 99.
  • Veillonella parvula strain A can be cultured according to methods known in the art. For example, Veillonella parvula strain A can be grown under anaerobic conditions in PM1 l+5g/L Na-L-lactate liquid medium supplemented with 0.05g/L FeSO4, and 0.5 g/L L-cysteine-HCL as reducing agent at 37 degrees C. See also WO 2019/157003.
  • the bacterial compositions comprise whole Veillonella parvula bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • the bacterial compositions comprise whole Veillonella parvula bacteria (e.g., gamma irradiated Veillonella parvula bacteria).
  • the bacterial composition (e.g., pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises about 1 x 10 10 total cells, about 2 x 10 10 total cells, about 3 x 10 10 total cells, about 4 x 10 10 total cells, about 4.5 x 10 10 total cells, about 5 x 10 10 total cells, about 6 x 10 10 total cells, about 7 x 10 10 total cells, about 8 x 10 10 total cells, about 9 x 10 10 total cells, about 1 x 10 11 total cells, about 1.5 x 10 11 total cells, about 2 x 10 11 total cells, about 3 x 10 11 total cells, about 4 x 10 11 total cells, about 5 x 10 11 total cells, about 6 x 10 11 total cells, about 7 x 10 11 total cells, about 7.5 x 10 11 total cells, about 8 x 10 11 total cells, about 9 x 10 11 total cells, about 1 x 10 12 total cells, about 1.5 x 10 12 total cells
  • the bacterial composition (e.g. , pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises at least 1 x 10 10 total cells (e.g., at least 1 x 10 10 total cells, at least 2 x 10 10 total cells, at least 3 x 10 10 total cells, at least 4 x 10 10 total cells, at least 4.5 x 10 10 total cells, at least 5 x 10 10 total cells, at least 6 x 10 10 total cells, at least 7 x 10 10 total cells, at least 8 x 10 10 total cells, at least 9 x 10 10 total cells, at least 1 x 10 11 total cells, at least 1.5 x 10 11 total cells, at least 2 x 10 11 total cells, at least 3 x 10 11 total cells, at least 4 x 10 11 total cells, at least 5 x 10 11 total cells, at least 6 x 10 11 total cells, at least 7 x 10 11 total cells, at least 7.5 x 10 11 total cells, at
  • the bacterial composition comprises about 3 x 10 10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 4.5 x 10 10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10 11 total cells of the Veillonella parvulci bacteria. In some embodiments, the bacterial composition comprises about 7.5 x 10 11 total cells the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10 12 total cells the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 3 x 10 10 to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria.
  • the bacterial composition comprises about 4.5 x 10 10 to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 3 x 10 10 to about 1.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 4.5 x 10 10 to about 1.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10 n to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10 n to about 7.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 7.5 x 10 n to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria.
  • the bacterial composition comprises about 3 x 10 10 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 4.5 x 10 10 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 1.5 x 10 11 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 7.5 x 10 n total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 1.5 x 10 12 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691). [178] In some embodiments, the bacterial composition comprises about 3 x 10 10 to about 1.5 x 10 12 total cells of Veillonella parvula, e.g., of Veillonella parvulci strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 4.5 x 10 10 to about 1.5 x 10 12 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 3 x 10 10 to about 1.5 x 10 11 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 4.5 x 10 10 to about 1.5 x 10 11 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 1.5 x 10 n to about 1.5 x 10 12 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 1.5 x 10 n to about 7.5 x 10 11 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition comprises about 7.5 x 10 n to about 1.5 x 10 12 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the Veillonella parvula bacteria may be quantified based on total cells, e.g., total cell count (TCC) (e.g., determined by Coulter counter).
  • TCC total cell count
  • the bacterial composition is administered orally. In some embodiments, the administration to the subject once daily. In some embodiments, the bacterial composition is administered in 2 or more doses (e.g., 3 or more, 4 or more or 5 or more doses).
  • the administration to the subject of the two or more doses are separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.
  • the bacterial composition is administered once daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
  • the bacterial composition is administered once daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for 8 weeks. In some embodiments, the bacterial composition is administered once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for at least 8 weeks.
  • the bacterial composition is formulated as a capsule or a tablet.
  • the bacterial formulation (e.g., composition) comprises an enteric coating or micro encapsulation.
  • the capsule is an enteric coated capsule.
  • the enteric coating allows release of the bacterial composition in the small intestine, e.g., in the upper small intestine, e.g., in the duodenum.
  • the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal (e.g., 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).
  • a non-human mammal e.g., 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 methods provided herein comprise use of bacterial compositions (e.g., pharmaceutical compositions) comprising Veillonella parvula bacteria provided herein.
  • the bacterial compositions comprise whole Veillonella parvula bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • the Veillonella parvula bacteria is non- viable.
  • the Veillonella parvula bacteria has been gamma irradiated (e.g., according to a method described herein).
  • the Veillonella parvula bacteria is live.
  • the bacterial composition e.g, pharmaceutical composition
  • the bacterial composition (e.g., pharmaceutical composition) comprises more than one strain of bacteria, e.g., Veillonella parvula, and the therapeutic effect caused by the bacterial composition is due to the presence of the Veillonella parvula bacteria (e.g., a therapeutically effective amount thereof) present in the composition.
  • At least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are of the Veillonella parvula strain.
  • 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are of the Veillonella parvula strain.
  • at least 99% of the bacteria in the bacterial composition are of the Veillonella parvula strain.
  • the bacteria in the composition are essentially (e.g., about 100%) of the Veillonella parvula strain.
  • the protein in the bacterial composition is Veillonella parvula strain bacteria protein.
  • the Veillonella parvula is Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the Veillonella parvula strain is a strain comprising at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial compositions e.g., pharmaceutical compositions
  • the bacterial compositions comprise whole Veillonella parvula bacteria (e.g., gamma irradiated Veillonella parvula bacteria).
  • the bacterial compositions (e.g., pharmaceutical compositions) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises about 1 x IO 10 total cells, about 2 x IO 10 total cells, about 3 x IO 10 total cells, about 4 x IO 10 total cells, about 4.5 x IO 10 total cells, about 5 x IO 10 total cells, about 6 x IO 10 total cells, about 7 x IO 10 total cells, about 8 x IO 10 total cells, about 9 x IO 10 total cells, about 1 x 10 11 total cells, about 1.5 x 10 11 total cells, about 2 x 10 11 total cells, about 3 x 10 11 total cells, about 4 x 10 11 total cells, about 5 x 10 11 total cells, about 6 x 10 11 total cells, about 7 x 10 11 total cells, about 7.5 x 10 11 total cells, about 8 x 10 11 total cells, about 9 x 10 11 total cells, about 1 x 10 12
  • the bacterial composition (e.g., pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises at least 1 x 10 10 total cells (e.g., at least 1 x 10 10 total cells, at least 2 x IO 10 total cells, at least 3 x IO 10 total cells, at least 4 x IO 10 total cells, at least 4.5 x IO 10 total cells, at least 5 x IO 10 total cells, at least 6 x IO 10 total cells, at least 7 x IO 10 total cells, at least 8 x IO 10 total cells, at least 9 x IO 10 total cells, at least 1 x 10 11 total cells, at least 1.5 x 10 11 total cells, at least 2 x 10 11 total cells, at least 3 x 10 11 total cells, at least 4 x 10 11 total cells, at least 5 x 10 11 total cells, at least 6 x 10 11 total cells, at least 7 x 10 11 total cells, at least 7.5
  • the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x IO 10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x IO 10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 7.5 x 10 11 total cells the Veillonella parvulci bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10 12 total cells the Veillonella parvula bacteria.
  • the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x 10 10 to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x 10 10 to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x 10 10 to about 1.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x 10 10 to about 1.5 x 10 11 total cells of the Veillonella parvula bacteria.
  • the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10 n to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10 n to about 7.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 7.5 x 10 n to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria.
  • the Veillonella parvula bacteria may be quantified based on total cells, e.g., total cell count (TCC) (e.g., determined by Coulter counter).
  • TCC total cell count
  • the bacterial composition (e.g., pharmaceutical composition) is administered orally. In some embodiments, the administration to the subject once daily. In some embodiments, the bacterial composition is administered in 2 or more doses (e.g. , 3 or more, 4 or more or 5 or more doses).
  • the administration to the subject of the two or more doses are separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.
  • the bacterial composition (e.g., pharmaceutical composition) is administered once daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
  • the bacterial composition (e.g, pharmaceutical composition) is administered twice daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
  • the bacterial composition (e.g, pharmaceutical composition) is administered once daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered once daily for 8 weeks. In some embodiments, the bacterial composition is administered once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for at least 8 weeks.
  • the bacterial composition (e.g, pharmaceutical composition) is administered twice daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered twice daily for 8 weeks. In some embodiments, the bacterial composition is administered twice daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered twice daily for at least 8 weeks.
  • the bacterial composition (e.g., pharmaceutical composition) is formulated as a capsule or a tablet.
  • the bacterial formulation (e.g., composition) comprises an enteric coating or micro encapsulation.
  • the capsule is an enteric coated capsule.
  • the enteric coating allows release of the bacterial composition in the small intestine, e.g., in the upper small intestine, e.g., in the duodenum.
  • the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal (e.g., 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).
  • a non-human mammal e.g., 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.
  • NTA nanoparticle tracking analysis
  • DLS dynamic light scattering
  • Combined results from Coulter counting and NTA can reveal the numbers of bacteria in a given sample.
  • Coulter counting reveals the numbers of particles with diameters of 0.7-10 um.
  • NTA reveals the numbers of particles with diameters of 50- 1400 nm.
  • the Coulter counter alone can reveal the number of bacteria in a sample.
  • the bacterial composition (e.g., pharmaceutical composition) comprises an enteric coating or micro encapsulation.
  • the enteric coating or micro encapsulation improves targeting to a desired region of the gastrointestinal tract.
  • the bacterial composition (e.g., pharmaceutical composition) comprises an enteric coating and/or microcapsules that dissolves at a pH associated with a particular region of the gastrointestinal tract.
  • the enteric coating and/or microcapsules dissolve at a pH of about 5.5 - 6.2 to release in the duodenum, at a pH value of about 7.2 - 7.5 to release in the ileum, and/or at a pH value of about 5.6 - 6.2 to release in the colon.
  • Exemplary enteric coatings and microcapsules are described, for example, in U.S. Pat. Pub. No. 2016/0022592, which is hereby incorporated by reference in its entirety.
  • bacterial compositions for administration subjects.
  • the bacterial 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 bacterial compositions are combined with an adjuvant such as an immuno-adjuvant (e.g., STING agonists, TLR agonists, NOD agonists).
  • an adjuvant such as an immuno-adjuvant (e.g., STING agonists, TLR agonists, NOD agonists).
  • the bacterial composition (e.g., pharmaceutical composition) comprises at least one carbohydrate.
  • 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 CnFEnOn.
  • a carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide.
  • the most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose.
  • Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose.
  • 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.
  • the bacterial composition (e.g., pharmaceutical composition) comprises at least one lipid.
  • 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).
  • 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), docosahexaenoic acid (22:6) (DHA), and
  • the bacterial composition (e.g., pharmaceutical 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 bacterial composition (e.g., pharmaceutical 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 B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin.
  • Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
  • the bacterial composition (e.g, pharmaceutical 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 bacterial composition (e.g, pharmaceutical 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 bacterial composition (e.g., pharmaceutical 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 bacterial composition (e.g, pharmaceutical 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 bacterial composition (e.g., pharmaceutical composition) comprises a disintegrant as an excipient.
  • the disintegrant is a non-effervescent disintegrant.
  • suitable non-effervescent disintegrants include starches such as com 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 bacterial 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 bacterial 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.
  • Dose forms comprising Veillonella parvulci bacteria are also provided herein, e.g., for use in methods provided herein, e.g., to induce an immune effect in a subject (e.g., a human subject).
  • a bacterial composition e.g., pharmaceutical composition
  • the solid dose form can comprise one or more excipients, e.g., pharmaceutically acceptable excipients.
  • the Veillonella parvula bacteria in the solid dose form can be isolated Veillonella parvula bacteria.
  • the Veillonella parvula bacteria in the solid dose form can be lyophilized.
  • the Veillonella parvula bacteria in the solid dose form are live.
  • the Veillonella parvula bacteria in the solid dose form are gamma irradiated.
  • the solid dose form can comprise a tablet, a minitablet, a capsule, a pill, or a powder; or a combination of these forms (e.g., minitablets comprised in a capsule).
  • the Veillonella parvula bacteria in the solid dose form can be in a powder (e.g., the powder comprises lyophilized Veillonella parvula bacteria).
  • the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide.
  • the powder further comprises mannitol, magnesium stearate, and colloidal silicon dioxide.
  • the powder further comprises sucrose and/or dextran.
  • the powder further comprises sucrose, dextran, and/or L-cysteine HC1.
  • the powder is resuspended in a solution.
  • the lyophilized Veillonella parvula bacteria is resuspended in a solution.
  • the bacterial composition (e.g., pharmaceutical composition) provided herein is prepared as a solid dosage form comprising Veillonella parvula bacteria and a pharmaceutically acceptable carrier.
  • the solid dosage form comprises a capsule.
  • the capsule can comprise an enteric coating.
  • the capsule can be a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule.
  • the capsule can comprise Veillonella parvula bacteria powder (e.g., lyophilized Veillonella parvula bacteria).
  • the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide.
  • the powder further comprises mannitol, magnesium stearate, and colloidal silicon dioxide.
  • the powder further comprises sucrose and/or dextran. In some embodiments, the powder further comprises sucrose, dextran, and/or L-cysteine HC1. In some embodiments, the capsule comprises excipients and the excipients include mannitol, colloidal silicon dioxide, magnesium stearate, hydroxypropyl methylcellulose, methacrylic acid ethyl acrylate copolymer, triethyl citrate, and/or talc.
  • the solid dosage form described herein can be, e.g., a tablet or a mini -tablet.
  • a plurality of mini-tablets can be in (e.g., loaded into) a capsule.
  • the solid dosage form comprises a tablet (> 4mm) (e.g., 5mm-17mm).
  • the tablet is a 5mm, 5.5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, or 18mm tablet.
  • the size refers to the diameter of the tablet, as is known in the art. As used herein, the size of the tablet refers to the size of the tablet prior to application of an enteric coating.
  • the solid dosage form comprises a mini-tablet.
  • the mini-tablet can be in the size range of lmm-4 mm range.
  • the mini-tablet can be a 1mm mini-tablet, 1.5 mm mini-tablet, 2mm mini-tablet, 3mm mini-tablet, or 4mm minitablet.
  • the size refers to the diameter of the mini-tablet, as is known in the art.
  • the size of the minitablet refers to the size of the mini-tablet prior to application of an enteric coating.
  • the mini-tablets 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 mini -tablets can comprise a single layer coating, e.g. , a non-enteric coating such as gelatin or HPMC.
  • the mini-tablets can be inside a capsule: the number of mini -tablets inside a capsule will depend on the size of the capsule and the size of the mini -tablets. As an example, a size 0 capsule can contain 31-35 (an average of 33) mini-tablets that are 3mm mini-tablets.
  • the solid dosage form (e.g., tablet or mini-tablet or capsule) described herein can be enterically coated.
  • the enteric coating comprises a polymethacrylate-based copolymer.
  • the enteric coating comprises a methacrylic acid ethyl acrylate (MAE) copolymer (1: 1).
  • the enteric coating comprises methacrylic acid ethyl acrylate (MAE) copolymer (1: 1) (such as Kollicoat MAE 100P).
  • the solid dose form can comprise a coating.
  • the solid dose form can comprise a single layer coating, e.g., enteric coating, e.g, a Eudragit-based coating, e.g., EUDRAGIT L30 D-55, triethylcitrate, and talc.
  • the solid dose form can comprise two layers of coating.
  • an inner coating can comprise, e.g., EUDRAGIT L30 D- 55, triethylcitrate, talc, citric acid anhydrous, and sodium hydroxide
  • an outer coating can comprise, e.g., EUDRAGIT L30 D-55, triethylcitrate, and talc.
  • 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.
  • Eudragits are amorphous polymers having glass transition temperatures between 9 to > 150°C. Eudragits are non-biodegradable, nonabsorbable, and nontoxic. Anionic Eudragit L dissolves at pH > 6 and is used for enteric coating, while Eudragit S, soluble at pH > 7 is used for colon targeting.
  • Eudragit RL and RS having quaternary ammonium groups, are water insoluble, but swellable/permeable polymers which are suitable for the sustained release fdm coating applications.
  • Cationic Eudragit E insoluble at pH > 5, can prevent drug release in saliva.
  • the solid dose form (e.g., a capsule) can comprise HPMC or gelatin.
  • a bacterial composition comprising Veillonella parvula bacteria can be formulated as a suspension, e.g., for oral administration or for injection. Administration by injection includes intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration.
  • IV intravenous
  • IM intramuscular
  • SC subcutaneous
  • Veillonella parvula bacteria 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 Veillonella parvula bacteria in the suspension can be isolated Veillonella parvula bacteria.
  • the Veillonella parvula bacteria in the suspension can be lyophilized.
  • the Veillonella parvula bacteria in the solid dose form are live.
  • the Veillonella parvula bacteria in the suspension can be gamma irradiated.
  • the dose of Veillonella parvulci bacteria can be, e.g., about 3 x 10 10 to about 1.5 x 10 12 particles, about 4.5 x 10 10 to about
  • the dose can be, e.g., about 1 x 10 10 particles, about 2 x 10 10 particles, about 3 x 10 10 particles, about 4 x 10 10 particles, about 4.5 x 10 10 particles, about 5 x 10 10 particles, about 6 x 10 10 particles, about 7 x 10 10 particles, about 8 x 10 10 particles, about 9 x 10 10 particles, about 1 x 10 11 particles, about
  • the dose can be, e.g., about 3x10 10 particles.
  • the dose can be, e.g., about 4.5xlO 10 particles.
  • the dose can be, e.g., about 1.5xlO n particles.
  • the dose can be, e.g., about 7.5xlO n particles.
  • the dose can be, e.g., about 1.5xl0 12 particles.
  • Particle count can be determined, e.g., by NTA.
  • the dose of Veillonella parvula bacteria can be, e.g., about 3 x 10 10 to about 1.5 x 10 12 total cells, about 4.5 x 10 10 to about
  • the dose can be, e.g., about 1 x 10 10 total cells, about 2 x 10 10 total cells, about 3 x 10 10 total cells, about 4 x 10 10 total cells, about 4.5 x 10 10 total cells, about 5 x 10 10 total cells, about 6 x 10 10 total cells, about 7 x 10 10 total cells, about 8 x 10 10 total cells, about 9 x 10 10 total cells, about 1 x 10 11 total cells, about 1.5 x 10 11 total cells, about 2 x 10 11 total cells, about 3 x 10 11 total cells, about 4 x 10 10 total cells, about 4.5 x 10 10 total cells, about 5 x 10 10 total cells, about 6 x 10 total cells, about 7 x 10 10 total cells, about 8 x 10 10 total cells, about 9 x 10 10 total cells, about 1 x 10 11 total cells, about 1.5 x 10 11 total cells, about 2 x 10 11 total cells, about 3 x 10 11 total cells, about 4 x 10 11 total cells, about 5 x 10 11 total cells
  • the bacterial composition (e.g., pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises about 1 x 10 10 total cells, about 2 x 10 10 total cells, about 3 x 10 10 total cells, about 4 x 10 10 total cells, about 4.5 x 10 10 total cells, about 5 x 10 10 total cells, about 6 x IO 10 total cells, about 7 x IO 10 total cells, about 8 x IO 10 total cells, about 9 x IO 10 total cells, about 1 x 10 11 total cells, about 1.5 x 10 11 total cells, about 2 x 10 11 total cells, about 3 x 10 11 total cells, about 4 x 10 11 total cells, about 5 x 10 11 total cells, about 6 x 10 11 total cells, about 7 x 10 11 total cells, about 7.5 x 10 11 total cells, about 8 x 10 11 total cells, about 9 x 10 11 total cells, about 1 x 10 12 total cells, about 1.5 x
  • the bacterial composition (e.g., pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises at least 1 x IO 10 total cells (e.g., at least 1 x IO 10 total cells, at least 2 x IO 10 total cells, at least 3 x IO 10 total cells, at least 4 x IO 10 total cells, at least 4.5 x IO 10 total cells, at least 5 x IO 10 total cells, at least 6 x IO 10 total cells, at least 7 x IO 10 total cells, at least 8 x IO 10 total cells, at least 9 x IO 10 total cells, at least 1 x 10 11 total cells, at least 1.5 x 10 11 total cells, at least 2 x 10 11 total cells, at least 3 x 10 11 total cells, at least 4 x 10 11 total cells, at least 5 x 10 11 total cells, at least 6 x 10 11 total cells, at least 7 x 10 11 total cells, at least
  • the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x IO 10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x IO 10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 7.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10 12 total cells of the Veillonella parvula bacteria.
  • the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x 10 10 to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x 10 10 to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x 10 10 to about 1.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x 10 10 to about 1.5 x 10 11 total cells of the Veillonella parvula bacteria.
  • the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10 n to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10 n to about 7.5 x 10 11 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 7.5 x 10 n to about 1.5 x 10 12 total cells of the Veillonella parvula bacteria.
  • the Veillonella parvula bacteria may be quantified based on total cells, e.g., total cell count (TCC) (e.g., determined by Coulter counter).
  • TCC total cell count
  • solid dosage forms comprising the Veillonella parvula bacteria.
  • the solid dosage form comprises an enteric coating.
  • the solid dosage form is a capsule, e.g., an enteric coated capsule.
  • each capsule comprises about 3 x 10 10 total cells of the Veillonella parvula bacteria.
  • each capsule comprises about 4.5 x IO 10 total cells of the Veillonella parvula bacteria.
  • each capsule comprises about 1.5 x 10 11 total cells of the Veillonella parvula bacteria.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 capsules are administered, e.g., once or twice daily to a subject.
  • 1 capsule e.g., comprising about 3 x IO 10 total cells
  • 2 capsules e.g., each comprising about 3 x IO 10 total cells
  • 4 capsules e.g., each comprising about 3 x IO 10 total cells
  • 10 capsules are administered, e.g., once or twice daily to a subject.
  • 1 capsule e.g., comprising about 4.5 x IO 10 total cells
  • 2 capsules e.g., each comprising about 4.5 x IO 10 total cells
  • 4 capsules e.g., each comprising about 4.5 x IO 10 total cells
  • 10 capsules are administered, e.g., once or twice daily to a subject.
  • 1 capsule e.g., comprising about 1.5 x 10 n total cells
  • 2 capsules e.g., each comprising about 1.5 x 10 11 total cells
  • 5 capsules e.g., each comprising about 1.5 x 10 11 total cells
  • 10 capsules are administered, e.g., once or twice daily to a subject.
  • the Veillonella parvulci bacteria in the capsule are lyophilized (e.g., in a powder). In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the Veillonella parvula bacteria of the capsule are gamma irradiated.
  • the solid dosage form comprises a capsule.
  • the capsule is an enteric coated capsule.
  • the capsule comprises about 3 x IO 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules).
  • the capsule comprises about 4.5 x IO 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules).
  • the capsule comprises about 1.5 x 10 11 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules).
  • the Veillonella parvula bacteria in the capsule are lyophilized (e.g., in a powder). In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the Veillonella parvula bacteria of the capsule are gamma irradiated.
  • the solid dosage form comprises a tablet.
  • the tablet is an enteric coated tablet.
  • the enteric coated tablet is from 5mm to 18mm in diameter.
  • the tablet comprises about 3 x IO 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets).
  • the tablet comprises about 4.5 x IO 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets).
  • the tablet comprises about 1.5 x 10 11 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets).
  • the Veillonella parvula bacteria in the tablet are lyophilized.
  • the Veillonella parvula bacteria of the tablet are gamma irradiated.
  • the solid dosage form comprises a mini-tablet.
  • the mini-tablet is enteric coated.
  • the mini- tablet is from 1mm to 4mm in diameter.
  • the mini-tablet e.g., enteric coated mini-tablet
  • the solid dosage form comprises mini-tablets that comprise about 3 x 10 10 total cells of the Veillonella parvulci bacteria (e.g., total dose of a plurality of mini -tablets).
  • the solid dosage form comprises mini-tablets that comprise about 4.5 x IO 10 total cells of the Veillonella parvula bacteria (e.g., total dose of a plurality of mini -tablets). In some embodiments, the solid dosage form comprises mini-tablets that comprise about 1.5 x 10 11 total cells of the Veillonella parvula bacteria (e.g., total dose of a plurality of minitablets). In some embodiments, the Veillonella parvula bacteria in the mini-tablets are lyophilized. In some embodiments, the Veillonella parvula bacteria in the mini-tablet are gamma irradiated.
  • the mini -tablets are contained in a capsule.
  • the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule.
  • the capsule comprises a non- enteric coating (e.g., gelatin) (e.g., is coated with a non-enteric coating).
  • the capsule comprises a non-enteric coating.
  • the capsule comprises gelatin.
  • the capsule comprises HPMC.
  • the mini-tablets e.g., enteric coated mini -tablets
  • the minitablets that comprise about 3 x 10 10 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC.
  • the minitablets e.g., enteric coated mini-tablets
  • the minitablets that comprise about 4.5 x 10 10 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC.
  • the mini-tablets e.g., enteric coated mini-tablets
  • the mini-tablets that comprise about 1.5 x 10 11 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC.
  • the Veillonella parvula bacteria of the mini-tablet are gamma irradiated.
  • the mini-tablets are contained in a capsule.
  • the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule.
  • the capsule comprises a non- enteric coating (e.g., gelatin) (e.g., is coated with a non-enteric coating).
  • the capsule comprises a non-enteric coating.
  • the capsule comprises gelatin or HPMC.
  • the mini-tablets e.g., enteric coated mini -tablets
  • the mini-tablets that comprise about 3 x 10 10 total cells of the Veillonella parvulci bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC.
  • the mini-tablets e.g., enteric coated mini -tablets
  • the mini-tablets that comprise about 4.5 x IO 10 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC.
  • the mini-tablets e.g., enteric coated mini-tablets
  • the mini-tablets that comprise about 1.5 x 10 11 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC.
  • the bacterial composition comprising Veillonella parvula bacteria is prepared as a powder (e.g., for resuspension or for use in a solid dose form (such as a capsule)) or as a solid dose form, such as a tablet, a mini-tablet, a capsule, a pill, or a powder; or a combination of these forms (e.g., mini -tablets comprised in a capsule).
  • the powder can comprise lyophilized bacteria.
  • the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide.
  • the powder further comprises sucrose and/or dextran.
  • the powder further comprises sucrose, dextran, and/or L-cysteine HC1.
  • the Veillonella parvula bacteria are gamma irradiated.
  • Powders e.g., of Veillonella parvula bacteria
  • Powders can be gamma-irradiated at 17.5 kGy radiation unit at ambient temperature.
  • Frozen biomasses e.g., of Veillonella parvula bacteria
  • the methods provided herein include the administration to a subject of a bacterial composition described herein either alone or in combination with an additional therapeutic.
  • the additional therapeutic is an immunosuppressant, or a steroid.
  • the additional therapeutic is an IL-6 antagonist.
  • the additional therapeutic is an anti-IL-6 antagonist antibody.
  • the Veillonella parvula bacteria is administered to the subject before the therapeutic 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).
  • the Veillonella parvula bacteria is administered to the subject after the therapeutic 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 Veillonella parvula bacteria and the therapeutic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other).
  • the subject is administered an antibiotic before the Veillonella parvula bacteria 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 before).
  • an antibiotic 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 before.
  • the subject is administered an antibiotic after the Veillonella parvula bacteria 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 Veillonella parvula bacteria and the antibiotic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other).
  • antibiotics can be selected based on their bactericidal or bacteriostatic properties.
  • Bactericidal antibiotics include mechanisms of action that disrupt the cell wall (e.g., [3-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.
  • Gram-negative bacteria such as Escherichia coli, Klebsiella, Pseudomonas aeruginosa, and Francisella tularensis
  • Aminoglycosides are believed to bind to the bacterial 30S 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 Grampositive 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 (MRS A). 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 Grampositive 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.
  • Quinolone s/Fluoroquinolone are effective, e.g., against Streptococcus and Neisseria.
  • Quinolone s/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, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, and Tetracycline. Tetracyclines are effective, e.g., against Gram-negative 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 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, cyclosporin, 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, lomoxicam, isoxicam, mefanamic acid, meclofenamic acid,
  • the additional therapeutic is an oral PDE4 inhibitor (such as apremilast). In some embodiments, the additional therapeutic is apremilast, etanercept, infliximab, adalimumab, ustekinumab, or secukinumab.
  • the 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, anticholinergic 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 inhibitors such
  • the additional therapeutic is cyclosporine.
  • the additional therapeutic is dupilumab.
  • the additional therapeutic is apremilast.
  • the additional therapeutic is etanercept, infliximab, adalimumab, ustekinumab, or secukinumab.
  • the additional therapeutic is an inhaled corticosteroid.
  • the additional therapeutic is a systemic corticosteroid.
  • the additional therapeutic is a monoclonal antibody targeting IL-4, IL4R or IL-5.
  • the bacterial composition is administered orally. In some embodiments, the administration to the subject is once daily. In some embodiments, the administration to the subject is twice daily. In some embodiments, the bacterial composition is administered in 2 or more doses (e.g., 3 or more, 4 or more or 5 or more doses).
  • the administration to the subject of the two or more doses are separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.
  • the bacterial composition is administered once daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
  • the bacterial composition (e.g., pharmaceutical composition) is administered twice daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
  • the bacterial composition (e.g, pharmaceutical composition) is administered once daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered once daily for 8 weeks. In some embodiments, the bacterial composition is administered once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for at least 8 weeks.
  • the bacterial composition (e.g., pharmaceutical composition) is administered twice daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered twice daily for 8 weeks. In some embodiments, the bacterial composition is administered twice daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered twice daily for at least 8 weeks. [294] In some embodiments, the bacterial composition is formulated as a capsule or a tablet. In some embodiments, the bacterial formulation comprises an enteric coating or micro encapsulation. In some embodiments, the capsule is an enteric coated capsule.
  • the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal (e.g., 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).
  • a non-human mammal e.g., 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 bacterial composition is administered in conjunction with the administration of an additional therapeutic.
  • the bacterial composition comprises Veillonella parvulci bacteria coformulated with the additional therapeutic.
  • the bacterial composition is co-administered with the additional therapeutic.
  • the additional therapeutic is administered to the subject before administration of the bacterial composition (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).
  • the additional therapeutic is administered to the subject after administration of the bacterial composition (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 are used to deliver both the bacterial composition and the additional therapeutic.
  • different modes of delivery are used to administer the bacterial composition and the additional therapeutic.
  • the bacterial composition is administered orally while the additional therapeutic is administered via injection (e.g., an intravenous, and/or intramuscular injection).
  • the bacterial compositions, dosage forms, and kits described herein can be administered in conjunction with any other conventional treatment. These treatments may be applied as necessary and/or as indicated and may occur before, concurrent with or after administration of the bacterial compositions, dosage forms, and kits described herein.
  • 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, and other compounds such as drugs being administered 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 the bacterial compositions 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 induce an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell).
  • an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell.
  • dosage will depend upon a variety of factors including the strength of the particular compound 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 compound and the desired physiological effect.
  • Suitable doses and dosage regimens can be determined by conventional rangefinding 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 (“MID”) of bioactive agent per kilogram weight. MID”) of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.
  • MID maximal tolerable dose
  • the dosages of the active 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.
  • 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 bacterial 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 and/or the time period for a subject to clear the bacteria from normal tissue.
  • 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 time period can be a function of the time period for a subject to clear the bacteria from normal tissue; for example, the time period can be more than the time period for a subject to clear the bacteria from normal tissue, such as more than about a day, more than about two days, more than about three days, more than about five days, or more than about a week.
  • the delivery of an additional therapeutic in combination with the bacterial composition described herein reduces the adverse effects and/or improves the efficacy of the additional therapeutic.
  • the effective dose of an additional therapeutic described herein is the amount of the therapeutic agent that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, with the least toxicity to the patient.
  • 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 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 patient being treated, and like factors well known in the medical arts.
  • an effective dose of an additional therapy will be the amount of the 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 therapy is the level of adverse effects experienced by the subject during and following treatment.
  • Adverse events associated with additional therapy toxicity 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, dyspepsia,
  • the methods and compositions described herein relate to inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) in a subject.
  • an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2
  • the immune effect comprises an increase in expression of an anti-inflammatory cytokine by an immune cell.
  • the antiinflammatory cytokine is IL- 10, or IL-27.
  • the anti-inflammatory cytokine is IL- 10.
  • the immune effect comprises a decrease in expression of a pro-inflammatory cytokine by an immune cell.
  • the pro-inflammatory cytokine is IL-ip, IL-6, and TNE-a, IL-5, IL-4, IL- 13, IL- 17, or IL-8.
  • TNE-a may also be referred to herein as “TNE.”
  • the pro- inflammatory cytokine is IL-5, IL-4, IL- 13, or IL- 17.
  • the immune cell is a peripheral blood mononuclear cell (PBMC), a dendritic cell, or a macrophage.
  • PBMC peripheral blood mononuclear cell
  • a dendritic cell or a macrophage.
  • the immune effect comprises elevating expression (e.g., in the small intestine) of a gene provided in Example 5.
  • the cell types found enriched amongst genes showing elevated expression are immune epithelial cells.
  • the cell types found enriched amongst genes showing elevated expression are immune cells (e.g., B cells; T cells; and/or myeloid cells).
  • the gene showing elevated expression is Spinkl, Tm4sf5, and/or Aocl.
  • the gene showing elevated expression is a gene provided in Table 2.
  • the gene showing elevated expression is associated with lymphocyte migration, gut-homing, and adhesion (e.g., Ccl25, CcrlO, Ccl22, Ccl24, Itgb7, Itgal, and/ or It gam).
  • the gene showing elevated expression is associated with T cell lineage maturation and activation (e.g., Cd69, Icos, Il2rg, Cd3d, Trbcl, Trbc2, Trac, Lat, Zap70, Lek, and/or Cd2).
  • the gene showing elevated expression is associated with B cell lineage maturation and activation (e.g., CD19, CD79a, CD79b, CD69, Ighd, Fcrll, Blk, Ikzfi, Tnfrsfl3c, Jchain, Iglcl, and/or Iglc2) .
  • the gene showing elevated expression is associated with immune modulation (e.g., Foxp3, Lag3, Traf3ip3, Slamf6, 1133, Cd5, Adamdecl, and/or Nrli3).
  • the gene showing elevated expression is associated with intestinal epithelial cell homeostasis (barrier, metabolic, absorption) (e.g., Gpx2, Gstm3, Aqp8, Guca2b, Adipoq, Dgatl, Dgat2, Slc23al, and/or Slc51 b).
  • the gene showing elevated expression is associated with host-protective pathways (e.g., Zgl6, Def5a, Reg3a, Retnlb, Reg3g, Cfd, Lypd8, and/or Casp6).
  • the subject has an immune disorder.
  • the immune disorder is atopic dermatitis, psoriasis, or asthma.
  • the atopic dermatitis is moderate atopic dermatitis.
  • the psoriasis is moderate psoriasis.
  • the asthma is moderate asthma.
  • the subject has a disease or disorder.
  • the disease or disorder is an inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis).
  • the disease or disorder is psoriasis (e.g., moderate psoriasis).
  • the disease or disorder is atopic dermatitis (e.g., moderate atopic dermatitis).
  • the disease or disorder is asthma (e.g., mild asthma).
  • the methods described herein can be used to induce an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) in any subject in need thereof.
  • an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2
  • a “subject in need thereof’ includes any subject that has a disease or disorder associated with a pathological immune response (psoriasis (e.g., moderate psoriasis) or atopic dermatitis (e.g., moderate atopic dermatitis) or asthma (e.g., mild asthma)), as well as any subject with an increased likelihood of acquiring such a disease or disorder.
  • psoriasis e.g., moderate psoriasis
  • atopic dermatitis e.g., moderate atopic dermatitis
  • asthma e.g., mild asthma
  • compositions described herein can be used, for example, as a bacterial composition for inducing an immune effect (e.g., an increase in expression of an antiinflammatory cytokine by an immune cell or a decrease in expression of a pro- inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) in a subject.
  • an immune effect e.g., an increase in expression of an antiinflammatory cytokine by an immune cell or a decrease in expression of a pro- inflammatory cytokine by an immune cell
  • elevating expression of a gene provided in Example 5 e.g., an increase in expression of an antiinflammatory cytokine by an immune cell or a decrease in expression of a pro- inflammatory cytokine by an immune cell
  • elevating expression of a gene provided in Example 5 e.g., an increase in expression of an antiinflammatory cytokine by an immune cell or a decrease in expression of a pro- inflammatory cyto
  • the subject has 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 bacterial composition for suppressing rejection in organ transplantation or other situations in which tissue rejection might occur; a supplement, food, or beverage for improving immune functions; or a reagent for suppressing the proliferation or function of immune cells.
  • an autoimmune disease such as chronic inflammatory bowel disease, systemic lupus erythematosus
  • the subject has inflammation.
  • the inflammation of any tissue and organs of the body including musculoskeletal inflammation, vascular inflammation, neural inflammation, digestive system inflammation, ocular inflammation, inflammation of the reproductive system, and other inflammation, as discussed below.
  • Immune disorders of the musculoskeletal system include, but are not limited, to those conditions affecting skeletal joints, including joints of the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle, and foot, and conditions affecting tissues connecting muscles to bones such as tendons.
  • immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis, tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis, myositis, and osteitis (including, for example, Paget's disease, osteitis pubis, and osteitis fibrosa cystic).
  • arthritis including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis
  • tendonitis synovitis, ten
  • Ocular immune disorders refers to an 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 which may be treated with the methods and 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 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 subject has an autoimmune condition having an inflammatory component.
  • autoimmune condition 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, good pasture'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
  • the subject has a T-cell mediated hypersensitivity disease having an inflammatory component.
  • a condition includes, but are not limited to, contact hypersensitivity, contact dermatitis (including that due to poison ivy), uticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dustmite allergy) and gluten-sensitive enteropathy (Celiac disease).
  • immune disorders that the subject may have include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, ulceris, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel, skin allografts, skin homografts, and heart valve xengrafts, sewrum sickness, and graft vs host disease), acute
  • Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosis, psoriasis, chronic obstructive pulmonary disease, and inflammation accompanying infectious conditions (e.g., sepsis).
  • bacterial compositions for use in inducing an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2 are disclosed.
  • a bacterial composition comprising Veillonella parvula, wherein the Veillonella parvulci is a strain comprising at least 85% sequence identity to the nucleotide sequence of the Veillonella parvula strain A (ATCC Deposit Number PTA-125691) for use in inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) is described herein.
  • an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2
  • a bacterial composition for the preparation of a medicament for inducing an immune effect e.g., an increase in expression of an antiinflammatory cytokine by an immune cell or a decrease in expression of a pro- inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2 are disclosed.
  • a bacterial composition for the preparation of a medicament for inducing an immune effect e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2
  • the bacterial composition comprises Veillonella parvula, wherein the Veillonella parvulci is a strain comprising at least 85% sequence identity to the nucleotide sequence of the Veillonella parvula strain A (ATCC Deposit Number PTA-125691) is described herein.
  • gut microbiota also called the “gut microbiota”
  • gut microbiota can have a significant impact on an individual’s health through microbial activity and influence (local and/or distal) on immune and other cells of the host
  • a healthy host-gut microbiome homeostasis is sometimes referred to as a “eubiosis” or “normobiosis,” whereas a detrimental change in the host microbiome composition and/or its diversity can lead to an unhealthy imbalance in the microbiome, or a “dysbiosis” (Hooks and O’Malley. Dysbiosis and its discontents. American Society for Microbiology. Oct 2017. Vol. 8. Issue 5. mBio 8:e01492-17. https://doi.org/10.1128/mBio.01492-17).
  • Dysbiosis, and associated local or distal host inflammatory or immune effects may occur where microbiome homeostasis is lost or diminished, resulting in: increased susceptibility to pathogens; altered host bacterial metabolic activity; induction of host proinflammatory activity and/or reduction of host anti-inflammatory activity.
  • Such effects are mediated in part by interactions between host immune cells (e.g., T cells, dendritic cells, mast cells, NK cells, intestinal epithelial lymphocytes (IEC), macrophages and phagocytes) and cytokines, and other substances released by such cells and other host cells.
  • host immune cells e.g., T cells, dendritic cells, mast cells, NK cells, intestinal epithelial lymphocytes (IEC), macrophages and phagocytes
  • a dysbiosis may occur within the gastrointestinal tract (a “gastrointestinal dysbiosis” or “gut dysbiosis”) or may occur outside the lumen of the gastrointestinal tract (a “distal dysbiosis”). Gastrointestinal dysbiosis is often associated with a reduction in integrity of the intestinal epithelial barrier, reduced tight junction integrity and increased intestinal permeability. Citi, S. Intestinal Barriers protect against disease, Science
  • a gastrointestinal dysbiosis can have physiological and immune effects within and outside the gastrointestinal tract.
  • dysbiosis has been associated with a wide variety of diseases and conditions including: infection, cancer, autoimmune disorders (e.g., systemic lupus erythematosus (SLE)) or inflammatory disorders (e.g., functional gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease), neuroinflammatory diseases (e.g., multiple sclerosis), transplant disorders (e.g., graft-versus-host disease), fatty liver disease, type I diabetes, rheumatoid arthritis, Sjogren’s syndrome, celiac disease, cystic fibrosis, chronic obstructive pulmonary disorder (COPD), and other diseases and conditions associated with immune dysfunction.
  • autoimmune disorders e.g., systemic lupus erythematosus (SLE)
  • inflammatory disorders e.g., functional gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease
  • neuroinflammatory diseases e.g.
  • Exemplary bacterial compositions 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, elevating expression of a gene provided in Example 5, and/or elevating expression of a gene provided in Table 2.
  • Exemplary bacterial compositions disclosed herein that are useful for treatment of disorders associated with a dysbiosis contain one or more types of immunomodulatory bacteria (e.g., anti-inflammatory bacteria). Such compositions are capable of affecting the recipient host’s immune function, in the gastrointestinal tract, and/or a systemic effect at distal sites outside the subject’s gastrointestinal tract.
  • immunomodulatory bacteria e.g., anti-inflammatory bacteria
  • Exemplary bacterial compositions disclosed herein that are useful for treatment of disorders associated with a dysbiosis contain a population of immunomodulatory bacteria of a single bacterial species (e.g., a single strain) (e.g., anti-inflammatory bacteria). Such compositions are capable of affecting the recipient host’s immune function, in the gastrointestinal tract, and /or a systemic effect at distal sites outside the subject’s gastrointestinal tract.
  • bacterial compositions containing an isolated population of immunomodulatory bacteria e.g., anti-inflammatory bacterial cells
  • 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 bacterial compositions can treat a gastrointestinal dysbiosis and one or more of its effects by modulating the recipient immune response via cellular and cytokine modulation to reduce gut permeability by increasing the integrity of the intestinal epithelial barrier.
  • the bacterial compositions 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 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 immunomodulatory bacteria, e.g., a single strain) capable of altering the relative proportions of immune cell subpopulations, e.g., T cell subpopulations, immune lymphoid cells, NK cells and other immune cells, or the function thereof, in the recipient subject.
  • a population of immunomodulatory bacteria e.g., a single strain
  • immune cell subpopulations e.g., T cell subpopulations, immune lymphoid cells, NK cells and other immune cells, or the function thereof, in the recipient subject.
  • the invention provides methods of treating a gastrointestinal dysbiosis and one or more of its effects by orally administering to a subject in need thereof a bacterial composition which alters the microbiome population existing at the site of the dysbiosis.
  • the bacterial composition can contain one or more types of immunomodulatory bacteria or a population of immunomodulatory bacteria of a single bacterial species (e.g., a single strain).
  • the invention provides methods of treating a distal dysbiosis and one or more of its effects by orally administering to a subject in need thereof a bacterial composition which alters the subject’s immune response outside the gastrointestinal tract.
  • the bacterial composition can contain one or more types of immunomodulatory bacteria or a population of immunomodulatory bacteria of a single bacterial species (e.g., a single strain).
  • bacterial compositions useful for treatment of disorders associated with a dysbiosis stimulate secretion of one or more antiinflammatory cytokines by host immune cells.
  • Anti-inflammatory cytokines include, but are not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGF(3, and combinations thereof.
  • bacterial compositions useful for treatment of disorders associated with a dysbiosis that decrease (e.g., inhibit) secretion of one or more pro- inflammatory cytokines by host immune cells.
  • Pro-inflammatory cytokines include, but are not limited to, IFNy, IL-I2p70, IL-Ia, IL-6, IL-8, MCP1, MIPla, MIP1 , TNFa, and combinations thereof.
  • Other exemplary cytokines are known in the art and are described herein.
  • bacterial compositions useful for treatment of disorders associated with a dysbiosis elevate expression (e.g., in the small intestine) of a gene provided in Example 5.
  • the cell types found enriched amongst genes showing elevated expression are immune epithelial cells.
  • the cell types found enriched amongst genes showing elevated expression are immune cells (e.g., B cells; T cells; and/or myeloid cells).
  • the gene showing elevated expression is Spinkl, Tm4sf5, and/or Aocl.
  • the gene showing elevated expression is a gene provided in Table 2.
  • the gene showing elevated expression is associated with lymphocyte migration, gut-homing, and adhesion (e.g., Ccl25, CcrlO, Ccl22, Ccl24, Itgb7, Itgal, and/or It gam).
  • the gene showing elevated expression is associated with T cell lineage maturation and activation (e.g., Cd69, Icos, Il2rg, Cd3d, Trbcl, Trbc2, Trac, Lat, Zap70, Lek, and/or Cd2).
  • the gene showing elevated expression is associated with B cell lineage maturation and activation (e.g., CD19, CD79a, CD79b, CD69, Ighd, Fcrll, Blk, Ikzfi, Tnfrsfl3c, Jchain, Iglcl, and/or Iglc2).
  • the gene showing elevated expression is associated with immune modulation (e.g., Foxp3, Lag3, Traf3ip3, Slamf6, 1133, Cd5, Adamdecl, and/or Nrli3).
  • the gene showing elevated expression is associated with intestinal epithelial cell homeostasis (barrier, metabolic, absorption) (e.g., Gpx2, Gstm3, Aqp8, Guca2b, Adipoq, Dgatl, Dgat2, Slc23al, and/or Slc51b).
  • the gene showing elevated expression is associated with host-protective pathways (e.g., Zgl6, Def 5a, Reg3a, Retnlb, Reg3g, Cfd, Lypd8, and/or Casp6).
  • 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 bacteria in an amount sufficient to alter the microbiome at a site of the dysbiosis, such that the disorder associated with the dysbiosis is treated.
  • a 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.
  • the Veillonella parvula is a strain comprising at least 99.9% sequence identity to the nucleotide sequence of the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the Veillonella parvula is the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
  • the bacterial composition is administered orally.
  • the bacterial composition is formulated as a capsule or a tablet.
  • the capsule is an enteric coated capsule.
  • the bacterial composition comprises about 3 x IO 10 total cells of Veillonella parvula. In some embodiments, the bacterial composition comprises about 1.5 x 10 11 total cells of Veillonella parvula. In some embodiments, the bacterial composition comprises about 7.5 x 10 11 total cells of Veillonella parvula. In some embodiments, the bacterial composition comprises about 1.5 x 10 12 total cells of Veillonella parvula. In some embodiments, the bacterial composition comprises from about 3 x IO 10 to about 1.5 x 10 12 total cells of Veillonella parvula. In some embodiments, the bacterial composition is administered at least once daily. In some embodiments, the bacterial composition is administered once daily. In some embodiments, the bacterial composition is administered once daily for at least 15 continuous days.
  • the bacterial composition is administered once daily for at least 28 continuous days. In some embodiments, the bacterial composition is administered once daily for at least 56 continuous days. In some embodiments, the psoriasis is moderate psoriasis. In some embodiments, the atopic dermatitis is moderate atopic dermatitis. In some embodiments, the asthma is mild asthma.
  • the cryoprotectant may contain, e.g., maltodextrin, sodium ascorbate, sodium glutamate, and/or calcium chloride.
  • the cryoprotectant may contain, e.g., sucrose, dextran, and/or L-cysteine HC1.
  • Powders are stored (e.g., in vacuum sealed bags) at 2-8 degrees C (e.g., at 4 degrees C), e.g., in a desiccator.
  • Powders are gamma-irradiated at 17.5 kGy radiation unit at ambient temperature.
  • Frozen biomasses are gamma-irradiated at 25 kGy radiation unit in the presence of dry ice.
  • Veillonella parvula strain A- G.I. (gamma irradiated) is a pharmaceutical preparation of a single strain of Veillonella parvula (Veillonella parvula strain A), originally isolated from a fresh ileostomy sample of an IBD patient in remission, which has been gamma-irradiated (G.I.).
  • the final drug product is therefore non-viable at the time of dosing. It has not been genetically modified. Because it is non-viable, it does not colonize the gut and has no detectable systemic exposure following oral dosing. It has dose dependent therapeutic effects in multiple pre-clinical models.
  • Veillonella parvula strain A has been deposited as ATCC Accession Number PTA-125691.
  • Veillonella strains are found in several niches of the human body including the mouth, lungs, gastrointestinal tract, and vagina, and is a normal component of a healthy human microbiota. Although there are sporadic reports of some Veillonella species that can act as opportunistic pathogens, recent data suggests that Veillonella may perform a protective role and aid in early childhood immune system development. Epidemiological studies of infants have demonstrated that presence of Veillonella is negatively correlated with asthma and bronchiolitis.
  • Veillonella parvula strain A- G.I. in human and mouse cellular assays and in vivo models support its use in the treatment of immunoinflammatory diseases, including atopic dermatitis, psoriasis, and asthma.
  • Veillonella parvula strain A- G.L increases secretion of anti-inflammatory cytokines from human immune cells, such as interleukin (IL)- 10, while inducing minimal production of pro-inflammatory cytokines such as TNFy and IFNy.
  • IL interleukin
  • Veillonella parvula strain A- G.I. Oral administration of Veillonella parvula strain A- G.I. to mice led to striking therapeutic effects on delayed-type hypersensitivity (DTH), imiquimod-induced skin inflammation, fluorescein isothiocyanate (FITC) cutaneous hypersensitivity, MC903- induced dermatitis, and experimental acute encephalomyelitis (EAE) in-vivo models.
  • DTH delayed-type hypersensitivity
  • FITC fluorescein isothiocyanate
  • MC903- induced dermatitis MC903- induced dermatitis
  • EAE experimental acute encephalomyelitis
  • Veillonella parvula strain A- G.I. drug product (DP) formulation is a blend of freeze-dried powder of V. parvula (DS) and excipients (mannitol, colloidal silicon dioxide, magnesium stearate, hydroxypropyl methylcellulose, poly(methacrylic acid, ethyl acrylate, 1: 1), triethyl citrate, and talc). Two active drug product strengths with matching placebo are manufactured to support the clinical studies.
  • Veillonella parvula strain A- G.I. finished product is manufactured as an enteric- coated capsule, designed to protect Veillonella parvula strain A- G.I. from stomach pH degradation and designed to release at pH > 5.5, which is approximately the pH in the small intestine.
  • APC antigen presenting cells
  • CD cluster of differentiation
  • CEF
  • Cytomegalovirus Epstein Bar virus and Flu viruses
  • CHS contact hypersensitivity
  • DC dendritic cell
  • DTH delayed-type hypersensitivity
  • EAE experimental acute encephalomyelitis
  • FITC Fluorescein isothiocyanate
  • GI gastrointestinal
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IFNy interferon gamma
  • IL interleukin
  • KLH keyhole limpet haemocyanin
  • LPS lipopolysaccharide
  • MLN mesenteric lymph node
  • PLP proteolipid protein
  • TH 17 T-helper 17
  • TNF tumor necrosis factor
  • Veillonella parvulci strain A is the identifier of the original parental strain from which Veillonella parvula strain A- G.I. was derived. Veillonella parvula strain A has been deposited as ATCC Accession Number PTA-125691.
  • Veillonella parvula strain A- G.I. is restricted to the gut, there are a limited number of immune cells types with which it can interact as it passes through the GI tract.
  • the immune cells that are most likely to directly encounter Veillonella parvula strain A- G.I. are located in the lamina basement underlying the intestinal epithelial cell layer, and are in the myeloid lineage, including dendritic cells and macrophages.
  • a series of in vitro studies was performed to examine the effects of the interaction of Veillonella parvula strain A- G.I. or parental bacterial Veillonella parvula strain A on human immune cells derived from healthy volunteer peripheral blood. The effect that a human dendritic cell exposed to Veillonella parvula strain A- G.I. might have on its ability to activate CD 8+ T cells, a major producer of the pro-inflammatory cytokine IFNy was examined.
  • Veillonella parvula strain A consistently induces high levels of IL- 10 from primary purified macrophages from multiple human donors.
  • Luminex technology was used to measure pro- and anti-inflammatory cytokine production including IL-12p70, TNF, CXCL10 (IP- 10), and IL-10.
  • IL-12p70 pro- and anti-inflammatory cytokine production
  • TNF TNF
  • CXCL10 CXCL10
  • IL-10 IL-10
  • the data show that of the >70 unique commensal strains evaluated, those of the genus Veillonella, including Veillonella parvulci strain A, were the strongest inducers of the anti-inflammatory cytokine IL- 10. Additionally, Veillonella parvula strain A did not induce high levels of pro-inflammatory cytokines CXCL10, IL-12p70, or TNF compared to other commensal anaerobic strains. Based on these data, the cytokine stimulating profile of parental strain Veillonella parvula strain A is attractive which is higher for anti-inflammatory IL- 10 compared to pro-inflammatory cytokines made from the same cell.
  • Isolated human PBMCs, DCs, and macrophages from whole blood were cultured with a range of doses from 10 4 - 5 x 10 6 total cell count (TCC) of gamma-irradiated Veillonella parvula strain A as described above. After 24 hours, supernatants were collected and IL- 10 was measured by Luminex. A dose-response relationship was observed between increasing TCC and production of IL- 10 from both PBMCs and dendritic cells ( Figures 1A and IB, respectively), are shown in Figure 1C.
  • Gamma-irradiated Veillonella parvula strain A does not induce induction of the inflammatory cytokine TNF from pro-inflammatory macrophages.
  • Macrophages are one of the major human cell types to produce and secrete the pro-inflammatory cytokine TNF in response to exposure to microbial products.
  • an in vitro assay with primary human myeloid cells skewed towards a pro-inflammatory phenotype was carried out.
  • CD 14+ PBMCs primary human CD 14+ PBMCs were purified from three healthy donors by Ficoll-Paque gradient centrifugation in combination with magnetic cell separation.
  • CD 14+ cells were differentiated to a mature APC phenotype for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the cells were skewed to an inflammatory phenotype with a cocktail of lipopolysaccharide (LPS) + IFNy, and subsequently co-cultured with 88 individual anaerobic microbial strains.
  • LPS lipopolysaccharide
  • Beckman Coulter robotics were used to co-culture the obligate anaerobes with mammalian cells in a 96-well format at a MOI of 1.
  • DCs Veillonella yarvula strain A-conditioned dendritic cells
  • CEF peptide pool is composed of peptides from Cytomegalovirus, Epstein-Bar virus, and Influenza virus, pathogens which the majority of the human population has been exposed to. Human donors were characterized prior to the assay and chosen based on their responsiveness to the peptide pool. After 24 hours of stimulation with CEF peptide, DC-CD8+ T cell supernatants were collected and Luminex technology was used to measure pro- and anti-inflammatory cytokines including IFNy, TNF, IL-10, and IL-27.
  • the delayed-type hypersensitivity (DTH) model is a mechanistic skin inflammation model that measures antigen-specific inflammation in the ear
  • the fluorescein isothiocyanate (FITC) induced contact dermatitis model is mediated by the TH2 pathway and exhibits many of the hallmarks of atopic dermatitis
  • MC903 -induced dermatitis is another model mediated by the TH2 pathway, and clinically relevant cytokine pathways that are pathogenic in atopic dermatitis.
  • the imiquimod (IMQ)-induced model of TH17-mediated skin inflammation which is a model of psoriasis based on immune mechanisms.
  • EAE experimental autoimmune encephalomyelitis
  • DTH Delayed-type hypersensitivity
  • the Mantoux Test is used as a classical cell-mediated immunity test to demonstrate previous exposure to pathogens and is induced by the intra-dermal injections of antigens.
  • the response represents a cutaneous T cell-mediated memory recall immune response, and in humans is typically used to determine immunity to tuberculosis.
  • the immunological mechanisms that drive the skin inflammatory response in the murine and human DTH responses are the same, and thus the DTH mouse model is considered a physiologically relevant model for predicting T cell-mediated responses in humans.
  • mice were immunized by subcutaneous injection with keyhole limpet haemocyanin (KLH) emulsified with Complete Freund’s Adjuvant (CFA). Eight days after sensitization, the previously sensitized mice were challenged by intradermal ear injection with KLH or a buffer control. The DTH response was evaluated 24 hours post— challenge, which represents the peak of disease in this model. Mice were dosed for 10 days daily from the day of sensitization through to the end of the study by oral gavage with vehicle, reconstituted Veillonella parvula strain A- G.I. at different doses, or a positive control.
  • KLH keyhole limpet haemocyanin
  • CFA Complete Freund’s Adjuvant
  • mice were dosed for 9 days daily from the day of sensitization through to the end of the study by oral gavage with vehicle, dexamethasone, reconstituted Veillonella parvula strain A- G.I. at different doses.
  • a range of 3 doses over 3 logs (1.5 x 10 8 , 1.5 x 10 9 , and 1.5 x IO 10 total cell count) of Veillonella parvula strain A- G.I. were explored.
  • KLH DTH 2 Veillonella parvula strain A- G.I. is well-tolerated in 30-day DTH
  • a 30-day murine DTH study with KLH was carried out to test the antiinflammatory properties of Veillonella parvula strain A- G.I. over an extended period of dosing. Mice were subcutaneously immunized with an emulsion of 50pL KLH in CFA on Day 0, and then received a KLH booster on Day 7. Mice received two intradermal antigen challenges with lOpg KLH in the ear on Days 15 and 29. Changes in ear swelling thickness vs. baseline were measured using a caliper at 24 after each ear challenge on Days 16 and Days 30. Three treatment groups were included in the study: Vehicle, Veillonella parvulci strain A- G.I.
  • mice were dosed orally Monday to Friday from Days 1 to 30. A group of age-matched naive mice that did not receive immunization, challenge, or treatment was also included in the study for comparison. Health observations and body weight were recorded daily. At study termination, various tissues were collected and preserved in formalin including spleen, colon, small intestine, mesenteric lymph node, cervical lymph node, liver and kidney. Organ weights for spleen and liver were recorded.
  • ALT alanine transaminase
  • AST aspartate transaminase
  • ALP alkaline phosphatase
  • GTT gamma-glutamyltransferase
  • Ear thickness caliper measurements showed that treatment with Veillonella parvula strain A- G.I. significantly reduced ear swelling at 24 hours after the first challenge on Day 15 and the second challenge on Day 29 (Figure 5).
  • mice treated with Veillonella parvula strain A- G.I. for up to 30 days were treated with vehicle.
  • Liver weight was slightly higher in mice treated with Veillonella parvula strain A- G.I. compared to mice treated with vehicle, but there was no significant difference compared to naive mice.
  • No notable changes in body weight were observed throughout the study. Some variability in weight was measured on Day 3, which was likely due to variability in stress responses at the beginning of the study. Body weights returned to normal thereafter. Daily health observations did not show any abnormalities in the health of the animals that were dosed with Veillonella parvula strain A- G.I. for up to 30 days.
  • FITC is used as an experimental hapten in mechanistic studies of contact allergy. Studies have demonstrated that topical exposure of mice to FITC results in the selective development of activated lymph node cells (LNC) expressing a preferential TH2 cytokine -secretion profile, with high levels of IL-4 and IL- 10, but low levels of IFNy.
  • LNC activated lymph node cells
  • mice were sensitized with FITC on the back on Day 1 and Day 2. Mice were gavaged daily from Day 1 to Day 6 with Veillonella parvula strain A- G.I. (1.5 x IO 10 TCC per dose), vehicle, or dexamethasone. They were then challenged on the ear with FITC on Day 6, and ear thickness was measured 24 hours later. The level of the TH2 cytokine IL-4 in the ear tissue was measured by ELISA.
  • the MC903 dermatitis model is a well-established murine model of atopic dermatitis (AD)-like disease. This model recapitulates many of the central features of AD including erythema (redness), scaling, blood eosinophilia, serum IgE elevation, itch behavior (pruritus), and histopathologic features of AD including acanthosis (epidermal thickening), hyperkeratosis (stratum comeum thickening), spongiosis (epidermal edema), and mixed dermal lymphocyte and eosinophil infiltration.
  • Topical application of the vitamin D3 analog MC903 induces changes in skin morphology and inflammation resembling immune perturbations observed in acute lesions of patients with AD.
  • mice were sensitized daily for 14 consecutive days with 2nmol of MC903 (calcipotriol; Tocris Bioscience,) in 20 uL of 100% EtOH on ears. Mice were gavaged daily from Day 1 to Day 6 with 10 mg of Veillonella parvula strain A- G.I. (1.5 x 10 10 TCC per dose), vehicle, or tofacitinib (20 mg/kg). Mice were also treated with dexamethasone daily (1 mg/kg intraperitoneally). Baseline ear measurements were taken prior to the first ear sensitization on day 1 using Digital Calipers (Fowler). On day 14, ear thickness was measured. Delta change in ear thickness was expressed as ear thickness at day 14 minus ear thickness at baseline.
  • mice showed significant reduction of ear thickness associated with MC903 -induced skin inflammation compared to vehicle-treated mice, with the area under the curve calculation for the complete time course showing a similar effect as both dexamethasone and tofacitinib ( Figure 7).
  • the key TH2 cytokine IL-4 was also reduced in both Veillonella parvula strain A- G.I. and tofacitinib treated mice ( Figure 8).
  • the imiquimod-induced psoriasis model is particularly translational into the clinic as it has many of the significant markers of human disease, including histopathology of lesions and strong activation of the immune system.
  • Clinically relevant psoriasis model in mice is through the topical application of 5% imiquimod (IMQ) cream.
  • IMQ imiquimod
  • This imiquimod- induced psoriasis models human plaque-type psoriasis in which the IL-23/IL-17 cytokine axis plays a pivotal role.
  • Study parameters include in-life clinical evaluation of skin, measurement of ear thickness, and analysis of cytokine expression in the skin and immune organs.
  • mice were sensitized on the shaved back and ear with imiquimod cream daily for 7 consecutive days. Mice were gavaged daily with 10 mg of Veillonella parvula strain A- G.I. (1.5 x IO 10 TCC per dose) or vehicle. Mice were also treated with dexamethasone (1 mg/kg intraperitoneally) or given an anti-IL-17 antibody or an anti-IL- 12/23p40 antibody (200 ug/mouse intraperitoneally on days 2, 4, and 6). The severity of inflammation of the back skin was evaluated using a lesion psoriasis severity scoring system.
  • mice were monitored and graded daily on the scale: 0 (no alteration), 1 (mild erythema), 2 (moderate to severe erythema and some plaques), 3 (marked erythema and plaques) and 4 (very marked erythema and plaques).
  • Ear measurements were taken daily using digital calipers and scores were reported as change in ear thickness calculated as ear score on day 8 minus baseline ear score on day 1.
  • skin samples were taken for cytokine expression and protein analysis, and cells from spleen and lymph node were stimulated in vitro to induce cytokine production.
  • Systemic dexamethasone, and two biologies which are highly efficacious in severe psoriasis - anti- IL-17A and anti -IL-23 - were used as comparators.
  • mice Veillonella parvula strain A- G.I. -treated mice showed visibly substantial reduction of erythema, scaling and thickening associated with IMQ-induced skin inflammation compared to vehicle -treated mice on par with anti-IL-17 treatment. Commensurate with reduction of ear inflammation, pro-inflammatory cytokine IL-17A protein was reduced. See figure 10.
  • EAE experimental autoimmune encephalomyelitis
  • EAE is a widely-used model of demyelinating diseases, which is induced in mice through activation immunization using a spinal cord antigen, proteolipid protein (PLP).
  • PLP proteolipid protein
  • PLP injected in Swiss Jim Lambert (SJL) mice together with pertussis toxin (PT) leads to remitting and relapsing demyelinating disease, which can be scored based on the following observations: Score 0 Normal mouse with no overt signs of disease; Score 1 Limp tail or hind limb weakness but not both; Score 2 Limp tail and hind limb weakness; Score 3 Partial hind limb paralysis, Score 4 Complete hind limb paralysis.
  • mice were monitored daily and given a weakness score.
  • EAE develops 10-15 days after immunization in 90-100% of immunized mice.
  • the first wave of EAE lasts several days and most mice fully or almost fully recover from this first wave. After a disease-free period, which can last from one day to several months, most mice relapse.
  • mice are enrolled into treatment groups at the time of EAE onset or at the time of recovery from the first wave of EAE. Mice are distributed into the various treatment groups in a balanced manner to achieve groups with similar time of EAE onset and similar first wave disease severity.
  • mice were injected subcutaneously at four sites in the back with the emulsion component (containing PLP139-151 and CFA). Two sites of injection were in the upper back approximately 1 cm caudal of the neck line. Two more sites were in the lower back approximately 2 cm cranial of the base of the tail. The injection volume was 0.05 mb at each site.
  • the pertussis toxin component of the kit was administered intraperitoneally. Mice were orally gavaged starting on Day 0 with vehicle or Veillonella parvulci strain A- G.I. (1.5 x IO 10 TCC per dose). Animals were monitored and scored daily starting from Day 7. All readouts were continued until termination of the study on Day 42.
  • mice treated with Veillonella parvula strain A- G.I. had significantly lower disease scores compared to vehicle-treated mice during both the initial acute phase, as well as the relapsing -remitting phase (Figure 11).
  • the overall reduction in disease score in mice treated with Veillonella parvula strain A- G.I. could be seen in the area under the curve calculations for the total study (Days 7 - 42), as well as for just the acute phase (Days 7 - 20) ( Figure 12).
  • a biodistribution study was designed to determine where Veillonella parvula strain A- G.I. bacteria are present in a mouse following oral treatment.
  • the study assessed Veillonella parvula strain A- G.I. distribution following a single oral dose using fluorescently labelled Veillonella parvula strain A- G.I. which was administered to naive mice by oral gavage, followed by imaging using the Licor imaging system at various time points after gavage.
  • Veillonella parvula strain A- G.I. led to a transient rise in the GI tract.
  • Veillonella parvula strain A- G.I. was only detected in the intestine and stool for up to 12 hours post-treatment, demonstrating that gamma-irradiated, non-live bacteria are unable to colonize the intestinal tract after a single dose.
  • Veillonella parvula strain A- G.I. was not detected outside of the GI tract at any time point above background fluorescence as determined by the free dye control.
  • Veillonella parvula strain A- G.I. is a single strain of the Veillonella genus (Veillonella parvula) that is being investigated for potential benefit in a range of immuno- inflammatory disorders including atopic dermatitis, and potentially psoriasis and asthma.
  • Veillonella parvulci strain A- G.I. has demonstrated specific pharmacological activity in in vitro and in vivo models of immuno-inflammatory disease and so is being developed as a medicinal product.
  • Example 4 A non-viable, non-colonizing form of Veillonella parvula requires lymphocyte homing to gut-associated lymphoid tissues to regulate systemic inflammation
  • V. parvula Veillonella parvula
  • V. parvula a strain of Veillonella parvula
  • a non-viable form of V. parvula was developed by gamma irradiating the live microbe (designated as Veillonella parvula Strain A).
  • Veillonella parvula Strain A all references to Veillonella parvula Strain A refer to this gamma irradiated form of the strain.
  • the strain has been deposited as ATCC Accession Number PTA-125691.
  • Fresh primary human CD1 lb + immune cells were purified from the peripheral blood mononuclear cells (PBMC) of 6 healthy human donors.
  • the purified human cells were individually co-cultured with a single bacterial obligate anaerobe strain at a multiplicity of infection (MOI) of 1.
  • MOI multiplicity of infection
  • the data show that of the >70 unique commensal strains evaluated, significant levels of IL- 10 were induced from all donors in response to stimulation with Veillonella parvula Strain A compared with several closely related species (Figure 13A).
  • Veillonella parvula Strain A did not induce high levels of pro- inflammatory cytokines IP- 10 or TNFa compared to other commensal anaerobic strains ( Figures 13B and 13C). Furthermore, isolated human PBMCs, DCs, and macrophages from whole blood were cultured with a range of doses from 10 4 - 5 x 10 6 total cell count (TCC) of Veillonella parvula Strain A for 24 hours, and IL- 10 was measured in supernatants. A dose-dependent response was observed between increasing TCC and production of IL-10 from both PBMCs, DCs and macrophages.
  • TCC total cell count
  • Veillonella parvula Strain A is gut restricted and transits rapidly through the intestine without alteration of the gut microbial flora
  • Veillonella parvula Strain A is an orally delivered, gut restricted agent that exerts its pharmacological effects through SINTAX.
  • a study was designed to determine the biodistribution of Veillonella parvula Strain A in a mouse following oral treatment and tracked transit through the gastrointestinal (GI) tract. Oral administration of Veillonella parvula Strain A led to a transient rise in the GI tract. Complete exposure of the small intestine occurred within 1 hour post oral administration of Veillonella parvula Strain A. Veillonella parvula Strain A was only detected in the intestine and stool for up to 12 hours post-treatment after a single dose (TCC - 1E+9) ( Figure 14A).
  • Veillonella parvula Strain A inhibits cutaneous inflammation in imiquimod driven model of psoriasis
  • mice were sensitized on the ear with 20 mg 5% imiquimod cream daily for 7 consecutive days and orally dosed daily with Veillonella parvula Strain A (TCC - 7.8E+11). Veillonella parvula Strain A treated mice showed substantially lower ear inflammation over the course of disease progression ( Figure 15A).
  • transcript levels in the ear tissue revealed reduction in III 7a, III 7f and Deft>3 levels upon treatment with Veillonella parvula Strain A in comparison to vehicle ( Figure 15B).
  • Veillonella parvula Strain A ameliorates inflammation in murine model of relapsingremitting multiple sclerosis
  • EAE autoimmune encephalomyelitis
  • mice began to display motor impairment by day 9 and reached peak clinical disease scores by day 15.
  • Veillonella parvula Strain A showed significant reduction in clinical score compared to vehicle treated animals over the course of the disease.
  • the treatment effects of Veillonella parvula Strain A were most pronounced in the relapsing phase of disease indicating increased recovery after acute phase of disease.
  • mice had an overall lower cumulative EAE score compared to control ( Figure 16A).
  • the effects of Veillonella parvula Strain A on cellular responses in vivo were also determined.
  • Sections of spinal cord tissue from cervical and lumbar regions of mice that received treatment with Veillonella parvula Strain A, a positive control drug fingolimod and a vehicle treated group were analyzed for inflammatory cell infiltration. Mice treated prophylactically with Veillonella parvula Strain A showed significantly reduced frequency of infiltrating inflammatory cells in the spinal cord compared to vehicle treated animals (Figure 16B).
  • Veillonella parvula Strain A reduces inflammation in FITC-driven contact hypersensitivity
  • Atopic dermatitis is a chronic inflammatory skin disease, driven by strong Th2 immune responses. It is a considered a primarily T cell-driven disease with a key role for cytokines IL-4, IL-5, IL-13, alarmin IL-33 (Hardman and Ogg, 2016; Ziegler, 2012) and IL-31, a cytokine associated with itch (Sonkoly et al., 2006) in AD pathogenesis.
  • Veillonella parvula Strain A was tested in a murine model of contact hypersensitivity, using a hapten fluorescein isothiocyanate (FITC) to drive cutaneous inflammation (Boehme et al., 2009; Takeshita et al., 2004).
  • FITC hapten fluorescein isothiocyanate
  • BALB/c mice were topically sensitized with 0.5% FITC on day 1 and 2, and 6 days later they were challenged with 0.5% FITC on the ear. Mice were dosed daily orally with vehicle or Veillonella parvula Strain A (TCC - 2. 16E+12). Ear inflammation was measured 24 h post ear challenge on day 7.
  • Veillonella parvula Strain A treatment leads to reduction of local and systemic pro inflammatory cytokines in vivo
  • the first step is the interaction between Veillonella parvula Strain A and cells in the small intestine, including epithelial cells and dendritic cells.
  • TLRs represent a major class (Price et al., 2018).
  • HEK-293 cells stably transfected with human TLR1/2/6, TLR2/6 and TLR1/6 and an NF-KB-inducible reporter gene were stimulated with different doses of Veillonella parvula Strain A was mostly detected by TLR2/6 with little to no detection by TLR1/6 ( Figure 19A).
  • simultaneous dosing with Veillonella parvula Strain A and a TLR2 blocking antibody resulted in reduced efficacy in controlling ear inflammation in comparison with Veillonella parvulci Strain A dosed with an isotype antibody ( Figure 19B).
  • T cells traffic through mLN to encounter gut resident DCs that could have interacted with Veillonella parvula Strain A. This leads to activation of effector T cells, which then enter the systemic circulation and migrate to peripheral tissues.
  • LPAM-1 and CD62L are expressed on T and B cells and act as intestinal homing receptors to mediate migration of lymphocytes into mLN. Blockade of a4[37 integrin and CD62L prevents lymphocytes from entering mLN and Peyer’s patches (Dutt et al., 2005).
  • This example describes the development of Veillonella parvula Strain A, an orally administered, gut-restricted strain of Veillonella parvula for the treatment of inflammatory diseases.
  • Animal models and human diseases tend to be arbitrarily defined in pathway specific terms Thl, Th2, Th 17, innate or adaptive.
  • induction of inflammation is not generally pathway specific.
  • existing therapeutic interventions do indicate some pathway biases in disease pathogenesis, such as TNF inhibitors in rheumatoid arthritis or IL 17 inhibitors in psoriasis
  • the data presented herein demonstrate that orally administered Veillonella parvula Strain A is effective in murine models of Thl, Th2 and Th 17 inflammation. This shows that the functional connections radiating from the small intestinal mucosa can induce systemic resolution of broad inflammatory mechanisms leading to restoration of immune homeostasis.
  • mice Female BALB/c and C57BL/6 mice (6-8 weeks old) were purchased from Taconic Farms. Animals were housed in specific pathogen-free conditions in a vivarium (5 mice per cage), and all experiments were performed under Institutional Animal Care and Use Committee (IACUC) approved protocols and vivarium guidelines.
  • Veillonella parvula Strain A was prepared in different forms- powders and frozen biomasses and was characterized by TCC method. Bacterial total cells count (TCC) was enumerated by Coulter Counter Multisizer 4e. In powders, TCC varied from 4.0e+l 1 to 2.2e+12 cells/g. In biomasses TCC varied from 2.8e+10 to 8.5e+10 cells/ml. Bacterial biomass identity was confirmed by 16S rDNA sequencing. Powder was produced following in-house developed fermentation and lyophilization protocols and stored in sealed mylar bags inside desiccator at 4°C.
  • Veillonella parvula Strain A aliquots were subjected to 25 kGy Gamma Irradiation treatment at Sterigenics U.S., LLC. Veillonella parvula Strain A aliquots were characterized by TCC and VCC methods before and after Gamma Irradiation. Total cell number did not change, while there were no viable cells left after the treatment.
  • Veillonella parvula Strain A Dosing with Veillonella parvula Strain A and controls in vivo. For each in vivo study, Veillonella parvula Strain A aliquots were distributed into plastic test tubes with caps and stored at 4°C. Mice were treated orally with Veillonella parvula Strain A (4.0E+11 to 2.2E+12 cells/g PO- specific TCC is noted in figure legends) or vehicle control (anaerobic sucrose, PO) for duration of different models as described in figure legends. Dexamethasone (1 mg/kg, i.p., Sigma) was used as a positive control unless otherwise specified. For EAE studies, fingolimod (1 mg/kg, PO, Tocris Biosciences) was dosed daily.
  • Veillonella parvula Strain A aliquots were distributed into plastic test tubes with caps and stored at 4°C. Mice were treated orally with Veillonella parvula Strain A (4.0E+11 to 2.2E+12 cells/g PO
  • mice were injected IP with 100 pL of either anti-IL-lOR (Bio X Cell; Clone 1B1.3A; Cat# BE0050) or Rat IgGl isotope control (Bio X Cell; Clone HRPN; Cat# BE0088) at 2 mg/mL. Mice were treated on days 0, 3, and 6.
  • mice were injected IP with 100 pL of either anti-TLR2 (Invivogen; Clone T2A; Cat# mab2-mtlr2) or Mouse IgGl isotope control (Bio X Cell; Clone MOPC-21; Cat# BE0083) at 2 mg/mL. Mice were treated days 0, 3, and 6.
  • mice were injected IP with 100 pL of either anti-CD62L (Bio X Cell; Clone Mel-14; Cat# BE0021) and anti-LPAM-1 (Bio X Cell; Clone DATK32; Cat# BE0034) prepared at 5 mg/mL each and mixed 1: 1 or Rat IgG2a isotope control (Bio X Cell; Clone 2A3; Cat# BE0089) at 5 mg/mL. Mice were treated days 1, 3, 5, and 7.
  • CD4 + T cells were isolated using EasySep Mouse CD4+ isolation kits (StemCell Technologies, Cat#19852). 1.5-2 x 10 7 cells resuspended in 200 pl of PBS were injected into recipient mice. 4 days after adoptive transfer of cells, recipient mice were challenged with KLH (20 pg/20 pl) intradermally in the ear. Ear measurements were recorded 24 hours post ear challenge.
  • mice Female SJL mice (8-10 weeks old) were subcutaneously injected at four sites with myelin proteolipid protein (PLP)i39- i5i in CFA emulsion (0.05 mL/injection site; ⁇ 0.5 mg PLP PLPi39-i5i/mL; Hooke Laboratories; EK-2120). Following immunization, EAE induction was completed by intraperitoneal injections of pertussis toxin (6 pg/mL; 0.1 mL/mouse) within 2 hours of immunization. Mice were randomized into groups and monitored for EAE clinical score over the course of 42 days. Disease progression was scored blinded of treatment or prior measurements.
  • PLP myelin proteolipid protein
  • mice Disease severity was scored using standard EAE criteria: 0 (normal); 1 (loss of tail tone); 2 (hind limb weakness); 3 (hind limb paralysis); 4 (hind limb paralysis and forelimb paralysis or weakness); 5 (morbidity /death). Mice were observed daily for clinical symptoms. Mice were euthanized if they had a score of 4 for 2 days, and a score of 5 was recorded for remainder of the study for these animals.
  • HRP based detection was performed online using the standard DABMAP Kit (Roche/Ventana 760- 124) followed by a Hematoxylin counterstain. Slides were then dehydrated through graded alcohols cleared in Xylene and cover slipped. Stained Slides were scanned on Nanaozoomer 2.0 HT (Hamamatsu) at 20X magnification and images were analyzed by a pathologist.
  • Count of inflammatory foci - spinal cord -Inflammatory foci of approximately 20 cells were counted in each H&E-stained section. When inflammatory infiltrates consisted of more than 20 cells, an estimate was made of how many foci of 20 cells were present.
  • Estimation of demyelinated area - spinal cord - The demyelination score represents an estimate of demyelinated area for each section as follows: 0 - no demyelination (less than 5% demyelinated area)
  • the size of the demyelinated area was estimated based on less intense brown staining of myelin.
  • FITC-induced allergic inflammation Backs of female BALB/c mice were shaved and on days 1 and 2 400 pl of 0.5% FITC solution (dissolved in acetone: dibutyl phthalate, 1: 1, v/v) was painted on the shaved skin. On day 6, baseline ear measurements were taken and then mice were challenged with 20 pl 0.5% FITC on the right ear. On day 7, ear thickness was measured 24 hours post FITC challenge using digital calipers (Fowler). Change in ear thickness was expressed as ear thickness at day 7 minus ear thickness at baseline.
  • Ear-draining cervical lymph nodes (CLNs), gut draining mesenteric lymph nodes (MLNs) and spleens were harvested at terminal time points from various studies and collected into 0.5 ml of cold, complete-RPMI (10% FBS, lx Glutamax, 1 mM sodium pyruvate, 100 mM HEPES, lx non-essential amino acids, lx beta-mercaptoethanol, lx antibiotic-antimycotic) (all reagents from Gibco). Single cell suspensions were prepared (spleens were RBC lysed with ACK lysing buffer) and 200,000 cells/well were plated.
  • Cells were stimulated ex vivo with either LPS (200 ng/ml, Invivogen) or PMA (eBioscience) for 48 hours, or KLH (50 pg/ml, Sigma) or OVA (50 pg/ml) for 72 hours at 37°C and 5% CO2.
  • Supernatants were collected at the end of stimulations and used for multiplex ELISAs of cytokine levels using Meso Scale Discovery (MSD) kits.
  • Ear tissues were dissociated in 250 pl T-PER buffer (Thermo Scientific) containing Halt Protease (Thermo Scientific) and protein was quantified with BCA kit (Thermo Scientific). 100 pg of protein was used to measure cytokine levels using MSD kits.
  • mice were place under a specific low fluorescence diet without chlorophyl (AIN-93G, Bio-Serv). Mice were randomized in groups of 3 animals and orally dosed with 100 pl PBS buffer containing 1 x 10 9 total Veillonellci parvulci Strain A cells covalently labeled with IRDye800, as well as an equimolar dye amount of IRDye680RD-Carboxylate (LICOR Biosciences) as a free-dye control.
  • mice were sacrificed using CO2 and cervical dislocation followed by careful removal of the complete Gastrointestinal Tract (GIT), mesenteric lymph nodes (MLN), liver, spleen, heart, and lungs. These were imaged in a tray using a whole-animal fluorescence imaging instrument (Pearl®, LI-COR Biosciences). Tissues were imaged using the 800 nm emission channel (Veillonellci parvulci Strain A), the 700 nm emission channel (free dye control), and the standard white epi-illumination channel (overall tissues). After imaging, the individual organ weights were recorded.
  • Fluorescence images were used to quantify the distribution of Veillonella parvula Strain A signal in the GIT as well as the different organs. Tissues were manually outlined, and the fluorescence signals quantified using image analysis software (Image Studio®, LICOR Biosciences). The fluorescence signal from different organs was divided by the recorded tissue weight in order to normalize the data. Data points corresponded to mean ⁇ standard deviation values.
  • HEK-TLR assay HEK293-SEAP reporter cells (Invivogen) expressing human TLR1, TLR2, and TLR6 combinations were plated at a final concentration of 20,000 cells per well in 96 well plates and cultured in appropriate selection media. After 48 hours selection media was washed out and replaced with complete media, and Veillonella parvula Strain A was added at the indicated concentrations per well. Cells were cultured in the presence of Veillonella parvula Strain A for 24 hours. Supernatant was collected and incubated with HEK-Blue reagent (Invivogen) for 1 hr, followed by reading absorbance at OD 630 nm for SEAP production to determine stimulation of TLR2 heterodimers.
  • HEK-Blue reagent Invivogen
  • the unlabeled cells were washed and labeled with a DC enrichment cocktail (100 pL per 10 8 cells) and the cell suspension was incubated at 4°C for 15 minutes. Following incubation, cells were washed and resuspended in MACS buffer and run through a magnetic MS column for a positive selection. Positively Isolated cells were further cultured at 30,000 dendritic cells/well in 100 pl and incubated at 37°C overnight. The following day, 75 pl of supernatant was removed and replaced with fresh antibiotic free media. Microbes were added in anaerobic conditions and flushed with 1% O2. Plates were incubated for 24 hrs in an anaerobic box at 37°C +5% CO2. After 24 hours, plates were centrifuged, and supernatants collected to assay cytokine levels using MSD assays.
  • a DC enrichment cocktail 100 pL per 10 8 cells
  • Example 5 Veillonella parvula Strain A acts on cells of the small intestine to resolve peripheral inflammation
  • EAE Experimental Autoimmune Encephalomyelitis SJL mouse model. EAE was induced in SJL mice by injection of PLP139-151 emulsified in complete Freund’s adjuvant (CFA) on day 0.
  • CFA complete Freund’s adjuvant
  • mice received a 10 mg oral daily dose of Veillonella parvula Strain A-G.I. or vehicle from day 0-42.
  • mice received Veillonella parvula Strain A- G.I. or vehicle starting on the second day of disease.
  • RNA from day 42 duodenal tissue was sequenced to a target depth of 20 million paired-end reads with Illumina 2x150 technology and processed using STAR and RSEM.
  • Data normalization used trimmed mean of M-values (TMM), with generalized linear models for variance estimation using functions from empirical analysis of gene expression. P-values were adjusted to control false discovery rate (FDR) in multiple testing (Benjamini and Hochberg method). Differential gene expression was defined as a fold change of >1.5 and FDR-adj. p ⁇ 0.05.
  • Enrichment and over-representation analyses were performed on datasets available through KEGG, gene ontology, EnrichR and MSigDB.
  • B & T cell Cd69 Gpr174 Ptprcap Cd52 H2rg Ltb Traf3ip3 Slamf6 Myolg Gmfg S1pr4 1116 Ikzfl Arhgap25 T cell: Trbcl Prf1 Cd3d Cd6 Gimap7 Cd5 Trac Zap70 Lat Trbc2 Lek Skapl Septin 1 Foxp3 Lag3 Tnfrsf18 Izumolr
  • Myeloid Cell CledOa Ncf2 Ptafr S100a8 Hk3 Itgam Csf2rb Tyrobp Spi1 Aif1 Lst1 Lrrc25 Trafl Pdcd1lg2 Rab30 Ryr1 Gpr132 Lgals3 Ccl22 Samsnl Ccl24 Slc7a7 1133 Srgn Gplbb Cyp2u1 Tmem71 B, T & Myeloid Cell:
  • Veillonella parvulci Strain A-G.I. elevated genes with known function in immune cell migration to the gut, immune modulation, and intestinal homeostasis are provided in Table 2.
  • mice groups were dosed for 4 or 8 days, respectively, with Veillonella Strain A-G.L PO. On day 0, all groups were immunized with KLH emulsified with Complete Freund’s Adjuvant. On day 5, “vehicle,” “dexamethasone,” and “Veillonella Strain A-G.I.
  • mice groups were immunized with KLH emulsified with Complete Freund’s Adjuvant and dosed with sucrose vehicle, dexamethasone (Img/kg), or Veillonella Strain A-G.I for 4 days.
  • baseline ear thickness was measured using calipers, then all mice were challenged by intradermal ear injection with KLH. After 24 hours, the change in ear thickness was evaluated and compared to baseline measurements.
  • Veillonella Strain A-G.I. powder was dosed in sucrose at 10 mg/dose.
  • Veillonella Strain A refers to Veillonella parvula Strain A, which has been deposited as ATCC Accession Number PTA-125691.
  • Veillonella Strain A-G.I. refers to the gamma-irradiated form of the strain. Results are shown in Figure 21.
  • CONCLUSION Pre-dosing a KLH DTH model with Veillonella Strain A-G.I. for 4 or 8 days shows no efficacy. This indicates that Veillonella Strain A-G.I. does not suppress the immune system in the absence of inflammation but instead resolves an aberrant inflammatory response.
  • Example 7 Induction of inflammation and subsequent dosing with Veillonella Strain A-G.L is sufficient to induce resolution of inflammation in a subsequent DTH response
  • mice were immunized by subcutaneous injection with PBS emulsified with Complete or Incomplete Freund’s Adjuvant (CFA or IFA, respectively) (see Petrovsky N. & Aguilar JC., 2004. Vaccine adjuvants: Current state and future trends. Immunol Cell Biol. 82(5): 488-96).
  • mice were dosed for 4 days with PBS or Veillonella Strain A-G.I. PO.
  • 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.
  • Veillonella Strain A-G.I. powder was dosed in sucrose at lOmg/dose.
  • Veillonella Strain A refers to Veillonella parvula Strain A which has been deposited as ATCC Accession Number PTA-125691.
  • Veillonella Strain A-G.I. refers to the gammairradiated form of the strain. Results are shown in Figure 22.
  • CONCLUSION CFA (containing TLR agonists and heat-killed Mycobacterium tuberculosis)' and IFA (containing TLR agonists) have been shown to drive Thl and Th2 immune responses, respectively. Moreover, IFA immunization induces an inflammatory response that is absent of any antigen.
  • Example 8 Transfer of CD4+ T cells from KLH-CFA immunized mice dosed with Veillonella Strain A-G.I. into untreated KLH-CFA immunized mice can reduce inflammation following ear challenge.
  • CD4+ T cells from KLH-CFA immunized and Veillonella Strain A-G.7. -dosed mice can confer immune resolution function.
  • mice were immunized with KLH emulsified with Complete Freund’s Adjuvant.
  • mice were dosed with sucrose vehicle or Veillonella Strain A-G.I. PO for 4 days and a second set of recipient mice were immunized with KLH emulsified with Complete Freund’s Adjuvant.
  • brachial, axillary and inguinal lymph nodes, and spleens were isolated from each group, pooled, 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 IxlO 8 cells/mL in PBS. After enrichment, IxlO 7 CD4+ T cells were then transferred IP into recipient mice. On day 12, 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.
  • Veillonella Strain A-G.I. powder was dosed in sucrose at 10 mg/dose.
  • Veillonella Strain A refers to Veillonella parvula Strain A which has been deposited as ATCC Accession Number PTA-125691.
  • Veillonella Strain A-G.I. refers to the gamma-irradiated form of the strain. Results are shown in Figure 23. [482] CONCLUSION: Transfer of CD4+ T cells from mice immunized with KLH-CFA and dosed with Veillonella Strain A-G.I. is sufficient for reducing inflammation in a KLH-DTH model in recipient mice that had not been dosed with Veillonella Strain A-G.I.
  • Example 9 Depletion of B cells during or post-dosing of Veillonella Strain A- G.L in the DTH model inhibits efficacy of immune resolution
  • mice were immunized with KLH emulsified with Complete Freund’s Adjuvant.
  • mice were also treated with 100 pL 2 mg/mL anti-CD20 antibody or isotype control by IP injection on days 0, 3, 6, and 9.
  • mice were treated for 4 days with sucrose vehicle PO, Veillonella Strain A-G.I. PO, or dexamethasone (1 mg/kg) IP.
  • 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.
  • Veillonella Strain A-G.I. powder was dosed in sucrose at 10 mg/dose.
  • Veillonella Strain A refers to Veillonella parvula Strain A which has been deposited as ATCC Accession Number PTA-125691.
  • Veillonella Strain A-G.I. refers to the gamma-irradiated form of the strain.
  • mice were immunized with KLH emulsified with Complete Freund’s Adjuvant.
  • mice were treated for 4 days with vehicle sucrose PO, Veillonella Strain A-G.I. PO, or dexamethasone (1 mg/kg) IP.
  • Mice were then treated with 100 pL 2 mg/mL anti- CD20 antibody or isotype control antibody by IP injection on days 9 and 11.
  • 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.
  • Veillonella Strain A-G.I. powder was dosed in sucrose at 10 mg/dose. Results are shown in Figure 24.
  • CONCLUSION Depletion of B cells inhibits efficacy of Veillonella Strain A-G.I. in the reduction of inflammation of the KLH-DTH response. Depletion of B cells either during the entire study or after dosing inhibits efficacy. This suggests that B cells are critical for the oral microbe-mediated resolution of inflammation.
  • Example 10 Combination of Veillonella Strain A-G.I. and anti-IL-6 increases inflammation resolution in a KLH-DTH model
  • mice were immunized with KLH emulsified with Complete Freund’s Adjuvant. Mice were also treated with 100 L anti-IL-6 antibodies (1 or 0.25 mg/mL) or isotype control (2 mg/mL) by IP injection on days 0, 3, and 6. On day 5, mice were treated for 4 days with vehicle sucrose PO, Veillonella Strain A-G.I. PO, or dexamethasone (1 mg/kg) IP. On day 8, baseline ear thickness was measured using calipers, then mice were challenged by intradermal ear injection with KLH.
  • Veillonella Strain A- G.I. powder was dosed in sucrose at 10 mg/dose.
  • Veillonella Strain A refers to Veillonella parvula Strain A which has been deposited as ATCC Accession Number PTA-125691.
  • Veillonella Strain A-G.I. refers to the gamma-irradiated form of the strain. Results are shown in Figure 25.
  • CONCLUSION Treatment with both Veillonella Strain A-G.I. and anti-IL-6 (1 mg/mL) decreases ear swelling more compared to monotherapy. This suggests that the combination of both Veillonella Strain A-G.I. and anti-IL-6 provides an additive reduction of inflammation in the KLH DTH model.

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Abstract

Provided herein are methods and compositions related to Veillonella parvula bacteria useful as therapeutic agents, e.g., for the induction of one or more of the disclosed immune effects.

Description

INDUCING IMMUNE EFFECTS USING VEILLONELLA PARVULA BACTERIA
CROSS-REFERENCE TO RELATED APPLICATIONS
[1] This application claims the benefit of the following U.S. Provisional Application Nos.: 63/110,761, filed November 6, 2020; 63/112,330, filed November 11, 2020; 63/237,818, filed August 27, 2021; 63/249,181, filed September 28, 2021; and 63/250,588, filed September 30, 2021, the entire contents of each are incorporated herein by reference.
SUMMARY
[2] In certain aspects, provided herein are bacterial compositions (e.g, pharmaceutical compositions) comprising Veillonella parvulci useful for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell and/or a decrease in expression of a pro-inflammatory cytokine by an immune cell) and methods of using such bacterial compositions (e.g., for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell)).
[3] In certain aspects, provided herein is a method of inducing an immune effect in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
[4] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula for the preparation of a medicament for inducing an immune effect in a subject (e.g., a human subject).
[5] In certain aspects, provided herein is a bacterial composition comprising bacteria of a Veillonella parvula strain for use in inducing an immune effect in a subject (e.g., a human subject). [6] In certain aspects, provided herein is use of a bacterial composition comprising bacteria of a Veillonella parvulci strain for the preparation of a medicament for inducing an immune effect in a subject (e.g., a human subject).
[7] In some embodiments, the bacterial compositions comprise whole Veillonella parvula bacteria. In some embodiments, the Veillonella parvula bacteria are gamma irradiated.
[8] In some embodiments, the immune effect comprises an increase in expression of an anti-inflammatory cytokine by an immune cell. In some embodiments, the anti-inflammatory cytokine is IL- 10 and/or IL-27. In some embodiments, the antiinflammatory cytokine is IL- 10.
[9] In some embodiments, the immune effect comprises a decrease in expression of a pro-inflammatory cytokine by an immune cell. In some embodiments, the pro-inflammatory cytokine is IL-1[3, IL-6, TNF-a, IL-5, IL-4, IL-13, IL-17, and/or IL-8. In some embodiments, the pro-inflammatory cytokine is IL-5, IL-4, IL-13, and/or IL-17.
[10] In some embodiments, the immune effect comprises a decrease in mRNA transcript levels of a pro-inflammatory cytokine by an immune cell. In some embodiments, the mRNA transcript levels are //.-///. IL-6, TNF-a, IL-5, IL-4, IL-13, IL- 17 A, and/or IL-8 transcript levels. In some embodiments, the mRNA transcript levels are IL-5, IL-4, IL-13, and/or IL- 17 A transcript levels. In some embodiments, mRNA transcript levels are III 7f and/or Defb3 transcript levels.
[11] In some embodiments, the immune cells in which the immune effect are induced comprise a peripheral blood mononuclear cell (PBMC), a dendritic cell, and/or a macrophage.
[12] In certain aspects, provided herein is a method of resolving an inflammatory response in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
[13] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula for the preparation of a medicament for resolving an inflammatory response in a subject (e.g., a human subject). [14] In certain aspects, provided herein is a bacterial composition comprising bacteria of a Veillonella parvulci strain for use in resolving an inflammatory response in a subject (e.g., a human subject).
[15] In certain aspects, provided herein is use of a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for resolving an inflammatory response in a subject (e.g., a human subject).
[16] In some embodiments, the inflammatory response comprises aThl and/or Th2 response.
[17] In certain aspects, provided herein are bacterial compositions (e.g, pharmaceutical compositions) comprising Veillonella parvula useful for inducing an immune response in a human subject, the method comprising administering to the subject a population of T cells (e.g., CD4+ T cells) obtained from a different human subject, wherein the different human subject was administered Veillonella parvula.
[18] In certain aspects, provided herein is a method of generating inflammation-resolving CD4+ T cells in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition ]) comprising a strain of Veillonella parvula.
[19] In certain aspects, provided herein is a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition ) comprising bacteria of a Veillonella parvula strain for use in generating inflammation-resolving CD4+ T cells in a subject (e.g., a human subject).
[20] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for generating inflammation-resolving CD4+ T cells in a subject (e.g., a human subject).
[21] In certain aspects, provided herein are bacterial compositions (e.g., pharmaceutical compositions) comprising Veillonella parvula useful for affecting T cells to mediate an effect on inflammation (e.g., resolve inflammation) in a subject (e.g., a human subject), e.g., as described herein.
[22] In certain aspects, provided herein is a method of affecting T cells to mediate an effect on inflammation (e.g., resolve inflammation) in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
[23] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula for the preparation of a medicament for affecting T cells to mediate an effect on inflammation (e.g., resolve inflammation) in a subject (e.g., a human subject).
[24] In certain aspects, provided herein is a bacterial composition comprising bacteria of a Veillonella parvula strain for use in affecting T cells to mediate an effect on inflammation (e.g., resolve inflammation) in a subject (e.g., a human subject).
[25] In certain aspects, provided herein is use of a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for affecting T cells to mediate an effect on inflammation (e.g., resolve inflammation) in a subject (e.g., a human subject).
[26] In certain aspects, provided herein is a method of instructing T cells to be less inflammatory in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of Veillonella parvula.
[27] In certain aspects, provided herein is a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising bacteria of a Veillonella parvula strain for use in instructing T cells to be less inflammatory in a subject (e.g., a human subject).
[28] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for instructing T cells to be less inflammatory in a subject (e.g., a human subject).
[29] In certain aspects, provided herein is use of a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for instructing T cells to be less inflammatory in a subject (e.g., a human subject). [30] In some embodiments, the T cells are instructed in mesenteric lymph nodes.
[31] In some embodiments, the Veillonella parvulci strain (a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition)) is orally administered (e.g., and travels to the small intestine), dendritic cells interact with the Veillonella parvula strain 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.
[32] In certain aspects, provided herein is a method of affecting T cells that traffic to mesenteric lymph nodes in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of Veillonella parvula.
[33] In certain aspects, provided herein is a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising bacteria of a Veillonella parvula strain for use in affecting T cells that traffic to mesenteric lymph nodes in a subject (e.g., a human subject).
[34] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for affecting T cells that traffic to mesenteric lymph nodes in a subject (e.g., a human subject).
[35] In certain aspects, provided herein is use of a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament or affecting T cells that traffic to mesenteric lymph nodes in a subject (e.g., a human subject).
[36] In some embodiments, the T cells are affected in mesenteric lymph nodes.
[37] In certain aspects, provided herein are bacterial compositions (e.g., pharmaceutical compositions) comprising Veillonella parvula useful for affecting B cells to mediate an effect on inflammation (e.g., resolve inflammation) in a subject (e.g., a human subject), e.g., as described herein. [38] In certain aspects, provided herein is a method of affecting B cells to mediate an effect on inflammation (e.g., to resolve inflammation) in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition ) comprising a strain of Veillonella parvula.
[39] In certain aspects, provided herein is a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition ) comprising bacteria of a Veillonella parvula strain for use in affecting B cells to mediate an effect on inflammation (e.g., to resolve inflammation) in a subject (e.g., a human subject).
[40] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for affecting B cells to mediate an effect on inflammation (e.g., to resolve inflammation) in a subject (e.g., a human subject).
[41] In certain aspects, provided herein is use of a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for affecting B cells to mediate an effect on inflammation (e.g., to resolve inflammation) in a subject (e.g., a human subject).
[42] In certain aspects, provided herein are methods of enhancing IL- 10 production, e.g., from a peripheral blood mononuclear cell (PBMC), a dendritic cell, or a macrophage, e.g., in a subject (e.g., a human subject).
[43] In certain aspects, provided herein is a method of enhancing IL- 10 production in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
[44] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula for the preparation of a medicament for enhancing IL- 10 production in a subject (e.g., a human subject). [45] In certain aspects, provided herein is a bacterial composition comprising bacteria of a Veillonella parvulci strain for use in enhancing IL- 10 production in a subject (e.g., a human subject).
[46] In certain aspects, provided herein is use of a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for enhancing IL-10 production in a subject (e.g., a human subject).
[47] In certain aspects, provided herein is a method of activating TLR2 in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
[48] In certain aspects, provided herein is a method of activating TLR2 in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
[49] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula for the preparation of a medicament for activating TLR2 in a subject (e.g., a human subject).
[50] In certain aspects, provided herein is a bacterial composition comprising bacteria of a Veillonella parvula strain for use in activating TLR2 in a subject (e.g., a human subject).
[51] In certain aspects, provided herein is use of a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for activating TLR2 in a subject (e.g., a human subject).
[52] In some embodiments, the TLR2 is activated in intestinal epithelial cells (IEC) in the subject. In some embodiments, the TLR2 is activated in immune cells in the lamina propria of the subject. In some embodiments, the activation of TLR2 results in increased expression of IL-10 by the subject.
[53] In certain aspects, provided herein is a method of activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
[54] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula for the preparation of a medicament for activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject).
[55] In certain aspects, provided herein is a bacterial composition comprising bacteria of a Veillonella parvula strain for use in activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject).
[56] In certain aspects, provided herein is use of a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject).
[57] In certain aspects, provided herein are methods of activating TLR2 in a subject (e.g., a human subject).
[58] In certain aspects, provided herein are methods of activating TLR1/2 and/or TLR2/6 heterodimers in a subject (e.g., a human subject).
[59] In certain aspects, provided herein are bacterial compositions (e.g., pharmaceutical compositions) comprising Veillonella parvula useful for elevating expression (e.g., in the small intestine) of a gene provided in Example 5.
[60] In certain aspects, provided herein is a method of elevating expression (e.g., in the small intestine) of a gene provided in Example 5 in a subject (e.g., a human subject) comprising administering (e.g., orally administering) to the subject a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula.
[61] In certain aspects, provided herein is use of a dose (e.g., a therapeutically effective dose) of a Veillonella parvula strain and/or a bacterial composition (e.g., a pharmaceutical composition) comprising a strain of a Veillonella parvula for the preparation of a medicament for elevating expression (e.g., in the small intestine) of a gene provided in Example 5 in a subject (e.g., a human subject). [62] In certain aspects, provided herein is a bacterial composition comprising bacteria of a Veillonella parvulci strain for use in elevating expression (e.g., in the small intestine) of a gene provided in Example 5 in a subject (e.g., a human subject).
[63] In certain aspects, provided herein is use of a bacterial composition comprising bacteria of a Veillonella parvula strain for the preparation of a medicament for elevating expression (e.g., in the small intestine) of a gene provided in Example 5 in a subject (e.g., a human subject).
[64] In some embodiments, the cell types found enriched amongst genes showing elevated expression are immune epithelial cells. In some embodiments, the cell types found enriched amongst genes showing elevated expression are immune cells (e.g., B cells; T cells; and/or myeloid cells). In some embodiments, the gene showing elevated expression is Spinkl, Tm4sf5, and/or Aocl. In some embodiments, the gene showing elevated expression is a gene provided in Table 2. In some embodiments, the gene showing elevated expression is associated with lymphocyte migration, gut-homing, and adhesion (e.g., Ccl25, CcrlO, Ccl22, Ccl24, Itgb7, Itgal, and/or Itgani). In some embodiments, the gene showing elevated expression is associated with T cell lineage maturation and activation (e.g., Cd69, Icos, Il2rg, Cd3d, Trbcl, Trbc2, Trac, Lat, Zap70, Lek, and/or Cd2). In some embodiments, the gene showing elevated expression is associated with B cell lineage maturation and activation (e.g., CD19, CD79a, CD79b, CD69, Ighd, Fcrll, Blk, Ikzf3, Tnfrsfl 3c, Jchain, Iglcl, and/or Iglc2) . In some embodiments, the gene showing elevated expression is associated with immune modulation (e.g., Foxp3, Lag3, Traf3ip3, Slamf6, 1133, Cd5, Adamdecl, and/or Nrli3). In some embodiments, the gene showing elevated expression is associated with intestinal epithelial cell homeostasis (barrier, metabolic, absorption) (e.g., Gpx2, Gstm3, Aqp8, Guca2b, Adipoq, Dgatl, Dgat2, Slc23al, and/or Slc51b). In some embodiments, the gene showing elevated expression is associated with host-protective pathways (e.g., Zgl6, Def 5a, Reg3a, Retnlb, Reg3g, Cfd, Lypd8, and/or Casp6).
[65] In some embodiments, the Veillonella parvula is Veillonella parvula strain A (ATCC Deposit Number PTA-125691). In some embodiments, the Veillonella parvula strain is a strain comprising at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Veillonella parvulci strain A.
[66] In some embodiments, the bacterial composition comprises one strain of bacteria, wherein the one strain of bacteria is a strain comprising at least 99.9% sequence identity to the nucleotide sequence of the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691). In some embodiments, the bacterial composition comprises one strain of bacteria, wherein the one strain of bacteria is the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[67] In some embodiments, the bacterial composition is administered in combination with an additional therapeutic. In some embodiments, the additional therapeutic is an IL-6 antagonist. In some embodiments, the additional therapeutic is an anti-IL-6 antagonist antibody.
[68] In some embodiments, the bacterial composition comprises at least about 3 x 1010 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[69] In some embodiments, the bacterial composition comprises about 3 x 1010 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[70] In some embodiments, the bacterial composition comprises about 4.5 x 1010total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[71] In some embodiments, the bacterial composition comprises about 1.5 x 10ntotal cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[72] In some embodiments, the bacterial composition comprises about 7.5 x 10ntotal cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[73] In some embodiments, the bacterial composition comprises about 1.5 x 1012total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691). [74] In some embodiments, the bacterial composition comprises about 3 x 1010 to about 1.5 x 1012 total cells of Veillonella parvula, e.g., of Veillonella parvulci strain A (ATCC Deposit Number PTA- 125691).
[75] In some embodiments, the bacterial composition comprises about 4.5 x 1010to about 1.5 x 1012 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[76] In some embodiments, the bacterial composition comprises about 3 x 1010 to about 1.5 x 1011 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[77] In some embodiments, the bacterial composition comprises about 4.5 x 1010to about 1.5 x 1011 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[78] In some embodiments, the bacterial composition comprises about 1.5 x 10nto about 1.5 x 1012 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[79] In some embodiments, the bacterial composition comprises about 1.5 x 10nto about 7.5 x 1011 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[80] In some embodiments, the bacterial composition comprises about 7.5 x 10nto about 1.5 x 1012 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[81] In certain embodiments, the bacterial composition (e.g. , pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises about 1 x 1010 total cells, about 2 x 1010 total cells, about 3 x 1010 total cells, about 4 x 1010 total cells, about 4.5 x 1010 total cells, about 5 x 1010 total cells, about 6 x 1010 total cells, about 7 x 1010 total cells, about 8 x 1010 total cells, about 9 x 1010 total cells, about 1 x 1011 total cells, about 1.5 x 1011 total cells, about 2 x 1011 total cells, about 3 x 1011 total cells, about 4 x 1011 total cells, about 5 x 1011 total cells, about 6 x 1011 total cells, about 7 x 1011 total cells, about 7.5 x 1011 total cells, about 8 x 1011 total cells, about 9 x 1011 total cells, about 1 x 1012 total cells, about 1.5 x 1012 total cells, about 2 x 1012 total cells of the Veillonella parvula bacteria. In certain embodiments, the bacterial composition (e.g., composition of the total dose administered, e.g., once or twice daily) comprises at least 1 x 1010 total cells (e.g., at least 1 x 1010 total cells, at least 2 x 1010 total cells, at least 3 x IO10 total cells, at least 4 x IO10 total cells, at least 4.5 x IO10 total cells, at least 5 x IO10 total cells, at least 6 x IO10 total cells, at least 7 x IO10 total cells, at least 8 x IO10 total cells, at least 9 x IO10 total cells, at least 1 x 1011 total cells, at least 1.5 x 1011 total cells, at least 2 x 1011 total cells, at least 3 x 1011 total cells, at least 4 x 1011 total cells, at least 5 x 1011 total cells, at least 6 x 1011 total cells, at least 7 x 1011 total cells, at least 7.5 x 1011 total cells, at least 8 x 1011 total cells, at least 9 x 1011 total cells, at least 1 x 1012 total cells, at least 1.5 x 1012 total cells, at least 2 x 1012 total cells) of the Veillonella parvulci bacteria. In some embodiments, the bacterial composition comprises about 3 x 1010total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 4.5 x 1010total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 7.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 3 x 1010to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 4.5 x 1010to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 3 x 1010to about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 4.5 x 1010to about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10n to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10n to about 7.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 7.5 x 1011 to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the Veillonella parvula bacteria are gamma irradiated.
[82] In certain embodiments, provided herein are solid dosage forms comprising the Veillonella parvula bacteria. In some embodiments, the solid dosage form comprises an enteric coating. In some embodiments, the solid dosage form is a capsule, e.g., an enteric coated capsule. In some embodiments, each capsule comprises about 3 x 1010 total cells of the Veillonella parvula bacteria. In some embodiments, each capsule comprises about 4.5 x IO10 total cells of the Veillonella parvula bacteria. In some embodiments, each capsule comprises about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 capsules are administered, e.g., once or twice daily to a subject. In some embodiments, 1 capsule (e.g., comprising about 3 x IO10 total cells) is administered, e.g., once or twice daily to a subject. In some embodiments, 2 capsules (e.g., each comprising about 3 x IO10 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 4 capsules (e.g., each comprising about 3 x IO10 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 10 capsules (e.g., each comprising about 3 x IO10 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 1 capsule (e.g., comprising about 4.5 x IO10 total cells) is administered, e.g., once or twice daily to a subject. In some embodiments, 2 capsules (e.g., each comprising about 4.5 x IO10 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 4 capsules (e.g., each comprising about 4.5 x IO10 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 10 capsules (e.g., each comprising about 4.5 x 1010 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 1 capsule (e.g., comprising about 1.5 x 10n total cells) is administered, e.g., once ortwice daily to a subject. In some embodiments, 2 capsules (e.g., each comprising about 1.5 x 1011 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 5 capsules (e.g., each comprising about 1.5 x 1011 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 10 capsules (e.g., each comprising about 1.5 x 1011 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized (e.g., in a powder). In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises sucrose and/or dextran. In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises sucrose, dextran, and/or L- cysteine HC1. In some embodiments, the capsule comprises excipients and the excipients include mannitol, colloidal silicon dioxide, magnesium stearate, hydroxypropyl methylcellulose, methacrylic acid ethyl acrylate copolymer, triethyl citrate, and/or talc. In some embodiments, the Veillonella parvula bacteria of the capsule are gamma irradiated.
[83] In some embodiments, the solid dosage form comprises a capsule. In some embodiments, the capsule is an enteric coated capsule. In some embodiments, the capsule comprises about 3 x IO10 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules). In some embodiments, the capsule comprises about 4.5 x IO10 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules). In some embodiments, the capsule comprises about 1.5 x 1011 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules). In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized (e.g., in a powder). In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises sucrose and/or dextran. In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises sucrose, dextran, and/or L-cysteine HC1. In some embodiments, the capsule comprises excipients and the excipients include mannitol, colloidal silicon dioxide, magnesium stearate, hydroxypropyl methylcellulose, methacrylic acid ethyl acrylate copolymer, triethyl citrate, and/or talc. In some embodiments, the Veillonella parvula bacteria of the capsule are gamma irradiated.
[84] In some embodiments, the solid dosage form comprises a tablet. In some embodiments, the tablet is an enteric coated tablet. In some embodiments, the enteric coated tablet is from 5mm to 18mm in diameter. In some embodiments, the enteric coated tablet is 5.5mm in diameter. In some embodiments, the enteric coated tablet is 18mm in diameter. In some embodiments, the tablet comprises about 3 x IO10 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets). In some embodiments, the tablet comprises about 4.5 x 1010 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets). In some embodiments, the tablet comprises about 1.5 x 1011 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets). In some embodiments, the Veillonella parvula bacteria in the tablet are lyophilized. In some embodiments, the Veillonella parvula bacteria of the tablet are gamma irradiated. [85] In certain embodiments, provided herein are solid dosage forms comprising the Veillonella parvulci bacteria. In some embodiments, the solid dosage form is a tablet, e.g., an enteric coated tablet. In some embodiments, the enteric coating comprises a polymethacrylate-based copolymer. In some embodiments, the enteric coating comprises a methacrylic acid ethyl acrylate (MAE) copolymer (1: 1). In some embodiments, the enteric coating comprises methacrylic acid ethyl acrylate (MAE) copolymer (1: 1) (such as Kollicoat MAE 100P).
[86] In some embodiments, each tablet comprises about 3 x IO10 total cells, 4.5 x 1010 total cells or about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tablets are administered, e.g., once or twice daily to a subject. In some embodiments, 1 tablet (e.g., comprising about 3 x 1010 total cells) is administered, e.g., once or twice daily to a subject. In some embodiments, 2 tablets (e.g., each comprising about 3 x 1010 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 5 tablets (e.g., each comprising about 3 x 1010 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 10 tablets (e.g., each comprising about 3 x 1010 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 1 tablet (e.g., comprising about 4.5 x 1010 total cells) is administered, e.g., once or twice daily to a subject. In some embodiments, 2 tablets (e.g., each comprising about 4.5 x 1010 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 5 tablets (e.g., each comprising about 4.5 x 1010 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 10 tablets (e.g., each comprising about 4.5 x 1010 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, each tablet comprises about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tablets are administered, e.g., once or twice daily to a subject. In some embodiments, 1 tablet (e.g., comprising about 1.5 x 1011 total cells) is administered, e.g., once or twice daily to a subject. In some embodiments, 2 tablets (e.g., each comprising about 1.5 x 10n total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 5 tablets (e.g., each comprising about 1.5 x 1011 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 10 tablets (e.g., each comprising about 1.5 x 1011 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, the Veillonella parvula bacteria in the tablet are lyophilized (e.g., in a powder). In some embodiments, the Veillonella parvula bacteria in the tablet are lyophilized in a powder, and the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the Veillonella parvula bacteria in the tablet are lyophilized in a powder, and the powder further comprises sucrose and/or dextran. In some embodiments, the Veillonella parvula bacteria in the tablet are lyophilized in a powder, and the powder further comprises sucrose, dextran, and/or L-cysteine HC1. In some embodiments, the Veillonella parvula bacteria of the tablet are gamma irradiated.
[87] In some embodiments, the solid dosage form comprises a mini-tablet. In some embodiments, the mini-tablet is enteric coated. In some embodiments, the minitablet is from 1 mm to 4 mm in diameter. In some embodiments, the mini -tablet (e.g., enteric coated mini-tablet) is a 1 mm mini-tablet, 1.5 mm mini-tablet, 2 mm mini-tablet, 3 mm mini-tablet, or 4mm mini-tablet. In some embodiments, the solid dosage form comprises mini -tablets that comprise about 3 x 1010 total cells of the Veillonella parvula bacteria (e.g., total dose of a plurality of mini -tablets). In some embodiments, the solid dosage form comprises mini-tablets that comprise about 4.5 x IO10 total cells of the Veillonella parvula bacteria (e.g., total dose of a plurality of mini -tablets). In some embodiments, the solid dosage form comprises mini-tablets that comprise about 1.5 x 1011 total cells of the Veillonella parvula bacteria (e.g., total dose of a plurality of minitablets). In some embodiments, the Veillonella parvula bacteria in the mini-tablets are lyophilized. In some embodiments, the Veillonella parvula bacteria of the mini-tablet are gamma irradiated.
[88] In some embodiments, the mini -tablets (e.g., enteric coated mini -tablets) are contained in a capsule. In some embodiments, the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule. In some embodiments, the capsule comprises a non- enteric coating (e.g., gelatin) (e.g., is coated with a non-enteric coating). In some embodiments, the capsule comprises a non-enteric coating. In some embodiments, the capsule comprises gelatin. In some embodiments, the capsule comprises HPMC. In some embodiments, the mini-tablets (e.g., enteric coated mini -tablets) that comprise about 3 x 1010 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC. In some embodiments, the minitablets (e.g., enteric coated mini-tablets) that comprise about 4.5 x 1010 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC. In some embodiments, the mini-tablets (e.g., enteric coated mini-tablets) that comprise about 1.5 x 1011 total cells of the Veillonella parvulci bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC. In some embodiments, the Veillonella parvula bacteria of the mini-tablet are gamma irradiated.
[89] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprising Veillonella parvula bacteria is prepared as a powder (e.g., for resuspension or for use in a solid dose form (such as a capsule)) or as a solid dose form, such as a tablet, a mini-tablet, a capsule, a pill, or a powder; or a combination of these forms (e.g., mini -tablets comprised in a capsule). The powder can comprise lyophilized bacteria. In some embodiments, the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the powder further comprises sucrose and/or dextran. In some embodiments, the powder further comprises sucrose, dextran, and/or L-cysteine HC1. In some embodiments, the Veillonella parvula bacteria are gamma irradiated.
[90] In some embodiments, the bacterial composition is administered orally. In some embodiments, the administration to the subject once daily. In some embodiments, the bacterial composition is administered in 2 or more doses (e.g., 3 or more, 4 or more or 5 or more doses). In some embodiments, the administration to the subject of the two or more doses are separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.
[91] In some embodiments, the bacterial composition is administered once daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days. In some embodiments, the bacterial composition is administered once daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for 8 weeks. In some embodiments, the bacterial composition is administered once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for at least 8 weeks.
[92] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises lyophilized Veillonella parvulci bacteria. In certain embodiments, the lyophilized Veillonella parvula bacteria is formulated into a solid dose form, such as a tablet, a mini-tablet, a capsule, a pill, or a powder. In some embodiments, the lyophilized Veillonella parvula bacteria is contained in a capsule. In some embodiments, the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the powder further comprises sucrose and/or dextran. In some embodiments, the powder further comprises sucrose, dextran, and/or L-cysteine HC1. In some embodiments, the powder is resuspended in a solution. In some embodiments, the lyophilized Veillonella parvula bacteria are resuspended in a solution.
[93] In some embodiments, the bacterial composition is formulated as a capsule or a tablet. In some embodiments, the bacterial formulation (e.g., composition) comprises an enteric coating or micro encapsulation. In some embodiments, the capsule is an enteric coated capsule. In some embodiments, the enteric coating allows the bacterial composition to be released in the upper small intestine, e.g., duodenum.
[94] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal (e.g., 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).
[95] In some embodiments, the subject (e.g., human subject) has an immunoinflammatory disorder. In some embodiments, the immunoinflammatory disorder is arthrosclerosis, arthritis (e.g., psoriatic 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 associated masses or lesions, primary sclerosing cholangitis, cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis, vulvodynia, acute disseminated alopecia universalise, Chagas' disease, chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, diabetes mellitus type 1, giant cell arteritis, good pasture's syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonlein purpura, Kawasaki's disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, Muckle-Wells syndrome, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmune haemolytic anemia, interstitial cystitis, Lyme disease, morphea, psoriasis, sarcoidosis, scleroderma, contact hypersensitivity, contact dermatitis (including that due to poison ivy), uticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dustmite allergy), appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, pericarditis, peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis, pyelonephritis, stomatisi, transplant rejection, acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, Sexary's syndrome, congenital adrenal hyperplasis, nonsuppurative thyroiditis, hypercalcemia associated with cancer, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensistivity reactions, allergic conjunctivitis, keratitis, herpes zoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonary tuberculosis chemotherapy, idiopathic thrombocytopenic purpura in adults, secondary thrombocytopenia in adults, acquired (autoimmune) haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia of childhood, regional enteritis, autoimmune vasculitis, chronic obstructive pulmonary disease, or sepsis.
[96] In some embodiments, the subject has an immune disorder. In some embodiments, the immune disorder is atopic dermatitis, psoriasis, or asthma. In some embodiments, the atopic dermatitis is moderate atopic dermatitis. In some embodiments, the psoriasis is moderate psoriasis. In some embodiments, the asthma is moderate asthma. [97] In some aspects, the disclosure provides a bacterial composition described herein (e.g., in an amount described herein) for use in inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell).
[98] In some aspects, the disclosure provides use of a bacterial composition described herein (e.g., in an amount described herein) for the preparation of a medicament for the induction of an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell).
BRIEF DESCRIPTION OF FIGURES
[99] Figures 1A-1C show that Veillonella parvulci strain A induces IL-10 production from human immune cells. Figure 1A shows IL-10 production in PBMC. Figure IB shows IL-10 production in Dendritic cells. Figure 1C shows IL-10 production in macrophages.
[100] Figure 2 shows stimulation of the production of TNF by pro-inflammatory macrophages. Average TNF (pg/ml) from 3 individual donors stimulated by Veillonella parvula strain A- G.I. in an assay with pro-inflammatory macrophages after 24 hours of cell-microbe co-culture. Error bars represent standard deviation (S.D.) of the mean.
[101] Figures 3A-3B show effect of Veillonella parvula strain A on cytokine expression in a human dendritic cell: T cell in vitro co-culture system. Antigen-specific IL-10 (Figure 3A) and IFNy (Figure 3B) production in 3 human DC-CD8 T cell co-cultures, with or without pre-conditioning of DCs with gamma-irradiated Veillonella parvula strain A. Cytokine is reported as pg/mL at 24 hours after addition of the CEF-peptide pool. Error bars on control treatments represent SD of duplicate wells.
[102] Figure 4 shows mouse KLH delayed-type hypersensitivity model. Shown are the 24-hour ear thickness change measurements. Error bars represent standard deviation. Statistical significance is measured from one-way ANOVA against vehicle. **** denotes pO.OOOL
[103] Figure 5 shows reduction in ear inflammation after challenge on days 15 and 29.
[104] Figure 6 shows mouse FITC model of cutaneous hypersensitivity. Results compiled for 4 individual experiments. Error bars represent standard deviation. Statistical significance is measured from one-way ANOVA against vehicle. * denotes p<0.05 *** denotes p<0.001, **** denotes p<0.0001. [105] Figure 7 shows Veillonella parvula strain A- G.I. (gamma-irradiated) reduces MC903 atopic dermatitis-like skin inflammation with efficacy similar to systemic dexamethasone and tofacitinib. Ear inflammation measured daily. Total area under the curve (AUC) of the ear swelling measurements for the time course from days 1-14 were calculated. Error bars represent standard error. Statistical significance is measured from one-way ANOVA against vehicle. * * * * denotes p<0.0001.
[106] Figure 8 shows treatment with Veillonella parvula strain A- G.I. results in decrease in protein and mRNA levels in the ear. IL-4 protein and mRNA measurement in ears of treated mice. Error bars represent standard deviation. Statistical significance is measured from one-way ANOVA against vehicle. * denotes p<0.05, ** denotes p<0.01.
[107] Figure 9 shows Veillonella parvula strain A- G.I. treatment reduces the production of TH2 and TH1 cytokines in the lymph node draining the site of inflammation. Ex vivo restimulation of cervical lymph nodes (cLN) and spleen using PMA and ionomycin. Error bars represent standard deviation. Statistical significance is measured from one-way ANOVA against vehicle. * denotes p<0.05, *** denotes p<0.001, **** denotes p<0.0001.
[108] Figure 10 shows Veillonella parvula strain A- G.I. treatment reduces both ear inflammation and tissue expression of IL-17A. Compiled data from 5 individual studies. Ear inflammation measured on Day 8. Error bars represent standard error. Statistical significance is measured from one-way ANOVA against vehicle. **** denotes pO.OOOI.
[109] Figure 11 shows disease scores in EAE model. Clinical weakness scores over 42 days of daily dosing with Veillonella parvula strain A- G.I. or vehicle. Error bars represent standard error.
[110] Figure 12 shows the area under the curve (AUC) for total study and acute phase. Area under the curve for total study and acute phase. Error bars represent standard deviation (S.D.) **** denotes p<0.0001 by unpaired t-test.
[111] Figures 13A-13G shows that Veillonella parvula Strain A induces cytokine production in vitro and resolves inflammation in vivo. Figure 13A shows IL-10 cytokine levels determined by performing a Meso Scale Discovery (MSD) assay of supernatants derived from human macrophages stimulated with different anaerobic bacterial strains for 24 hours and flushed with 1% oxygen. Data shown represent collective data from 6 independent human donors. Figure 13B shows CXCL10/IP-10 levels determined by performing a MSD assay of supernatants derived from stimulated with different anaerobic bacterial strains for 24h and flushed with 1% oxygen. Data shown represent collective data from 6 independent human donors. Figure 13C shows TNFa levels determined by performing a MSD assay of supernatants derived from stimulated with different anaerobic bacterial strains for 24h and flushed with 1% oxygen Figure 13D shows human PBMCs, dendritic cells and macrophages stimulated with Veillonella parvula Strain A bacterial strains for 24 hours and flushed with 1% O2. Supernatants were collected to test for cytokine levels by MSD. Data shown represent collective data from 6 independent human donors DTH response to KLH. C57BL/6 mice were immunized with KLH and CFA on day 0 s.c. and challenged in the ear i.d. 9 days later with KLH. Mice were orally dosed daily from the day after immunization through ear challenge with vehicle or Veillonella parvula Strain A; TCC-2.84E+10. Ear inflammation was measured on day 9. Figure 13E shows the change in ear thickness (n = 5 mice/group) for groups dosed with Veillonella parvula Strain A; TCC -7.8E+11 and other non-replicating Veillonella strains (TCC- 3.8E+11- 1.03E+12). Figure 13F shows dose dependent effects of Veillonella parvula Strain A (TCC 2. 16E+12). Figure 13G shows that Veillonella parvula Strain A acts through IL-10R pathway to reduce ear inflammation. Mice in various groups were treated with IL-10R blocking antibody on days 2, 4, and 6 as indicated (Veillonella parvula Strain A TCC 2. 16E+12). This is a representative figure from n = 2 experiments with 5 mice/group in each experiment. All data show mean ± SEM. **p < 0.01, ****p < 0.0001, ns: not significant, as determined by unpaired Student's t-test. DTH - delayed-type hypersensitivity, KLH - keyhole limpet hemocyanin, CFA- complete Freund’s adjuvant, TCC- total cell count, s.c. - subcutaneous, i.p.- intraperitoneal, i.d. - intradermal.
[112] Figures 14A-14B show that Veillonella parvula Strain A is gut restricted and transits through GI tract within 24 hours. Figure 14A is a graph showing the reduction of signal indicating the presence of Veillonella parvula Strain A in the GI tract over time. Figure 14B comprises graphs of the signal detected indicating the presence of Veillonella parvula Strain A in various tissues.
[113] Figures 15A-15B show that Veillonella parvula Strain A alleviates skin inflammation in imiquimod-induced psoriasis. Figure 15 A shows ear inflammation over the course of 7 days and area under the curve. Figure 15B shows mRNA transcript levels for III 7a, III 7f and Defb3 measured by qPCR. Data are representative from 2 experiments with n = 5/group. All data show mean ± SEM. *p < 0.05, ***p < 0.0005, ****p < 0.0001, ns: not significant, as determined by unpaired Student's t-test.
[114] Figures 16A-16E show that Veillonella parvulci Strain A displays efficacy when treating neuroinflammation in a model for relapsing remitting MS. EAE was induced in SJL mice by immunization with PLP 139-151 in CFA on day 0, hour 0 and PTX was administered on day 0, hour 2. Figure 16A shows cumulative EAE scores of mice after for 41 days of oral prophylactic dosing with vehicle, Veillonella parvula Strain A (TCC- 8.46E+10), and fingolimod (1 mg/kg). Clinical scores were assessed daily forthe duration of the experiment. ****p < 0.00005 by Unpaired t-test with Welch’s correction was used to calculate p-value in cumulative AUC forthe EAE score. Figure 16B shows inflammation levels of the brain of spinal cords of mice treated with Veillonella parvula Strain A, vehicle or fingolimod. Figure 16C shows the cumulative EAE scores of mice after day 10 to day 41 of oral therapeutic dosing with vehicle, Veillonella parvula Strain A (TCC- 8.46E+10), and fingolimod (1 mg/kg). Clinical scores were assessed daily forthe duration of the experiment. ****p < 0.00005 by Unpaired t-test with Welch’s correction was used to calculate p-value in cumulative AUC forthe EAE score. Figure 16D is graph showing the inflammatory loci observed in the spinal cord cells. Figure 16E is a graph showing demyelination of the spinal cords of mice treated with Veillonella parvula Strain A, vehicle, or fingolimod. Data for Fig 16E are representative from n=15 mice per group. *p < 0.05 by unpaired Student’s t-test. PLP- proteolipid protein, EAE - experimental autoimmune encephalomyelitis, PTX - pertussis toxin, CFA-complete Freund’s adjuvant.
[115] Figures 17A-17B show that Veillonella parvula Strain A resolves inflammation in a Th2 cell driven atopic dermatitis model. Figure 17A shows the change in ear thickness in FITC- driven atopic dermatitis (AD). Figure 17B shows cytokine levels measured from supernatants and homogenates by multiplex ELISA derived from cells from mesenteric lymph nodes and cervical lymph nodes that had been restimulated with PMA for 48 hours. Data are representative of 3 independent experiments (n = 5 mice/group). *p < 0.05, ***p < 0.0005, ****p < 0.0001, ns: not significant, as determined by unpaired Student's t-test.
[116] Figures 18A-18B show that Veillonella parvula Strain A resolves inflammation in a delayed type hypersensitivity T cell driven disease model in vivo. Figure 18A shows the change in ear thickness (n = 10 mice/group). Figure 18B comprises graphs showing cytokine levels, as measured by multiplex ELISA, in supernatants derived from total cells from mesenteric lymph nodes and the spleen after restimulation with PMA for 48 hours and total cells from ear draining lymph nodes after restimulation with KLH for 72 hours. Data are representative from 2 experiments with n = 10/group. All data show mean ± SEM. *p < 0.05, **p < 0.001, ***p < 0.0005, ****p < 0.0001, ns: not significant, as determined by unpaired Student's t-test
[117] Figures 19A-19D show that Veillonella parvulci Strain is TLR2 dependent and TLR4 independent. Figure 19A shows that Veillonella parvula Strain A stimulates both human TLR1/2 and TLR2/6 heterodimers, with greater potency observed for TLR2/6 heterodimer. KLH-DTH was induced as previously described. Mice were orally dosed with vehicle or V. parvula Strain A (TCC- 2. 16E+12) from day 5 through 8. Ear inflammation was measured on day 9. In Figure 19B, mice were treated with anti-TLR2 blocking antibody on days 2, 4, and 6 as indicated and change in ear thickness was measured. In Figure 19C, change in ear thickness (n = 5 mice/group) in C3HEJ (TLR4- deficient) and C3HEN (wild type) mice dosed with V. parvula Strain A (TCC- 7.8E+10) All studies have n = 5 mice/group. Representative figure from n = 1- 2 experiments. All data show mean ± SEM. **p < 0.01, ****p < 0.0001, ns: not significant as determined by ordinary One-Way ANOVAFigure 19D shows that Veillonella parvula Strain A requires trafficking of gut immune cells to periphery to impact efficacy. All studies have n = 5 mice/group. Representative figure from n = 1- 2 experiments. All data show mean ± SEM. **p < 0.01, ****p < 0.0001, ns: not significant as determined by ordinary One- Way ANOVA.
[118] Figure 20 shows that adoptively transferred Veillonella parvula Strain A-treated CD4 T cells mediate efficacy in DTH. Representative figure from n = 2 experiments with 5mice/group in each experiment; ****p < 0.0001, as determined by Ordinary one-way ANOVA.
[119] Figure 21 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following dosing pre- or -post-immunization.
[120] Figure 22 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following dosing with vehicle (PBS) or Veillonella Strain A-G. I after immunization with PBS-CFA or PBS-IFA prior to immunization with KLH-CFA and a KLH ear challenge. [121] Figure 23 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following adoptive transfer of CD4+ T cells from vehicle or Veillonella Strain A-G.I. treated mice into untreated KLH-CFA-immunized recipients.
[122] Figure 24 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following treatment with vehicle or Veillonella Strain A-G.I. and depletion of B cells during the indicated time points.
[123] Figure 25 is a graph showing changes in ear thickness 24 hours after KLH ear challenge following treatment with vehicle or Veillonella Strain A-G.I. in combination with anti -IL-6.
DETAILED DESCRIPTION
[124] Veillonella parvula strain A- G.I. is a pharmaceutical preparation of a single strain of Veillonella parvula, originally isolated from a fresh ileostomy sample of an IBD patient in remission, which has been gamma-irradiated.
[125] Veillonella parvula strain A- G.I. can be manufactured by anaerobic fermentation, followed by lyophilization, gamma-irradiation, and encapsulation in a capsule with an enteric coating which protects the drug substance from low pH in the stomach. Because it is non-viable, it does not colonize the gut and has no detectable systemic exposure following oral dosing. In non-clinical studies its therapeutic effects are dose-dependent.
[126] Veillonella parvula strain A- G.I falls into a new pharmacological class termed non -live bacterial pharmaceutical products. This is a unique positioning as an oral medicine which modulates the small intestinal axis (SINTAX) for systemic pharmacological effects, with no systemic exposure. Since the lumen of the gut is topologically on the outside of the body, this is effectively a topical drug with systemic activity. This is made possible by the discovery of the systemic control network that emanates from the gut, and the small intestine in particular.
[127] In vivo preclinical models show Veillonella parvula strain A- G.I. to have potent anti-inflammatory effects in disease models covering multiple pathways of TH1, TH2 and TH 17 inflammation. Efficacy in these models is comparable to biologic treatments, including antibodies to IL-6, IL- 17, IL 12/23, VLA4 and small molecule drugs including tofacitinib, fmgolimod and dexamethasone. These results show that the gut-restricted effects of Veillonella parvula strain A- G.I. linking intestinal mucosal immune modulation to systemic immunity compares favorably with well proven systemic drugs. [128] Results from these preclinical studies suggest that Veillonella parvula strain A- G.I. has the potential to benefit patients with a wide range of conditions that have inflammation as an underlying driver. And the preclinical observation that these effects are achieved without broadly inhibiting the ability of the immune system to mount defensive responses indicates possible suitability of its safe use in large numbers of patients at all stages of disease with a placebo-like safety and tolerability profile.
[129] Preclinical studies using Veillonella parvula strain A- G.I. have been carried out across a range of human and mouse in vitro assays as well as key in vivo models of human disease, including delayed type hypersensitivity (DTH), imiquimod-induced skin inflammation, fluorescein isothiocyanate (FITC) cutaneous hypersensitivity, MC903- induced dermatitis, and experimental acute encephalomyelitis (EAE) in-vivo models. It has dose dependent therapeutic effects. Data from the in-vivo models support the use of Veillonella parvula strain A- G.I. in the treatment of Th2 -mediated (e.g., atopic dermatitis, asthma) and Thl7-mediated (e.g., psoriasis) immunoinflammatory diseases.
[130] As predicted based on the mechanism of action, no potentially related adverse effects were seen in the animals used in these experiments with daily dosing up to 6 weeks. In addition, while ex vivo immunophenotyping in these models shows decreases in pro-inflammatory cytokines such as IL-6, IL- 13, TNFa, 11-17, TSLP and KC (murine IL- 8), Veillonella parvula strain A- G.I. does not suppress the expression of interferon gamma (IFNy) in these experiments, suggesting that the broad spectrum of antiinflammatory effects is achieved without damaging mechanisms of immune surveillance critical for the prevention of malignancy and response to pathogens such as viral or bacterial infections.
Definitions
[131] “Adjuvant” or “Adjuvant therapy” broadly refers to an agent that affects an immunological or physiological response in a patient or subject. For example, an adjuvant might increase the presence of an antigen over time or help absorb an antigen presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines. By changing an immune response, 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. For example, an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent. [132] “Administration” broadly refers to a route of administration of a composition to a subject. Examples of 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. The bacterial compositions 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. In preferred embodiments, the bacterial compositions described herein are administered orally, rectally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously. In some preferred embodiments, the bacterial compositions described herein are administered orally.
[133] As used herein, 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). 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), singlechain antibodies and antigen-binding antibody fragments.
[134] The terms “antigen binding fragment” and “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. Examples of 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.
[135] “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.
[136] ‘ ‘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. “Operational taxonomic units,” “OTU” (or plural, “OTUs”) 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. In some embodiments the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence. In other embodiments, the entire genomes of two entities are sequenced and compared. In another embodiment, select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared. In 16S embodiments, OTUs that share 1197% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU (see e.g. Claesson M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ros R P, and O'Toole P W. 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 K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Land B Biol Sci 361: 1929- 1940.). In embodiments involving the complete genome, MLSTs, specific genes, or sets of genes OTUs that share 1195% 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 K T, Ramette A, and Tiedje J M. 2006. The bacterial species definition in the genomic era. Philos Trans R SocLond B Biol Sci 361: 1929-1940.). OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. 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. Such characterization employs, e.g., WGS data or a whole genome sequence.
[137] A “combination” of two or more monoclonal microbial strains includes the physical co-existence of the two monoclonal microbial strains, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the monoclonal microbial strains.
[138] The term “decrease” or “deplete” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to 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)). A “decrease” refers to a decrease as compared to amount produced (e.g., mRNA and/or protein) in the absence of the bacterial composition).
[139] “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.
[140] As used herein, “engineered bacteria” are any bacteria that have been genetically altered from their natural state by human intervention 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.
[141] The term “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.
[142] The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. The term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
[143] “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)).
[144] As used herein, 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., 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 environmental allergies).
[145] “Immunotherapy” is treatment that uses a subject’s immune system to treat disease (e.g., immune disease) and includes, for example, checkpoint inhibitors, cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.
[146] The term “increase” means a change, 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, 10A3 fold, 10A4 fold, 10A5 fold, 10A6 fold, and/or 10A7 fold greater after treatment when compared to a pre-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). An “increase” refers to an increase as compared to amount produced (e.g., mRNA and/or protein) in the absence of the bacterial composition).
[147] ‘ ‘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, STING-cGAS Pathway components, inflammasome complexes. For example, 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. Examples of STING 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). Examples of TLR agonists include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR1O and TLRI 1. Examples of 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).
[148] The term “isolated” or “enriched” encompasses a microbe, bacteria 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 microbes 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. In some embodiments, isolated microbes 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. The terms “purify,” “purifying,” and “purified” refer to a microbe 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. A microbe or a microbial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial 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 “isolated.” In some embodiments, purified microbes or microbial population 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. In the instance of microbial compositions provided herein, the one or more microbial types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type. Microbial compositions and the microbial components thereof are generally purified from residual habitat products.
[149] ‘ ‘Metabolite” as used herein refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or microbial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or microbial metabolic reaction.
[150] ‘ ‘Microbe” refers to any natural or engineered organism characterized as a bacterium, fungus, microscopic alga, protozoan, and the stages of development or life cycle stages (e.g., vegetative, spore (including sporulation, dormancy, and germination), latent, biofdm) associated with the organism.
[151] ‘ ‘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 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). In some aspects, the microbiome occurs at a mucosal surface. In some aspects, the microbiome is a gut microbiome.
[152] 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. In some embodiments, 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. In some embodiments, 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 in a sample. In some embodiments, the microbiome profile indicates the relative or absolute amount of each bacterial strain detected in the sample.
[153] ‘ ‘Modified” in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form. Examples of bacterial modifications include genetic modification, gene expression, phenotype modification, formulation, 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 that increase or decrease virulence.
[154] As used 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. Similarly, 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.
[155] The terms “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. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), micro RNA (miRNA), silencing RNA (siRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. 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.
[156] “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. In some embodiments the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence. In other embodiments, the entire genomes of two entities are sequenced and compared. In another embodiment, select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared. For 16S, 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. 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 Uond B Biol Sci 361: 1929-1940. OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. 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.
[157] As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying” and “purified” refer to a microbe 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. A microbe 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 microbial 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.” In some embodiments, purified microbes 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. Bacterial compositions and the microbial components thereof are, e.g., purified from residual habitat products.
[158] ‘ ‘Residual habitat products” refers to material derived from the habitat for microbiota within or on a subject. For example, 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 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 human or animal and that only microbial cells are detectable. In one embodiment, substantially free of residual habitat products may also mean that the microbial composition contains no detectable viral (including microbial viruses (e.g., phage)), fungal, mycoplasmal contaminants. In another embodiment, it means that fewer than 1x10-2%, 1x10-3%, 1x10-4%, 1x10-5%, 1x10-6%, 1x10-7%, 1x10-8% 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. Thus, 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. Alternatively, reduction of contamination can be accomplished by multiple rounds of serial dilutions to single desired cells (e.g., a dilution of 10-8 or 10-9), 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. Other methods for confirming adequate purity include genetic analysis (e.g., PCR, DNA sequencing), serology and antigen analysis, enzymatic and metabolic analysis, and methods using instrumentation such as flow cytometry with reagents that distinguish desired constituents from contaminants.
[159] As used herein, “specific 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. Typically, an antibody or polypeptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about KF7 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 non-specific and unrelated antigen/binding partner (e.g., BSA, casein). Alternatively, 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.
[160] The terms “subject” or “patient” refers to any animal. A subject or a patient described as “in need thereof’ refers to one in need of a treatment for a disease. Mammals (i.e., mammalian animals) include humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents). For example, 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. In some embodiments, the subject is a human subject. [161] ‘ ‘Strain” refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species. The genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic signatures between different strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite , respectively .
[162] As used herein, the term “treating” a disease in a subject or “treating” a subject having or suspected of having a disease refers to subjecting 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. Thus, in one embodiment, “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.
[163] As used herein, a “type” of bacteria may be distinguished from other bacteria by: genus, species, sub-species, strain or by any other taxonomic categorization, whether based on morphology, physiology, genotype, protein expression or other characteristics known in the art.
Bacteria
[164] In certain aspects, provided herein are bacterial compositions (e.g., pharmaceutical compositions) comprising Veillonella parvulci useful for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell) and methods of using such bacterial compositions (e.g., for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell)), e.g., in a subject, e.g., in a human subject. In some embodiments, the bacterial compositions comprise whole Veillonella parvula bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In some embodiments, the Veillonella parvula bacteria are gamma irradiated. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises only one strain of bacteria, e.g., Veillonella parvula.
[165] Under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure, the Veillonella parvula strain A was deposited on January 25, 2019, with the American Type Culture Collection (ATCC) of 10801 University Boulevard, Manassas, Va. 20110-2209 USA and was assigned ATCC Accession Number PTA-125691.
[166] Applicant represents that the ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited plasmid, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicant acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
[167] In some embodiments, the Veillonella parvula is Veillonella parvula strain A (ATCC Deposit Number PTA-125691) (also referred to as “Veillonella parvula strain A”). In some embodiments, the Veillonella parvula strain is a strain comprising at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[168] Veillonella parvula strain A can be cultured according to methods known in the art. For example, Veillonella parvula strain A can be grown under anaerobic conditions in PM1 l+5g/L Na-L-lactate liquid medium supplemented with 0.05g/L FeSO4, and 0.5 g/L L-cysteine-HCL as reducing agent at 37 degrees C. See also WO 2019/157003.
[169] In some embodiments, the bacterial compositions comprise whole Veillonella parvula bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
[170] In some embodiments, the bacterial compositions comprise whole Veillonella parvula bacteria (e.g., gamma irradiated Veillonella parvula bacteria).
[171] In certain embodiments, the bacterial composition (e.g., pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises about 1 x 1010 total cells, about 2 x 1010 total cells, about 3 x 1010 total cells, about 4 x 1010 total cells, about 4.5 x 1010 total cells, about 5 x 1010 total cells, about 6 x 1010 total cells, about 7 x 1010 total cells, about 8 x 1010 total cells, about 9 x 1010 total cells, about 1 x 1011 total cells, about 1.5 x 1011 total cells, about 2 x 1011 total cells, about 3 x 1011 total cells, about 4 x 1011 total cells, about 5 x 1011 total cells, about 6 x 1011 total cells, about 7 x 1011 total cells, about 7.5 x 1011 total cells, about 8 x 1011 total cells, about 9 x 1011 total cells, about 1 x 1012 total cells, about 1.5 x 1012 total cells, about 2 x 1012 total cells of the Veillonella parvula bacteria.
[172] In certain embodiments, the bacterial composition (e.g. , pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises at least 1 x 1010 total cells (e.g., at least 1 x 1010 total cells, at least 2 x 1010 total cells, at least 3 x 1010 total cells, at least 4 x 1010 total cells, at least 4.5 x 1010 total cells, at least 5 x 1010 total cells, at least 6 x 1010 total cells, at least 7 x 1010 total cells, at least 8 x 1010 total cells, at least 9 x 1010 total cells, at least 1 x 1011 total cells, at least 1.5 x 1011 total cells, at least 2 x 1011 total cells, at least 3 x 1011 total cells, at least 4 x 1011 total cells, at least 5 x 1011 total cells, at least 6 x 1011 total cells, at least 7 x 1011 total cells, at least 7.5 x 1011 total cells, at least 8 x 1011 total cells, at least 9 x 1011 total cells, at least 1 x 1012 total cells, at least 1.5 x 1012 total cells, at least 2 x 1012 total cells) of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 3 x 1010total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 4.5 x 1010total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 1011 total cells of the Veillonella parvulci bacteria. In some embodiments, the bacterial composition comprises about 7.5 x 1011 total cells the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 1012total cells the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 3 x 1010to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 4.5 x 1010to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 3 x 1010to about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 4.5 x 1010to about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10n to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 1.5 x 10n to about 7.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition comprises about 7.5 x 10n to about 1.5 x 1012 total cells of the Veillonella parvula bacteria.
[173] In some embodiments, the bacterial composition comprises about 3 x 1010 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[174] In some embodiments, the bacterial composition comprises about 4.5 x 1010total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[175] In some embodiments, the bacterial composition comprises about 1.5 x 1011 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[176] In some embodiments, the bacterial composition comprises about 7.5 x 10ntotal cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[177] In some embodiments, the bacterial composition comprises about 1.5 x 1012total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691). [178] In some embodiments, the bacterial composition comprises about 3 x 1010 to about 1.5 x 1012 total cells of Veillonella parvula, e.g., of Veillonella parvulci strain A (ATCC Deposit Number PTA- 125691).
[179] In some embodiments, the bacterial composition comprises about 4.5 x 1010to about 1.5 x 1012 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[180] In some embodiments, the bacterial composition comprises about 3 x 1010 to about 1.5 x 1011 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[181] In some embodiments, the bacterial composition comprises about 4.5 x 1010to about 1.5 x 1011 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[182] In some embodiments, the bacterial composition comprises about 1.5 x 10nto about 1.5 x 1012 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[183] In some embodiments, the bacterial composition comprises about 1.5 x 10nto about 7.5 x 1011 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[184] In some embodiments, the bacterial composition comprises about 7.5 x 10nto about 1.5 x 1012 total cells of Veillonella parvula, e.g., of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
[185] In some embodiments, the Veillonella parvula bacteria may be quantified based on total cells, e.g., total cell count (TCC) (e.g., determined by Coulter counter).
[186] In some embodiments, the bacterial composition is administered orally. In some embodiments, the administration to the subject once daily. In some embodiments, the bacterial composition is administered in 2 or more doses (e.g., 3 or more, 4 or more or 5 or more doses). In some embodiments, the administration to the subject of the two or more doses are separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days. [187] In some embodiments, the bacterial composition is administered once daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
[188] In some embodiments, the bacterial composition is administered once daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for 8 weeks. In some embodiments, the bacterial composition is administered once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for at least 8 weeks.
[189] In some embodiments, the bacterial composition is formulated as a capsule or a tablet. In some embodiments, the bacterial formulation (e.g., composition) comprises an enteric coating or micro encapsulation. In some embodiments, the capsule is an enteric coated capsule. In some embodiments, the enteric coating allows release of the bacterial composition in the small intestine, e.g., in the upper small intestine, e.g., in the duodenum.
[190] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal (e.g., 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).
Bacterial Compositions
[191] In certain embodiments, the methods provided herein comprise use of bacterial compositions (e.g., pharmaceutical compositions) comprising Veillonella parvula bacteria provided herein.
[192] In some embodiments, the bacterial compositions (e.g., pharmaceutical compositions) comprise whole Veillonella parvula bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In some embodiments, the Veillonella parvula bacteria is non- viable. In some embodiments, the Veillonella parvula bacteria has been gamma irradiated (e.g., according to a method described herein). In some embodiments, the Veillonella parvula bacteria is live. [193] In some embodiments, the bacterial composition (e.g, pharmaceutical composition) comprises only one strain of bacteria, e.g., Veillonella parvula.
[194] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises more than one strain of bacteria, e.g., Veillonella parvula, and the therapeutic effect caused by the bacterial composition is due to the presence of the Veillonella parvula bacteria (e.g., a therapeutically effective amount thereof) present in the composition.
[195] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are of the Veillonella parvula strain. E.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are of the Veillonella parvula strain. In some embodiments, at least 99% of the bacteria in the bacterial composition are of the Veillonella parvula strain. In some embodiments, the bacteria in the composition are essentially (e.g., about 100%) of the Veillonella parvula strain.
[196] In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,
25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,
40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,
55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the protein in the bacterial composition is Veillonella parvula strain bacteria protein.
[197] In some embodiments, the Veillonella parvula is Veillonella parvula strain A (ATCC Deposit Number PTA- 125691). In some embodiments, the Veillonella parvula strain is a strain comprising at least at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691). [198] In some embodiments, the bacterial compositions (e.g., pharmaceutical compositions) comprise whole Veillonella parvulci bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
[199] In some embodiments, the bacterial compositions (e.g., pharmaceutical compositions) comprise whole Veillonella parvula bacteria (e.g., gamma irradiated Veillonella parvula bacteria).
[200] In certain embodiments, the bacterial compositions (e.g., pharmaceutical compositions) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises about 1 x IO10 total cells, about 2 x IO10 total cells, about 3 x IO10 total cells, about 4 x IO10 total cells, about 4.5 x IO10 total cells, about 5 x IO10 total cells, about 6 x IO10 total cells, about 7 x IO10 total cells, about 8 x IO10 total cells, about 9 x IO10 total cells, about 1 x 1011 total cells, about 1.5 x 1011 total cells, about 2 x 1011 total cells, about 3 x 1011 total cells, about 4 x 1011 total cells, about 5 x 1011 total cells, about 6 x 1011 total cells, about 7 x 1011 total cells, about 7.5 x 1011 total cells, about 8 x 1011 total cells, about 9 x 1011 total cells, about 1 x 1012 total cells, about 1.5 x 1012 total cells, about 2 x 1012 total cells of the Veillonella parvula bacteria.
[201] In certain embodiments, the bacterial composition (e.g., pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises at least 1 x 1010 total cells (e.g., at least 1 x 1010 total cells, at least 2 x IO10 total cells, at least 3 x IO10 total cells, at least 4 x IO10 total cells, at least 4.5 x IO10 total cells, at least 5 x IO10 total cells, at least 6 x IO10 total cells, at least 7 x IO10 total cells, at least 8 x IO10 total cells, at least 9 x IO10 total cells, at least 1 x 1011 total cells, at least 1.5 x 1011 total cells, at least 2 x 1011 total cells, at least 3 x 1011 total cells, at least 4 x 1011 total cells, at least 5 x 1011 total cells, at least 6 x 1011 total cells, at least 7 x 1011 total cells, at least 7.5 x 1011 total cells, at least 8 x 1011 total cells, at least 9 x 1011 total cells, at least 1 x 1012 total cells, at least 1.5 x 1012 total cells, at least 2 x 1012 total cells) of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x IO10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x IO10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 7.5 x 1011 total cells the Veillonella parvulci bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 1012 total cells the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x 1010to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x 1010to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x 1010to about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x 1010to about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10n to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10n to about 7.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 7.5 x 10n to about 1.5 x 1012 total cells of the Veillonella parvula bacteria.
[202] In some embodiments, the Veillonella parvula bacteria may be quantified based on total cells, e.g., total cell count (TCC) (e.g., determined by Coulter counter).
[203] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered orally. In some embodiments, the administration to the subject once daily. In some embodiments, the bacterial composition is administered in 2 or more doses (e.g. , 3 or more, 4 or more or 5 or more doses). In some embodiments, the administration to the subject of the two or more doses are separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.
[204] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered once daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
[205] In some embodiments, the bacterial composition (e.g, pharmaceutical composition) is administered twice daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
[206] In some embodiments, the bacterial composition (e.g, pharmaceutical composition) is administered once daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered once daily for 8 weeks. In some embodiments, the bacterial composition is administered once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for at least 8 weeks.
[207] In some embodiments, the bacterial composition (e.g, pharmaceutical composition) is administered twice daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered twice daily for 8 weeks. In some embodiments, the bacterial composition is administered twice daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered twice daily for at least 8 weeks.
[208] In some embodiments, the bacterial composition (e.g, pharmaceutical composition) is formulated as a capsule or a tablet. In some embodiments, the bacterial formulation (e.g., composition) comprises an enteric coating or micro encapsulation. In some embodiments, the capsule is an enteric coated capsule. In some embodiments, the enteric coating allows release of the bacterial composition in the small intestine, e.g., in the upper small intestine, e.g., in the duodenum.
[209] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal (e.g., 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). [210] In some embodiments, to quantify the numbers of Veillonella parvula bacteria present in a bacterial sample, electron microscopy (e.g., EM of ultrathin frozen sections) can be used to visualize the bacteria and count their relative numbers. Alternatively, combinations of nanoparticle tracking analysis (NTA), Coulter counting, and dynamic light scattering (DLS) or a combination of these techniques can be used. NTA and the Coulter counter count particles and show their sizes. DLS gives the size distribution of particles, but not the concentration. Bacteria frequently have diameters of 1-2 um. The full range is 0.2-20 um. Combined results from Coulter counting and NTA can reveal the numbers of bacteria in a given sample. Coulter counting reveals the numbers of particles with diameters of 0.7-10 um. NTA reveals the numbers of particles with diameters of 50- 1400 nm. For most bacterial samples, the Coulter counter alone can reveal the number of bacteria in a sample.
[2H] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises an enteric coating or micro encapsulation. In certain embodiments, the enteric coating or micro encapsulation improves targeting to a desired region of the gastrointestinal tract. For example, in certain embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises an enteric coating and/or microcapsules that dissolves at a pH associated with a particular region of the gastrointestinal tract. In some embodiments, the enteric coating and/or microcapsules dissolve at a pH of about 5.5 - 6.2 to release in the duodenum, at a pH value of about 7.2 - 7.5 to release in the ileum, and/or at a pH value of about 5.6 - 6.2 to release in the colon. Exemplary enteric coatings and microcapsules are described, for example, in U.S. Pat. Pub. No. 2016/0022592, which is hereby incorporated by reference in its entirety.
[212] In certain aspects, provided are bacterial compositions (e.g., pharmaceutical compositions) for administration subjects. In some embodiments, the bacterial 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. In some embodiments, the bacterial compositions are combined with an adjuvant such as an immuno-adjuvant (e.g., STING agonists, TLR agonists, NOD agonists).
[213] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises at least one carbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars. The terms “saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide” may be used interchangeably. 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 CnFEnOn. A carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide. The most basic carbohydrate is a monosaccharide, such as glucose, sucrose, 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.
[214] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises at least one lipid. As used herein 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). In some embodiments, the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16: 1), margaric acid (17:0), heptadecenoic acid (17: 1), stearic acid (18:0), oleic acid (18: 1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20: 1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EP A), docosanoic acid (22:0), docosenoic acid (22: 1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0). In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises at least one modified lipid, for example a lipid that has been modified by cooking.
[215] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises at least one supplemental mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
[216] In some embodiments, the bacterial composition (e.g., pharmaceutical 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 B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
[217] In some embodiments, the bacterial composition (e.g, pharmaceutical composition) comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
[218] In some embodiments, the excipient is a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
[219] In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
[220] In some embodiments, the bacterial composition (e.g, pharmaceutical composition) comprises a binder as an excipient. Non-limiting examples of 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.
[221] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises a lubricant as an excipient. Non-limiting examples of 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. [222] In some embodiments, the bacterial composition (e.g, pharmaceutical 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.
[223] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises a disintegrant as an excipient. In some embodiments the disintegrant is a non-effervescent disintegrant. Non-limiting examples of suitable non- effervescent disintegrants include starches such as com 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. In some embodiments the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
[224] In some embodiments, the bacterial 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. Specific examples of 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. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies. [225] In some embodiments, the bacterial 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.
Dose Forms
[226] Dose forms comprising Veillonella parvulci bacteria are also provided herein, e.g., for use in methods provided herein, e.g., to induce an immune effect in a subject (e.g., a human subject). A bacterial composition (e.g., pharmaceutical composition) comprising Veillonella parvula bacteria can be formulated as a solid dose form, e.g., for oral administration. The solid dose form can comprise one or more excipients, e.g., pharmaceutically acceptable excipients. The Veillonella parvula bacteria in the solid dose form can be isolated Veillonella parvula bacteria. Optionally, the Veillonella parvula bacteria in the solid dose form can be lyophilized. Optionally, the Veillonella parvula bacteria in the solid dose form are live. Optionally, the Veillonella parvula bacteria in the solid dose form are gamma irradiated. The solid dose form can comprise a tablet, a minitablet, a capsule, a pill, or a powder; or a combination of these forms (e.g., minitablets comprised in a capsule).
[227] The Veillonella parvula bacteria in the solid dose form can be in a powder (e.g., the powder comprises lyophilized Veillonella parvula bacteria). In some embodiments, the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the powder further comprises mannitol, magnesium stearate, and colloidal silicon dioxide. In some embodiments, the powder further comprises sucrose and/or dextran. In some embodiments, the powder further comprises sucrose, dextran, and/or L-cysteine HC1.
[228] In some embodiments, the powder is resuspended in a solution.
[229] In some embodiments, the lyophilized Veillonella parvula bacteria is resuspended in a solution.
[230] In certain embodiments, the bacterial composition (e.g., pharmaceutical composition) provided herein is prepared as a solid dosage form comprising Veillonella parvula bacteria and a pharmaceutically acceptable carrier. [231] In some embodiments, the solid dosage form comprises a capsule. The capsule can comprise an enteric coating. The capsule can be a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule. The capsule can comprise Veillonella parvula bacteria powder (e.g., lyophilized Veillonella parvula bacteria). In some embodiments, the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the powder further comprises mannitol, magnesium stearate, and colloidal silicon dioxide. In some embodiments, the powder further comprises sucrose and/or dextran. In some embodiments, the powder further comprises sucrose, dextran, and/or L-cysteine HC1. In some embodiments, the capsule comprises excipients and the excipients include mannitol, colloidal silicon dioxide, magnesium stearate, hydroxypropyl methylcellulose, methacrylic acid ethyl acrylate copolymer, triethyl citrate, and/or talc.
[232] In some embodiments, the solid dosage form described herein can be, e.g., a tablet or a mini -tablet. In some embodiments, a plurality of mini-tablets can be in (e.g., loaded into) a capsule.
[233] In some embodiments, the solid dosage form comprises a tablet (> 4mm) (e.g., 5mm-17mm). For example, the tablet is a 5mm, 5.5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, or 18mm tablet. The size refers to the diameter of the tablet, as is known in the art. As used herein, the size of the tablet refers to the size of the tablet prior to application of an enteric coating.
[234] In some embodiments, the solid dosage form comprises a mini-tablet. The mini-tablet can be in the size range of lmm-4 mm range. E.g., the mini-tablet can be a 1mm mini-tablet, 1.5 mm mini-tablet, 2mm mini-tablet, 3mm mini-tablet, or 4mm minitablet. The size refers to the diameter of the mini-tablet, as is known in the art. As used herein, the size of the minitablet refers to the size of the mini-tablet prior to application of an enteric coating.
[235] The mini-tablets 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 mini -tablets can comprise a single layer coating, e.g. , a non-enteric coating such as gelatin or HPMC. The mini-tablets can be inside a capsule: the number of mini -tablets inside a capsule will depend on the size of the capsule and the size of the mini -tablets. As an example, a size 0 capsule can contain 31-35 (an average of 33) mini-tablets that are 3mm mini-tablets.
[236] The solid dosage form (e.g., tablet or mini-tablet or capsule) described herein can be enterically coated. In some embodiments, the enteric coating comprises a polymethacrylate-based copolymer. In some embodiments, the enteric coating comprises a methacrylic acid ethyl acrylate (MAE) copolymer (1: 1). In some embodiments, the enteric coating comprises methacrylic acid ethyl acrylate (MAE) copolymer (1: 1) (such as Kollicoat MAE 100P).
[237] The solid dose form can comprise a coating. The solid dose form can comprise a single layer coating, e.g., enteric coating, e.g, a Eudragit-based coating, e.g., EUDRAGIT L30 D-55, triethylcitrate, and talc. The solid dose form can comprise two layers of coating. For example, an inner coating can comprise, e.g., EUDRAGIT L30 D- 55, triethylcitrate, talc, citric acid anhydrous, and sodium hydroxide, and an outer coating can comprise, e.g., EUDRAGIT L30 D-55, triethylcitrate, and talc. 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. Eudragits are amorphous polymers having glass transition temperatures between 9 to > 150°C. Eudragits are non-biodegradable, nonabsorbable, and nontoxic. Anionic Eudragit L dissolves at pH > 6 and is used for enteric coating, while Eudragit S, soluble at pH > 7 is used for colon targeting. Eudragit RL and RS, having quaternary ammonium groups, are water insoluble, but swellable/permeable polymers which are suitable for the sustained release fdm coating applications. Cationic Eudragit E, insoluble at pH > 5, can prevent drug release in saliva.
[238] The solid dose form (e.g., a capsule) can comprise HPMC or gelatin.
[239] A bacterial composition (e.g., pharmaceutical composition) comprising Veillonella parvula bacteria can be formulated as a suspension, e.g., for oral administration or for injection. Administration by injection includes intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration. For a suspension, Veillonella parvula bacteria 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 Veillonella parvula bacteria in the suspension can be isolated Veillonella parvula bacteria. Optionally, the Veillonella parvula bacteria in the suspension can be lyophilized. Optionally, the Veillonella parvula bacteria in the solid dose form are live. Optionally, the Veillonella parvula bacteria in the suspension can be gamma irradiated. Dosage
[240] For oral administration to a human subject, the dose of Veillonella parvulci bacteria can be, e.g., about 3 x 1010to about 1.5 x 1012 particles, about 4.5 x 1010to about
1.5 x 1012 particles, about 3 x 1010to about 1.5 x 1011 particles, about 4.5 x 1010to about
1.5 x 1011 particles, about 1.5 x 10n to about 1.5 x 1012 particles, 1.5 x 10n to about 7.5 x 1011 particles, about 7.5 x 10n to about 1.5 x 1012 particles. The dose can be, e.g., about 1 x 1010 particles, about 2 x 1010 particles, about 3 x 1010 particles, about 4 x 1010 particles, about 4.5 x 1010 particles, about 5 x 1010 particles, about 6 x 1010 particles, about 7 x 1010 particles, about 8 x 1010 particles, about 9 x 1010 particles, about 1 x 1011 particles, about
1.5 x 1011 particles, about 2 x 1011 particles, about 3 x 1011 particles, about 4 x 1011 particles, about 5 x 1011 particles, about 6 x 1011 particles, about 7 x 1011 particles, about
7.5 x 1011 particles, about 8 x 1011 particles, about 9 x 1011 particles, about 1 x 1012 particles, about 1.5 x 1012 particles, about 2 x 1012 particles. The dose can be, e.g., about 3x1010 particles. The dose can be, e.g., about 4.5xlO10 particles. The dose can be, e.g., about 1.5xlOn particles. The dose can be, e.g., about 7.5xlOn particles. The dose can be, e.g., about 1.5xl012 particles. Particle count can be determined, e.g., by NTA.
[241] For oral administration to a human subject, the dose of Veillonella parvula bacteria can be, e.g., about 3 x 1010to about 1.5 x 1012 total cells, about 4.5 x 1010to about
1.5 x 1012 total cells, about 3 x 1010to about 1.5 x 10n total cells, about 4.5 x 1010to about
1.5 x 10n total cells, about 1.5 x 10n to about 1.5 x 1012total cells, 1.5 x 10n to about 7.5 x 1011 total cells, about 7.5 x 10n to about 1.5 x 1012total cells. The dose can be, e.g., about 1 x 1010 total cells, about 2 x 1010 total cells, about 3 x 1010 total cells, about 4 x 1010 total cells, about 4.5 x 1010 total cells, about 5 x 1010 total cells, about 6 x 1010 total cells, about 7 x 1010 total cells, about 8 x 1010 total cells, about 9 x 1010 total cells, about 1 x 1011 total cells, about 1.5 x 1011 total cells, about 2 x 1011 total cells, about 3 x 1011 total cells, about 4 x 1011 total cells, about 5 x 1011 total cells, about 6 x 1011 total cells, about 7 x 1011 total cells, about 7.5 x 1011 total cells, about 8 x 1011 total cells, about 9 x 1011 total cells, about 1 x 1012 total cells, about 1.5 x 1012 total cells, about 2 x 1012 total cells of the Veillonella parvula bacteria.
[242] In certain embodiments, the bacterial composition (e.g., pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises about 1 x 1010 total cells, about 2 x 1010 total cells, about 3 x 1010 total cells, about 4 x 1010 total cells, about 4.5 x 1010 total cells, about 5 x 1010 total cells, about 6 x IO10 total cells, about 7 x IO10 total cells, about 8 x IO10 total cells, about 9 x IO10 total cells, about 1 x 1011 total cells, about 1.5 x 1011 total cells, about 2 x 1011 total cells, about 3 x 1011 total cells, about 4 x 1011 total cells, about 5 x 1011 total cells, about 6 x 1011 total cells, about 7 x 1011 total cells, about 7.5 x 1011 total cells, about 8 x 1011 total cells, about 9 x 1011 total cells, about 1 x 1012 total cells, about 1.5 x 1012 total cells, about 2 x 1012 total cells of the Veillonella parvulci bacteria.
[243] In certain embodiments, the bacterial composition (e.g., pharmaceutical composition) (e.g., composition of the total dose administered, e.g., once or twice daily) comprises at least 1 x IO10 total cells (e.g., at least 1 x IO10 total cells, at least 2 x IO10 total cells, at least 3 x IO10 total cells, at least 4 x IO10 total cells, at least 4.5 x IO10 total cells, at least 5 x IO10 total cells, at least 6 x IO10 total cells, at least 7 x IO10 total cells, at least 8 x IO10 total cells, at least 9 x IO10 total cells, at least 1 x 1011 total cells, at least 1.5 x 1011 total cells, at least 2 x 1011 total cells, at least 3 x 1011 total cells, at least 4 x 1011 total cells, at least 5 x 1011 total cells, at least 6 x 1011 total cells, at least 7 x 1011 total cells, at least 7.5 x 1011 total cells, at least 8 x 1011 total cells, at least 9 x 1011 total cells, at least 1 x 1012 total cells, at least 1.5 x 1012 total cells, at least 2 x 1012 total cells) of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x IO10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x IO10 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 7.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 1012total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x 1010to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x 1010to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 3 x 1010to about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 4.5 x 1010to about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10n to about 1.5 x 1012 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 1.5 x 10n to about 7.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprises about 7.5 x 10n to about 1.5 x 1012 total cells of the Veillonella parvula bacteria.
[244] In some embodiments, the Veillonella parvula bacteria may be quantified based on total cells, e.g., total cell count (TCC) (e.g., determined by Coulter counter).
[245] In certain embodiments, provided herein are solid dosage forms comprising the Veillonella parvula bacteria. In some embodiments, the solid dosage form comprises an enteric coating. In some embodiments, the solid dosage form is a capsule, e.g., an enteric coated capsule. In some embodiments, each capsule comprises about 3 x 1010 total cells of the Veillonella parvula bacteria. In some embodiments, each capsule comprises about 4.5 x IO10 total cells of the Veillonella parvula bacteria. In some embodiments, each capsule comprises about 1.5 x 1011 total cells of the Veillonella parvula bacteria. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 capsules are administered, e.g., once or twice daily to a subject. In some embodiments, 1 capsule (e.g., comprising about 3 x IO10 total cells) is administered, e.g., once or twice daily to a subject. In some embodiments, 2 capsules (e.g., each comprising about 3 x IO10 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 4 capsules (e.g., each comprising about 3 x IO10 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 10 capsules (e.g., each comprising about 3 x IO10 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 1 capsule (e.g., comprising about 4.5 x IO10 total cells) is administered, e.g., once or twice daily to a subject. In some embodiments, 2 capsules (e.g., each comprising about 4.5 x IO10 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 4 capsules (e.g., each comprising about 4.5 x IO10 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 10 capsules (e.g., each comprising about 4.5 x 1010 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 1 capsule (e.g., comprising about 1.5 x 10n total cells) is administered, e.g., once ortwice daily to a subject. In some embodiments, 2 capsules (e.g., each comprising about 1.5 x 1011 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 5 capsules (e.g., each comprising about 1.5 x 1011 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, 10 capsules (e.g., each comprising about 1.5 x 1011 total cells) are administered, e.g., once or twice daily to a subject. In some embodiments, the Veillonella parvulci bacteria in the capsule are lyophilized (e.g., in a powder). In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the Veillonella parvula bacteria of the capsule are gamma irradiated.
[246] In some embodiments, the solid dosage form comprises a capsule. In some embodiments, the capsule is an enteric coated capsule. In some embodiments, the capsule comprises about 3 x IO10 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules). In some embodiments, the capsule comprises about 4.5 x IO10 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules). In some embodiments, the capsule comprises about 1.5 x 1011 total cells of the Veillonella parvula bacteria (e.g., total dose of a capsule or plurality of capsules). In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized (e.g., in a powder). In some embodiments, the Veillonella parvula bacteria in the capsule are lyophilized in a powder, and the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the Veillonella parvula bacteria of the capsule are gamma irradiated.
[247] In some embodiments, the solid dosage form comprises a tablet. In some embodiments, the tablet is an enteric coated tablet. In some embodiments, the enteric coated tablet is from 5mm to 18mm in diameter. In some embodiments, the tablet comprises about 3 x IO10 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets). In some embodiments, the tablet comprises about 4.5 x IO10 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets). In some embodiments, the tablet comprises about 1.5 x 1011 total cells of the Veillonella parvula bacteria (e.g., total dose of a tablet or plurality of tablets). In some embodiments, the Veillonella parvula bacteria in the tablet are lyophilized. In some embodiments, the Veillonella parvula bacteria of the tablet are gamma irradiated.
[248] In some embodiments, the solid dosage form comprises a mini-tablet. In some embodiments, the mini-tablet is enteric coated. In some embodiments, the mini- tablet is from 1mm to 4mm in diameter. In some embodiments, the mini-tablet (e.g., enteric coated mini-tablet) is a 1mm mini-tablet, 1.5 mm mini-tablet, 2mm mini-tablet, 3mm mini-tablet, or 4mm mini-tablet. In some embodiments, the solid dosage form comprises mini-tablets that comprise about 3 x 1010 total cells of the Veillonella parvulci bacteria (e.g., total dose of a plurality of mini -tablets). In some embodiments, the solid dosage form comprises mini-tablets that comprise about 4.5 x IO10 total cells of the Veillonella parvula bacteria (e.g., total dose of a plurality of mini -tablets). In some embodiments, the solid dosage form comprises mini-tablets that comprise about 1.5 x 1011 total cells of the Veillonella parvula bacteria (e.g., total dose of a plurality of minitablets). In some embodiments, the Veillonella parvula bacteria in the mini-tablets are lyophilized. In some embodiments, the Veillonella parvula bacteria in the mini-tablet are gamma irradiated.
[249] In some embodiments, the mini -tablets (e.g., enteric coated mini -tablets) are contained in a capsule. In some embodiments, the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule. In some embodiments, the capsule comprises a non- enteric coating (e.g., gelatin) (e.g., is coated with a non-enteric coating). In some embodiments, the capsule comprises a non-enteric coating. In some embodiments, the capsule comprises gelatin. In some embodiments, the capsule comprises HPMC. In some embodiments, the mini-tablets (e.g., enteric coated mini -tablets) that comprise about 3 x 1010 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC. In some embodiments, the minitablets (e.g., enteric coated mini-tablets) that comprise about 4.5 x 1010 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC. In some embodiments, the mini-tablets (e.g., enteric coated mini-tablets) that comprise about 1.5 x 1011 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC. In some embodiments, the Veillonella parvula bacteria of the mini-tablet are gamma irradiated.
[250] In some embodiments, the mini-tablets (e.g., enteric coated mini-tablets) are contained in a capsule. In some embodiments, the capsule is a size 00, size 0, size 1, size 2, size 3, size 4, or size 5 capsule. In some embodiments, the capsule comprises a non- enteric coating (e.g., gelatin) (e.g., is coated with a non-enteric coating). In some embodiments, the capsule comprises a non-enteric coating. In some embodiments, the capsule comprises gelatin or HPMC. In some embodiments, the mini-tablets (e.g., enteric coated mini -tablets) that comprise about 3 x 1010 total cells of the Veillonella parvulci bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC. In some embodiments, the mini-tablets (e.g., enteric coated mini -tablets) that comprise about 4.5 x IO10 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC. In some embodiments, the mini-tablets (e.g., enteric coated mini-tablets) that comprise about 1.5 x 1011 total cells of the Veillonella parvula bacteria are contained in a capsule(s), wherein optionally the capsule comprises gelatin or HPMC.
[251] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) comprising Veillonella parvula bacteria is prepared as a powder (e.g., for resuspension or for use in a solid dose form (such as a capsule)) or as a solid dose form, such as a tablet, a mini-tablet, a capsule, a pill, or a powder; or a combination of these forms (e.g., mini -tablets comprised in a capsule). The powder can comprise lyophilized bacteria. In some embodiments, the powder further comprises mannitol, magnesium stearate, and/or colloidal silicon dioxide. In some embodiments, the powder further comprises sucrose and/or dextran. In some embodiments, the powder further comprises sucrose, dextran, and/or L-cysteine HC1. In some embodiments, the Veillonella parvula bacteria are gamma irradiated.
Gamma-irradiation
[252] Powders (e.g., of Veillonella parvula bacteria) can be gamma-irradiated at 17.5 kGy radiation unit at ambient temperature.
[253] Frozen biomasses (e.g., of Veillonella parvula bacteria) can be gamma-irradiated at 25 kGy radiation unit in the presence of dry ice.
Therapeutic Agents
[254] In certain aspects, the methods provided herein include the administration to a subject of a bacterial composition described herein either alone or in combination with an additional therapeutic. In some embodiments, the additional therapeutic is an immunosuppressant, or a steroid. In some embodiments, the additional therapeutic is an IL-6 antagonist. In some embodiments, the additional therapeutic is an anti-IL-6 antagonist antibody. [255] In some embodiments the Veillonella parvula bacteria is administered to the subject before the therapeutic 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, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before). In some embodiments the Veillonella parvula bacteria is administered to the subject after the therapeutic 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). In some embodiments, the Veillonella parvula bacteria and the therapeutic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other). In some embodiments, the subject is administered an antibiotic before the Veillonella parvula bacteria 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 before).
[256] In some embodiments, the subject is administered an antibiotic after the Veillonella parvula bacteria 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). In some embodiments, the Veillonella parvula bacteria and the antibiotic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other).
[257] In some aspects, antibiotics can be selected based on their bactericidal or bacteriostatic properties. Bactericidal antibiotics include mechanisms of action that disrupt the cell wall (e.g., [3-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. Furthermore, while 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. In certain treatment conditions, bacteriostatic antibiotics inhibit the activity of bactericidal antibiotics. Thus, in certain embodiments, bactericidal and bacteriostatic antibiotics are not combined. [258] 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.
[259] 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 30S or 50S ribosomal subunit thereby inhibiting bacterial protein synthesis.
[260] 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.
[261] Carbacephems include, but are not limited to, Loracarbef. Carbacephems are believed to inhibit bacterial cell wall synthesis.
[262] Carbapenems include, but are not limited to, Ertapenem, Doripenem, Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal for both Grampositive and Gram-negative bacteria as broad-spectrum antibiotics. Carbapenems are believed to inhibit bacterial cell wall synthesis.
[263] 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. Selected 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 (MRS A). Cephalosporins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
[264] Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin, and Telavancin. Glycopeptides are effective, e.g., against aerobic and anaerobic Grampositive 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.
[265] 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.
[266] 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.
[267] 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.
[268] 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.
[269] Nitrofurans include, but are not limited to, Furazolidone and Nitrofurantoin.
[270] Oxazolidonones include, but are not limited to, Linezolid, Posizolid, Radezolid, and Torezolid. Oxazolidonones are believed to be protein synthesis inhibitors.
[271] 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.
[272] Penicillin combinations include, but are not limited to, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, and Ticarcillin/clavulanate.
[273] 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.
[274] 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. Quinolone s/Fluoroquinolone are effective, e.g., against Streptococcus and Neisseria. Quinolone s/Fluoroquinolone are believed to inhibit the bacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNA replication and transcription.
[275] 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.
[276] Tetracyclines include, but are not limited to, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, and Tetracycline. Tetracyclines are effective, e.g., against Gram-negative bacteria. Tetracyclines are believed to bind to the bacterial 30S ribosomal subunit thereby inhibiting bacterial protein synthesis.
[277] Anti-mycobacterial compounds include, but are not limited to, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, and Streptomycin.
[278] 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, oleandomycin, ostreogrycin, piperacillin/tazobactam, pristinamycin, ramoplanin, ranalexin, reuterin, rifaximin, rosamicin, rosaramicin, spectinomycin, spiramycin, staphylomycin, streptogramin, streptogramin A, synergistin, taurolidine, teicoplanin, telithromycin, ticarcillin/clavulanic acid, triacetyloleandomycin, tylosin, tyrocidin, tyrothricin, vancomycin, vemamycin, and virginiamycin. [279] In some embodiments, the additional therapeutic 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. Representative agents include, but are not limited to, cyclosporin, 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, lomoxicam, isoxicam, mefanamic acid, meclofenamic acid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, etodolac, indomethacin, aspirin, ibuprophen, firocoxib, methotrexate (MTX), antimalarial drugs (e.g., hydroxychloroquine and chloroquine), sulfasalazine, Leflunomide, azathioprine, cyclosporin, gold salts, minocycline, cyclophosphamide, D -penicillamine, minocycline, auranofin, tacrolimus, myocrisin, chlorambucil, TNF alpha antagonists (e.g., TNF alpha antagonists or TNF alpha receptor antagonists), 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®), integrin antagonists (TYSABRI® (natalizumab)), IL-1 antagonists (ACZ885 (Haris)), Anakinra (Kineret®)), CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6 antagonists, BLyS antagonists (e.g., Atacicept, Benlysta®/ LymphoStat-B® (belimumab)), p38 Inhibitors, CD20 antagonists (Ocrelizumab, Ofatumumab (Arzerra®)), interferon gamma antagonists (Fontolizumab), prednisolone, Prednisone, dexamethasone, Cortisol, cortisone, hydrocortisone, methylprednisolone, betamethasone, triamcinolone, beclometasome, fludrocortisone, deoxycorticosterone, aldosterone, Doxycycline, vancomycin, pioglitazone, SBI-087, SCIO-469, Cura- 100, Oncoxin + Viusid, TwHF, Methoxsalen, Vitamin D - ergocalciferol, Milnacipran, Paclitaxel, rosig tazone, Tacrolimus (Prograf®), RADOO1, rapamune, rapamycin, fostamatinib, Fentanyl, XOMA 052, Fostamatinib disodium, rosightazone, Curcumin (Longvida™), Rosuvastatin, Maraviroc, ramipnl, Milnacipran, Cobiprostone, somatropin, tgAAC94 gene therapy vector, MK0359, GW856553, esomeprazole, everolimus, trastuzumab, JAK1 and JAK2 inhibitors, pan JAK inhibitors, e.g., tetracyclic pyridone 6 (P6), 325, PF-956980, denosumab, IL-6 antagonists, CD20 antagonists, CTLA4 antagonists, IL-8 antagonists, IL-21 antagonists, IL-22 antagonist, integrin antagonists (Tysarbri® (natalizumab)), VGEF antagnosits, CXCL antagonists, MMP antagonists, defensin antagonists, IL-1 antagonists (including IL-1 beta antagonsits), and IL-23 antagonists (e.g., receptor decoys, antagonistic antibodies, etc.).
[280] In some embodiments, the additional therapeutic is an oral PDE4 inhibitor (such as apremilast). In some embodiments, the additional therapeutic is apremilast, etanercept, infliximab, adalimumab, ustekinumab, or secukinumab.
[281] In some embodiments, the agent is an immunosuppressive agent. Examples of 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, anticholinergic 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 inhibitors such as infliximab, adalimumab, certolizumab pegol, golimumab, or etanercept, iand combinations thereof.
[282] In some embodiments, the additional therapeutic is cyclosporine.
[283] In some embodiments, the additional therapeutic is dupilumab.
[284] In some embodiments, the additional therapeutic is apremilast.
[285] In some embodiments, the additional therapeutic is etanercept, infliximab, adalimumab, ustekinumab, or secukinumab.
[286] In some embodiments, the additional therapeutic is an inhaled corticosteroid.
[287] In some embodiments, the additional therapeutic is a systemic corticosteroid.
[288] In some embodiments, the additional therapeutic is a monoclonal antibody targeting IL-4, IL4R or IL-5.
Administration
[289] In some embodiments, the bacterial composition is administered orally. In some embodiments, the administration to the subject is once daily. In some embodiments, the administration to the subject is twice daily. In some embodiments, the bacterial composition is administered in 2 or more doses (e.g., 3 or more, 4 or more or 5 or more doses). In some embodiments, the administration to the subject of the two or more doses are separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.
[290] In some embodiments, the bacterial composition is administered once daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
[291] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered twice daily for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 45 days, 48 days, 52 days, or 56 days.
[292] In some embodiments, the bacterial composition (e.g, pharmaceutical composition) is administered once daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered once daily for 8 weeks. In some embodiments, the bacterial composition is administered once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered once daily for at least 8 weeks.
[293] In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered twice daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition (e.g., pharmaceutical composition) is administered twice daily for 8 weeks. In some embodiments, the bacterial composition is administered twice daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In some embodiments, the bacterial composition is administered twice daily for at least 8 weeks. [294] In some embodiments, the bacterial composition is formulated as a capsule or a tablet. In some embodiments, the bacterial formulation comprises an enteric coating or micro encapsulation. In some embodiments, the capsule is an enteric coated capsule.
[295] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal (e.g., 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).
[296] In some embodiments of the methods provided herein, the bacterial composition is administered in conjunction with the administration of an additional therapeutic. In some embodiments, the bacterial composition comprises Veillonella parvulci bacteria coformulated with the additional therapeutic. In some embodiments, the bacterial composition is co-administered with the additional therapeutic. In some embodiments, the additional therapeutic is administered to the subject before administration of the bacterial composition (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). In some embodiments, the additional therapeutic is administered to the subject after administration of the bacterial composition (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). In some embodiments, the same mode of delivery are used to deliver both the bacterial composition and the additional therapeutic. In some embodiments, different modes of delivery are used to administer the bacterial composition and the additional therapeutic. For example, in some embodiments the bacterial composition is administered orally while the additional therapeutic is administered via injection (e.g., an intravenous, and/or intramuscular injection).
[297] In certain embodiments, the bacterial compositions, dosage forms, and kits described herein can be administered in conjunction with any other conventional treatment. These treatments may be applied as necessary and/or as indicated and may occur before, concurrent with or after administration of the bacterial compositions, dosage forms, and kits described herein.
[298] 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, and other compounds such as drugs being administered concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art. In the present methods, appropriate minimum dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate. The dose of the bacterial compositions 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. For example, 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.
[299] In some embodiments, the dose administered to a subject is sufficient to induce an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell). One skilled in the art will recognize that dosage will depend upon a variety of factors including the strength of the particular compound 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 compound and the desired physiological effect.
[300] Suitable doses and dosage regimens can be determined by conventional rangefinding 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 (“MID”) of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.
[301] In accordance with the above, in therapeutic applications (e.g., for treatment and/or prevention), the dosages of the active 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.
[302] Separate administrations can include any number of two or more administrations, including two, three, four, five or six administrations. One skilled in the art can readily determine the number of administrations to perform or the desirability of performing one or more additional administrations according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein. Accordingly, the methods provided herein include methods of providing to the subject one or more administrations of a bacterial 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.
[303] 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 and/or the time period for a subject to clear the bacteria from normal tissue. In one example, the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be 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. In another example, the time period can be a function of the time period for a subject to clear the bacteria from normal tissue; for example, the time period can be more than the time period for a subject to clear the bacteria from normal tissue, such as more than about a day, more than about two days, more than about three days, more than about five days, or more than about a week.
[304] In some embodiments, the delivery of an additional therapeutic in combination with the bacterial composition described herein reduces the adverse effects and/or improves the efficacy of the additional therapeutic.
[305] The effective dose of an additional therapeutic described herein is the amount of the therapeutic agent that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, with the least toxicity to the patient. 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 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 patient being treated, and like factors well known in the medical arts. In general, an effective dose of an additional therapy will be the amount of the 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.
[306] The toxicity of an additional therapy is the level of adverse effects experienced by the subject during and following treatment. Adverse events associated with additional therapy toxicity 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, dyspepsia, dyspnea, edema, electrolyte imbalance, esophagitis, fatigue, loss of fertility, fever, flatulence, flushing, gastric reflux, gastroesophageal reflux disease, genital pain, granulocytopenia, gynecomastia, glaucoma, hair loss, hand-foot syndrome, headache, hearing loss, heart failure, heart palpitations, heartbum, hematoma, hemorrhagic cystitis, hepatotoxicity, hyperamylasemia, hypercalcemia, hyperchloremia, hyperglycemia, hyperkalemia, hyperlipasemia, hypermagnesemia, hypernatremia, hyperphosphatemia, hyperpigmentation, hypertriglyceridemia, hyperuricemia, hypoalbuminemia, hypocalcemia, hypochloremia, hypoglycemia, hypokalemia, hypomagnesemia, hyponatremia, hypophosphatemia, impotence, infection, injection site reactions, insomnia, iron deficiency, itching, joint pain, kidney failure, leukopenia, liver dysfunction, memory loss, menopause, mouth sores, mucositis, muscle pain, myalgias, myelosuppression, myocarditis, neutropenic fever, nausea, nephrotoxicity, neutropenia, nosebleeds, numbness, ototoxicity, pain, palmar-plantar erythrodysesthesia, pancytopenia, pericarditis, peripheral neuropathy, pharyngitis, photophobia, photosensitivity, pneumonia, pneumonitis, proteinuria, pulmonary embolus, pulmonary fibrosis, pulmonary toxicity, rash, rapid heart beat, rectal bleeding, restlessness, rhinitis, seizures, shortness of breath, sinusitis, thrombocytopenia, tinnitus, urinary tract infection, vaginal bleeding, vaginal dryness, vertigo, water retention, weakness, weight loss, weight gain, and xerostomia. In general, toxicity is acceptable if the benefits to the subject achieved through the therapy outweigh the adverse events experienced by the subject due to the therapy.
Immune effects
[307] In some embodiments, the methods and compositions described herein relate to inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) in a subject.
[308] In some embodiments, the immune effect comprises an increase in expression of an anti-inflammatory cytokine by an immune cell. In some embodiments, the antiinflammatory cytokine is IL- 10, or IL-27. In some embodiments, the anti-inflammatory cytokine is IL- 10. In some embodiments, the immune effect comprises a decrease in expression of a pro-inflammatory cytokine by an immune cell. In some embodiments, the pro-inflammatory cytokine is IL-ip, IL-6, and TNE-a, IL-5, IL-4, IL- 13, IL- 17, or IL-8. “TNE-a” may also be referred to herein as “TNE." In some embodiments, the pro- inflammatory cytokine is IL-5, IL-4, IL- 13, or IL- 17. In some embodiments, the immune cell is a peripheral blood mononuclear cell (PBMC), a dendritic cell, or a macrophage.
[309] In some embodiments, the immune effect comprises elevating expression (e.g., in the small intestine) of a gene provided in Example 5. In some embodiments, the cell types found enriched amongst genes showing elevated expression are immune epithelial cells. In some embodiments, the cell types found enriched amongst genes showing elevated expression are immune cells (e.g., B cells; T cells; and/or myeloid cells). In some embodiments, the gene showing elevated expression is Spinkl, Tm4sf5, and/or Aocl. In some embodiments, the gene showing elevated expression is a gene provided in Table 2. In some embodiments, the gene showing elevated expression is associated with lymphocyte migration, gut-homing, and adhesion (e.g., Ccl25, CcrlO, Ccl22, Ccl24, Itgb7, Itgal, and/ or It gam). In some embodiments, the gene showing elevated expression is associated with T cell lineage maturation and activation (e.g., Cd69, Icos, Il2rg, Cd3d, Trbcl, Trbc2, Trac, Lat, Zap70, Lek, and/or Cd2). In some embodiments, the gene showing elevated expression is associated with B cell lineage maturation and activation (e.g., CD19, CD79a, CD79b, CD69, Ighd, Fcrll, Blk, Ikzfi, Tnfrsfl3c, Jchain, Iglcl, and/or Iglc2) . In some embodiments, the gene showing elevated expression is associated with immune modulation (e.g., Foxp3, Lag3, Traf3ip3, Slamf6, 1133, Cd5, Adamdecl, and/or Nrli3). In some embodiments, the gene showing elevated expression is associated with intestinal epithelial cell homeostasis (barrier, metabolic, absorption) (e.g., Gpx2, Gstm3, Aqp8, Guca2b, Adipoq, Dgatl, Dgat2, Slc23al, and/or Slc51 b). In some embodiments, the gene showing elevated expression is associated with host-protective pathways (e.g., Zgl6, Def5a, Reg3a, Retnlb, Reg3g, Cfd, Lypd8, and/or Casp6).
[310] In some embodiments, the subject has an immune disorder. In some embodiments, the immune disorder is atopic dermatitis, psoriasis, or asthma. In some embodiments, the atopic dermatitis is moderate atopic dermatitis. In some embodiments, the psoriasis is moderate psoriasis. In some embodiments, the asthma is moderate asthma.
[3H] In some embodiments, the subject has a disease or disorder. In some embodiments, the disease or disorder is an inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis). In some embodiments, the disease or disorder is psoriasis (e.g., moderate psoriasis). In some embodiments, the disease or disorder is atopic dermatitis (e.g., moderate atopic dermatitis). In some embodiments, the disease or disorder is asthma (e.g., mild asthma). [312] The methods described herein can be used to induce an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) in any subject in need thereof. As used herein, a “subject in need thereof’ includes any subject that has a disease or disorder associated with a pathological immune response (psoriasis (e.g., moderate psoriasis) or atopic dermatitis (e.g., moderate atopic dermatitis) or asthma (e.g., mild asthma)), as well as any subject with an increased likelihood of acquiring such a disease or disorder.
[313] The compositions described herein can be used, for example, as a bacterial composition for inducing an immune effect (e.g., an increase in expression of an antiinflammatory cytokine by an immune cell or a decrease in expression of a pro- inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) in a subject. In some embodiments, the subject has 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 bacterial composition for suppressing rejection in organ transplantation or other situations in which tissue rejection might occur; a supplement, food, or beverage for improving immune functions; or a reagent for suppressing the proliferation or function of immune cells.
[314] In some embodiments, the subject has inflammation. In certain embodiments, the inflammation of any tissue and organs of the body, including musculoskeletal inflammation, vascular inflammation, neural inflammation, digestive system inflammation, ocular inflammation, inflammation of the reproductive system, and other inflammation, as discussed below.
[315] Immune disorders of the musculoskeletal system include, but are not limited, to those conditions affecting skeletal joints, including joints of the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle, and foot, and conditions affecting tissues connecting muscles to bones such as tendons. Examples of such immune disorders, which may be treated with the methods and compositions described herein include, but are not limited to, arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis, tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis, myositis, and osteitis (including, for example, Paget's disease, osteitis pubis, and osteitis fibrosa cystic).
[316] Ocular immune disorders refers to an immune disorder that affects any structure of the eye, including the eye lids. Examples of ocular immune disorders which may be treated with the methods and compositions described herein include, but are not limited to, blepharitis, blepharochalasis, conjunctivitis, dacryoadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis.
[317] 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.
[318] Examples of digestive system immune disorders which may be treated with the methods and 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. Several major forms of inflammatory bowel diseases are known, with Crohn's disease (regional bowel disease, e.g., inactive and active forms) and ulcerative colitis (e.g., inactive and active forms) the most common of these disorders. In addition, the inflammatory bowel disease encompasses irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis. Other less common forms of IBD include indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet’s disease, sarcoidosis, scleroderma, IBD-associated dysplasia, dysplasia associated masses or lesions, and primary sclerosing cholangitis. [319] Examples of reproductive system immune disorders which may be treated with the methods and 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.
[320] In some embodiments, the subject has an autoimmune condition 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, good pasture's syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonlein purpura, Kawasaki's disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, Muckle-Wells syndrome, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmune haemolytic anemia, interstitial cystitis, Lyme disease, morphea, psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.
[321] In some embodiments, the subject has a T-cell mediated hypersensitivity disease having an inflammatory component. Such a condition includes, but are not limited to, contact hypersensitivity, contact dermatitis (including that due to poison ivy), uticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dustmite allergy) and gluten-sensitive enteropathy (Celiac disease).
[322] Other immune disorders that the subject may have include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel, skin allografts, skin homografts, and heart valve xengrafts, sewrum sickness, and graft vs host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, Sexary's syndrome, congenital adrenal hyperplasis, nonsuppurative thyroiditis, hypercalcemia associated with cancer, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensistivity reactions, allergic conjunctivitis, keratitis, herpes zoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonary tuberculosis chemotherapy, idiopathic thrombocytopenic purpura in adults, secondary thrombocytopenia in adults, acquired (autroimmine) haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia of childhood, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, solid organ transplant rejection, sepsis. Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosis, psoriasis, chronic obstructive pulmonary disease, and inflammation accompanying infectious conditions (e.g., sepsis).
[323] In some aspects, bacterial compositions for use in inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) are disclosed. In some aspects, a bacterial composition comprising Veillonella parvula, wherein the Veillonella parvulci is a strain comprising at least 85% sequence identity to the nucleotide sequence of the Veillonella parvula strain A (ATCC Deposit Number PTA-125691) for use in inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) is described herein.
[324] In some aspects, uses of a bacterial composition for the preparation of a medicament for inducing an immune effect (e.g., an increase in expression of an antiinflammatory cytokine by an immune cell or a decrease in expression of a pro- inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) are disclosed. In some aspects, use of a bacterial composition for the preparation of a medicament for inducing an immune effect (e.g., an increase in expression of an anti-inflammatory cytokine by an immune cell or a decrease in expression of a pro-inflammatory cytokine by an immune cell; elevating expression of a gene provided in Example 5; and/or elevating expression of a gene provided in Table 2) wherein the bacterial composition comprises Veillonella parvula, wherein the Veillonella parvulci is a strain comprising at least 85% sequence identity to the nucleotide sequence of the Veillonella parvula strain A (ATCC Deposit Number PTA-125691) is described herein.
Dysbiosis
[325] In recent years, it has become increasingly clear that the gut microbiome (also called the “gut microbiota”) can have a significant impact on an individual’s health through microbial activity and influence (local and/or distal) on immune and other cells of the host (Walker, W.A., Dysbiosis. The Microbiota in Gastrointestinal Pathophysiology. Chapter 25. 2017; Weiss and Thierry, Mechanisms and consequences of intestinal dysbiosis. Cellular and Molecular Life Sciences. (2017) 74(16):2959-2977. Zurich Open Repository and Archive, doi.org/10.1007/s00018-017-2509-x)).
[326] A healthy host-gut microbiome homeostasis is sometimes referred to as a “eubiosis” or “normobiosis,” whereas a detrimental change in the host microbiome composition and/or its diversity can lead to an unhealthy imbalance in the microbiome, or a “dysbiosis” (Hooks and O’Malley. Dysbiosis and its discontents. American Society for Microbiology. Oct 2017. Vol. 8. Issue 5. mBio 8:e01492-17. https://doi.org/10.1128/mBio.01492-17). Dysbiosis, and associated local or distal host inflammatory or immune effects, may occur where microbiome homeostasis is lost or diminished, resulting in: increased susceptibility to pathogens; altered host bacterial metabolic activity; induction of host proinflammatory activity and/or reduction of host anti-inflammatory activity. Such effects are mediated in part by interactions between host immune cells (e.g., T cells, dendritic cells, mast cells, NK cells, intestinal epithelial lymphocytes (IEC), macrophages and phagocytes) and cytokines, and other substances released by such cells and other host cells.
[327] A dysbiosis may occur within the gastrointestinal tract (a “gastrointestinal dysbiosis” or “gut dysbiosis”) or may occur outside the lumen of the gastrointestinal tract (a “distal dysbiosis”). Gastrointestinal dysbiosis is often associated with a reduction in integrity of the intestinal epithelial barrier, reduced tight junction integrity and increased intestinal permeability. Citi, S. Intestinal Barriers protect against disease, Science
359: 1098-99 (2018); Srinivasan et al., TEER measurement techniques for in vitro barrier model systems. J. Lab. Autom. 20: 107-126 (2015). A gastrointestinal dysbiosis can have physiological and immune effects within and outside the gastrointestinal tract.
[328] The presence of a dysbiosis has been associated with a wide variety of diseases and conditions including: infection, cancer, autoimmune disorders (e.g., systemic lupus erythematosus (SLE)) or inflammatory disorders (e.g., functional gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease), neuroinflammatory diseases (e.g., multiple sclerosis), transplant disorders (e.g., graft-versus-host disease), fatty liver disease, type I diabetes, rheumatoid arthritis, Sjogren’s syndrome, celiac disease, cystic fibrosis, chronic obstructive pulmonary disorder (COPD), and other diseases and conditions associated with immune dysfunction. Lynch et al., The Human Microbiome in Health and Disease, N. Engl. J. Med .375:2369- 79 (2016), Carding et al., Dysbiosis of the gut microbiota in disease. Microb. Ecol. Health Dis. (2015); 26: 10: 3402/mehd.v26.2619; Levy et al, Dysbiosis and the Immune System, Nature Reviews Immunology 17:219 (April 2017)
[329] Exemplary bacterial compositions disclosed herein can treat a dysbiosis and its effects by modifying the immune activity present at the site of dysbiosis. As described herein, such compositions can modify a dysbiosis via effects on host immune cells, resulting in, e.g., an increase in secretion of anti-inflammatory cytokines and/or a decrease in secretion of pro-inflammatory cytokines, reducing inflammation in the subject recipient or via changes in metabolite production, elevating expression of a gene provided in Example 5, and/or elevating expression of a gene provided in Table 2.
[330] Exemplary bacterial compositions disclosed herein that are useful for treatment of disorders associated with a dysbiosis contain one or more types of immunomodulatory bacteria (e.g., anti-inflammatory bacteria). Such compositions are capable of affecting the recipient host’s immune function, in the gastrointestinal tract, and/or a systemic effect at distal sites outside the subject’s gastrointestinal tract.
[331] Exemplary bacterial compositions disclosed herein that are useful for treatment of disorders associated with a dysbiosis contain a population of immunomodulatory bacteria of a single bacterial species (e.g., a single strain) (e.g., anti-inflammatory bacteria). Such compositions are capable of affecting the recipient host’s immune function, in the gastrointestinal tract, and /or a systemic effect at distal sites outside the subject’s gastrointestinal tract. [332] In one embodiment, bacterial compositions containing an isolated population of immunomodulatory bacteria (e.g., anti-inflammatory bacterial cells) 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.
[333] In another embodiment, bacterial 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.
[334] In another embodiment, the bacterial compositions can treat a gastrointestinal dysbiosis and one or more of its effects by modulating the recipient immune response via cellular and cytokine modulation to reduce gut permeability by increasing the integrity of the intestinal epithelial barrier.
[335] In another embodiment, the bacterial 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.
[336] Other exemplary bacterial compositions are useful for treatment of disorders associated with a dysbiosis, which compositions contain one or more types of bacteria 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.
[337] Other exemplary bacterial compositions are useful for treatment of disorders associated with a dysbiosis, which compositions contain a population of immunomodulatory bacteria, e.g., a single strain) capable of altering the relative proportions of immune cell subpopulations, e.g., T cell subpopulations, immune lymphoid cells, NK cells and other immune cells, or the function thereof, in the recipient subject.
[338] In one embodiment, the invention provides methods of treating a gastrointestinal dysbiosis and one or more of its effects by orally administering to a subject in need thereof a bacterial composition which alters the microbiome population existing at the site of the dysbiosis. The bacterial composition can contain one or more types of immunomodulatory bacteria or a population of immunomodulatory bacteria of a single bacterial species (e.g., a single strain). [339] In one embodiment, the invention provides methods of treating a distal dysbiosis and one or more of its effects by orally administering to a subject in need thereof a bacterial composition which alters the subject’s immune response outside the gastrointestinal tract. The bacterial composition can contain one or more types of immunomodulatory bacteria or a population of immunomodulatory bacteria of a single bacterial species (e.g., a single strain).
[340] In exemplary embodiments, bacterial compositions useful for treatment of disorders associated with a dysbiosis stimulate secretion of one or more antiinflammatory cytokines by host immune cells. Anti-inflammatory cytokines include, but are not limited to, IL-10, IL-13, IL-9, IL-4, IL-5, TGF(3, and combinations thereof. In other exemplary embodiments, bacterial compositions useful for treatment of disorders associated with a dysbiosis that decrease (e.g., inhibit) secretion of one or more pro- inflammatory cytokines by host immune cells. Pro-inflammatory cytokines include, but are not limited to, IFNy, IL-I2p70, IL-Ia, IL-6, IL-8, MCP1, MIPla, MIP1 , TNFa, and combinations thereof. Other exemplary cytokines are known in the art and are described herein.
[341] In exemplary embodiments, bacterial compositions useful for treatment of disorders associated with a dysbiosis elevate expression (e.g., in the small intestine) of a gene provided in Example 5. In some embodiments, the cell types found enriched amongst genes showing elevated expression are immune epithelial cells. In some embodiments, the cell types found enriched amongst genes showing elevated expression are immune cells (e.g., B cells; T cells; and/or myeloid cells). In some embodiments, the gene showing elevated expression is Spinkl, Tm4sf5, and/or Aocl. In some embodiments, the gene showing elevated expression is a gene provided in Table 2. In some embodiments, the gene showing elevated expression is associated with lymphocyte migration, gut-homing, and adhesion (e.g., Ccl25, CcrlO, Ccl22, Ccl24, Itgb7, Itgal, and/or It gam). In some embodiments, the gene showing elevated expression is associated with T cell lineage maturation and activation (e.g., Cd69, Icos, Il2rg, Cd3d, Trbcl, Trbc2, Trac, Lat, Zap70, Lek, and/or Cd2). In some embodiments, the gene showing elevated expression is associated with B cell lineage maturation and activation (e.g., CD19, CD79a, CD79b, CD69, Ighd, Fcrll, Blk, Ikzfi, Tnfrsfl3c, Jchain, Iglcl, and/or Iglc2). In some embodiments, the gene showing elevated expression is associated with immune modulation (e.g., Foxp3, Lag3, Traf3ip3, Slamf6, 1133, Cd5, Adamdecl, and/or Nrli3). In some embodiments, the gene showing elevated expression is associated with intestinal epithelial cell homeostasis (barrier, metabolic, absorption) (e.g., Gpx2, Gstm3, Aqp8, Guca2b, Adipoq, Dgatl, Dgat2, Slc23al, and/or Slc51b). In some embodiments, the gene showing elevated expression is associated with host-protective pathways (e.g., Zgl6, Def 5a, Reg3a, Retnlb, Reg3g, Cfd, Lypd8, and/or Casp6).
[342] In another aspect, the invention provides a method of treating or preventing a disorder associated with a dysbiosis in a subject in need thereof, comprising administering (e.g., orally administering) to the subject a therapeutic composition in the form of a probiotic or medical food comprising bacteria in an amount sufficient to alter the microbiome at a site of the dysbiosis, such that the disorder associated with the dysbiosis is treated.
[343] In another embodiment, 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.
Other Aspects
[344] Numerous embodiments are further provided that can be applied to any aspect of the present invention described herein. For example, in some embodiments, the Veillonella parvula is a strain comprising at least 99.9% sequence identity to the nucleotide sequence of the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691). In some embodiments, the Veillonella parvula is the Veillonella parvula strain A (ATCC Deposit Number PTA- 125691). In some embodiments, the bacterial composition is administered orally. In some embodiments, the bacterial composition is formulated as a capsule or a tablet. In some embodiments, the capsule is an enteric coated capsule. In some embodiments, the bacterial composition comprises about 3 x IO10 total cells of Veillonella parvula. In some embodiments, the bacterial composition comprises about 1.5 x 1011 total cells of Veillonella parvula. In some embodiments, the bacterial composition comprises about 7.5 x 1011 total cells of Veillonella parvula. In some embodiments, the bacterial composition comprises about 1.5 x 1012 total cells of Veillonella parvula. In some embodiments, the bacterial composition comprises from about 3 x IO10 to about 1.5 x 1012 total cells of Veillonella parvula. In some embodiments, the bacterial composition is administered at least once daily. In some embodiments, the bacterial composition is administered once daily. In some embodiments, the bacterial composition is administered once daily for at least 15 continuous days. In some embodiments, the bacterial composition is administered once daily for at least 28 continuous days. In some embodiments, the bacterial composition is administered once daily for at least 56 continuous days. In some embodiments, the psoriasis is moderate psoriasis. In some embodiments, the atopic dermatitis is moderate atopic dermatitis. In some embodiments, the asthma is mild asthma.
EXAMPLES
Example 1: Powder Preparation Sample Protocol
After desired level of bacterial culture growth is achieved, centrifuge cultures, discard the supernatant, leaving the pellet as dry as possible. Resuspend pellet in desired cryoprotectant solution to create a formulated cell paste. The cryoprotectant may contain, e.g., maltodextrin, sodium ascorbate, sodium glutamate, and/or calcium chloride. The cryoprotectant may contain, e.g., sucrose, dextran, and/or L-cysteine HC1. Load the formulated cell paste onto stainless steel trays and load into a freeze drier, e.g., operating in automated mode with defined cycle parameters. The freeze dried product is fed into a milling machine and the resulting powder is collected.
Powders are stored (e.g., in vacuum sealed bags) at 2-8 degrees C (e.g., at 4 degrees C), e.g., in a desiccator.
Example 2: Gamma-Irradiation: Sample Protocol:
Powders are gamma-irradiated at 17.5 kGy radiation unit at ambient temperature. Frozen biomasses are gamma-irradiated at 25 kGy radiation unit in the presence of dry ice.
Example 3: Preclinical studies of Veillonella parvula Strain A
[345] Veillonella parvula strain A- G.I. (gamma irradiated) is a pharmaceutical preparation of a single strain of Veillonella parvula (Veillonella parvula strain A), originally isolated from a fresh ileostomy sample of an IBD patient in remission, which has been gamma-irradiated (G.I.). The final drug product is therefore non-viable at the time of dosing. It has not been genetically modified. Because it is non-viable, it does not colonize the gut and has no detectable systemic exposure following oral dosing. It has dose dependent therapeutic effects in multiple pre-clinical models. Veillonella parvula strain A has been deposited as ATCC Accession Number PTA-125691. [346] Veillonella strains are found in several niches of the human body including the mouth, lungs, gastrointestinal tract, and vagina, and is a normal component of a healthy human microbiota. Although there are sporadic reports of some Veillonella species that can act as opportunistic pathogens, recent data suggests that Veillonella may perform a protective role and aid in early childhood immune system development. Epidemiological studies of infants have demonstrated that presence of Veillonella is negatively correlated with asthma and bronchiolitis.
[347] In vitro studies of Veillonella parvula strain A- G.I. in human and mouse cellular assays and in vivo models support its use in the treatment of immunoinflammatory diseases, including atopic dermatitis, psoriasis, and asthma. Veillonella parvula strain A- G.L increases secretion of anti-inflammatory cytokines from human immune cells, such as interleukin (IL)- 10, while inducing minimal production of pro-inflammatory cytokines such as TNFy and IFNy.
[348] Oral administration of Veillonella parvula strain A- G.I. to mice led to striking therapeutic effects on delayed-type hypersensitivity (DTH), imiquimod-induced skin inflammation, fluorescein isothiocyanate (FITC) cutaneous hypersensitivity, MC903- induced dermatitis, and experimental acute encephalomyelitis (EAE) in-vivo models. This consistency of effect and dose shows that Veillonella parvula strain A- G.I. is coordinately resolving systemic inflammation across TH1, TH2 and TH17 pathways. This suggests the potential for clinical benefit across multiple conditions and populations. No potentially related adverse effects were seen in the animals used in these experiments with daily dosing for up to 6 weeks. Ex vivo immunophenotyping in these models shows decreases in pro-inflammatory cytokines such as IL-6, IL-4, IL-13, TNF, IL-17, and KC (murine IL-8). Veillonella parvula strain A- G.I. does not suppress the expression of circulating cell production of interferon gamma (IFNy) in these ex vivo experiments, suggesting that the broad spectrum of anti-inflammatory effects is achieved without damaging mechanisms of immune surveillance critical for the prevention of malignancy and response to pathogens. The effects on immune parameters have been observed both within and outside of the GI tract, which demonstrates that host-microbe interactions in the gut can affect the immune response in peripheral tissues.
Formulation
[349] Veillonella parvula strain A- G.I. drug product (DP) formulation is a blend of freeze-dried powder of V. parvula (DS) and excipients (mannitol, colloidal silicon dioxide, magnesium stearate, hydroxypropyl methylcellulose, poly(methacrylic acid, ethyl acrylate, 1: 1), triethyl citrate, and talc). Two active drug product strengths with matching placebo are manufactured to support the clinical studies.
[350] Veillonella parvula strain A- G.I. finished product is manufactured as an enteric- coated capsule, designed to protect Veillonella parvula strain A- G.I. from stomach pH degradation and designed to release at pH > 5.5, which is approximately the pH in the small intestine.
Preclinical Pharmacology
[351] In vitro studies of Veillonella parvulci strain A- G.I. or the parental strain Veillonella parvulci strain A have been carried out across a range of human and mouse assays and 5 key in vivo models have been used to support its use in the treatment of immuno-inflammatory diseases.
[352] A summary of these studies is provided in Table 1.
Table 1: Tabular Summaries of Key Preclinical Studies
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
[353] APC = antigen presenting cells; CD = cluster of differentiation; CEF =
Cytomegalovirus, Epstein Bar virus and Flu viruses; CHS = contact hypersensitivity; DC = dendritic cell; DTH = delayed-type hypersensitivity; EAE = experimental acute encephalomyelitis; FITC = Fluorescein isothiocyanate; GI = gastrointestinal; GM-CSF = granulocyte-macrophage colony-stimulating factor; IFNy = interferon gamma; IL = interleukin; KLH = keyhole limpet haemocyanin; LPS = lipopolysaccharide; MLN = mesenteric lymph node; PLP = proteolipid protein; TH 17 = T-helper 17; TNF = tumor necrosis factor
[354] * Veillonella parvulci strain A is the identifier of the original parental strain from which Veillonella parvula strain A- G.I. was derived. Veillonella parvula strain A has been deposited as ATCC Accession Number PTA-125691.
In vitro assays to determine anti- and pro-inflammatory potential of Veillonella parvula strain A- G.I. or parent strain Veillonella parvula strain A on primary human immune cells
[355] Because orally administered Veillonella parvula strain A- G.I. is restricted to the gut, there are a limited number of immune cells types with which it can interact as it passes through the GI tract. The immune cells that are most likely to directly encounter Veillonella parvula strain A- G.I. are located in the lamina propria underlying the intestinal epithelial cell layer, and are in the myeloid lineage, including dendritic cells and macrophages. A series of in vitro studies was performed to examine the effects of the interaction of Veillonella parvula strain A- G.I. or parental bacterial Veillonella parvula strain A on human immune cells derived from healthy volunteer peripheral blood. The effect that a human dendritic cell exposed to Veillonella parvula strain A- G.I. might have on its ability to activate CD 8+ T cells, a major producer of the pro-inflammatory cytokine IFNy was examined.
Effect Veillonella parvula strain A on primary purified macrophages from multiple human donors
[356] Veillonella parvula strain A consistently induces high levels of IL- 10 from primary purified macrophages from multiple human donors.
[357] An in vitro assay with primary human macrophages was carried out to evaluate the pro- and anti-inflammatory properties of >70 unique human commensal bacterial strains, including Veillonella parvula strain A. All the strains were gamma-irradiated. Fresh primary human CD1 lb+ immune cells were purified from the peripheral blood mononuclear cells (PBMC) of 6 healthy human donors by Ficoll-Paque gradient centrifugation in combination with magnetic cell separation. The purified human cells were individually co-cultured with a single bacterial strain. Beckman Coulter robotics were used to co-culture the obligate anaerobes with mammalian cells in a 96-well format at a multiplicity of infection (MOI) of 1. After 24 hours of mammalian cell-microbe coculture, cell supernatants were collected and Luminex technology was used to measure pro- and anti-inflammatory cytokine production including IL-12p70, TNF, CXCL10 (IP- 10), and IL-10. The data show that of the >70 unique commensal strains evaluated, those of the genus Veillonella, including Veillonella parvulci strain A, were the strongest inducers of the anti-inflammatory cytokine IL- 10. Additionally, Veillonella parvula strain A did not induce high levels of pro-inflammatory cytokines CXCL10, IL-12p70, or TNF compared to other commensal anaerobic strains. Based on these data, the cytokine stimulating profile of parental strain Veillonella parvula strain A is attractive which is higher for anti-inflammatory IL- 10 compared to pro-inflammatory cytokines made from the same cell.
Dose-response of Veillonella parvula strain A on the induction of IL-10 from primary purified peripheral blood monocytes, dendritic cells, and macrophages from human donors
[358] Gamma-irradiated Veillonella parvula strain A stimulates the production of the anti-inflammatory cytokine IL- 10 from purified human peripheral blood monocytes (PBMC) and dendritic cells from six individual donors in a dose-dependent manner.
[359] Isolated human PBMCs, DCs, and macrophages from whole blood were cultured with a range of doses from 104 - 5 x 106 total cell count (TCC) of gamma-irradiated Veillonella parvula strain A as described above. After 24 hours, supernatants were collected and IL- 10 was measured by Luminex. A dose-response relationship was observed between increasing TCC and production of IL- 10 from both PBMCs and dendritic cells (Figures 1A and IB, respectively), are shown in Figure 1C. These results demonstrate the pharmacological activity of gamma-irradiated Veillonella parvula strain A on immune cells, and establishes EC50 of 5.2 log TCC for PBMCs, 4.8 log TCC for DCs, and 4.9 log TCC for macrophages.
Effect of Veillonella parvula strain A on pro-inflammatory human macrophages
[360] Gamma-irradiated Veillonella parvula strain A does not induce induction of the inflammatory cytokine TNF from pro-inflammatory macrophages. [361] Macrophages are one of the major human cell types to produce and secrete the pro-inflammatory cytokine TNF in response to exposure to microbial products. To determine whether exposure to gamma-irradiated Veillonella parvulci strain A might induce pro-inflammatory cytokines from already inflamed macrophages, an in vitro assay with primary human myeloid cells skewed towards a pro-inflammatory phenotype was carried out. Briefly, primary human CD 14+ PBMCs were purified from three healthy donors by Ficoll-Paque gradient centrifugation in combination with magnetic cell separation. CD 14+ cells were differentiated to a mature APC phenotype for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF). The cells were skewed to an inflammatory phenotype with a cocktail of lipopolysaccharide (LPS) + IFNy, and subsequently co-cultured with 88 individual anaerobic microbial strains. Beckman Coulter robotics were used to co-culture the obligate anaerobes with mammalian cells in a 96-well format at a MOI of 1. After 24 hours of APC-microbe co-culture, cell supernatants were collected and Luminex technology was used to measure the pro- inflammatory cytokine TNF. The results are shown in Figure 2. The data demonstrate that gamma-irradiated Veillonella parvula strain A does not enhance the production of the pro-inflammatory cytokine TNF. Other common commensal strains examined include those in the genera Prevotella, Parabacteroides, and Blautia.
Effect of Veillonella yarvula strain A-conditioned dendritic cells (DCs) on CD8 T cells
[362] Veillonella parvula strain A-conditioned dendritic cells do not stimulate pro- inflammatory T cell responses in vitro.
[363] An in vitro assay with primary human DCs and autologous CD 8+ T cells was carried out to measure the capacity of gamma-irradiated Veillonella parvula strain A to modulate antigen (Ag)-specific CD8+ T cell responses. Primary human DCs from 3 healthy donors were differentiated in vitro for 7 days. DCs were pre-conditioned with gamma-irradiated Veillonella parvula strain A for 24 hours in vitro. Beckman Coulter robotics were used to co-culture the obligate anaerobes with mammalian cells in a 96- well format at a MOI of 1. After 24 hours of microbe conditioning, the Veillonella parvula strain A was removed from the DC culture and autologous human CD8+ T cells and CEF Class I peptide pool was added. CEF peptide pool is composed of peptides from Cytomegalovirus, Epstein-Bar virus, and Influenza virus, pathogens which the majority of the human population has been exposed to. Human donors were characterized prior to the assay and chosen based on their responsiveness to the peptide pool. After 24 hours of stimulation with CEF peptide, DC-CD8+ T cell supernatants were collected and Luminex technology was used to measure pro- and anti-inflammatory cytokines including IFNy, TNF, IL-10, and IL-27. All three DC-CD8+ T cell co-cultures produced IFNy in response to CEF peptide (Figure 3B). When human DCs were pre-conditioned with gammairradiated Veillonella parvulci strain A, the IFNy response to CEF was not affected (neither enhanced nor decreased). The anti-inflammatory cytokine IL- 10, which was not present in unconditioned DC-T cell cultures, was induced by Veillonella parvula strain A (Figure 3A). These data show that while gamma-irradiated Veillonella parvula strain A does not affect human T cell Ag-specific pro-inflammatory cytokine production, it has the capacity to induce anti-inflammatory responses.
In vivo Murine Disease Models
[364] Five models of immuno-inflammatory disease have been used to generate efficacy and safety information on Veillonella parvula strain A- G.E with the potential to treat allergic and atopic inflammation, as well as other immuno-inflammatory diseases.
• The delayed-type hypersensitivity (DTH) model is a mechanistic skin inflammation model that measures antigen-specific inflammation in the ear
• The fluorescein isothiocyanate (FITC) induced contact dermatitis model is mediated by the TH2 pathway and exhibits many of the hallmarks of atopic dermatitis
• MC903 -induced dermatitis is another model mediated by the TH2 pathway, and clinically relevant cytokine pathways that are pathogenic in atopic dermatitis.
• The imiquimod (IMQ)-induced model of TH17-mediated skin inflammation, which is a model of psoriasis based on immune mechanisms.
• The experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell- mediated autoimmune disease characterized by perivascular CD4+ T cell and mononuclear cell inflammation and subsequent primary demyelination of axonal tracks in the central nervous system (CNS), leading to progressive hind-limb paralysis.
[365] In addition to the models of inflammation, a bio-distribution study in naive mice was also performed using fluorescently labelled Veillonella parvula strain A- G.E to establish the kinetics of passage through the gastrointestinal tract (GT), as well as to confirm that Veillonella parvulci strain A- G.I. exposure was restricted to the GT following oral administration.
Delayed-type hypersensitivity (DTH)
[366] In humans, the Mantoux Test is used as a classical cell-mediated immunity test to demonstrate previous exposure to pathogens and is induced by the intra-dermal injections of antigens. The response represents a cutaneous T cell-mediated memory recall immune response, and in humans is typically used to determine immunity to tuberculosis. The immunological mechanisms that drive the skin inflammatory response in the murine and human DTH responses are the same, and thus the DTH mouse model is considered a physiologically relevant model for predicting T cell-mediated responses in humans.
[367] Methods: Mice were immunized by subcutaneous injection with keyhole limpet haemocyanin (KLH) emulsified with Complete Freund’s Adjuvant (CFA). Eight days after sensitization, the previously sensitized mice were challenged by intradermal ear injection with KLH or a buffer control. The DTH response was evaluated 24 hours post— challenge, which represents the peak of disease in this model. Mice were dosed for 10 days daily from the day of sensitization through to the end of the study by oral gavage with vehicle, reconstituted Veillonella parvula strain A- G.I. at different doses, or a positive control.
[368] Results: KLH DTH 1 : In this study, mice were dosed for 9 days daily from the day of sensitization through to the end of the study by oral gavage with vehicle, dexamethasone, reconstituted Veillonella parvula strain A- G.I. at different doses. A range of 3 doses over 3 logs (1.5 x 108, 1.5 x 109, and 1.5 x IO10 total cell count) of Veillonella parvula strain A- G.I. were explored.
[369] A shallow dose-response relationship between total cells and reduction in ear inflammation was observed (Figure 4). While the highest dose (1.5 x IO10 total cells) had the highest maximal efficacy (Emax) all doses tested significantly reduced ear swelling compared to vehicle. Based on this study and other dose-response studies, the minimal efficacious dose was determined to lie between 1.5 x 108 and 1.5 x 109 TCC.
KLH DTH 2: Veillonella parvula strain A- G.I. is well-tolerated in 30-day DTH
[370] A 30-day murine DTH study with KLH was carried out to test the antiinflammatory properties of Veillonella parvula strain A- G.I. over an extended period of dosing. Mice were subcutaneously immunized with an emulsion of 50pL KLH in CFA on Day 0, and then received a KLH booster on Day 7. Mice received two intradermal antigen challenges with lOpg KLH in the ear on Days 15 and 29. Changes in ear swelling thickness vs. baseline were measured using a caliper at 24 after each ear challenge on Days 16 and Days 30. Three treatment groups were included in the study: Vehicle, Veillonella parvulci strain A- G.I. 10 mg (1.5 x 1010 TCC), and Veillonella parvulci strain A- G.I. 1.78 mg (2.7 x 109 TCC). Mice were dosed orally Monday to Friday from Days 1 to 30. A group of age-matched naive mice that did not receive immunization, challenge, or treatment was also included in the study for comparison. Health observations and body weight were recorded daily. At study termination, various tissues were collected and preserved in formalin including spleen, colon, small intestine, mesenteric lymph node, cervical lymph node, liver and kidney. Organ weights for spleen and liver were recorded. Serum was collected to measure alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), total bilirubin, cholesterol and albumin.
[371] Ear thickness caliper measurements showed that treatment with Veillonella parvula strain A- G.I. significantly reduced ear swelling at 24 hours after the first challenge on Day 15 and the second challenge on Day 29 (Figure 5).
[372] At the termination of the study on Day 30, there was no difference in spleen weights observed for mice treated with Veillonella parvula strain A- G.I. for up to 30 days compared to mice treated with vehicle. Liver weight was slightly higher in mice treated with Veillonella parvula strain A- G.I. compared to mice treated with vehicle, but there was no significant difference compared to naive mice. No notable changes in body weight were observed throughout the study. Some variability in weight was measured on Day 3, which was likely due to variability in stress responses at the beginning of the study. Body weights returned to normal thereafter. Daily health observations did not show any abnormalities in the health of the animals that were dosed with Veillonella parvula strain A- G.I. for up to 30 days.
[373] At study termination on Day 30, blood was collected from the animals and serum was obtained. A liver chemistry assessment was carried out on serum to measure liver enzymes and other factors that indicate toxicity and liver damage. The assay included ALP, ALT, AST, GGT, bilirubin (Tbil), and cholesterol (Choi). The results demonstrated that all analytes were within the expected normal range and no significant fluctuations were observed for animals treated with Veillonella parvulci strain A- G.I. versus vehicle.
FITC Cutaneous Hypersensitivity
[374] Due to its skin-sensitizing properties, FITC is used as an experimental hapten in mechanistic studies of contact allergy. Studies have demonstrated that topical exposure of mice to FITC results in the selective development of activated lymph node cells (LNC) expressing a preferential TH2 cytokine -secretion profile, with high levels of IL-4 and IL- 10, but low levels of IFNy.
[375] Methods: Mice were sensitized with FITC on the back on Day 1 and Day 2. Mice were gavaged daily from Day 1 to Day 6 with Veillonella parvula strain A- G.I. (1.5 x IO10 TCC per dose), vehicle, or dexamethasone. They were then challenged on the ear with FITC on Day 6, and ear thickness was measured 24 hours later. The level of the TH2 cytokine IL-4 in the ear tissue was measured by ELISA.
[376] Results: FITC sensitization induced robust ear swelling. Treatment with dexamethasone and Veillonella parvula strain A- G.I. significantly inhibited ear swelling compared to the vehicle-treated group. Both dexamethasone and Veillonella parvula strain A- G.I. inhibited levels of IL-4 in the ear tissue (Figure 6). These results demonstrate that Veillonella parvula strain A- G.I. can inhibit inflammation mediated by the TH2 pathway and supports the development in TH2 -mediated atopic diseases.
MC903 model of atopic dermatitis
[377] The MC903 dermatitis model is a well-established murine model of atopic dermatitis (AD)-like disease. This model recapitulates many of the central features of AD including erythema (redness), scaling, blood eosinophilia, serum IgE elevation, itch behavior (pruritus), and histopathologic features of AD including acanthosis (epidermal thickening), hyperkeratosis (stratum comeum thickening), spongiosis (epidermal edema), and mixed dermal lymphocyte and eosinophil infiltration. Topical application of the vitamin D3 analog MC903 induces changes in skin morphology and inflammation resembling immune perturbations observed in acute lesions of patients with AD.
[378] Methods: Mice were sensitized daily for 14 consecutive days with 2nmol of MC903 (calcipotriol; Tocris Bioscience,) in 20 uL of 100% EtOH on ears. Mice were gavaged daily from Day 1 to Day 6 with 10 mg of Veillonella parvula strain A- G.I. (1.5 x 1010 TCC per dose), vehicle, or tofacitinib (20 mg/kg). Mice were also treated with dexamethasone daily (1 mg/kg intraperitoneally). Baseline ear measurements were taken prior to the first ear sensitization on day 1 using Digital Calipers (Fowler). On day 14, ear thickness was measured. Delta change in ear thickness was expressed as ear thickness at day 14 minus ear thickness at baseline. At the end of the study, single cell suspensions were made from ear-draining cLN and spleens, and stimulated ex vivo using PMA and ionomycin for 2 days. Cytokines in the supernatants were measured by electrochemiluminescence .
[379] Results: Veillonella parvulci strain A- G.I. -treated mice showed significant reduction of ear thickness associated with MC903 -induced skin inflammation compared to vehicle-treated mice, with the area under the curve calculation for the complete time course showing a similar effect as both dexamethasone and tofacitinib (Figure 7). Commensurate with reduction of ear inflammation, the key TH2 cytokine IL-4 was also reduced in both Veillonella parvula strain A- G.I. and tofacitinib treated mice (Figure 8). Furthermore, ex vivo re-stimulation of cells from the lymph nodes draining the site of inflammation (cervical lymph nodes, cLN) with PMA/Ionomycin showed a decreased production of TH2 cytokines IL-5 and IL-13, and TH1 cytokine IFNy which is also increased in this model, on Day 14 (Figure 9), demonstrating the systemic effect of gut- restricted Veillonella parvula strain A- G.I. Notably, the effect of Veillonella parvula strain A- G.I. on these lymph node cytokines was much greater than the effect of both tofacitinib and dexamethasone. Furthermore, ex vivo stimulation of the spleen from mice treated with Veillonella parvula strain A- G.I. demonstrated that immune cells in circulation were not impaired in their ability to make IFNy while those cells from spleens of mice treated with tofacitinib did show impairment of IFNy. These results show that Veillonella parvula strain A- G.I. reduces inflammation in the tissues, but does not impair systemic responses to viruses or pathogens.
Imiquimod (IMQ)-induced model of TH17-mediated skin inflammation
[380] The imiquimod-induced psoriasis model is particularly translational into the clinic as it has many of the significant markers of human disease, including histopathology of lesions and strong activation of the immune system. Clinically relevant psoriasis model in mice is through the topical application of 5% imiquimod (IMQ) cream. This imiquimod- induced psoriasis models human plaque-type psoriasis in which the IL-23/IL-17 cytokine axis plays a pivotal role. Study parameters include in-life clinical evaluation of skin, measurement of ear thickness, and analysis of cytokine expression in the skin and immune organs.
[381] Methods: Mice were sensitized on the shaved back and ear with imiquimod cream daily for 7 consecutive days. Mice were gavaged daily with 10 mg of Veillonella parvula strain A- G.I. (1.5 x IO10 TCC per dose) or vehicle. Mice were also treated with dexamethasone (1 mg/kg intraperitoneally) or given an anti-IL-17 antibody or an anti-IL- 12/23p40 antibody (200 ug/mouse intraperitoneally on days 2, 4, and 6). The severity of inflammation of the back skin was evaluated using a lesion psoriasis severity scoring system. Mice were monitored and graded daily on the scale: 0 (no alteration), 1 (mild erythema), 2 (moderate to severe erythema and some plaques), 3 (marked erythema and plaques) and 4 (very marked erythema and plaques). Ear measurements were taken daily using digital calipers and scores were reported as change in ear thickness calculated as ear score on day 8 minus baseline ear score on day 1. On day 8 study termination, skin samples were taken for cytokine expression and protein analysis, and cells from spleen and lymph node were stimulated in vitro to induce cytokine production. Systemic dexamethasone, and two biologies which are highly efficacious in severe psoriasis - anti- IL-17A and anti -IL-23 - were used as comparators.
[382] Results: Veillonella parvula strain A- G.I. -treated mice showed visibly substantial reduction of erythema, scaling and thickening associated with IMQ-induced skin inflammation compared to vehicle -treated mice on par with anti-IL-17 treatment. Commensurate with reduction of ear inflammation, pro-inflammatory cytokine IL-17A protein was reduced. See figure 10.
PLP-induced relapsing/remitting experimental autoimmune encephalomyelitis (EAE) model of central nervous system inflammation
[383] EAE is a widely-used model of demyelinating diseases, which is induced in mice through activation immunization using a spinal cord antigen, proteolipid protein (PLP). PLP is a major protein component of CNS myelin. PLP injected in Swiss Jim Lambert (SJL) mice together with pertussis toxin (PT) leads to remitting and relapsing demyelinating disease, which can be scored based on the following observations: Score 0 Normal mouse with no overt signs of disease; Score 1 Limp tail or hind limb weakness but not both; Score 2 Limp tail and hind limb weakness; Score 3 Partial hind limb paralysis, Score 4 Complete hind limb paralysis. Mice were monitored daily and given a weakness score. In this model, EAE develops 10-15 days after immunization in 90-100% of immunized mice. The first wave of EAE lasts several days and most mice fully or almost fully recover from this first wave. After a disease-free period, which can last from one day to several months, most mice relapse. In the therapeutic dosing model, mice are enrolled into treatment groups at the time of EAE onset or at the time of recovery from the first wave of EAE. Mice are distributed into the various treatment groups in a balanced manner to achieve groups with similar time of EAE onset and similar first wave disease severity.
[384] Methods: Mice were injected subcutaneously at four sites in the back with the emulsion component (containing PLP139-151 and CFA). Two sites of injection were in the upper back approximately 1 cm caudal of the neck line. Two more sites were in the lower back approximately 2 cm cranial of the base of the tail. The injection volume was 0.05 mb at each site. Within 2 hours of the injection of emulsion, the pertussis toxin component of the kit was administered intraperitoneally. Mice were orally gavaged starting on Day 0 with vehicle or Veillonella parvulci strain A- G.I. (1.5 x IO10 TCC per dose). Animals were monitored and scored daily starting from Day 7. All readouts were continued until termination of the study on Day 42.
[385] Results: Mice treated with Veillonella parvula strain A- G.I. had significantly lower disease scores compared to vehicle-treated mice during both the initial acute phase, as well as the relapsing -remitting phase (Figure 11). The overall reduction in disease score in mice treated with Veillonella parvula strain A- G.I. could be seen in the area under the curve calculations for the total study (Days 7 - 42), as well as for just the acute phase (Days 7 - 20) (Figure 12).
Summary of preclinical studies:
[386] Taken together, the in vitro and in vivo data support the selection of Veillonella parvula strain A- G.I. as a unique strain of Veillonella parvula that:
• Induces the production of anti-inflammatory cytokines, and does not induce significant levels of pro-inflammatory cytokines from human immune cells, including PBMCs, macrophages, dendritic cells, or T cells
• Inhibits skin inflammation and effector cytokine (IL-4, IL-5, and IL-13) production in two different models of TH2 -driven atopic/allergic inflammation.
• Additionally inhibits TH1- and TH17-mediated skin inflammation
• Inhibits TH17-mediated central nervous system inflammation and is well- tolerated after 42 days of once daily dosing. • Remains gut-restricted after oral dosing
[387] Given the effects of Veillonella parvulci strain A- G.I. on skin inflammation preclinically in both TH2- and TH17-driven models, human atopic inflammatory disorders such as atopic dermatitis and asthma, as well as psoriasis may be responsive to treatment with Veillonella parvula strain A- G.I. The equivalent of safety pharmacology testing has been performed in vitro, demonstrating no chemokine signature suggestive of a pathogenic organism.
Pharmacokinetics and Product Metabolism in Animals
[388] A biodistribution study was designed to determine where Veillonella parvula strain A- G.I. bacteria are present in a mouse following oral treatment. The study assessed Veillonella parvula strain A- G.I. distribution following a single oral dose using fluorescently labelled Veillonella parvula strain A- G.I. which was administered to naive mice by oral gavage, followed by imaging using the Licor imaging system at various time points after gavage. The kinetics of Veillonella parvula strain A- G.I. passage through the stomach, small intestine, and colon, were determined following high, medium and low doses (109, 108, and 107, respectively) of Veillonella parvula strain A- G.I. In addition, dissemination from the intestine was examined by measuring fluorescence levels in mesenteric lymph nodes, spleen, liver, heart, kidney, and lungs, 10 minutes, 1 hour, 6 hours, 12 hours, and 24 hours post-gavage. A free dye control was administered and measured at the same time points in order to establish a baseline for background fluorescence.
[389] Oral administration of Veillonella parvula strain A- G.I. led to a transient rise in the GI tract. Veillonella parvula strain A- G.I. was only detected in the intestine and stool for up to 12 hours post-treatment, demonstrating that gamma-irradiated, non-live bacteria are unable to colonize the intestinal tract after a single dose. Importantly, Veillonella parvula strain A- G.I. was not detected outside of the GI tract at any time point above background fluorescence as determined by the free dye control. These data demonstrate that Veillonella parvula strain A- G.I. is luminally restricted with undetectable systemic exposure following oral dosing.
[390] Summary
[391] Veillonella parvula strain A- G.I. is a single strain of the Veillonella genus (Veillonella parvula) that is being investigated for potential benefit in a range of immuno- inflammatory disorders including atopic dermatitis, and potentially psoriasis and asthma. Veillonella parvulci strain A- G.I. has demonstrated specific pharmacological activity in in vitro and in vivo models of immuno-inflammatory disease and so is being developed as a medicinal product.
[392] Preclinical studies have shown that Veillonella parvula strain A- G.I. increases secretion of anti-inflammatory cytokines and induces minimal production of pro- inflammatory cytokines, thereby reducing immune activation and inflammation overall. Evidence of a positive effect has also been seen in the DTH, FITC, MC903, and EAE in vivo models, suggesting the potential for positive clinical benefit in a broad range of clinical scenarios.
Example 4: A non-viable, non-colonizing form of Veillonella parvula requires lymphocyte homing to gut-associated lymphoid tissues to regulate systemic inflammation
Small intestine derived bacterial strain Veillonella parvula induces IL- 10 in vitro and resolves inflammation in vivo
[393] The central premise of this study is that single strains of bacteria isolated from human intestinal tissues can be developed as therapeutics with well-defined pharmacological activities. To demonstrate this, a strain of Veillonella parvula (V. parvula) was isolated from a fresh ileostomy sample of an IBD patient in remission. V. parvula is a gram-negative, obligate anaerobe that belongs to the Negativicutes class and is a natural commensal found in the oral cavity, gastrointestinal and genitourinary tracts. A non-viable form of V. parvula was developed by gamma irradiating the live microbe (designated as Veillonella parvula Strain A). In this example, all references to Veillonella parvula Strain A refer to this gamma irradiated form of the strain. The strain has been deposited as ATCC Accession Number PTA-125691.
[394] Fresh primary human CD1 lb+ immune cells were purified from the peripheral blood mononuclear cells (PBMC) of 6 healthy human donors. The purified human cells were individually co-cultured with a single bacterial obligate anaerobe strain at a multiplicity of infection (MOI) of 1. After 24 hours of mammalian cell -microbe coculture, cell supernatants were collected, and pro- and anti-inflammatory cytokine production was measured. The data show that of the >70 unique commensal strains evaluated, significant levels of IL- 10 were induced from all donors in response to stimulation with Veillonella parvula Strain A compared with several closely related species (Figure 13A). Veillonella parvula Strain A did not induce high levels of pro- inflammatory cytokines IP- 10 or TNFa compared to other commensal anaerobic strains (Figures 13B and 13C). Furthermore, isolated human PBMCs, DCs, and macrophages from whole blood were cultured with a range of doses from 104 - 5 x 106 total cell count (TCC) of Veillonella parvula Strain A for 24 hours, and IL- 10 was measured in supernatants. A dose-dependent response was observed between increasing TCC and production of IL-10 from both PBMCs, DCs and macrophages. These results demonstrate pharmacological activity of Veillonella parvula Strain A on immune cells, and establish EC50 of 5.2 log TCC for PBMCs, 4.8 log TCC for DCs, and 4.9 log TCC for macrophages in comparison with an unrelated microbe from a different genus like Clostridium cadaveris (Figure 13D).
[395] To determine efficacy of Veillonella parvula Strain A as a therapeutic agent in vivo, it was tested in a DTH mouse model. Mice were sensitized by subcutaneous injection with keyhole limpet hemocyanin (KLH) emulsified with Complete Freund’s Adjuvant (CFA). Eight days after sensitization, the mice were challenged by intradermal ear injection with KLH. DTH response was evaluated 24 hours post-challenge, which represents the peak of disease in this model. Mice were orally dosed daily for 8 days. Veillonella parvula Strain A was the most efficacious strain in lowering ear inflammation compared to other closely related Veillonella species (Figure 13E) and displayed dose dependent efficacy (Figure 13F). The anti-inflammatory effect of Veillonella parvula Strain A in vivo was evaluated by blocking the IL- 10 signaling pathway during DTH. Coadministration with anti -IL- 1 OR antibody impaired the effect of Veillonella parvula Strain A in resolution of ear inflammation suggesting that Veillonella parvula Strain A exerts its anti-inflammatory activity in part through the IL- 10 pathway (Figure 13G).
[396] Taken together, a non-viable form of Veillonella parvula Strain A demonstrates effectiveness as an anti-inflammatory therapeutic agent.
Orally administered Veillonella parvula Strain A is gut restricted and transits rapidly through the intestine without alteration of the gut microbial flora
[397] Veillonella parvula Strain A is an orally delivered, gut restricted agent that exerts its pharmacological effects through SINTAX. A study was designed to determine the biodistribution of Veillonella parvula Strain A in a mouse following oral treatment and tracked transit through the gastrointestinal (GI) tract. Oral administration of Veillonella parvula Strain A led to a transient rise in the GI tract. Complete exposure of the small intestine occurred within 1 hour post oral administration of Veillonella parvula Strain A. Veillonella parvula Strain A was only detected in the intestine and stool for up to 12 hours post-treatment after a single dose (TCC - 1E+9) (Figure 14A). Exposure of the small intestine occurs by 10 mins post-administration and complete exposure in 1 hour. By 6 hours, Veillonella parvula Strain A is incorporated into fecal pellets, and by 24 hours signal in the GI tract diminishes, suggesting complete excretion of the microbe with no detectable persistence. No systemic exposure is observed at any timepoint and >99% of the total signal remains in the GI tract. Importantly, Veillonella parvula Strain A was not detected outside the GI tract at any time point above background fluorescence as determined by the free dye control (Figure 14B). Since Veillonella parvula Strain A is non-viable, lack of persistence in the GI tract suggests that it does not require colonization to achieve its pharmacological activity. In an experiment to determine the effects of Veillonella parvula Strain A on the microbiome in vivo, it was confirmed that there were no discernible changes to the microbiome composition at various time points after dosing.
[398] Together, these data demonstrate that Veillonella parvula Strain A is luminally restricted with undetectable systemic exposure following oral dosing and does not alter the microbiome.
Veillonella parvula Strain A inhibits cutaneous inflammation in imiquimod driven model of psoriasis
[399] Efficacy in a T cell mediated model like DTH suggested an anti-inflammatory role for Veillonella parvula Strain A. Thus, the efficacy of Veillonella parvula Strain A was tested in disease models with a strong T cell component. The imiquimod-induced psoriasis model where Thl7 cell associated IL-23/IL-17 cytokine axis plays a pivotal role was chosen for this study. This model recapitulates aspects of human disease, including clinical and histological characteristics similar to human psoriasis, such as epidermal thickening, scaling and erythema, as well as infiltrates of T cells, neutrophils and dendritic cells (van der Fits et al., 2009). BALB/c mice were sensitized on the ear with 20 mg 5% imiquimod cream daily for 7 consecutive days and orally dosed daily with Veillonella parvula Strain A (TCC - 7.8E+11). Veillonella parvula Strain A treated mice showed substantially lower ear inflammation over the course of disease progression (Figure 15A). At termination of study, transcript levels in the ear tissue revealed reduction in III 7a, III 7f and Deft>3 levels upon treatment with Veillonella parvula Strain A in comparison to vehicle (Figure 15B).
Veillonella parvula Strain A ameliorates inflammation in murine model of relapsingremitting multiple sclerosis
[400] The role for Veillonella parvula Strain A in alleviating a second Thl7-driven disease as also investigated by assessing its effects in a murine experimental autoimmune encephalomyelitis (EAE) model of relapsing remitting multiple sclerosis. EAE, a widely used model of demyelinating diseases, is induced by immunization with a spinal cord antigen, proteolipid protein (PLP), a major protein component of CNS myelin. PLP injected in mice together with pertussis toxin leads to remitting and relapsing demyelinating disease. Mice were scored daily for disease onset and progression as per the standard EAE scoring criteria, reflecting the degree of motor deficit. Mice began to display motor impairment by day 9 and reached peak clinical disease scores by day 15. In a prophylactic treatment regimen where dosing started on day 1, Veillonella parvula Strain A showed significant reduction in clinical score compared to vehicle treated animals over the course of the disease. The treatment effects of Veillonella parvula Strain A were most pronounced in the relapsing phase of disease indicating increased recovery after acute phase of disease. By the end of the study at day 42, Veillonella parvula Strain A treated mice had an overall lower cumulative EAE score compared to control (Figure 16A). The effects of Veillonella parvula Strain A on cellular responses in vivo were also determined. Sections of spinal cord tissue from cervical and lumbar regions of mice that received treatment with Veillonella parvula Strain A, a positive control drug fingolimod and a vehicle treated group were analyzed for inflammatory cell infiltration. Mice treated prophylactically with Veillonella parvula Strain A showed significantly reduced frequency of infiltrating inflammatory cells in the spinal cord compared to vehicle treated animals (Figure 16B).
[401] Next, Veillonella parvula Strain A was tested in a therapeutic dosing regimen where dosing started after the peak of disease in the acute phase was reached on day 10. Significantly reduced cumulative disease scores were observed at study termination compared to vehicle (Figure 16C). While lymphocytic cell infiltration was not as different as seen with prophylactic dosing (Figure 16D), Veillonella parvula Strain A treated mice had significant reduced demyelination of the spinal cord. Significantly, Veillonella parvula Strain A was better at protecting from demyelination than a human dose equivalent of fingolimod (Figure 16E). This suggests that while Veillonella parvula Strain A did not block migration of cells to the CNS in the therapeutic model, immune cell infdtrates in the CNS were unable to cause severe inflammation.
[402] Collectively, these data demonstrate efficacy of Veillonella parvula Strain A in reducing pathology in two distinct IL-23/IL-17A axis driven inflammation models.
Veillonella parvula Strain A reduces inflammation in FITC-driven contact hypersensitivity
[403] Since inflammation resolving activity was observed for Veillonella parvula Strain A in Th 17 driven psoriasis and neuroinflammation, Veillonella parvula Strain A was probed for efficacy in Th2 pathway driven diseases. Atopic dermatitis (AD) is a chronic inflammatory skin disease, driven by strong Th2 immune responses. It is a considered a primarily T cell-driven disease with a key role for cytokines IL-4, IL-5, IL-13, alarmin IL-33 (Hardman and Ogg, 2016; Ziegler, 2012) and IL-31, a cytokine associated with itch (Sonkoly et al., 2006) in AD pathogenesis. Veillonella parvula Strain A was tested in a murine model of contact hypersensitivity, using a hapten fluorescein isothiocyanate (FITC) to drive cutaneous inflammation (Boehme et al., 2009; Takeshita et al., 2004). In this model, BALB/c mice were topically sensitized with 0.5% FITC on day 1 and 2, and 6 days later they were challenged with 0.5% FITC on the ear. Mice were dosed daily orally with vehicle or Veillonella parvula Strain A (TCC - 2. 16E+12). Ear inflammation was measured 24 h post ear challenge on day 7. This model has similarities to human AD with a strong CD4+ T helper cell component and pathology associated with disease recapitulates features of acute AD lesions. FITC sensitization induced robust ear swelling. Oral treatment with Veillonella parvula Strain A significantly inhibited ear swelling compared to the vehicle-treated group (Figure 17A). It was investigated whether Veillonella parvula Strain A impacts production of cytokines driving inflammation. Ex vivo restimulation of cells from gut draining mesenteric lymph nodes (mLN) with PMA/Ionomycin showed decreased production of Th2 cytokines like IL-4, IL-5, IL-13, IL-31 and IL-33. Furthermore, ex vivo re-stimulation of cells from cervical lymph nodes (cLN), the draining lymph node for the site of inflammation, resulted in reduced production of IL-5 and IL- 13, and homogenates made from ear tissue had reduced IL-4 in Veillonella parvula Strain A treated animals demonstrating the systemic effect of gut- restricted Veillonella parvula Strain A (Figure 17B). These findings confirm that Veillonella parvula Strain A has an immune resolving effect in inflammation by modulating levels of proinflammatory Th2 cytokines.
Veillonella parvula Strain A treatment leads to reduction of local and systemic pro inflammatory cytokines in vivo
[404] Given that Veillonella parvula Strain A exerts its effects locally in the gut to contain systemic inflammation, the impact of Veillonella parvula Strain A on downstream effectors such as cytokines and chemokines in the inflammatory milieu was investigated. A compressed tablet formulation of Veillonella parvula Strain A, dosed daily for 9 days, was tested in the KLH DTH model as previously described. Veillonella parvula Strain A showed efficacy in reducing ear inflammation in comparison to a group that received a placebo formulation (Figure 18A). Ex vivo analyses restimulation of cells from spleen and mLN taken from mice 24 h post-ear challenge revealed significantly reduced production of proinflammatory cytokines including the Thl cytokines IFNy, TNFa, IL-12p70, IL-6, and the Thl7 cytokines GMCSF and IL-17A. This suggested that treatment with Veillonella parvula Strain A had an anti-inflammatory effect on cells in the mLN as well as cells in periphery represented by splenic cells. (Figure 17B). Ex vivo restimulation with KLH to mimic antigenic stimulation of cells from cLN showed a similar trend of reduced pro-inflammatory cytokines TNFa, IL-6, IL-12p70, GMCSF, IL-17A and IFNy in mice treated with Veillonella parvula Strain A, compared to a control group treated with vehicle (Figure 18B).
The anti-inflammatory effects driven by Veillonella parvula Strain A require trafficking of immune cells from the gut to periphery to resolve inflammation
[405] To better understand the immunomodulatory effects of Veillonella parvula Strain A on SINTAX, a systematic approach was employed to dissect the steps in the process. The first step is the interaction between Veillonella parvula Strain A and cells in the small intestine, including epithelial cells and dendritic cells. Several pattern recognition receptors are located on cell surfaces and of these, TLRs represent a major class (Price et al., 2018). HEK-293 cells stably transfected with human TLR1/2/6, TLR2/6 and TLR1/6 and an NF-KB-inducible reporter gene were stimulated with different doses of Veillonella parvula Strain A was mostly detected by TLR2/6 with little to no detection by TLR1/6 (Figure 19A). When exploring the role for TLR2 signaling in vivo in the KLH DTH model as previously described, simultaneous dosing with Veillonella parvula Strain A and a TLR2 blocking antibody resulted in reduced efficacy in controlling ear inflammation in comparison with Veillonella parvulci Strain A dosed with an isotype antibody (Figure 19B).
[406] In C3HEJ mice that are genetically deficient for TLR4 (Akeson et al., 2006), induced with KLH-DTH, treatment with Veillonella parvula Strain A was efficacious in lowering ear inflammation (Figure 19C). This suggested recognition of Veillonella parvula Strain A was dependent on signaling via TLR2 receptor cascade and independent of TLR4 signaling.
[407] In the second step, T cells traffic through mLN to encounter gut resident DCs that could have interacted with Veillonella parvula Strain A. This leads to activation of effector T cells, which then enter the systemic circulation and migrate to peripheral tissues. Expression of specific adhesion molecules and chemokine receptors on lymphocytes, in concert with spatial and temporal expression of specific ligands by cells in tissues, is responsible for lymphocyte migration. LPAM-1 and CD62L are expressed on T and B cells and act as intestinal homing receptors to mediate migration of lymphocytes into mLN. Blockade of a4[37 integrin and CD62L prevents lymphocytes from entering mLN and Peyer’s patches (Dutt et al., 2005). The efficacy of Veillonella parvula Strain A was observed to be contingent on the engagement of CD62L and LPAM-1 to drive lymphocyte homing to the gut. Simultaneous blockade of both receptors prevented lymphocyte homing to the gut and resulted in loss of efficacy with Veillonella parvula Strain A treatment in comparison with a control group that received Veillonella parvula Strain A with an isotype antibody (Figure 19D).
[408] Taken together, these data illustrate, in part, mechanisms of action for Veillonella parvula Strain A acting through SINTAX to drive systemic efficacy for inflammation resolution.
Adoptive transfer of CD4+ T cells from Veillonella parvula Strain A treated donors leads to inflammation resolution in vivo
[409] Based on the data presented herein, which confirm a role for Veillonella parvula Strain A in inflammation resolution, the specific role for CD4+ T cells in this context was investigated. Using an adoptive cell transfer model, CD4+ T cells from Veillonella parvula Strain A treated KLH-immunized donor mice were transferred into KLH- immunized recipient mice that were not dosed with Veillonella parvula Strain A. Four days post adoptive transfer, recipient mice were challenged with KLH intradermally in the ear to elicit local inflammation. Recipient mice that received CD4+ T cells from Veillonella parvulci Strain A treated donors had significantly reduced ear inflammation in comparison with recipients that received CD4+T cells from vehicle treated mice (Figure 20). These data suggest that treatment with Veillonella parvula Strain A might confer anti-inflammatory functions to the CD4+ T cells that are subsequently transferred and that these cells are sufficient to drive an anti-inflammatory response in the recipient mice.
[410] This example describes the development of Veillonella parvula Strain A, an orally administered, gut-restricted strain of Veillonella parvula for the treatment of inflammatory diseases. Animal models and human diseases tend to be arbitrarily defined in pathway specific terms Thl, Th2, Th 17, innate or adaptive. However, induction of inflammation is not generally pathway specific. While existing therapeutic interventions do indicate some pathway biases in disease pathogenesis, such as TNF inhibitors in rheumatoid arthritis or IL 17 inhibitors in psoriasis, the data presented herein demonstrate that orally administered Veillonella parvula Strain A is effective in murine models of Thl, Th2 and Th 17 inflammation. This shows that the functional connections radiating from the small intestinal mucosa can induce systemic resolution of broad inflammatory mechanisms leading to restoration of immune homeostasis.
[4H] The systematic analyses described herein identify immunomodulatory effects that are strain dependent. Hundreds of individual bacterial strains were screened across a wide range of genera for their effects on cytokine secretion patterns from human macrophages. Each strain has an individual phenotype with different effects on macrophage response. Here, the specific immunomodulatory activity of a single strain of Veillonella parvula, Veillonella parvula Strain A was demonstrated in depth. Various other species of Veillonella that were tested did not alter systemic immune responses to the same extent.
[412] Colonization of the intestine by Veillonella parvula Strain A is not required for its pharmacological activity. A rapid transit through the gastrointestinal tract within hours of administration and treatment with Veillonella parvula Strain A was observed and caused no changes in the microbiome. The mechanism of action of Veillonella parvula Strain A is dependent on direct interactions with host intestinal cells, in part mediated through TLR-2. This reinforces the central premise that microbe viability is not a necessity, as this gamma irradiated microbe is able to exert its pharmacological activity through structural interactions with SINTAX. This is distinct from reports of live bacterial therapeutics altering the ecology of colonic microbiota (Atarashi et al., 2013). While previous studies use a reductionist approach to identify immune effects of bacterial species in germ free animals (Geva-Zatorsky et al., 2017), all experiments described herein were done in specific pathogen-free animals with intact intestinal microbiomes to represent the complex relationships in the gut- microbial environment. The dose-dependent effects of Veillonella parvulci Strain A were superimposed on this microbial background.
[413] The dependence on IL- 10 for the efficacy of Veillonella parvula Strain A in the delayed-type hypersensitivity model is a clue to the differences between the effects mediated by SINTAX and established therapeutics based on inhibition of pro- inflammatory mediators. Systemic administration of IL- 10 as an anti-inflammatory therapy has not been successful to date, despite the long-standing awareness that it is a critical mediator of the resolution of inflammation. The pharmacological demonstration of the role that IL- 10 plays in the activity of Veillonella parvula Strain A shows an alternative way to harness its anti-inflammatory effects. It further bolsters the fact that local gut effects in response to specific immunomodulatory bacterial strains can regulate immune responses at distal sites in concordance with previous reports (Mangalam et al., 2017). This approach reveals the versatility of the platform disclosed herein to develop microbes as drugs with the ability to immunomodulate relevant disease specific pathways.
[414] There are two main conclusions from this work. The first is that the small intestinal mucosa, distinct from the colonic flora, is a central controller of systemic inflammation, operating at a distance in response to signals generated by substances passing through the gut. The effects appear to represent broad-based resolution of inflammation that re-establishes normal homeostatic inflammatory status across multiple pathways.
[415] The second is that it suggests a therapeutic approach for common inflammatory diseases suffered by millions of patients. Resolution of inflammation by a non-absorbed oral agent acting via SINTAX has the potential to create a new class of safe and effective medicines that can be manufactured at reasonable cost for the treatment of inflammatory diseases suffered by millions of patients around the globe. These results support a series of clinical studies currently ongoing with Veillonella parvula Strain A. MATERIALS AND METHODS
[416] Mice. Female BALB/c and C57BL/6 mice (6-8 weeks old) were purchased from Taconic Farms. Animals were housed in specific pathogen-free conditions in a vivarium (5 mice per cage), and all experiments were performed under Institutional Animal Care and Use Committee (IACUC) approved protocols and vivarium guidelines. Female SJL mice (8-10 weeks old) were purchased from Jackson Labs for EAE experiments which were performed under IACUC approved protocols at Hooke Laboratories (Lawrence, MA). Mice were allowed to acclimate in the vivarium for 1-2 weeks prior to the start of experiments. PicoLab Rodent Diet 20 was provided and autoclaved water via sipper bottle, given ad libitum and checked daily.
[417] Strains of microorganisms. Three individual strains of Veillonella species were obtained for this study. All strains were purified via single colony isolation method. A strain’s identity was confirmed by 16S rDNA sequence comparisons and by the whole genome sequencing. Veillonella parvula (Veillonella parvula Strain A) and Veillonella tobetsuensis were isolated from an ileal pouch sample and a pre-colonoscopy sample of two IBD patients in remission. Veillonella atypica was isolated from a fresh subgingival sample of a healthy volunteer.
[418] Microbial biomass growth conditions. All strains were grown in in-house developed semi-defined Soy Peptone-Y east Extract medium supplemented with Na-L- Lactate as a major carbon source, mineral elements, and L-cysteine-HCl as a reducing agent. All microbial cultures were incubated under anaerobic conditions at 37°C for 16-24 hours before harvesting. Bacterial biomass was concentrated by centrifugation at 7000 g for 20 min at 10°C, resuspended in anaerobic glycerol or yeast extract-sucrose solutions and distributed into 1.8 ml cryovials at 1.2-1.5 ml volume under anaerobic conditions. Cryovials were immediately frozen in liquid N2 and stored at -80°C.
[419] Veillonella parvula Strain A was prepared in different forms- powders and frozen biomasses and was characterized by TCC method. Bacterial total cells count (TCC) was enumerated by Coulter Counter Multisizer 4e. In powders, TCC varied from 4.0e+l 1 to 2.2e+12 cells/g. In biomasses TCC varied from 2.8e+10 to 8.5e+10 cells/ml. Bacterial biomass identity was confirmed by 16S rDNA sequencing. Powder was produced following in-house developed fermentation and lyophilization protocols and stored in sealed mylar bags inside desiccator at 4°C. [420] Veillonella parvula Strain A aliquots were subjected to 25 kGy Gamma Irradiation treatment at Sterigenics U.S., LLC. Veillonella parvula Strain A aliquots were characterized by TCC and VCC methods before and after Gamma Irradiation. Total cell number did not change, while there were no viable cells left after the treatment.
[421] Dosing with Veillonella parvula Strain A and controls in vivo. For each in vivo study, Veillonella parvula Strain A aliquots were distributed into plastic test tubes with caps and stored at 4°C. Mice were treated orally with Veillonella parvula Strain A (4.0E+11 to 2.2E+12 cells/g PO- specific TCC is noted in figure legends) or vehicle control (anaerobic sucrose, PO) for duration of different models as described in figure legends. Dexamethasone (1 mg/kg, i.p., Sigma) was used as a positive control unless otherwise specified. For EAE studies, fingolimod (1 mg/kg, PO, Tocris Biosciences) was dosed daily.
[422] For blockade of IL-10R, mice were injected IP with 100 pL of either anti-IL-lOR (Bio X Cell; Clone 1B1.3A; Cat# BE0050) or Rat IgGl isotope control (Bio X Cell; Clone HRPN; Cat# BE0088) at 2 mg/mL. Mice were treated on days 0, 3, and 6.
[423] For blockade of TLR2, mice were injected IP with 100 pL of either anti-TLR2 (Invivogen; Clone T2A; Cat# mab2-mtlr2) or Mouse IgGl isotope control (Bio X Cell; Clone MOPC-21; Cat# BE0083) at 2 mg/mL. Mice were treated days 0, 3, and 6.
[424] To inhibit gut homing of lymphocytes, mice were injected IP with 100 pL of either anti-CD62L (Bio X Cell; Clone Mel-14; Cat# BE0021) and anti-LPAM-1 (Bio X Cell; Clone DATK32; Cat# BE0034) prepared at 5 mg/mL each and mixed 1: 1 or Rat IgG2a isotope control (Bio X Cell; Clone 2A3; Cat# BE0089) at 5 mg/mL. Mice were treated days 1, 3, 5, and 7.
[425] Delayed Type Hypersensitivity mouse model. Mice were immunized with 50 pl of emulsion of keyhole limpet hemocyanin (KLH) in Complete Freund’s Adjuvant (CFA) on four sites on the back. 8 days later, recipient mice were challenged with KLH (10 pg/ 10 pl) intradermally in the ear. Ear measurements were recorded 24 hours post ear challenge using digital calipers. Change in ear thickness was expressed as ear thickness at day 7 minus ear thickness at baseline.
[426] For the adoptive transfer DTH, cells were isolated from spleens and all lymph nodes of C57BL/6 mice and single cell resuspensions were made. CD4+ T cells were isolated using EasySep Mouse CD4+ isolation kits (StemCell Technologies, Cat#19852). 1.5-2 x 107 cells resuspended in 200 pl of PBS were injected into recipient mice. 4 days after adoptive transfer of cells, recipient mice were challenged with KLH (20 pg/20 pl) intradermally in the ear. Ear measurements were recorded 24 hours post ear challenge.
[427] Imiquimod-induced psoriasis-like skin inflammation protocol. Mice were sensitized topically with 20 mg imiquimod cream (Aldara; 3M Pharmaceuticals, St Paul, MN, USA) on ears daily for 7 consecutive days. Ear measurements were taken daily using digital calipers and scores were reported as change in ear thickness calculated as ear score daily minus baseline ear score on day 1 prior to sensitization with imiquimod.
[428] Experimental Autoimmune Encephalomyelitis. Female SJL mice (8-10 weeks old) were subcutaneously injected at four sites with myelin proteolipid protein (PLP)i39- i5i in CFA emulsion (0.05 mL/injection site; ~0.5 mg PLP PLPi39-i5i/mL; Hooke Laboratories; EK-2120). Following immunization, EAE induction was completed by intraperitoneal injections of pertussis toxin (6 pg/mL; 0.1 mL/mouse) within 2 hours of immunization. Mice were randomized into groups and monitored for EAE clinical score over the course of 42 days. Disease progression was scored blinded of treatment or prior measurements. Disease severity was scored using standard EAE criteria: 0 (normal); 1 (loss of tail tone); 2 (hind limb weakness); 3 (hind limb paralysis); 4 (hind limb paralysis and forelimb paralysis or weakness); 5 (morbidity /death). Mice were observed daily for clinical symptoms. Mice were euthanized if they had a score of 4 for 2 days, and a score of 5 was recorded for remainder of the study for these animals.
[429] End point tissue collection and histology. After euthanasia at the end of the study, EAE mice were perfused with 5-10 mb PBS and the spinal column was extracted from the base of the skull to the beginning of the pelvic bone. Spinal columns were then drop-fixed in 10% neutral buffered formalin and stored horizontally for 48 hours. After fixation, spinal columns were treated in mild formic acid decalcification solution (Immunocal-Statlab, Fisher Scientific, #141432) overnight (12-24 hours) at room temperature. Spinal columns were then trimmed into 4 mm-thick cervical, thoracic, and lumbar segments and processed using a Sakura Tissue Tek VIP 5 by graded alcohol dehydration, cleared in xylene, and finally infiltrated with paraffin. After processing, spinal column segments were embedded into paraffin blocks. Paraffin blocks were then sectioned at 4 pm on charged slides, air-dried overnight and stained with Hematoxylin and Eosin according to standard automated H&E protocol (Tissue-Tek Prisma) and then cover slipped (Tissue-Tek Glass). Prepared tissue sections were then imaged using a NanoZoomer 2.0 HT (Hamamatsu) at 20X magnification. [430] Slides were also stained with Anti-MBP immunohistochemistry performed using the Ventana Discovery XT (Roche/Ventana, Tucson AZ) automated Platform. Slides were deparaffmized and rehydrated. Endogenous Biotin blocking was performed online 4 min followed by 32 min incubation in the anti-MBP primary (Abeam. Ab40390 (1 pg/ml) LotGR3264120-l) at a 1: 1000 dilution. The biotinylated GtxRb secondary (Invitrogen 65-6140 LotTD268284) was incubated for 32 minutes at a 1: 1000 dilution. HRP based detection was performed online using the standard DABMAP Kit (Roche/Ventana 760- 124) followed by a Hematoxylin counterstain. Slides were then dehydrated through graded alcohols cleared in Xylene and cover slipped. Stained Slides were scanned on Nanaozoomer 2.0 HT (Hamamatsu) at 20X magnification and images were analyzed by a pathologist.
[431] Histological analysis of spines. For each spine, one anti-MBP-stained slide and one H&E-stained slide was prepared and analyzed. Each slide contained a section with samples from lumbar, thoracic, and cervical spinal cord (3 samples). Histological analysis was performed blind to groups and included count of inflammatory foci, count of apoptotic cells, and demyelination score. All analysis was performed by a pathologist blinded to the experimental groups and all clinical readouts.
Count of inflammatory foci - spinal cord -Inflammatory foci of approximately 20 cells were counted in each H&E-stained section. When inflammatory infiltrates consisted of more than 20 cells, an estimate was made of how many foci of 20 cells were present. Estimation of demyelinated area - spinal cord - The demyelination score represents an estimate of demyelinated area for each section as follows: 0 - no demyelination (less than 5% demyelinated area)
1 - 5 to 20% demyelinated area
2 - 20 to 40% demyelinated area
3 - 40 to 60% demyelinated area
4 - 60 to 80% demyelinated area
5 - 80 to 100% demyelinated area
For anti-MBP-stained slides, the size of the demyelinated area was estimated based on less intense brown staining of myelin.
[432] FITC-induced allergic inflammation. Backs of female BALB/c mice were shaved and on days 1 and 2 400 pl of 0.5% FITC solution (dissolved in acetone: dibutyl phthalate, 1: 1, v/v) was painted on the shaved skin. On day 6, baseline ear measurements were taken and then mice were challenged with 20 pl 0.5% FITC on the right ear. On day 7, ear thickness was measured 24 hours post FITC challenge using digital calipers (Fowler). Change in ear thickness was expressed as ear thickness at day 7 minus ear thickness at baseline.
[433] Ex vivo re-stimulation assays. Ear-draining cervical lymph nodes (CLNs), gut draining mesenteric lymph nodes (MLNs) and spleens were harvested at terminal time points from various studies and collected into 0.5 ml of cold, complete-RPMI (10% FBS, lx Glutamax, 1 mM sodium pyruvate, 100 mM HEPES, lx non-essential amino acids, lx beta-mercaptoethanol, lx antibiotic-antimycotic) (all reagents from Gibco). Single cell suspensions were prepared (spleens were RBC lysed with ACK lysing buffer) and 200,000 cells/well were plated. Cells were stimulated ex vivo with either LPS (200 ng/ml, Invivogen) or PMA (eBioscience) for 48 hours, or KLH (50 pg/ml, Sigma) or OVA (50 pg/ml) for 72 hours at 37°C and 5% CO2. Supernatants were collected at the end of stimulations and used for multiplex ELISAs of cytokine levels using Meso Scale Discovery (MSD) kits. Ear tissues were dissociated in 250 pl T-PER buffer (Thermo Scientific) containing Halt Protease (Thermo Scientific) and protein was quantified with BCA kit (Thermo Scientific). 100 pg of protein was used to measure cytokine levels using MSD kits.
[434] Veillonella parvula Strain A Fluorescent Labeling Fluorescent Labeling.
Samples were kept in ice at all times unless specified. In order to remove excipients, 200 mg of Veillonella parvula Strain A drug substance (powder formulation) were resuspended and extensively washed by repeated centrifugation/resuspension (4 min, 9000 x g, 4°C) using sterile labeling buffer (2x PBS buffer pH 8.3). Final microbial cell pellets where resuspended in 1 mb final volume using labeling buffer. The fluorescent labeling reaction was started by addition of IRDye800-NHS Ester (LICOR Biosciences) at 50 pM final concentration (from 10 mM dye stock dissolved in DMSO). The reaction was allowed to continue for 1.5 hours at 22 0 C with gentle agitation, followed by overnight incubation at 4°C. Non-reacted dye was removed by repeated steps of centrifugation/resuspension in sterile PBS buffer until no fluorescence was detected in the supernatant. Fluorescently labeled microbial cells were resuspended in sterile PBS buffer and cell concentration was quantified using coulter counter instrument (Beckman). The molar concentration of IRDye800® covalently attached to microbial cells was quantified by UV-Vis absorbance and using the dye’s molar extinction coefficient 240.000 M^cm’1 at 778 nm in PBS buffer (LICOR Biosciences). A suspension of unlabeled bacterial cells was used to subtract any light scatter contributions.
[435] Biodistribution Studies. One week prior to experiments, Balb/C female mice were place under a specific low fluorescence diet without chlorophyl (AIN-93G, Bio-Serv). Mice were randomized in groups of 3 animals and orally dosed with 100 pl PBS buffer containing 1 x 109 total Veillonellci parvulci Strain A cells covalently labeled with IRDye800, as well as an equimolar dye amount of IRDye680RD-Carboxylate (LICOR Biosciences) as a free-dye control. At each time point within each experimental replicate, mice were sacrificed using CO2 and cervical dislocation followed by careful removal of the complete Gastrointestinal Tract (GIT), mesenteric lymph nodes (MLN), liver, spleen, heart, and lungs. These were imaged in a tray using a whole-animal fluorescence imaging instrument (Pearl®, LI-COR Biosciences). Tissues were imaged using the 800 nm emission channel (Veillonellci parvulci Strain A), the 700 nm emission channel (free dye control), and the standard white epi-illumination channel (overall tissues). After imaging, the individual organ weights were recorded. Fluorescence images were used to quantify the distribution of Veillonella parvula Strain A signal in the GIT as well as the different organs. Tissues were manually outlined, and the fluorescence signals quantified using image analysis software (Image Studio®, LICOR Biosciences). The fluorescence signal from different organs was divided by the recorded tissue weight in order to normalize the data. Data points corresponded to mean ± standard deviation values.
[436] HEK-TLR assay. HEK293-SEAP reporter cells (Invivogen) expressing human TLR1, TLR2, and TLR6 combinations were plated at a final concentration of 20,000 cells per well in 96 well plates and cultured in appropriate selection media. After 48 hours selection media was washed out and replaced with complete media, and Veillonella parvula Strain A was added at the indicated concentrations per well. Cells were cultured in the presence of Veillonella parvula Strain A for 24 hours. Supernatant was collected and incubated with HEK-Blue reagent (Invivogen) for 1 hr, followed by reading absorbance at OD 630 nm for SEAP production to determine stimulation of TLR2 heterodimers.
[437] In vitro assays. For in vitro assays thawed bacterial biomass was serially diluted in RPMI degassed medium inside anaerobic chamber (Coy Lab Products, USA) to reach approximately 2E+6 bacterial cells/ml. 100,000 bacterial cells were added to 200,000 purified human immune cells per each 96-well manually or by using automated Liquid handler Biomek 4000 (Beckman Coulter) inside custom-built Coy Anaerobic chamber (Coy Lab Products, USA). The co-cultures were incubated for 24 hrs under micro-oxic conditions (1% O2, 5% CO2, balanced by N2). After incubation cell supernatants were collected and Luminex or MSD technology was used to measure pro-and antiinflammatory cytokine production.
[438] Human PBMC assay. Freshly isolated PBMCs from 6 different human donors were cultured at 100,000 PBMCs/well in 100 pl and incubated at 37°C overnight. The following day 75 pl of supernatant was removed and replaced with fresh antibiotic free media. Microbes were added in anaerobic conditions and flushed with 1% oxygen. Plates were incubated for 24hrs in an anaerobic box at 37°C +5% CO2. After 24 hours, plated were centrifuged and supernatants collected to assay cytokine levels using MSD assays.
[439] Human Macrophage assay. Freshly isolated PBMCs from 6 different human donors were used for isolation of CD1 lb+ macrophages. PBMCs were washed in 10 ml MACS buffer, spun down and resuspended at a concentration of 107 total cells per 80 pl. Anti-CDl lb+ beads were added (20 pl per 107 cells) and the cell suspension was incubated at 4°C for 15 minutes. Following incubation, cells were washed and resuspended in MACS buffer and CD1 lb+ cells were isolated using magnetic separation as per manufacturer’s protocol (Miltenyi). Isolated cells were further cultured at 100,000 CDl lb+ cells/well in 100 pl and incubated at 37°C overnight. The following day, contents in the well were mixed to bring non-adherent cells into suspension and 75 pl of supernatant was removed and replaced with fresh antibiotic free media. Microbes were added in anaerobic conditions and flushed with 1% O2. Plates were incubated for 24 hrs in an anaerobic box at 37°C +5% CO2. After 24 hours, plates were centrifuged, and supernatants collected to assay cytokine levels using MSD assays.
[440] Human Dendritic assay. Freshly isolated PBMCs from 6 different human donors were used for isolation of blood dendritic cells. PBMCs were washed in 10ml MACS buffer, spun down, and resuspended at a concentration of 108 total cells per 300 pl. FcR blocking reagent and non-DC Depletion cocktail were added (100 pl per 108 cells) and the cell suspension was incubated at 4°C for 15 minutes. Following incubation, cells were washed and resuspended in MACS buffer and run through the magnetic LD column for a depletion separation. The non-DC cells were retained in the magnetic column whereas the unlabeled cells (DCs) were collected as flow through. The unlabeled cells (DCs) were washed and labeled with a DC enrichment cocktail (100 pL per 108 cells) and the cell suspension was incubated at 4°C for 15 minutes. Following incubation, cells were washed and resuspended in MACS buffer and run through a magnetic MS column for a positive selection. Positively Isolated cells were further cultured at 30,000 dendritic cells/well in 100 pl and incubated at 37°C overnight. The following day, 75 pl of supernatant was removed and replaced with fresh antibiotic free media. Microbes were added in anaerobic conditions and flushed with 1% O2. Plates were incubated for 24 hrs in an anaerobic box at 37°C +5% CO2. After 24 hours, plates were centrifuged, and supernatants collected to assay cytokine levels using MSD assays.
[441] Statistical analysis. The data were expressed as mean ± standard deviation. Statistical significance between groups was compared using the one-way ANOVA compared with sucrose-treated control. For statistical analyses of EAE data, the following tests were used for each readout: EAE incidence, Chi-square test; Mean day of EAE onset, 2-tailed Student’s t-test; Median day of EAE onset, Wilcoxon’s survival test;
Average clinical score, 2-tailed Student’s t-test; Average end clinical score, Wilcoxon’s non-parametric test; Mean maximum score (MMS), Wilcoxon’s non-parametric test;
Average weight gain/loss, 2-tailed Student’s t-test; End weight gain/loss, 2-tailed Student’s t-test; Incidence of EAE relapse, Chi-square test; MMS of relapses, Wilcoxon’s non-parametric test; MMS of relapse period, Wilcoxon’s non-parametric test.
Significance was assigned at p < 0.05, All statistical tests were performed using Prism 8 (GraphPad Software, San Diego, CA, USA).
References for Example 4 and Materials and Methods
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Example 5: Veillonella parvula Strain A acts on cells of the small intestine to resolve peripheral inflammation
[466] Transcriptional profiling of duodenal tissue revealed that Veillonella parvula Strain A-G.I. upregulated genes in lymphocytic pathways that resolve inflammation as well as genes associated with intestinal homeostasis.
[467] Orally-administered Veillonella parvula Strain A-G.I. was tested in the proteolipid protein (PLP)-induced relapsing-remitting Experimental Autoimmune Encephalomyelitis (EAE) SJL mouse model. EAE was induced in SJL mice by injection of PLP139-151 emulsified in complete Freund’s adjuvant (CFA) on day 0. To test prophylactic treatment mice received a 10 mg oral daily dose of Veillonella parvula Strain A-G.I. or vehicle from day 0-42. To test therapeutic treatment mice received Veillonella parvula Strain A- G.I. or vehicle starting on the second day of disease. Clinical score was assessed daily from day 9-42 and statistical analyses of mean maximum score (MMS), and relapse incidence were performed. Histological analysis was carried out to assess inflammation and demyelination in spinal cord. Small intestine tissue was assessed by RNA sequencing and gene set enrichment analysis (GSEA). Veillonella parvula Strain A has been deposited as ATCC Accession Number PTA-125691.
[468] RNA from day 42 duodenal tissue (from therapeutic treatment mice) was sequenced to a target depth of 20 million paired-end reads with Illumina 2x150 technology and processed using STAR and RSEM. Data normalization used trimmed mean of M-values (TMM), with generalized linear models for variance estimation using functions from empirical analysis of gene expression. P-values were adjusted to control false discovery rate (FDR) in multiple testing (Benjamini and Hochberg method). Differential gene expression was defined as a fold change of >1.5 and FDR-adj. p<0.05. Enrichment and over-representation analyses were performed on datasets available through KEGG, gene ontology, EnrichR and MSigDB.
[469] Cell types found enriched amongst genes showing elevated expression with Veillonella parvula Strain A vs. vehicle: [470] Intestinal epithelial cell (IEC):
Ihh Zg16 Defa5 Gpx2 Reg3a Slc13a2 Retn lb Smim22 Fabpl Mepla Mogat2 Guca2b Spinkl Tm4sf5 Acsl5 Reg3g Mptxl Adipoq Aoc1 Cfd Adamded Nr1i3 Rbp2 Fabp2 Tmem82 Dgatl Fahdl Pla2g12b Dgat2 Gstm3 Ccl25 Enpp7 Slc39a5 Anpep Casp6 Khk Lypd8 Aqp8 Slc23a1 Smlrl Slc51b Cfb
[471] Immune cell:
B cells:
Eaf2 Cd19 Tnfrsf13b Tnfrsf17 Stapl Cd79a Ighd Cd79b Mbl2 Map4k1 Fcrll Blk Ikzf3 Corol a T nfrsf 13c Vpreb3 Hvcnl Pou2af1 Klhie Tnfrsf9 Fcrla Dok3 Mzb1 Derl3 Irf4 Ccr10 Itgb7 Jchain Igld Igic2 Abcb9 Zbp1
B & T cell: Cd69 Gpr174 Ptprcap Cd52 H2rg Ltb Traf3ip3 Slamf6 Myolg Gmfg S1pr4 1116 Ikzfl Arhgap25 T cell:
Figure imgf000121_0001
Trbcl Prf1 Cd3d Cd6 Gimap7 Cd5 Trac Zap70 Lat Trbc2 Lek Skapl Septin 1 Foxp3 Lag3 Tnfrsf18 Izumolr
Myeloid Cell: CledOa Ncf2 Ptafr S100a8 Hk3 Itgam Csf2rb Tyrobp Spi1 Aif1 Lst1 Lrrc25 Trafl Pdcd1lg2 Rab30 Ryr1 Gpr132 Lgals3 Ccl22 Samsnl Ccl24 Slc7a7 1133 Srgn Gplbb Cyp2u1 Tmem71 B, T & Myeloid Cell:
Cytip
Cd48
Itgal
Cd53
Parvg
Dok2
Bin2
Sash3
[472] Veillonella parvulci Strain A-G.I. elevated genes with known function in immune cell migration to the gut, immune modulation, and intestinal homeostasis are provided in Table 2.
Table 2: Summary of Gene Set Enrichment Analysis for Veillonella parvula
Strain A-G.I. vs. Vehicle
Figure imgf000122_0001
Example 6: Oral dosing of Veillonella Strain A-G.L does not inhibit the immune system under non-inflammatory conditions
[473] Oral dosing prior to or following KLH DTH response: On day -9 or -5, "Veillonella Strain A-G.L 8 day pre -immunization” and "Veillonella Strain A-G.L 4 day pre-immunization,” respectively, mice groups were dosed for 4 or 8 days, respectively, with Veillonella Strain A-G.L PO. On day 0, all groups were immunized with KLH emulsified with Complete Freund’s Adjuvant. On day 5, “vehicle,” “dexamethasone,” and "Veillonella Strain A-G.I. post-immunization” mice groups were immunized with KLH emulsified with Complete Freund’s Adjuvant and dosed with sucrose vehicle, dexamethasone (Img/kg), or Veillonella Strain A-G.I for 4 days. On day 8, baseline ear thickness was measured using calipers, then all mice were challenged by intradermal ear injection with KLH. After 24 hours, the change in ear thickness was evaluated and compared to baseline measurements. Veillonella Strain A-G.I. powder was dosed in sucrose at 10 mg/dose. Veillonella Strain A refers to Veillonella parvula Strain A, which has been deposited as ATCC Accession Number PTA-125691. Veillonella Strain A-G.I. refers to the gamma-irradiated form of the strain. Results are shown in Figure 21.
[474] CONCLUSION: Pre-dosing a KLH DTH model with Veillonella Strain A-G.I. for 4 or 8 days shows no efficacy. This indicates that Veillonella Strain A-G.I. does not suppress the immune system in the absence of inflammation but instead resolves an aberrant inflammatory response.
[475] Example 7: Induction of inflammation and subsequent dosing with Veillonella Strain A-G.L is sufficient to induce resolution of inflammation in a subsequent DTH response
[476] Dosing following induction of inflammation is sufficient to induce inflammatory resolution in a subsequent KLH DTH challenge.
[477] On day -5, ‘IFA-CFA’ and ‘CFA-IFA’ mice were immunized by subcutaneous injection with PBS emulsified with Complete or Incomplete Freund’s Adjuvant (CFA or IFA, respectively) (see Petrovsky N. & Aguilar JC., 2004. Vaccine adjuvants: Current state and future trends. Immunol Cell Biol. 82(5): 488-96). On Day 5, mice were dosed for 4 days with PBS or Veillonella Strain A-G.I. PO. 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. Veillonella Strain A-G.I. powder was dosed in sucrose at lOmg/dose. Veillonella Strain A refers to Veillonella parvula Strain A which has been deposited as ATCC Accession Number PTA-125691. Veillonella Strain A-G.I. refers to the gammairradiated form of the strain. Results are shown in Figure 22.
[478] CONCLUSION: CFA (containing TLR agonists and heat-killed Mycobacterium tuberculosis)' and IFA (containing TLR agonists) have been shown to drive Thl and Th2 immune responses, respectively. Moreover, IFA immunization induces an inflammatory response that is absent of any antigen. These data show that dosing IFA- or CFA- inflamed mice with Veillonella Strain A-G.I. prior to KLH-CFA immunization and ear challenge reduces KLH-specific inflammatory responses.
[479] Example 8: Transfer of CD4+ T cells from KLH-CFA immunized mice dosed with Veillonella Strain A-G.I. into untreated KLH-CFA immunized mice can reduce inflammation following ear challenge.
[480] CD4+ T cells from KLH-CFA immunized and Veillonella Strain A-G.7. -dosed mice can confer immune resolution function.
[481] On day 0, donor mice were immunized with KLH emulsified with Complete Freund’s Adjuvant. On day 5, mice were dosed with sucrose vehicle or Veillonella Strain A-G.I. PO for 4 days and a second set of recipient mice were immunized with KLH emulsified with Complete Freund’s Adjuvant. On day 9, brachial, axillary and inguinal lymph nodes, and spleens were isolated from each group, pooled, 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 IxlO8 cells/mL in PBS. After enrichment, IxlO7 CD4+ T cells were then transferred IP into recipient mice. On day 12, 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. Veillonella Strain A-G.I. powder was dosed in sucrose at 10 mg/dose. Veillonella Strain A refers to Veillonella parvula Strain A which has been deposited as ATCC Accession Number PTA-125691. Veillonella Strain A-G.I. refers to the gamma-irradiated form of the strain. Results are shown in Figure 23. [482] CONCLUSION: Transfer of CD4+ T cells from mice immunized with KLH-CFA and dosed with Veillonella Strain A-G.I. is sufficient for reducing inflammation in a KLH-DTH model in recipient mice that had not been dosed with Veillonella Strain A-G.I.
[483] Example 9: Depletion of B cells during or post-dosing of Veillonella Strain A- G.L in the DTH model inhibits efficacy of immune resolution
[484] Depletion of B cells during a DTH model dosed with Veillonella Strain A-G.I. : On day 0, mice were immunized with KLH emulsified with Complete Freund’s Adjuvant.
Mice were also treated with 100 pL 2 mg/mL anti-CD20 antibody or isotype control by IP injection on days 0, 3, 6, and 9. On day 5, mice were treated for 4 days with sucrose vehicle PO, Veillonella Strain A-G.I. PO, or dexamethasone (1 mg/kg) IP. On day 11, 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. Veillonella Strain A-G.I. powder was dosed in sucrose at 10 mg/dose. Veillonella Strain A refers to Veillonella parvula Strain A which has been deposited as ATCC Accession Number PTA-125691. Veillonella Strain A-G.I. refers to the gamma-irradiated form of the strain.
[485] Depletion of B cells in a DTH model after dosing with Veillonella Strain A-G.I:. On day 0, mice were immunized with KLH emulsified with Complete Freund’s Adjuvant. On day 5, mice were treated for 4 days with vehicle sucrose PO, Veillonella Strain A-G.I. PO, or dexamethasone (1 mg/kg) IP. Mice were then treated with 100 pL 2 mg/mL anti- CD20 antibody or isotype control antibody by IP injection on days 9 and 11. On day 11, 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. Veillonella Strain A-G.I. powder was dosed in sucrose at 10 mg/dose. Results are shown in Figure 24.
[486] CONCLUSION: Depletion of B cells inhibits efficacy of Veillonella Strain A-G.I. in the reduction of inflammation of the KLH-DTH response. Depletion of B cells either during the entire study or after dosing inhibits efficacy. This suggests that B cells are critical for the oral microbe-mediated resolution of inflammation. [487] Example 10: Combination of Veillonella Strain A-G.I. and anti-IL-6 increases inflammation resolution in a KLH-DTH model
[488] Treatment of a KLH-DTH model with Veillonella Strain A-G.I. and anti -IL-6: On day 0, mice were immunized with KLH emulsified with Complete Freund’s Adjuvant. Mice were also treated with 100 L anti-IL-6 antibodies (1 or 0.25 mg/mL) or isotype control (2 mg/mL) by IP injection on days 0, 3, and 6. On day 5, mice were treated for 4 days with vehicle sucrose PO, Veillonella Strain A-G.I. PO, or dexamethasone (1 mg/kg) IP. 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. Veillonella Strain A- G.I. powder was dosed in sucrose at 10 mg/dose. Veillonella Strain A refers to Veillonella parvula Strain A which has been deposited as ATCC Accession Number PTA-125691. Veillonella Strain A-G.I. refers to the gamma-irradiated form of the strain. Results are shown in Figure 25.
[489] CONCLUSION: Treatment with both Veillonella Strain A-G.I. and anti-IL-6 (1 mg/mL) decreases ear swelling more compared to monotherapy. This suggests that the combination of both Veillonella Strain A-G.I. and anti-IL-6 provides an additive reduction of inflammation in the KLH DTH model.
Incorporation by Reference
[490] Each publication and patent mentioned herein is hereby incorporated by reference in its entirety. In case of conflict, the present specification, including any definitions herein, will control.
Equivalents
[491] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:
1. A method of inducing an immune effect in a human subject, the method comprising orally administering to the human subject a dose of a bacterial composition comprising about 3 x IO10 to about 1.5 x 1012 total cells of a Veillonella parvulci strain, wherein the Veillonella parvula strain is a strain comprising at least 95% sequence identity to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of Veillonella parvula strain A (ATCC Deposit Number PTA-125691).
2. A method of treating dysbosis, the method comprising orally administering to the human subject a dose of about 3 x IO10 to about 1.5 x 1012 total cells of a Veillonella parvula strain, wherein the Veillonella parvula strain is a strain comprising at least 95% sequence identity to the nucleotide sequence (e.g., genomic sequence, 16S sequence, CRISPR sequence) of Veillonella parvula strain A (ATCC Deposit Number PTA- 125691).
3. The method of claim 1 or 2, wherein the Veillonella parvula are Veillonella parvula strain A (ATCC Deposit Number PTA-125691).
4. The method of any one of claims 1-3, wherein the Veillonella parvula are formulated in one or more capsules or tablets or mini -tablets.
5. The method of any one of claims 1-4, wherein the capsules or tablets or minitablets are enteric coated.
6. The method of any one of claims 1-5, wherein the bacterial composition comprises about 3 x IO10 total cells of Veillonella parvula.
7. The method of any one of claims 1-6, wherein the bacterial composition comprises about 4.5 x IO10 total cells of Veillonella parvula.
8. The method of any one of claims 1-6, wherein the bacterial composition comprises about 1.5 x 1011 total cells of Veillonella parvula.
9. The method of any one of claims 1-6, wherein the bacterial composition comprises about 7.5 x 1011 total cells of Veillonella parvula.
10. The method of any one of claims 1-6, wherein the bacterial composition comprises about 1.5 x 1012 total cells of Veillonella parvula.
11. The method of any one of claims 1-10, wherein the bacterial composition is administered at least once daily.
12. The method of any one of claims 1-11, wherein the bacterial composition is administered once daily.
13. The method of claim 1-11, wherein the bacterial composition is administered twice daily.
14. The method of any one of claims 1-13, wherein the immune effect comprises an increase in expression of an anti-inflammatory cytokine by an immune cell.
15. The method of claim 14, wherein the anti-inflammatory cytokine is IL- 10 or IL- 27.
16. The method of claim 14, wherein the anti-inflammatory cytokine is IL-10.
17. The method of any one of claims 1-13, wherein the immune effect comprises a decrease in expression of a pro-inflammatory cytokine by an immune cell.
18. The method of claim 17, wherein the pro-inflammatory cytokine is a Th 1 cytokine.
19. The method of claim 17, wherein the pro-inflammatory cytokine is a Th2 cytokine.
20. The method of any one of claims 17-19, wherein the pro-inflammatory cytokine is IL-ip, IL-6, TNF-a, IL-5, IL-4, IL-13, IL-17, and/or IL-8.
21. The method of claim 20, wherein the pro-inflammatory cytokine is IL-5, IL-4, IL- 13, or IL-17.26. The method of any one of claims 18-25, wherein the immune cell is a peripheral blood mononuclear cell (PBMC), a dendritic cell, or a macrophage.
22. The method of any one of claims 1-13, wherein the immune effect comprises elevating expression of a gene provided in Example 5.
23. The method of claim 22, wherein gene is Spinkl, Tm4sf5, and/or Aocl .
24. The method of any one of claims 1-13, wherein the immune effect comprises elevating expression of a gene provided in Table 2.
25. The method of claim 24, wherein the gene showing elevated expression is associated with lymphocyte migration, gut-homing, and adhesion (e.g., Ccl25, CcrlO, Ccl22, Ccl24, Itgb7, Itgal, and/or Itgam).
26. The method of claim 24, wherein the gene showing elevated expression is associated with T cell lineage maturation and activation (e.g., Cd69, Icos, Il2rg, Cd3d, Trbcl, Trbc2, Trac, Lat, Zap70, Lek, and/or Cd2).
27. The method of claim 24, wherein the the gene showing elevated expression is associated with B cell lineage maturation and activation (e.g., CD19, CD79a, CD79b, CD69, Ighd, Fcrll, Blk, Ikzf3, Tnfrsfl3c, Jchain, Iglcl, and/or Iglc2) . T
28. The method of claim 24, wherein the gene showing elevated expression is associated with immune modulation (e.g., Foxp3, Lag3, Traf3ip3, Slamf6, 1133, Cd5, Adamdecl, and/or Nrli3).
29. The method of claim 24, wherein the gene showing elevated expression is associated with intestinal epithelial cell homeostasis (barrier, metabolic, absorption) (e.g., Gpx2, Gstm3, Aqp8, Guca2b, Adipoq, Dgatl, Dgat2, Slc23al, and/or Slc51 b).
30. The method of claim 24, wherein the gene showing elevated expression is associated with host-protective pathways (e.g., Zgl6, Def5a, Reg3a, Retnlb, Reg3g, Cfd, Lypd8, and/or Casp6).
31. The method of any one of claims 1-30, wherein the subject has an immune disorder.
32. The method of claim 31, wherein the immune disorder is atopic dermatitis, psoriasis, or asthma.
33. The method of any one of claims 1-32, wherein the Veillonella parvula are non- viable.
34. The method of any one of claims 1-32, wherein the Veillonella parvula are gamma irradiated.
35. The method of any one of claims 1-32, wherein the immune effect comprises a reduced frequency of infiltrating inflammatory cells.
129
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