Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS OF TREATING A TH2-MEDIATED
CONDITION USING PREVOTELLA
RELATED APPLICATIONS
[1] This application claims a right of priority to and the benefit of the
filing date of
U.S. Provisional Application No. 62/864,886, filed on June 21, 2019, and U.S.
Provisional
Application No. 62/910,920, filed on October 4, 2019, both of which are hereby
incorporated by
reference in their entirety.
BACKGROUND
[2] Helper T (TH) cells (also referred to as CD4+ T cells) coordinate
adaptive immune
responses through the secretion of cytokines that influence the activities of
other immune cells,
including B cells, other T cells, eosinophils, basophils, mast cells, and
macrophages.
[3] When a naive TH cell becomes activated it differentiates into one of
various TH
cell subsets, each of which is responsible for coordinating an aspect of the
immune response
through the secretion of a certain set of cytokines. For example, TH1 cells
secrete IFN-y and are
responsible for enhancing cell-mediated immune responses against viruses and
intracellular
bacteria. TH2 cells, on the other hand, secrete IL-4, IL-5, IL-9, IL-10, IL-
13, and IL-25 and are
responsible, inter alia, for enhancing a humoral immune response against
extracellular parasites,
such as helminths. TH17 cells secrete IL-17 and mediate inflammatory
responses, such as
responses against fungi (among other pathogens).
[4] The TH subset into which a TH cell differentiates upon activation is
determined, in
part, by the presence of certain polarizing cytokines. For example, if a naive
TH cell is activated
in the presence of IFN-y, it will likely differentiate into a TH1 cell. On the
other hand, if a naive
TH cell is activated in the presence of IL-4, it will likely differentiate
into a TH2 cell. Thus,
secretion of IFN-y by activated TH1 cells can lead to other TH cells to
polarize to the TH1 subset,
whereas secretion of IL-4 by activated TH2 cells can lead to other TH cells to
polarize to the TH2
subset. Because of this cascading effect, TH cell differentiation can have
widespread effects to a
person's global immune response. Indeed, a number of diseases and disorders
have been
associated with TH2 immune responses, including (but not limited to) allergies
(e.g., food
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allergies, insect-induced allergies, drug allergies, or allergic rhinitis),
asthma, and atopic
dermatitis.
[5] Thus, there is a need for new compositions and methods for promoting a
TH1
immune response and/or inhibiting a TH2 immune response in order to treat such
TH2-mediated
conditions.
SUMMARY
[6] As disclosed herein, pharmaceutical compositions including bacteria of
the genus
Prevotella have therapeutic effects and are useful for the treatment and/or
prevention (i.e., for
treatment, for prevention, or for both treatment and prevention) of one or
more TH2-mediated
conditions. Such TH2-mediated conditions include, but are not limited to,
allergic rhinitis,
asthma, atopic dermatitis, urticaria, angioedema, allergy (such as food
allergy, insect-induced
allergy, drug allergy), anaphylaxis, and eosinophilia.
[7] In some aspects, provided herein are methods for treating and/or
preventing a
TH2-mediated condition of a subject. In certain embodiments, the methods
include administering
to the subject a therapeutically effective amount of a pharmaceutical
composition that includes
bacteria of the genus Prevotella.
[8] In certain aspects, provided herein are pharmaceutical compositions
including
bacteria of the genus Prevotella that are useful for the treatment or
prevention of one or more
TH2-mediated conditions. Thus, in certain aspects, provided herein are
pharmaceutical
compositions including bacteria of the genus Prevotella for use in the
treatment or prevention of
a TH2-mediated condition.
[9] In certain aspects, provided herein are uses of a pharmaceutical
composition
including bacteria of the genus Prevotella for the preparation of a medicament
for the treatment
or prevention of a TH2-mediated condition.
[10] In certain embodiments, the TH2-mediated condition is an allergy,
atopic
dermatitis, asthma, urticaria, angioedema, anaphylaxis, eosinophilia, or a
combination thereof.
[11] In some embodiments TH2-mediated condition is an allergy. In certain
embodiments, the allergy is allergic rhinitis, a food allergy, an insect-
induced allergy, or a drug
allergy. In some embodiments, the allergy is a food allergy. In some
embodiments, the food
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allergy can include an allergy to eggs, milk, peanuts, tree nuts (e.g.,
walnuts), fish, shellfish,
wheat, soy, or a combination thereof.
[12] In some embodiments of any one of these aspects, the subject is a
human. In some
embodiments, the subject is a non-human animal.
[13] In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 99% of the total microbial content of the pharmaceutical
composition is bacteria of
the genus Prevotella or is biological material derived from bacteria of the
genus Prevotella.
[14] In certain embodiments of any one of the aspects disclosed herein, the
bacteria of
the genus Prevotella includes at least one protein having an amino acid
sequence selected from
SEQ ID NOs: 1 to 41. Additionally, or separately, in some embodiments, the
bacteria of the
genus Prevotella are substantially free of at least one or more of each
protein having an amino
acid sequence selected from SEQ ID NOs: 42 to 69.
[15] In some embodiments of any one of the aspects disclosed herein, the
bacteria of
the genus Prevotella include bacteria of the species Prevotella albensis,
Prevotella amnii,
Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella
bryantii, Prevotella buccae,
Prevotella buccahs, Prevotella copri, Prevotella dentalis, Prevotella
denticola, Prevotella
disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa,
Prevotella marshii,
Prevotella melaninogenica, Prevotella micans, Prevotella multiformis,
Prevotella nigrescens,
Prevotella orahs, Prevotella oris, Prevotella oulorum, Prevotella pa/lens,
Prevotella salivae,
Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella
jejuni, Prevotella
aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis,
Prevotella den tasini,
Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella
heparinolytica, Prevotella
loescheii, Prevotella muhisaccharivorax, Prevotella nanceiensis, Prevotella
oryzae, Prevotella
paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella
saccharolytica, Prevotella
scopos, Prevotella shahii, Prevotella zoogleoformans, Prevotella verorahs, or
a combination
thereof. In some embodiments, the bacteria of the genus Prevotella can include
bacteria of the
species Prevotella histicola.
[16] In some embodiments, the bacteria of the genus Prevotella can includes
a strain
that has 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% (e.g., at least 99.1%, at
least 99.2%, at least
99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at
least 99.8%, at least
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99.9%, or 100%) genomic, 16S ribosomal ribonucleic acid, and/or clustered
regularly
interspaced short palindromic repeats sequence identity with Prevotella
histicola strain B 50329
(NRRL accession: B 50329). In some embodiments, the bacteria of the genus
Prevotella include
Prevotella histicola strain B 50329 (NRRL accession: B 50329).
[17] In some embodiments, the Prevotella bacteria is a strain of Prevotella
bacteria
comprising a protein listed in Table 1 and/or a gene encoding a protein listed
in Table 1. In some
embodiments, the Prevotella bacteria is a strain of Prevotella bacteria free
or substantially free
of a protein listed in Table 2 and/or a gene encoding a protein listed in
Table 2. In some
embodiments, the Prevotella histicola bacteria is a strain of Prevotella
histicola bacteria
comprising a protein listed in Table 1 and/or a gene encoding a protein listed
in Table 1. In some
embodiments, the Prevotella histicola bacteria is a strain of Prevotella
histicola bacteria free or
substantially free of a protein listed in Table 2 and/or a gene encoding a
protein listed in Table 2.
[18] In certain embodiments, the pharmaceutical composition comprises a
single
species of Prevotella bacteria. In some embodiments, the pharmaceutical
composition comprises
a plurality of species of Prevotella bacteria (e.g., 2, 3, 4, 5 species). In
certain embodiments, the
pharmaceutical composition comprises a single strain of Prevotella bacteria.
In some
embodiments, the pharmaceutical composition comprises a plurality of strains
of Prevotella
bacteria (e.g., 2, 3, 4, 5 strains). In certain embodiments, the
pharmaceutical composition
comprises a single strain of Prevotella histicola bacteria. In some
embodiments, the
pharmaceutical composition comprises a plurality of strains of Prevotella
histicola bacteria (e.g.,
2, 3, 4, 5 strains).
[19] In some embodiments, the bacteria of the genus Prevotella of the
pharmaceutical
composition include live, killed, or attenuated bacteria. In some embodiments,
the bacteria of the
genus Prevotella of the pharmaceutical composition include live bacteria. In
some embodiments,
the pharmaceutical composition includes gamma-irradiated, UV irradiated, heat
inactivated (e.g.,
at 50 C for two hours, at 90 C for two hours), acid treated, or oxygen sparged
(e.g., at 0.1 vvm
for two hours) bacteria. In certain embodiments, the bacteria of the genus
Prevotella of the
pharmaceutical composition includes lyophilized bacteria. In some embodiments,
the
pharmaceutical composition further includes a pharmaceutically acceptable
excipient.
[20] In some embodiments of any one of the aspects disclosed herein, the
administration of the pharmaceutical composition to a subject can cause
decrease in the level of a
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TH2-protein and/or an mRNA encoding a TH2-protein in the subject (e.g., in a
mesenteric lymph
node or cervical lymph node thereof). For instance, in certain embodiments,
upon administration
of the pharmaceutical composition, the level of the TH2-protein and/or an mRNA
encoding a
TH2-protein becomes lower in the subject as compared the level of the TH2-
protein and/or an
mRNA encoding a TH2-protein before the administration of the pharmaceutical
composition. In
certain embodiments, the TH2-protein is interleukin-4 (IL-4), interleukin-5,
interleukin-13,
interleukin-19, interleukin-21, interleukin-31, interleukin-33, thymic stromal
lymphopoietin,
immunoglobulin Gl, immunoglobulin E, immunoglobulin A, or a combination
thereof. In certain
embodiments, the TH2-protein is IL-4, IL-13, IL-5, IL-31, IL-33 or a
combination thereof. In
certain embodiments, the TH2-protein is IL-13, IL-5, IL-31 or a combination
thereof. In certain
embodiments, the TH2-protein is IL-13. In certain embodiments, the TH2-protein
is IL-31. In
certain embodiments, the TH2-mRNA is 114, 115, IL 31, 1133, Tslp, Cc19, Ccr4
or a combination
thereof. In certain embodiments, the TH2-mRNA is 114, 115, 1133, 1117a, Il lb,
Tslp or a
combination thereof. In certain embodiments, the TH2-mRNA is 114, 115, Tslp,
Ccr4, 1119 or a
combination thereof. In certain embodiments, the TH2-mRNA is 115, 1131, and
Ccr4 or a
combination thereof. In certain embodiments, the TH2-mRNA is 115.
[21] In some embodiments of any one of the aspects disclosed herein, the
administration of the pharmaceutical composition to a subject can cause a
decrease in the level of
a mast cell related protein and/or an mRNA encoding a mast cell related
protein in the subject
(e.g., in the jejunum thereof). In certain embodiments, the mast cell mRNA is
Mcptl . For
instance, in certain embodiments, upon administration of the pharmaceutical
composition, the
level of the mast cell related protein and/or an mRNA encoding the mast cell
related protein
becomes lower in the subject as compared the level of the mast cell related
protein and/or an
mRNA encoding the mast cell related protein before the administration of the
pharmaceutical
composition.
[22] In some embodiments of any one of the aspects disclosed herein, the
administration of the pharmaceutical composition to a subject can cause an
increase in the level
of IL-10 in the subject (e.g., in the spleen or a mesenteric lymph node
thereof). For instance, in
certain embodiments, upon administration of the pharmaceutical composition,
the level of IL-10
and/or mRNA encoding IL-10 becomes higher in the subject (e.g., in the spleen
or a mesenteric
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lymph node thereof) as compared the level of IL-10 and/or mRNA encoding IL-10
before the
administration of the pharmaceutical composition.
[23] In some embodiments of any one of the aspects disclosed herein, an
additional
therapeutic agent is administered to the subject. In some embodiments, the
additional agent is an
anti-inflammatory agent. In certain embodiments the additional therapeutic
agent is a
corticosteroid, an antihistamine, a mast cell stabilizer, a decongestant, a
leukatriene receptor
antagonist, an antibody, or a combination thereof For example, the additional
therapeutic agent
can be acrivastine, albuterol, alimemazine, ANB020, andbenralizumab,
antazoline, AS1517499,
AS1810722, astemizole, azathioprine, AZD1981, azelastine, beclomethasone,
bepotastine,
betamethasone, BI671800, bilastine, bromazine, brompheniramine, budesonide,
budesonide-
formoterol, carbinoxamine, cetirizine, chlorcyclizine, chloropyramine,
chlorphenamine,
ciclesonide, ciclosporin, cimetidine, clemastine, cortisone, crisaborole.,
cromolyn, cyclizine,
cyproheptadine, CYT003., dapsone, desloratadine, dexamethasone,
dexbrompheniramine,
dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine,
dupilumab,
ebastine, ephedrine, epinephrine, famotidine, fexofenadine, flunisolide,
fluticasone, fluticasone
furoate, fluticasone propionate, fluticasone-salmeterol, formoterol,
formoterol-mometasone,
GSK3772847, hydroxychloroquine, hydroxyzine, imiquimod, interferongamma-lb,
ipratropium,
ketotifen, lebrikizumab, levalbuterol, levocabastine, levocetirizine,
levomethamphetamine,
loratadine, meclizine, meclofenamate sodium, mepolizumab, mepyramine,
methotrexate,
methylprednisolone, mizolastine, MK-886, mometasone, montelukast,
mycophenolate,
mycophenolatemofetil, naphazoline, 00000459, olopatadine, omalizumab,
orphenadrine,
oxymetazoline, pheniramine, phenylephrine, phenylpropanolamine, pimecrolimus,
prednisolone,
prednisone, promethazine, propylhexedrine, pseudoephedrine, quifenadine,
ranitidine,
resiquimod, resulizumab, RG6149/AMG282, rupatadine, salmeterol, SB010,
sirolimus,
sulfasalazine, synephrine, tacrolimus, terfenadine, tetryzoline, theophylline,
tixocortol,
tralokinumab, tramazoline, triamcinolone, triamcinolone acetonide,
tripelennamine, triprolidine,
vitaminD, xylometazoline, YM-341619, zafirlukast, zileuton, or a combination
thereof. In certain
embodiments, the additional therapeutic agent can be included as a part of the
pharmaceutical
composition or it can be separate.
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[24] In some embodiments, the pharmaceutical composition is administered by
oral,
intravenous, subcutaneous, intradermal, or intraperitoneal administration. In
some embodiments,
the pharmaceutical composition is administered by oral administration.
[25] In certain embodiments, the pharmaceutical composition can include a
total
protein amount of at least 5 mg (e.g., at least 10 mg, 20 mg, 30 mg, 40 mg, 50
mg, 60 mg, 70
mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170
mg, 180
mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg,
280 mg, 290
mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg,
390 mg, 400
mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg,
500 mg, 510
mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg,
610 mg, 620
mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg,
720 mg, 730
mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg,
830 mg, 840
mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, or 900 mg,) and no more than 900
mg (e.g., no
more than 890 mg, 880 mg, 870 mg, 860 mg, 850 mg, 840 mg, 830 mg, 820 mg, 810
mg, 800
mg, 790 mg, 780 mg, 770 mg, 760 mg, 750 mg, 740 mg, 730 mg, 720 mg, 710 mg,
700 mg, 690
mg, 680 mg, 670 mg, 660 mg, 650 mg, 640 mg, 630 mg, 620 mg, 610 mg, 600 mg,
590 mg, 580
mg, 570 mg, 560 mg, 550 mg, 540 mg, 530 mg, 520 mg, 510 mg, 500 mg, 490 mg,
480 mg, 470
mg, 460 mg, 450 mg, 440 mg, 430 mg, 420 mg, 410 mg, 400 mg, 390 mg, 380 mg,
370 mg, 360
mg, 350 mg, 340 mg, 330 mg, 320 mg, 310 mg, 300 mg, 290 mg, 280 mg, 270 mg,
260 mg, 250
mg, 240 mg, 230 mg, 220 mg, 210 mg, 200 mg, 190 mg, 180 mg, 170 mg, 160 mg,
150 mg, 140
mg, 130 mg, 120 mg, 110 mg, 100 mg, 90 mg, 80 mg, 70 mg, 60 mg, 50 mg, 40 mg,
30 mg, 20
mg, or 10 mg) (e.g., as determined by a Bradford assay, as determined by a BCA
assay). In
certain embodiments, the pharmaceutical composition can include a total
protein amount of
about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg,
about 70 mg,
about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130
mg, about 140
mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg,
about 200 mg,
about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about
260 mg, about
270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg,
about 330
mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg,
about 390 mg,
about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about
450 mg, about
460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg,
about 520
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mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg,
about 580 mg,
about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about
640 mg, about
650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg,
about 710
mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg,
about 770 mg,
about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about
830 mg, about
840 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg,
or about 900
mg (e.g., as determined by a Bradford assay, or as determined by a BCA assay).
[26] In some embodiments, the pharmaceutical composition can include a
total amount
of bacteria of the genus Prevotella of at least 5 mg (e.g., at least 6 mg, at
least 7 mg, at least 8
mg, at least 9 mg, at least 10 mg, at least 11 mg, at least 12 mg, at least 13
mg, at least 14 mg, at
least 15 mg, at least 16 mg, at least 17 mg, at least 18 mg, at least 19 mg,
or at least 20 mg) and
no more than 20 mg (e.g., no more than 19 mg, no more than 18 mg, no more than
17 mg, no
more than 16 mg, no more than 15 mg, no more than 14 mg, no more than 13 mg,
no more than
12 mg, no more than 11 mg, no more than 10 mg, no more than 9 mg, no more than
8 mg, no
more than 7 mg, no more than 6 mg, no more than 5 mg) (e.g., as determined by
a Bradford
assay, or as determined by a BCA assay). In some embodiments, the
pharmaceutical composition
can include a total amount of bacteria of the genus Prevotella of about 5 mg,
about 6 mg, about 7
mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13
mg, about 14
mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about
20 mg (e.g., as
determined by a Bradford assay, or as determined by a BCA assay).
[27] In certain embodiments, the 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 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 of the Prevotella
bacteria. In some embodiments, the pharmaceutical composition comprises no
more than 9 x
1011 total cells (e.g., no more than 1 x 1010 total cells, no more than 2 x
1010 total cells, no more
than 3 x 1010 total cells, no more than 4 x 1010 total cells, no more than 5 x
1010 total cells, no
more than 6 x 1010 total cells, no more than 7 x 1010 total cells, no more
than 8 x 1010 total cells,
no more than 9 x 1010 total cells, no more than 1 x 1011 total cells, no more
than 2 x 1011 total
cells, no more than 3 x 1011 total cells, no more than 4 x 1011 total cells,
no more than 5 x 1011
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total cells, no more than 6 x 1011 total cells, no more than 7 x 1011 total
cells, no more than 8 x
1011 total cells) of the Prevotella bacteria. In some embodiments, the
pharmaceutical
composition comprises about 6 x 109 total cells of the Prevotella bacteria. In
some embodiments,
the pharmaceutical composition comprises about 1.6 x 101 total cells of the
Prevotella bacteria.
In some embodiments, the pharmaceutical composition comprises about 8 x 1010
total cells of the
Prevotella bacteria. In some embodiments, the pharmaceutical composition
comprises about 1.6
x 1011 total cells the Prevotella bacteria. In some embodiments, the
pharmaceutical composition
comprises about 3.2 x 1011 total cells the Prevotella bacteria. In some
embodiments, the
pharmaceutical composition comprises about 8 x 1011 total cells of the
Prevotella bacteria. In
some embodiments, the pharmaceutical composition comprises about 1.6 x 1010 to
about 8 x 1011
total cells of the Prevotella bacteria. In some embodiments, the
pharmaceutical composition
comprises about 1.6 x 1010 to about 1.6 x 1011 total cells of the Prevotella
bacteria. In some
embodiments, the pharmaceutical composition comprises about 8 x 1010 to about
8 x 1011 total
cells of the Prevotella bacteria. In some embodiments, the pharmaceutical
composition
comprises about 1.6 x 1011 to about 8 x 1011 total cells of the Prevotella
bacteria.
[28] In certain embodiments, provided herein are solid dosage forms
comprising the
Prevotella 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 8 x 1010 total cells of the
Prevotella 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 8 x
1010 total cells) is
administered, e.g., once or twice daily to a subject. In some embodiments, 2
capsules (e.g., each
comprising about 8 x 1010 total cells) are administered, e.g., once or twice
daily to a subject. In
some embodiments, 4 capsules (e.g., each comprising about 8 x 1010 total
cells) are administered,
e.g., once or twice daily to a subject. In some embodiments, 10 capsules
(e.g., each comprising
about 8 x 1010 total cells) are administered, e.g., once or twice daily to a
subject. In some
embodiments, the Prevotella bacteria in the capsule are lyophilized (e.g., in
a powder).
[29] In some embodiments, the solid dosage form comprises a capsule. In
some
embodiments, the capsule is an enteric coated tablet. In some embodiments, the
capsule
comprises about 8 x 1010 total cells of the Prevotella bacteria (e.g., total
dose of a capsule or
plurality of capsules). In some embodiments, the capsule comprises about 1.6 x
1011 total cells of
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the Prevotella bacteria (e.g., total dose of a capsule or plurality of
capsules). In some
embodiments, the capsule comprises about 3.2 x 1011 total cells of the
Prevotella bacteria (e.g.,
total dose of a capsule or plurality of capsules). In some embodiments, the
capsule comprises
about 8 x 1011 total cells of the Prevotella bacteria (e.g., total dose of a
capsule or plurality of
capsules). In some embodiments, the Prevotella bacteria in the capsule are
lyophilized (e.g., in a
powder).
[30] 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 17mm in diameter. In some embodiments, the tablet
comprises about 8 x
1010 total cells of the Prevotella bacteria (e.g., total dose of a tablet or
plurality of tablets). In
some embodiments, the tablet comprises about 1.6 x 1011 total cells of the
Prevotella bacteria
(e.g., total dose of a tablet or plurality of tablets). In some embodiments,
the tablet comprises
about 3.2 x 1011 total cells of the Prevotella bacteria (e.g., total dose of a
tablet or plurality of
tablets). In some embodiments, the tablet comprises about 8 x 1011 total cells
of the Prevotella
bacteria (e.g., total dose of a tablet or plurality of tablets). In some
embodiments, the Prevotella
bacteria in the tablet are lyophilized.
[31] 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
1 mm to 4mm in diameter. In some embodiments, the mini-tablet (e.g., enteric
coated mini-tablet)
is a 1 mm 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 8
x 1010 total cells of the Prevotella bacteria (e.g., total dose of a plurality
of mini-tablets). In some
embodiments, the solid dosage form comprises mini-tablets that comprise about
1.6 x 1011 total
cells of the Prevotella bacteria (e.g., total dose of a plurality of mini-
tablets). In some
embodiments, the solid dosage form comprises mini-tablets that comprise about
3.2 x 1011 total
cells of the Prevotella bacteria (e.g., total dose of a plurality of mini-
tablets). In some
embodiments, the solid dosage form comprises mini-tablets that comprise about
8 x 1011 total
cells of the Prevotella bacteria (e.g., total dose of a plurality of mini-
tablets). In some
embodiments, the Prevotella bacteria in the mini-tablets are lyophilized.
[32] 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
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3, size 4, or size 5 capsule. In some embodiments, the capsule comprises a non-
enteric coating
(e.g., HPMC (hydroxyl propyl methyl cellulose) or 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 hydroxyl propyl methyl cellulose (HPMC). In
some
embodiments, the capsule comprises gelatin. In some embodiments, the mini-
tablets (e.g., enteric
coated mini-tablets) that comprise about 8 x 10" total cells of the Prevotella
bacteria are
contained in a capsule(s), wherein optionally the capsule comprises HPMC.
[33] In some embodiments, the pharmaceutical composition comprising
Prevotella
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 minitablet, a capsule,
a pill, or a powder; or a
combination of these forms (e.g., minitablets comprised in a capsule). In
certain embodiments,
the powder can comprise lyophilized bacteria.
[34] In certain embodiments of any one of the aspects disclosed herein, the
pharmaceutical composition is formulated as a solid dose form. In some
embodiments, the
pharmaceutical composition has a dose form selected from a tablet, a capsule,
a minitablet, a
powder, or a combination thereof. In some embodiments, the pharmaceutical
composition is
formulated as a suspension (e.g., in PBS). In some embodiments, pharmaceutical
composition
formulated for oral, intravenous, subcutaneous, intradermal, and/or
intraperitoneal
administration. In some embodiments, pharmaceutical composition is formulated
for oral
administration.
[35] In some embodiments, the pharmaceutical composition comprises
lyophilized
Prevotella bacteria. In certain embodiments, the lyophilized Prevotella
bacteria is formulated
into a solid dose form, such as a tablet, a minitablet, a capsule, a pill, or
a powder. In some
embodiments, the lyophilized Prevotella bacteria is contained in a capsule. In
some
embodiments, the lyophilized Prevotella bacteria is resuspended in a solution.
[36] In certain embodiments, provided herein are methods of treating a
subject who
has a TH2-mediated condition, the method comprising administering to the
subject a
pharmaceutical composition described herein.
[37] In certain embodiments, provided herein are methods of treating a
subject who
has atopic dermatitis, the method comprising administering to the subject a
pharmaceutical
composition described herein.
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[38] In certain embodiments, provided herein are methods of treating a
subject who
has a food allergy, the method comprising administering to the subject a
pharmaceutical
composition described herein.
[39] In some embodiments, the methods provided herein further comprise
administering to the subject an antibiotic. In some embodiments, the methods
further comprise
administering to the subject one or more other therapies for the condition. In
some embodiments,
the methods further comprise the administration of another therapeutic
bacterium.
BRIEF DESCRIPTION OF THE FIGURES
[40] Figure 1A is a graph showing that oral administration of Prevotella
histicola
significantly reduced ear swelling at 8h post FITC ear challenge in a FITC-
driven contact
hypersensitivity model. (Ordinary one-way ANOVA; p<0.001 for dexamethasone; p:
0.0031 for
P. histicola.)
[41] Figure 1B is a series of graphs showing that Prevotella histicola
treatment
reduced gene expression for 114, 115, 1133, 1117a, Il lb, and Tslp in the
ears.
[42] Figure 2 is a graph showing that oral administration of Prevotella
histicola
significantly reduced ear swelling at 8h post FITC ear challenge in a FITC-
driven contact
hypersensitivity model. (Ordinary one-way ANOVA; Strain 1 is a strain of
another species of
Prevotella; and Strain 2 is a strain of a different genus than Prevotella.)
[43] Figure 3A shows a study design for a model of atopic dermatitis-
associated food
allergy.
[44] Figure 3B is a graph showing that oral administration of Prevotella
histicola
significantly reduced ear inflammation in an atopic dermatitis-associated food
allergy model
using ovalbumin (OVA) food allergen model. (Ordinary one-way ANOVA; p: 0.0009
for
tofacitinib; p <0.0001 for P. histicola; p: 0.0047 for Strain B; Strain A is a
strain of a different
genus than Prevotella; and Stain B is another strain of Prevotella histicola.)
[45] Figure 3C is a pair of graphs showing IgG1 and IgE levels in mice
administered
vehicle, tofacitinib, Prevotella histicola, or two other bacterial species
("Strain A" and "Strain
B") in an atopic dermatitis-associated food allergy model using ovalbumin
(OVA) food allergen
(Strain A is a strain of a different genus than Prevotella; and Stain B is
another strain of
Prevotella histicola.).
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[46] Figure 3D is a series of graphs showing IL-4, IL-5, IL-13, IL-10, IL-
31 and IL-33
levels in mice administered vehicle, tofacitinib, Prevotella histicola, or two
other bacterial
species ("Strain A" and "Strain B") in an atopic dermatitis-associated food
allergy model using
ovalbumin(OVA) food allergen (Strain A is a strain of a different genus than
Prevotella; and
Stain B is another strain of Prevotella histicola.).
[47] Figure 4A shows a study design for a mouse model testing an atopic
dermatitis-
associated food allergy model using ovalbumin (OVA) or CPE (complete peanut
extract) as food
allergen.
[48] Figure 4B is a graph showing that oral administration of Prevotella
histicola
significantly reduced ear inflammation in both an OVA-driven and a peanut
allergen (CPE)-
driven atopic dermatitis-associated food allergy model. (Ordinary one-way
ANOVA; p: <0.0001
for tofacitinib; p: 0.0024 for anti-IL-4; p: 0.0163 for Strain A; p<0.0001 for
P. histicola (OVA
group); p: <0.0001 for CPE group (vehicle versus P. histicola); Strain A is a
strain of a different
genus than Prevotella.)
[49] Figure 4C is a pair of graphs showing anti-OVA IgG1 and anti-OVA IgE
levels
in mice administered vehicle, tofacitinib, anti-IL-4, Prevotella histicola, or
another bacterial
species ("Strain A") in an atopic dermatitis-associated food allergy model
using
ovalbumin(OVA) food allergen (Strain A is a strain of a different genus than
Prevotella).
[50] Figure 4D is a series of graphs showing IL-4, IL-5, IL-13, IL-10 and
IL-31 levels
in mice administered vehicle, tofacitinib, anti-IL-4, Prevotella histicola, or
another bacterial
species ("Strain A") in an atopic dermatitis-associated food allergy model
using
ovalbumin(OVA) food allergen and vehicle and Prevotella histicola in a peanut
allergen (CPE)-
driven contact hypersensitivity model (Strain A is a strain of a different
genus than Prevotella).
[51] Figure 4E is a series of graphs showing ear IL-4, IL-5, TSLP, CCR4 and
IL-19
RNA levels in mice administered vehicle, tofacitinib, anti-IL-4, Prevotella
histicola, or another
bacterial species ("Strain A") in an atopic dermatitis-associated food allergy
model using
ovalbumin(OVA) food allergen and vehicle and Prevotella histicola in an atopic
dermatitis-
associated food allergy model using peanut allergen (CPE) as the food allergen
(Strain A is a
strain of a different genus than Prevotella).
[52] Figure 5 shows an MC903 model of type 2 skin inflammation and food
allergy
induction.
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[53] Figure 6A shows a study design for a cutaneous sensitization peanut
allergy
model.
[54] Figure 6B shows a study design for a cutaneous sensitization peanut
allergy
model.
[55] Figure 6C shows a study design for a cutaneous sensitization peanut
allergy
model.
[56] Figure 6D is a graph showing that oral administration of Prevotella
histicola
significantly reduced ear swelling in a cutaneous sensitization peanut allergy
model.
[57] Figure 6E is a pair of graphs showing total IgE and anti-peanut IgE
levels on the
day of challenge in mice administered vehicle, tofacitinib ("tofa"), or
Prevotella histicola in a
cutaneous sensitization peanut allergy model.
[58] Figure 6F is a pair of graphs showing total IgG1 and anti-peanut IgG1
levels on
the day of challenge in mice administered vehicle, tofacitinib ("tofa"), or
Prevotella histicola in a
cutaneous sensitization peanut allergy model.
[59] Figure 6G is a pair of graphs showing total IgE and anti-peanut IgE
levels 2
weeks post-challenge in mice administered vehicle, tofacitinib ("tofa"), or
Prevotella histicola in
a cutaneous sensitization peanut allergy model.
[60] Figure 6H is a pair of graphs showing total IgG1 and anti-peanut IgG1
levels 2
weeks post-challenge in mice administered vehicle, tofacitinib ("tofa"), or
Prevotella histicola in
a cutaneous sensitization peanut allergy model.
[61] Figure 61 is a pair of graphs showing total IgA and anti-peanut IgGA
levels 2
weeks post-challenge in mice administered vehicle, tofacitinib ("tofa"), or
Prevotella histicola in
a cutaneous sensitization peanut allergy model.
[62] Figure 7A is a schematic showing a FITC-CHS model experimental design.
[63] Figure 7B is a series of graphs showing IL-13, IL-4, IL-5, IL-31, and
IL-33 levels
in ex vivo re-stimulated mesenteric lymph node (mLN) cells from mice that were
administered
vehicle, dexamethasone or Prevotella histicola in the FITC-CHS model.
[64] Figure 7C is a series of graphs showing IL-13, IL-4, IL-5, IL-31, and
IL-33 levels
in ex vivo re-stimulated ear draining cervical lymph node (cLN) cells from
mice that were
administered vehicle, dexamethasone or Prevotella histicola in the FITC-CHS
model.
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[65] Figure 8A is a schematic showing the MC903 driven atopic dermatitis
model
experimental design.
[66] Figure 8B is a pair of graphs showing ear measurements over time (left
panel)
and ear inflammation on day 14 (right panel) in mice treated with vehicle,
tofacitinib, or
Prevotella histicola in the MC903 driven atopic dermatitis model.
[67] Figure 8C is a series of graphs showing Tslp, 115, 1131, 114, Cc119,
and Ccr4
transcript levels in the ear tissue from mice treated with vehicle,
tofacitinib, or Prevotella
histicola in the MC903 driven atopic dermatitis model.
[68] Figure 8D is a graph showing mast cell related gene (Mcpt 1)
transcript levels in
the jejunum from mice treated with vehicle, tofacitinib, or Prevotella
histicola in the MC903
driven atopic dermatitis model.
[69] Figure 8E is a pair of graphs showing IL-10 levels in mesenteric lymph
nodes
(left panel) and spleen (right panel) in mice treated with vehicle,
tofacitinib, or Prevotella
histicola in the MC903-driven atopic dermatitis model.
DETAILED DESCRIPTION
[70] As disclosed herein, pharmaceutical compositions including bacteria of
the genus
Prevotella have therapeutic effects and are useful for the treatment or
prevention (i.e., for
treatment, for prevention, or for both treatment and prevention) of one or
more TH2-mediated
conditions. Such TH2-mediated conditions include, but are not limited to,
allergic rhinitis,
asthma, atopic dermatitis, urticaria, angioedema, allergy (e.g. food allergy,
insect-induced
allergy, or drug allergy), anaphylaxis, and eosinophilia. The pharmaceutical
compositions can
include Prevotella histicola bacteria.
Definitions
[71] "Adjuvant" or "Adjuvant therapy" broadly refers to an agent that
affects an
immunological or physiological response in a subject (e.g., human). For
example, an adjuvant
might increase the presence of an antigen over time or to an area of interest,
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
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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.
[72] "Administration" broadly refers to a route of administration of a
composition
(e.g., a pharmaceutical 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. A pharmaceutical composition described herein can be
administered in any
form by any effective route, including but not limited to oral, parenteral,
enteral, intravenous,
intraperitoneal, topical, transdermal (e.g., using any standard patch),
intradermal, ophthalmic,
(intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous,
intramuscular, buccal,
sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal,
transmucosal (e.g., sublingual,
lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and
perivaginally), implanted,
intravesical, intrapulmonary, intraduodenal, intragastrical, and
intrabronchial. In preferred
embodiments, a pharmaceutical composition described herein is administered
orally, rectally,
topically, intravesically, by injection into or adjacent to a draining lymph
node, intravenously, by
inhalation or aerosol, or subcutaneously. In another preferred embodiment, a
pharmaceutical
composition described herein is administered orally or intravenously.
[73] 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 Vii) 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 Vu 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 Vu and VL is
composed of three
CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the
following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and
light chains
contain a binding domain that interacts with an antigen. The term "antibody"
includes, for
example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies,
humanized
antibodies, human antibodies, multispecific antibodies (e.g., bispecific
antibodies), single-chain
antibodies and antigen-binding antibody fragments.
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[74] The terms "antigen binding fragment" and "antigen-binding portion" of
an
antibody, as used herein, refer to one or more fragments of an antibody that
retain the ability to
bind to an antigen. 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.
[75] 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
CnH2nOn. 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.
[76] "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 (e.g., pharmaceutical composition) and not present in the
composition itself. Cells
that augment the environment include immune cells, stromal cells, bacterial
and fungal cells.
[77] "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
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terminal leaves in the phylogenetic tree that is a distinct monophyletic
evolutionary unit and that
share some extent of sequence similarity.
[78] A "combination" of microbes (e.g., bacteria) from two or more
microbial strains
includes the physical co-existence of the microbes are obtained, either in the
same material or
product or in physically connected products, as well as the temporal co-
administration or co-
localization of the two or more strains. In some embodiments, one of the
strains is from
Prevotella bacteria, e.g., Prevotella histicola bacteria.
[79] 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)).
[80] The term "ecological consortium" is a group of bacteria which trades
metabolites
and positively co-regulates one another, in contrast to two bacteria which
induce host synergy
through activating complementary host pathways for improved efficacy.
[81] As used herein, "engineered bacteria" are any bacteria that have been
genetically
altered from their natural state by human activities, and the progeny of any
such bacteria.
Engineered bacteria include, for example, the products of targeted genetic
modification, the
products of random mutagenesis screens and the products of directed evolution.
[82] 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.
[83] 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.
[84] "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
"FAS TA" program, using for example, the default parameters as in Pearson et
al. (1988) Proc.
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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, Mrtin 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.)).
[85] "Immunotherapy" is treatment that uses a subject's immune system to
treat
disease (e.g., immune disease, inflammatory disease, metabolic disease,
cancer) and includes, for
example, checkpoint inhibitors, cancer vaccines, cytokines, cell therapy, CAR-
T cells, and
dendritic cell therapy.
[86] 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, 101\3 fold, 10A4 fold, 10A5 fold, 101\6 fold, and/or 101\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).
[87] "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, TLR10 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).
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[88] The "internal transcribed spacer" or "ITS" is a piece of non-
functional RNA
located between structural ribosomal RNAs (rRNA) on a common precursor
transcript often used
for identification of eukaryotic species in particular fungi. The rRNA of
fungi that forms the core
of the ribosome is transcribed as a signal gene and consists of the 8S, 5.8S
and 28S regions with
ITS4 and 5 between the 8S and 5.8S and 5.8S and 28S regions, respectively.
These two
intercistronic segments between the 18S and 5.8S and 5.8S and 28S regions are
removed by
splicing and contain significant variation between species for barcoding
purposes as previously
described (Schoch et al Nuclear ribosomal internal transcribed spacer (ITS)
region as a universal
DNA barcode marker for Fungi. PNAS 109:6241-6246. 2012). 18S rDNA is
traditionally used
for phylogenetic reconstruction however the ITS can serve this function as it
is generally highly
conserved but contains hypervariable regions that harbor sufficient nucleotide
diversity to
differentiate genera and species of most fungus.
[89] The term "isolated" or "enriched" encompasses a microbe 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
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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.
[90] 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).
[91] The term "LPS mutant or lipopolysaccharide mutant" broadly refers to
selected
bacteria that comprises loss of LPS. Loss of LPS might be due to mutations or
disruption to
genes involved in lipid A biosynthesis, such as 1pxA, 1pxC , and 1pxD.
Bacteria comprising LPS
mutants can be resistant to aminoglycosides and polymyxins (polymyxin B and
colistin).
[92] "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.
[93] "Microbe" refers to any natural or engineered organism characterized
as an
archaeaon, parasite, bacterium, fungus, microscopic alga, protozoan, and the
stages of
development or life cycle stages (e.g., vegetative, spore (including
sporulation, dormancy, and
germination), latent, biofilm) associated with the organism. In some
embodiments, the microbes
are bacteria. Examples of gut microbes include: Actinomyces graevenitzii,
Actinomyces
odontolyticus, Akkermansia mucimphila, Bacteroides caccae, Bacteroides
fragilis, Bacteroides
putredinis, Bacteroides thetaiotaomicron, Bacteroides vultagus,
Bifidobacterium adolescentis,
Bifidobacterium bifidum, Bilophila wadsworthia, Blautia, Butyrivibrio,
Campylobacter gracilis,
Clostridia cluster III, Clostridia cluster IV, Clostridia cluster IX
(Acidaminococcaceae group),
Clostridia cluster XI, Clostridia cluster XIII (Peptostreptococcus group),
Clostridia cluster XIV,
Clostridia cluster XT", Collinsella aerofaciens, Coprococcus, Corynebacterium
sunsvallense,
Desulfomonas pigra, Dorea formicigenerans, Dorea longicatena, Escherichia
coli, Eubacterium
hadrum, Eubacterium rectale, Faecalibacteria prausnitzii, Gemella,
Lactococcus, Lanchnospira,
Mollicutes cluster XVI, Mollicutes cluster XVIII, Prevotella, Rothia
mucilaginosa, Ruminococcus
callidus, Ruminococcus gnavus, Ruminococcus torques, and Streptococcus.
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[94] "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.
[95] 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.
[96] "Modified" in reference to a bacteria broadly refers to a bacteria
that has
undergone a change from its wild-type form. Bacterial modification can result
from engineering
bacteria. Examples of bacterial modifications include genetic modification,
gene expression
modification, phenotype modification, formulation modification, chemical
modification, and
dose or concentration. Examples of improved properties are described
throughout this
specification and include, e.g., attenuation, auxotrophy, homing, or
antigenicity. Phenotype
modification might include, by way of example, bacteria growth in media that
modify the
phenotype of a bacterium such that it increases or decreases virulence.
[97] "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
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of the 16S are considered the same OTU. See e.g., Claesson MJ, Wang Q,
O'Sullivan 0, 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 JIM. 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
JIM. 2006. The bacterial species definition in the genomic era. Philos Trans R
Soc Lond B Biol
Sci 361: 1929-1940. OTUs are frequently defined by comparing sequences between
organisms.
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.
[98] 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.
[99] 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
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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.
[100] As used herein, a "biomarker" includes molecules (e.g., mRNA or
protein) that
have an increase or a decrease in their levels due to a TH2-mediated pathway.
For instance, the
levels of one or more such biomarkers may increase in TH2-meidated conditions
as compared to
their levels in a healthy subject. Some examples of such protein biomarkers
include
interleukin-4, interleukin-5, interleukin-13, interleukin-19, interleukin-21,
interleukin-31,
interleukin-33, thymic stromal lymphopoietin, immunoglobulin G1 ,
immunoglobulin E,
immunoglobulin A, and combinations thereof. Some examples of such mRNA
biomarkers
include interleukin-4, interleukin-5, interleukin-13, interleukin-19,
interleukin-21, interleukin-31,
interleukin-33, thymic stromal lymphopoietin, and combinations thereof.
[101] As used herein, a substance is "pure" if it is substantially free of
other
components. The terms "purify," "purifying" and "purified" refer to Prevotella
bacteria
preparation or other material that has been separated from at least some of
the components with
which it was associated either when initially produced or generated (e.g.,
whether in nature or in
an experimental setting), or during any time after its initial production. A
Prevotella bacteria
preparation or composition 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 Prevotella bacteria 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. Prevotella bacteria
compositions or
preparations are, e.g., purified from residual habitat products.
[102] As used herein, the term "purified Prevotella bacteria" or
"Prevotella bacteria
composition" refers to a preparation that includes Prevotella bacteria that
have been separated
from at least one associated substance found in a source material (e.g.,
separated from at least
one other bacterial strain) or any material associated with the Prevotella
bacteria in any process
used to produce the preparation. It also refers to a composition that has been
significantly
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enriched or concentrated. In some embodiments, the Prevotella bacteria are
concentrated by 2
fold, 3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or
more than 10,000 fold.
In certain embodiments, the Prevotella bacteria composition can be a
pharmaceutical
composition.
[103] "Residual habitat products" refers to material derived from the
habitat for
microbiota within or on a subject. For example, fermentation cultures of
microbes can contain
contaminants, e.g., other microbe strains or forms (e.g., bacteria, virus,
mycoplasm, and/or
fungus). 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 culture or human or animal subject and
is 100% free, 99%
free, 98% free, 97% free, 96% free, or 95% free of any contaminating
biological matter
associated with the microbial community. Residual habitat products can include
abiotic materials
(including undigested food) or it can include unwanted microorganisms.
Substantially free of
residual habitat products may also mean that the microbial composition
contains no detectable
cells from a culture contaminant or a human or animal and that only microbial
cells are
detectable. In one embodiment, substantially free of residual habitat products
may also mean that
the microbial composition contains no detectable viral (including bacteria,
viruses (e.g., phage)),
fungal, mycoplasmal contaminants. In another embodiment, it means that fewer
than 1x102%,
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
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using instrumentation such as flow cytometry with reagents that distinguish
desired constituents
from contaminants.
[104] 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 10 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.
[105] "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.
[106] The terms "subject" or "patient" refers to any mammal. A subject or a
patient
described as "in need thereof- refers to one in need of a treatment (or
prevention) for a disease.
Mammals (i.e., mammalian animals) include humans, laboratory animals (e.g.,
primates, rats,
mice), livestock (e.g., cows, sheep, goats, pigs), and household pets (e.g.,
dogs, cats, rodents).
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The subject may be a human. The subject may be a non-human mammal including
but not
limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel,
a mouse, a rat, a guinea
pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee. The subject may be
healthy, or may
be suffering from a condition at any developmental stage, wherein any of the
stages are either
caused by or opportunistically supported of a condition-associated or
causative pathogen, or may
be at risk of developing a condition, or transmitting to others a condition-
associated or condition-
causative pathogen. In some embodiments, a subject has a TH2-mediated
condition. In some
embodiments, a subject has atopic dermatitis. In some embodiments, a subject
has asthma. In
some embodiments, a subject has a food allergy. In some embodiments, the
subject has
undergone a therapy for their condition.
[107] As used herein, the term "treating" a disease in a subject or
"treating" a subject
having or suspected of having a condition refers to subjecting the subject to
a pharmaceutical
treatment (e.g., a pharmaceutical composition), e.g., the administration of
one or more agents
(e.g., pharmaceutical compositions), such that at least one symptom of the
condition 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.
Bacteria
[108] In certain aspects, provided herein are bacterial compositions, such
as
pharmaceutical compositions, comprising certain bacteria and methods of using
such bacterial
compositions, such as pharmaceutical compositions to treat and/or prevent TH2-
mediated
conditions.
[109] In certain embodiments, the pharmaceutical compositions provided
herein
comprise bacteria of the genus Prevotella. In some embodiments, the bacteria
of the genus
Prevotella are of the species Prevotella albensis, Prevotella amnii,
Prevotella bergensis,
Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae,
Prevotella buccahs,
Prevotella copri, Prevotella dentalis, Prevotella dent/cola, Prevotella
disiens, Prevotella
histicola, Prevotella melanogenica, Prevotella intermedia, Prevotella
maculosa, Prevotella
marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis,
Prevotella
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nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella
pa/lens, Prevotella
salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis,
Prevotella jejuni,
Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella
corporis, Prevotella
dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca,
Prevotella heparinolytica,
Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis,
Prevotella oryzae,
Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola,
Prevotella saccharolytica,
Prevotella scopos, Prevotella shahii, Prevotella zoo gleoformans, Prevotella
veroralis, and/or a
combination thereof.
[110] In some embodiments, the bacteria of the genus Prevotella is of the
species
Prevotella histicola.
[111] In some embodiments, the bacteria of the genus Prevotella is a strain
comprising
at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at
least 97%, at least 98%, or at least 99% sequence identity (e.g., at least
99.1% sequence identity,
at least 99.2% sequence identity, at least 99.3% sequence identity, at least
99.4% sequence
identity, at least 99.5% sequence identity, at least 99.6% sequence identity,
at least 99.7%
sequence identity, at least 99.8% sequence identity, at least 99.9% sequence
identity) to a
nucleotide sequence (e.g., the genomic sequence, the 16S sequence, and/or the
CRISPR
sequence) of the Prevotella Strain B 50329. In some embodiments, the bacteria
of the genus
Prevotella is Prevotella Strain B 50329 (NRRL accession number B 50329). As
used herein, the
term "Prevotella Strain B 50329" includes both the strain deposited under NRRL
accession
number B 50329 and strains cloned and/or derived from the deposited strain.
[112] Prevotella histicola Strain B can be cultured according to methods
known in the
art. For example, Prevotella histicola can be grown in ATCC Medium 2722, ATCC
Medium
1490, or other medium using methods disclosed, for example in Caballero et
al., 2017.
"Cooperating Commensals Restore Colonization Resistance to Vancomycin-
Resistant
Enterococcus faecium" Cell Host & Microbe 21:592-602, which is hereby
incorporated by
reference in its entirety.
[113] In some embodiments, the Prevotella bacteria is a strain of
Prevotella bacteria
comprising one or more (e.g., 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more) proteins
listed in Table 1 and/or one
or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25,
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26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more) genes encoding proteins listed
in Table 1. In some
embodiments, the Prevotella bacteria comprises all of the proteins listed in
Table 1 and/or all of
the genes encoding the proteins listed in Table 1.
Table 1: Exemplary Prevotella proteins
Seq. Name Uniprot ID
Amino Acid Sequence
ID. No.
MNLKTFTKTVLCFALFAVSAITAKAADHLAIVGEAVWGGWD
LVKATAMVKSPNNPDVFMATVHLNAGKGFKFLTEREWGKL
EYRSGASDVVLKSGIRYKLYASIGASEDGKFKVSESANYEIICDL
ARKTVEVKKVAYQAKEIRYAALWMIGDATAGDWDYNNGVL
LSQDSGNPTCYTATVELKEGEFKFTTNKQWGYDHSVYIFRDV
NDQNKIVFGGEDNKWRITEDGMYNVTVDVPTKTISIKQIDD
PAGHKPQFGNDVILVGDATIAGWNLDNAIYLEHTGQAGRVF
KTTTYLEAGKGFKFLSMLSYDDIDYRPANNTVLNPGVPGTFV
Cluster: PSLPSSTDTKFSVERSGNYDIVCNMNNRTVVVTLSENQVLVN
1 Uncharacterized G6ADE1
YPALWLIGSATSAGWNPGKAVELKRSEADPAVYTARVQLKK
protein GEFKILTSKNVGFDQPTYYRDSTNEHRIVFGVDGDEVAKKDC
KWTLSENAEGTYDVTVDIEAMTIFCDKVNMDEPSVESTDKEL
ILIGDATYSAWDLPKSIVMTPVGPTTFKAVTHLEAGKEFKFLT
ELAWKRYEYRAESLRKELQEGSMSMLVPYRYTNDKDDKDHD
FKFVVKESGNYEIVCDLYIPALIIRKVRYQDTPVTYSSLWIVGSA
TPGGWTIERGIKMTQDENYPTKFTAKANLVPGELKFATNKFA
DFTQDFFFRGKDDYTAVLGGNDNKWNITEAGTYSVTIDVAS
KRVTITKPARNAPTGISTVDSSDEAPAEYFTLNGIKVTTPSSGIY
IKRQGGRTTKVVMK
MDTYQILDIIGCIVGLIYIYQEYKASIWLWMTGIIMPVIYMFVY
Nicotinamide_ribo YEAGLYADFGMQIYYTLAAIYGYLYWKLGKKKGTEDKEI PITH
2 side_transporter_ P24520
FPRRYIIPAIIVFFVLWIALYYILICFTNSTVPVLDSFGNALSFIGL
PnuC WALAKKYLEQWWIWIVVDAELSALYIYKGIPFTAMLYALYTVI
AVAGYFKWRRYIKQQK
MRVRLYKNILLFLFLWVNTLACVSADTSRTVESQPIENGLIITE
SKGWLETIYAKWKPVAEADGYYVYVKGGQYADYSKVDSELIR
Pectate trisacchar VYNGYVRVDIPGLKAGTYSLKIVAVKGGKETQSSEVTGLKVLN
3
ide-Iyase Q8GCB2 YVREGFAHKNYSGVGAYNDDGTLKSGAVVIYVNKDNAKTVS
AHLGKTTFIGLQAILNAYQKGNITTPLSVRILGLLRNGDTDTFG
SSTEGIQIKGKQADSEMNITIEGIGEDASIYGFGFLVRNAKSVE
FRNLGIMRAMDDGVSLDTNNSNIWIHHMDLFYGKASGGD
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HIKGDGSIDVKTDSKYVTIDNCHFWDTGKTSMCGMKKETGP
NYITYHHNWFDHSDSRHARVRTMSVHLWNNYYDGCAKYGI
GATMGCSVFSENNYFRATKNPILISKQGSDAKGTGKFSGEPG
GMVKEYGSLFTEKGAESTYTPISYADNNSSFDFYHAISRNEKV
PASVKTLNGGNIYNNFDTDAALMYSYTPDATALVPSQVTGFY
GAGRLNHGSLQFKFNNAVEDTNSTPIPALEALIDAYSGK
MKYNIAYCIEGFYNHGGMERILSVCANLLSDIYSITIIVANQRG
REHAYNLAQNVNVVDLGVSCKNYKEEYKKSLTRYLQDHQFS
VVISLAGLELFFLPQIKDGSKKVMWFHFAFDVSKMFLSERFH
GWKLNLLYYIHTIRRIYFAKKFDTIVVLSKSDCDSWSRFCNNVK
Glycosyltransferas
4 Q9AET5 YIYNPITIDRKVISNLSEESVIAVGRLGWQKGFDFLIDSWVLVD
eGtfl
_ DKHPDWHLDIFGEGPDRLELQHQIDRKGLHDKVRLCGVTKQ
IEEEYGKHSIYVMSSRAEGFPLALLEASSCGLPMISFNCHQGP
NEIIQEGENGFLVDKVGDIYTLSDRICKLIEDNNLRNMMGKK
ALDSSFRFEGEVIKKDWISLLKQLI
MKRLFFMFLFLGTITMNSLAQEEKPIKYETKNFSLPDKMPLYP
Cluster: Protein GGDGALRAFLSLNLHYPEKAQAFGVEGRSLMKFCVSSDGSIK
A0A09613759
TonB DISAVDCKITNYNRTEFNKLPLSKQESLKKECAKAFAKEAARVI
RLMPKWEPAELNGKKMNVYYSLPFTFKLR
MNYPLFIARKIYNGGDRTRKVSKPAIRIATIGVAIGLAVMIISV
GVVLGFKHTIRNKVVGFGSDITVANFLTLQSSEQYPIQITDSLV
KSLQITPGIKHVQRYDYTQGILKTDNDFLGVLLKGVGPDFDST
FIHENMVEGSLPHFHDNESQQKIVISKTIADKLNLKVGQRIFA
Cluster:
YFINKQGVRTRKFTITGIYATNMKQFDSQICFTDIYTTNKLNG
6 Uncharacterized G6AEN6
WEPDQYSGAELQVDNFSQLTPISMRVLNKVKNTVDHYGGT
protein
YSSENIIEQNPQIFSWLDLMDMNVWIILALMISVAGVTMISG
LLIIILERTQMIGILKALGSRNRQIRHIFLWFATFIIGKGLLWGNI
IGLGCILFQSWTGLVKLDPQTYYVNTVPVEINIPLIIALNMVT
MLVCLVILIAPSYLISHIHPAKSMHYE
MEDKFIYTDKERKLSYQILDELKDTLDKSFLENDLPMLQVQLK
DSVAKNTIHRNVFGLNPILCSLQTAAIAVKDIGLKRDSVIAILLH
QSVQDGYITLEDIDNRFGKSVAKIIHGLIRIQTLYQKNPIIESEN
Bifunctional_(p)pp FRNLLLSFAEDMRVILIMIADRVNLMRQIRDAEDKEAQHKVA
7 Gpp_synthase/hy P9WHG9 EEASYLYAPLAHKLGLYQLKRELEDLSLKYLEHDAYYLIKDKLN
drolase_RelA ATKASRDAYINQFIAPVRERLTAGGLRFHIKGRTKSIHSIWQK
MKKQKCGFEGIYDLFAIRIILDAPLEKEKIQCWQAYSIVTDMY
QPNPKRLRDWLSVPKSNGYECLHITVLGPEKKWVEVQIRTER
MDEIAEHGLAAHWRYKGIKEEGGLDDWLASIRAALEAGDNL
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EVMDQFKSDLYEKEIYVFTPKGDLLKFPKGATILDFAYHIHSKV
GNQCVGGKINAKNVSLRTELHSGDTVEILTSATQKPKAEWLK
IVKSSRAKAKIRLALKETQIKDGLYAKELLERRFKNKKIEIEESTM
GHLLRKLGFKEVSEFYKQVADEKLDPNYIIEEYQKVYNHDHNL
NQPKETESAENFEFENPTNEFLKKNDDVLVIDKNLKGLDFSLA
KCCHPIYGDPVFGFVTVNGGIKIHRTDCPNAPEMRKRFGYRI
VKARWSGKGSSQYAITLRVIGNDDIGIVSNITNVISKDEKIVM
RSINIDSHDGLFSGNLVVLLDDNSKLNMLIKKLRTVKGVKQVT
RI
MKRRIFLFVALSVSIVILFGLNLIIGSVHIPLSDILTILSGSFTGKES
WRFIIWDSRLPQALTAMLCGSSLAVCGLMLQTAFRNPLAGP
Vitamin_B12_imp DVFGISSGASLGVALVMLLLGGTVETSMFTASGFLAILIVAFA
ort_system_perm GAILVTAFILFLSSVVRNSVLLLIVGIMVGYVASSAVTLLNFFSS
8 P06609
ease_protein_Btu EDGVKGYIVWGMGNFGGVSMSHIPLFAFLCLAGIIASFLLVK
C
PLNILLLGPQYAESLGISIRRIRNILLVVVGILTAVTTAFCGPISFI
GLAAPHVARLLFRTENHQKLLPGTLLVGTVVALLCNLICFLPRE
SGMIPLNAVTPLIGAPIIIYVIMKRH
MKLENKEFGFDSFATEMARLKNEKHFDYLVTVVGEDFGTEE
GLGCIYILENTSTHERCSVKQLAKKVGEEFVIPSVIKLWADADL
LEREVYDFYGIKFLGHPDMRRLFLRNDFKGYPLRKDYDMDPA
KNMYTTEDDVELDTTTEWNLDKNGELVGTQHALFTDDNFV
VNIGPQHPSTHGVLRLQTVLDGETVTNIYPHLGYIHRGIEKLC
NADH-
EQFTYPQTLALTDRMNYLSAMMNRHALVGVIEEGMGIELSE
quinone_oxidored
9 P33599 RILYIRTIMDELQRIDNHLLYTACCAQDLGALTAFLYGMRDRE
uctase_subunit_C
HVLNVMEETTGGRLIQNYYRIGGLQADIDPNFVSNVKELCKY
/D
LRPMIQEYVDVFGDNVITHQRFEGVGVMDEKDCISYGVTGP
AGRASGWKNDVRKYHPYAMYDKVNFEEITLTNGDSMDRYF
CHIKEIYQSLNIIEQLIDNIPEGEFYIKQKPIIKVPEGQWYFSVEG
ASGEFGAYLDSRGDKTAYRLKFRPMGLTLVGAMDKMLRGQ
KIADLVTTGAALDFVIPDIDR
FKBP- MRTSTQSKDMGKKQEYKLRNEEFLHNISKKDSIKTLPHGIFYEI
type_peptidyl- P45523 IKEGSGEGTVQPRSIVICNYRGSLISGQVFDDSWQKPTPEAFR
prolyl_cis- LNELITGLQIALCAMHKGDSWRIYIPYQEGYGSKRNADIPAFS
trans_isomerase TLIFDIELINIA
Putative_acetolact
MADNKIAKESVKREVIAGERLYTLLVYSENVAGVLNQIAAVFT
11 ate_synthase_sma P9WKJ3
RRQVNIESLNVSASSIEGIHKYTITAWSDAATIEKITKQVEKKID
Il_subunit
VIKADYYEDSDLFIHEVGLYKIATPILLENAEVSRAIRKRNARM
31
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MEVNPTYSTVLLAGMTDEVTALYHDLKNFDCLLQYSRSGRV
AVTRGFSEPVSDFLKSEEESSVL
MKKKVKIGLLPRVIIAILLGIFFGYFMPTPLARVFLTFNGIFSQFL
GFMIPLIIIGLVTPAIADIGKGAGKLLLVTVIIAYVDTVVAGGLA
YGTGLCLFPSMIASTGGAMPHIDKATELAPYFSINIPAMADV
MSGLVFSFMLGLGIAYGGLTATKNIFNEFKYVIEKVIAKAIIPLL
Serine/threonine
12 ¨ POAGE4
PLYIFGVFLNMAHNGQAQQILLVFSQIIIVILVLHVFILVYQFCI
transporter_SstT
AGAIIRRNPFRLLWNMMPAYLTALGTSSSAATIPVTLEQTMK
NGVGKEIAGFVVPLCATIHLSGSAMKITACALTICLLVGLPHDP
ALFIYFILMLSIIMVAAPGVPGGAIMAALAPLASILGFNSEAQA
LMIALYIAMDSFGTACNVTGDGAIALVVNKMFGKKER
MKKLLLLVCAAVMSLSASAQAGDKALGAQLVFGSETNSLGF
Cluster:
GVKGQYYFTDHIRGEGSFDYFLKNKGISMWDINANVHYLFD
13 Uncharacterized G6AJO7
VADKFKVYPLAGLGYTNWSYKYEYAGAPVVEGSDGRLAVNL
protein
GGGVEYELTKNLNVNAEAKYQIISNYNQLVLGVGVAYKF
Heterocyst_differ
114 entiation_ATP- P22638 MHFYCTKSSLDTMSERYVKRMIAKLASQGKTVISIAHRFSTIM
binding_protein DAKHIILLAKGKVVAEGTHQELLKTSEDYRKLWSDQNDEID
MKNVYFLSDAHLGSLAIAHRRTQERRLVRFLDSIKHKASAVYL
LGDMFDFWDEYKYVVPKGFTRFLGKVSELTDMGVEVHFFTG
UDP-2,3- NHDLWTYGYLEEECGVILHRKPVTMEIYGKVFYLAHGDGLGD
15 diacylglucosamine Q912V0
PDPMFQFLRKVFHNRVCQRLLNFFHPWWGMQLGLNWAK
_hydrolase KSRLKRADGKEMPYLGEDKEYLVRYTKDYMRSHKDIDYYIYG
HRHIELDLTLSGKVRMULGDWIWQFTYAVFDGEHMFLEEYI
EGESKP
MNSKQNDNYDVIIIGGGITGAGTARDCALRGLKVLLVEKFDF
TNGATGRNHGLLHSGARYAVTDPESATECIKENMVLRRIAKH
CIEETDGLFITLPEDDINYQKTFVEACARAGISANIISPEEALRL
Anaerobic_glycero DPSVNPDLLGAVRVPDASVDPFHLTTANVLDARQHGADVLT
1-3-
YHEVVAILTSNGRVEGVRLRNNHTGEEIEKHAVLVINAAGIW
16 phosphate_dehyd P0A9C0
GHDIAKMADIKINMFPAKGTLLVFGHRVNKMVINRCRKPAN
rogenase ADILVPDDAVCVIGTTSDRVPYDTVDNLKITSEEVDTLIREGEK
LAPSLATTRILRAYAGVRPLVAADNDPTGRSISRGIVCLDHEKR
DGLTGMITITGGKMMTYRLMAEQATDLACKKLGINKTCETA
TTPLPGTAGKDSDNPHHTYSTAHKAAKGRQGNRVKEIDERT
EDDRALICECEEVSVGEAKYAIEELHVHDLLNLRRRTRVGMGT
32
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CQGELCACRAAGVMCENGVKVDKAMTDLTKFINERWKGM
RPVAWGSTLDEAQLTTIIYQGLCGLGI
MRYDTIIIGGGLSGLTAGITLAKAGQKVCIVSAGQSSLHFHSG
SFDLLGYDADGEVVTHPLQAIADLKAEHPYSKIGISNIEHLASQ
AKTLLCEAGISVMGNYEQNHYRVTPLGTLKPAWLTTEGYAMI
Anaerobic_glycero DDPEILPWKKVELLNIQGFMDFPTQFIAENLRMMGVECQIK
1-3- TFTTDELSTARQSPTEMRATNIAKVLANKDALSKVSERINAIS
17 P13033
phosphate_dehyd GDPDALLLPAVLGFSNAESLDEMKQWIKKPVQYIATLPPSVS
rogenase GVRTTILLKRLFAQAGGTLLIGDSATTGQFSGNHLVSITTDHLP
DEKLYADHFILASGSFMSHGIRSNYAGVYEPVFKLDVDAAEK
RDDWSVTNAFEAQPYMEFGVHTDKDFHATKDGKNIENLYAI
GSVLSGHNSIKHADGTGVSLLTALYVAKKITGKG
MAEGIQLKNISGNNLEQCLKCSICTAYCPVSAVEPKYPGPKQS
GPDQERYRLKDSKFFDEALKMCLNCKRCEVACPSGVRIADIIQ
ASRITYSTHRPIPRDIMLANTDFVGTMANMVAPIVNATLGLK
Anaerobic_glycero PVKAVLHGVMGIDKHRTFPAYSSQKFETWYKRMAAKKQDS
1-3- YSKHVSYFHGCYVNYNFPQLGKDLVKIMNAVGYGVHLLEKEK
18 P0A996
phosphate_dehyd CCGVALIANGLSGQARRQGKVNIRSIRKAAEQNRIVLTTSSTC
rogenase TFTMRDEYEHLLDIKTDDVRENITLATRFLYRLIEKGDIKLAFRK
DFKMRTAYHSACHMEKMGWIlYSTELLKMIPGLELIMLDSQ
CCGIAGTYGFKKENYQRSQEIGEGLFKQIKELNPDCVSTDCET
CKWQIEMSTGYEVKNPISILADALDVEETIKLNQ
MMIKNIVLSIPISLIIYLNHLIMEYSMTTQFLMELIGTLILVLFGD
GVCACVTLNKSKGQKAGWVVITIAWGLAVCMGVLVAGPYT
GAHLNPAVSIGLAVAGMFPWSSVPYYIVAQMIGGFLGGLLV
Glycerol_uptake_f
19 P18156 WFFYKDHYDATDDEAAKLGTFCTSPAIRNYKMNFLSEVIATL
acilitator_protein
VLVFIIISFSVDGNTGDAEHFKFGLAALGPIPVTLLIIALGMSLG
GTTGYAMNPARDLSPRLAHAVCMKGDNDWSYSWIPVLGPI
IGAIIAGFCGAALLLV
MSEKIIPSNEPAQAASEPIKASYTEYTVIPSQGYCQFVKCKKGD
QPVVLKGLKEAYRERVLLRNALKREFKQCQRLNHPGIVRYQG
Serine/threonine-
LVDVEGYGLCIEEEYVDGRTLQAYLKESHTDDEKITIVNQIADA
20 protein_kinase_St Q97PA9
LRYAHQQGVAHRNLKPSNILITKQGDHVKLIDFNVLSLDDVK
kP PTADTTRFMAPELKDETMTADGTADIYSLGTIMKVMGLTLA
YSEVIKRCCAFKRSDRYSDIDEFLADFNHDGSSFSMPKIGKGT
VVIGFIAVVVIALAALAYNYGGALVDQVGKIDVTSIFKSDAETA
PEDSAMVKSVEQNNNDSVADEAPATGKLAFMNTMKPALYK
33
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DLDRLFAKHSDDRAKLNRAIKVYYRGLIQANDTLDNEQRAEL
DRVFGNYVKQKKAALK
Cluster: D-alanyl-
21 D-alanine G6AHI1 MLVAQLFVGVLQAQKPVQNRRQAVGQSMERQGLVNVKAV
dipeptidase VPSIKVALMYARTDNFCHRMALS
MITGLVIIQLLIVLALIFIGARVGGIGLGIYGMIGVFILVYGFGLA
PGSAPIDVMMIIVAVITAASALQASGGLEYLVGVAAKFLQKH
PDHITYFGPITCWLFCVVAGTAHTSYSLMPIIAEIAQTNKIRPE
RPLSLSVIAASLGITCSPVSAATAALISQDLLGAKGIELGTVLMI
Anaerobic_C4- CIPTAFISILVAAFVENHIGKELEDDPEYKRRVAAGLINPEAACE
22 dicarboxylate_tra POABN5
EVQKAENEHDPSAKHAVWAFLFGVALVILFGFLPQLRPEGVS
nsporter_DcuA MSQTIEMIMMSDAALILLVGKGKVGDAVNGNIFKAGMNAV
VAIFGIAWMGNTFYVGNEKILDAALSSMISSTPILFAVALFLLSI
MLFSQAATVTTLYPVGIALGINPLLLIAMFPACNGYFFLPNYP
TEVAAIDFDRTGTTRVGKYVINHSFQIPGFITTIVSILLGVLMV
QFFR
MRILKITFVTVLALVMSTVVFAQKPKIRIIATGGTIAGVSASAT
SSAYGAGQVGVQTLIDAVPQIKDIADVSGEQLVNIGSQDMN
DEVWLKLAKRINDLLNKEGYDGVLITHGTDTMEETAYFLSLTV
HTDKPVVMVGSMRPSTAISADGPANLYNGICTLVDPSSKGH
23 L-asparaginase_2 P00805 GVMVCMNNELFEAKSVIKTHTTDVSTFKGGLYGEMGYVYN
GKPYFLHKPVAKQGLTSEFNVDNLTSLPKVGIVYGYANCSPLP
IQAFVNAKFDGIVLAGVGDGNFYKDVFDVALKAQNSGIQIVR
SSRVPFGPTNLNGEVDDAKYHFVASLNLNPQKARVLLMLALT
KTKDWQKIQQYFNEY
MALACAMTMSASAQMGTNPKWLGDAIFYQIYPSSYMDTD
GNGIGDLPGITQKLDYIKSLGVNAIWLNPVFESGWFDGGYDV
IDFYKIDPRFGTNTDMVNLVKEAHKRGIKVCLDLVAGHTSTK
CPWFKESANGDRNSRYSDYFIWTDSISEADKKEIAERHKEAN
PASSTHGRYVEMNAKRGKYYEKNFFECQPALNYGFAKPDPN
24
Trehalose_synthas P9WQ19 QPWEQPVTAPGPQAVRREMRNIMAFWFDKGVDGFRVDM
e/amylase_TreS ASSLVKNDWGKKEVSKLWNEMREWKDKNYPECVLISEWSD
PAVAIPAGFNIDFMIHFGIKGYPSLFFDRNTPWGKPWPGQDI
SKDYKFCYFDKAGKGEVKEFVDNFSEAYNATKNLGYIAIPSAN
HDYQRPNIGTRNTPEQLKVAMTFFLTMPGVPFIYYGDEIGM
KYQMDLPSKEGSNERAGTRTPMQWTSGPTAGFSTCNPSQL
YFPVDTEKGKLTVEAQQNDPRSLLNYTRELTRLRHSQPALRG
34
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NGEWILVSKESQPYPMVYKRTSGGETVVVAINPSDKKVSANI
AHLGKAKSLIMTGKASYKTGKTEDAVELNGVSAAVFKIAE
MNIAVIFAGGSGLRMHTKSRPKQFLDLNGKPIIIYTLELFDNH
PGIDAIVVACIESWIPFLEKQLRKFEINKVVKIVPGGESGQASIY
Ribito1-5-
NGLCAAEAYIKSKNVASEDTTVLIHDGVRPLITEETITDNINKV
25 phosphate_cytidyl .. Q720Y7
AEVGSCITCIPATETLVVKQHDGSLEIPSRADSLIARAPQSFLLS
yltransferase
DILTAHRRAIDEKKNDFIDSCTMMSHYGYRLGTIIGPMENI KIT
TPTDFFVLRAMVKVHEDQQIFGL
MTEKKSVSIVLCTYNGTKYLQEQLDSILAQTYPLHEIIIQDDGS
TDNTWQILEKYEEKYPLIHIYHNEGTHGVNANFLSAMHRTTG
UDP-Glc:alpha-D- DFIAIADQDDIWETDKIANQMTTIGNKLLCSGLTRPFSSDGSF
G1cNAc- AYFDNRPRNVSIFRMMFLGLPGHTMLFRRELLRMMPPVTH
26 B5L3F2
diphosphoundeca SFFNVSLYDAALSILAASHDSIAFCNKVLVNFRRHADATTYND
prenol YSRSLPSWQNGLYELLWGLRHYHQARSIALPIYRGKLALMEGI
TTNYHDFIEAKAIMRLETQKGLWAFLRLQYLLTKNHQRLFQT
SGGSFIKMIRAWLYPVMQLYMYHHALRRCK
MESFIIEGGHRLSGTIAPQGAKNEALEVICATLLTTEEVIIRNIP
NILDVNNLIKLLQDIGVKVKKLGANDFSFQADEVKLDYLESIDF
VKKCSSLRGSVLMIGPLLGRFGKATIAKPGGDKIGRRRLDTHF
LGFKNLGARFVRIEDRDVYEIQADKLVGDYMLLDEASVTGTA
NIIMSAVMAEGTTTIYNAACEPYIQQLCHLLNAMGAKITGIAS
UDP-N-
27 P33038 NLITIEGVTSLHGAEHRILPDMIEVGSFIGMAAMVGDGVRIK
acetylglucosamine
DVSIPNLGULDTFRRLGVQIIEDEDDLIIPRQDHYVIDSFIDGTI
MTISDAPWPGLTPDLISVLLVVATQAQGSVLFHQKMFESRLF
FVDKLIDMGAQIILCDPHRAVVVGHDHAKKLRAGRMSSPDI
RAGIALLIAALTAEGTSRIDNIAQIDRGYENIEGRLNALGAKVQ
RVEIC
MERSGNFYKAIRLGYILISILIGCMAYNSLYEWQEIEALELGNK
KIDELRKEINNINIQMIKFSLLGETILEWNDKDIEHYHARRMA
MDSMLCRFKATYPAERIDSVRHLLEDKERQMCQIVQILEQQ
QAINDKITSQVPVIVQKSVQEQPKKSKRKGFLGIFGKKEEAKP
Sensor_protein_E
TVTTTMHRSFNRNMRTEQQAQSRRLSVHADSLAARNAELN
28 vgS P30855 RQLQGLVVQIDGKVQTDLQKREAEITAMRERSFIQIGGLTGF
VILLLVISYIIIHRNANRIKRYKQETADLIERLQQMAKRNEALITS
RKKAVHTITHELRTPLTAITGYAGLIQKNFNADKTGMYIRNIQ
QSSDRMREMLNTLLSFFRLDDGKEQPNFSTCRISSIAHTLESE
FMPIAINKGLALTVTNHTDAVVLTDKERILQIGNNLLSNAIKFT
ENGAVSLTMGYDNGMLKLIVKDTGSGMTEEEQQRVFGAFE
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RLSNAAAKDGFGLGLSIVQRIVTMLGGTIQLKSEKGKGSRFTV
EIPMQSAEELPERINKTQIHHNRTLHDIVAIDNDKVLLLMLKE
MYAQEGIHCDTCTNAAELMEMIRRKEYSLLLTDLNMPDING
FELLELLRTSNVGNSRIIPIIVTTASGSCNREELLERGFSDCLLKP
FSISELMEVSDKCAMKGKQNEKPDFSSLLSYGNESVMLDKLI
AETEKEMQSVRDGEQRKDFQELDALTHHLRSSWEILRADQP
LRELYKQLHGSAVPDYEALNNAVTAVLDKGSEIIRLAKEERRK
YENG
MKRSRFYITVGLILSLTLLMSACGQKKAKDGRTDTPTSGTIKFA
SDESFSPIVEELLQNYQFRYPQAHLLPIYTDDNTGMKLLLDQK
VNLFITSHAMTKGEDAILRGKGPIPEVFPIGYDGIAFIVNRSNP
Phosphate-
DSCITVDDVKKILQGKIAKWNQLNPKNNRGSIEVVFDNKASA
29 binding_protein_P Q7A5Q2
TLHYVVDSILGGKNIKSENIVAAKNSKSVIDYVNKTPNAIGVIG
stS
SNWLNDHRDTTNTTFKKDVTVASISKATVASPSNSWQPYQA
YLLDGRYPFVRTIYALLADPHKALPYAFANYIANPIGQMIIFKA
GLLPYRGNINIREVEVKNQ
MAGTKRIKTALISVFHKDGLDDLLKKLDEEGVQFLSTGGTQQ
Bifunctional_purin FIESLGYECQKVEDVTSYPSILGGRVKTLHPKIFGGILARRDNEE
30 e_biosynthesis_pr P9WHM7 DQKQMVEYTIPAIDLVIVDLYPFEQTVASGASAQDIIEKIDIGG
otein_PuTH ISLIRAGAKNFKDVVIVPSKAEYPVLLQLLNTKGAETEIEDRKM
FAERAFGVSSHYDTAIHSWFAAE
MEEEKGGRIGQRPYILKIITERNYIIIIDMKKAKILLFVTALVAVL
TSCGGGQKGLPTSDEYPVITIGASNAQLKTTYPATIKGVQDVE
VRPKVSGFITKLNIHEGEYVHAGQVLFVIDNSTYQAAVRQAQ
AQVNSAQSAVAQAKANVVQANASLNSANAQAATSRLTYN
NSQNLYNNKVIGDYELQSAKNTYETAQASVRQAQSGIASAQ
Multidrug_efflux_
AAVKQAEAGVRQAQAMLSTAKDNLGFCYVKSPASGYVGSLP
31 pump_subunit_Ac POAE06
FKEDALVSASSAQPVTTISNTSTIEVYFSMTEADVLKLSRTDDG
TA
LSNAIKKFPAVSLLLADGSTYNHEGAIVKTSGMIDATTGTINVI
ARFPNPEHLLKSGGSGKIVIAKNNNRALLIPQEAVTQVQNKM
FVYKVDAKDKVHYSEITVDPQNDGINYIVTSGLKMGERIVSK
GVSSLEDGAKIKALTPAEYEEAIKKAEKLGENQSSASGFLKTM
KGDSK
MAKRRNKARSHHSLQVVTLCISTAMVLILIGMVVLTVFTSRN
Cell division_prot LSSYVKENLTVTMILQPDMSTEESAALCQRIRSLHYINSLNFIS
_
32 ein FtsX Q81X30 KEQALKEGTRELGANPAEFAGQNPFTGEIELQLKANYANNDS
_
IKNIERELRTYRGVSDITYPQNLVESVNHTLGKISLVLLVIAILLTI
VSFSLMNNTIRLSIYARRFSIHTMKLVGASWGFIRAPFLRRAV
36
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MEGLVSALLAIAVLGVGLCLLYDYEPDITKVLSWDVLVITAGV
MLAFGVLIATFCSWLSVNKFLRMKAGDLYKI
MKLSDLKTGETGVIVKVLGHGGFRKRIIEMGFIQGKQVEVLL
NAPLRDPVKYKIMGYEVSLRHSEADQIEVISAEEARQLEQAKA
DNEPQQGALSNNIPDESDHALTPFELTDAANRKSKVINVALV
GNPNCGKTSLFNFASGAHERVGNYSGVTVDAKVGRANYEG
YEFHLVDLPGTYSLSAYSPEELYVRKQLVEKTPDVVINVIDASN
LERNLYLTTQLIDMHVRMVCALNMFDETEQRGDNIDYQKIS
ELFGIPMVPTVFTNGRGVKELFHQVIAVYEGKEDETSQFRHIH
INHGHELEGGIKNIQEHLRAYPDICQRYSTRYLAIKLLEHDKDV
EELIKPLKDSDEIFKHRDIAAQRVKEETGNESETAIMDAKYGFI
Fe(2+)_transporte HGALEEADYSTGQKKDTYQTTHFIDQILTNKYFGFPIFFLILFI
33 Q9PMQ9
r_FeoB MFTATFVIGQYPMDWIDGGVSWLGDFISSNMPDGPVKDM
LVDGIIGGVGAVIVFLPQILILYFFISYMEDSGYMARAAFIMDK
LMHKMGLHGKSFIPLIMGFGCNVPAVMATRTIESRRSRLVT
MLILPLMSCSARLPIYVMITGSFFALKYRSLAMLSLYVIGILMS
VIMSRVFSRFLVKGEDTPFVMELPPYRFPTWKAIGRHTWEK
GKQYLKKMGGIILVASIIVWALGYFPLPDKPDMGQQERQEH
SFIGQIGHAVEPVFRPQGFNWKLDVGLLAGVGAKEIVASTM
GVLYSNDDSFKDDNSFSSEGGKYVKLHKQITQDVANLHGVSY
NEAEPIATLTAFCFLLFVLLYFPCIATIAAIKGETGSWGWALFA
AGYTTLLAWVVSAIVFQVGMLFIG
MKKNLLKAVLPASLALFAVTFGSCSQDGQLTGTKEDTGERVL
DNTREIQNYLRTLPLAPMMSRASDPVPSDDGTTVPVDEGTS
KTEEKGVLNGIPGSWVKTTRRYKMTQAFDESFLFDPTSDIVY
PGCVLKGGTIANGTYAIITSHETGDVTFSINLSPANPQEARETS
ATVHNIRKSEYQEVWNKWANMQWKESPITTIESVEKINSQE
ELATKLGVAVNSPVANGSLNFGFNFNKKKNHILARLIQKYFSV
STDAPKKGNIFESIDKEALDGYQPVYISNINYGRIIYLSVESDED
34 Pneumolysin Q04IN8
EKVVDEAINFAMNQIKGVDVSVSADQSLHYRKVLANCDIRIT
VLGGGQTIQKEVLKGDIDSFQRFLNADIPMEQMSPISFSLRYA
VDNSQARVVTSNEFTVTQRDFVPEFKKVRMQLQVLGFSGTN
TGPFPNLDREAGLWGSISLSLNGQDNELVKISQSNPFFFNYRE
KKETMHPIGFGGIVTVEFDKDPNESLEDFVDHQKMTFVSDL
HSTRSIYNYNFGRTTFTHTLGTLYTKYKGDDPIFVLESNNKNV
KIHTYVKVLDMKFFN
Cluster:
MTKFIYAMSLFLLAAISIKAQPIQKTSGCLLHGSVVSSTDATAI
35 Uncharacterized G6AG77
AGATVRLYQLKKLVGGTVSDASGNFDVKCPSSGSLQLRITAV
protein
GFKEVDTTLNVPTVTPLSIYMRAGKHAMDEVTVTASEKRGM
37
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TSTTVIGQTAMEHLQPSSFADLLALLPGGMTKIPALGSANVIT
LREAGPPSSQYATSSLGTKFVIDGQAIGTDANMQYIAGSFQG
DADNSRNHVSYGVDMREIPTDNIEKVEVVRGIPSVKYGELTS
GLINITRKRSQSPLLLRLKADEYGKLVSVGKGFLLSGKWNLNV
DGGLLDARKEPRNRFETYRRLTFSARLRRKWNLGERYVLEWS
GATDYSLNIDNVKTDPEIQIHREDSYRSSYLKMGMNHRLLLR
RKALVGLQSVSLAYSASLASDRIHQTEAVALQRDYVVPLAYEG
GEYDGLFLPMQYLCDYRVEGKPFYSTLRGETEWLARTSFISH
HITAGGEFLLNKNYGRGQIFDITKPLHASTARRPRSYKDIPATD
ILSFYAEDKATMPIGKHQLTVMAGLRTTQMLNIPASYAVHGK
LFTDTRVNVQWDFPSFLGFKSFVSGGLGMMTKMPTVLDLY
PDYVYKDITEMNYWDIRPAYKRIHIRTYKLNQVNPDLRPARN
KKWEIRLGMDKGAHHFSVTYFHEDMKDGFRSTTTMRPFIYK
RYDTSVINPSALTGPPSLASLPVVTDTLLDGYGRTENGSRITKQ
GIEFQYSSPRIPVIQTRITVNGAWFRTLYENSIPLFRSAPNVVV
GTVAIADRYAGYYMSTDKYDKQIFTSNFIFDSYVDKLGLILSAT
AECFWMSNTKRPATSSTPMGYMDITGTVHPYVEADQSDPY
LRWLVLTGTAGQDMDYRERSYMLVNFKATKRFGRHLSLSFF
ADRVFYVAPDYEVNGFIVRRTFSPYFGMEIGLKI
MLIDFKKVNIYQDERLILKDIDFQATEGEFIYLIGRVGSGKSSLL
KTFYGELDIDQEDAEKAEVLGESVLDIKQKRIPALRRQMGIIFQ
Cell_division_ATP-
DFQLLHDRSVAKNLKFVLQATGWKDKEKIKQRIKEVLEQVG
36 binding_protein_F P0A9R7
MIDKAAKMPSELSGGEQQRIAIARAFLNNPKIILADEPTGNLD
tsE
PETASNIVSILKDTCKNGTTVIMSTHNINLLSQFPGKVYRCME
QALVPVTNEAQTKDLEEDSTSVEPLIEPVLEEEAQAEDSKE
MFENQPKALYALALANTGERFGYYTMIAVFALFLRANFGLEP
GTAGLIYSIFLGLVYFLPLIGGIMADKFGYGKMVTIGIIVM FAG
YLFLSVPLGGGTVAFGAMLAALLLISFGTGLFKGNLQVMVGN
LYDTPELASKRDSAFSIFYMAINIGALFAPTAAVKIKEWAETSL
GYAGNDAYHFSFAVACVSLIVSMGIYYAFRSTFKHVEGGTKK
Di-
TEKAAAAAVEELTPQQTKERIVALCLVFAVVIFFWMAFHQN
37 /tripeptide_transp POC2U3
GLTLTYFADEFVSPTSTGVQSMAFDVVNLVMIVFIVYSIMALF
orter
QSKTTKAKGIACAVILAAIAVLAYKYMNVNGQVEVSAPIFQQ
FNPFYVVALTPISMAIFGSLAAKGKEPSAPRKIAYGMIVAGCA
YLLMVLASQGLLTPHEQKLAKAAGETVPFASANWLIGTYLVL
TFGELLLSPMGISFVSKVAPPKYKGAMMGGWFVATAIGNILV
SVGGYLWGDLSLTVVWTVFIVLCLVSASFMFLMMKRLEKVA
38
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MKKILIFVAGLCMSLAASAQIQRPKLVVGLVVDQMRWDYLY
YYYNEYGTDGLRRLVDNGFSFENTHINYAPTVTAIGHSSVYTG
SVPAITGIAGNYFFQDDKNVYCCEDPNVKSVGSDSKEGQMS
PHRLLASTIGDELQISNDFRSKVIGVALKDRASILPAGHAADA
AYWWDTSAGHFVTSTFYTDHLPQWVIDFNEKNHTAPNFNI
Calcium-
KTSTQGVTMTFKMAEAALKNENLGKGKETDMLAVSISSTDAI
38 transporting_ATPa Q47910
GHVYSTRGKENHDVYMQLDKDLAHFLKTLDEQVGKGNYLLF
se
LTADHGAAHNYNYMKEHRIPAGGWDYRQSVKDLNGYLQG
KFGIAPVMAEDDYQFFLNDSLIAASGLKKQQIIDESVEYLKKD
PRYLYVFDEERISEVTMPQWIKERMINGYFRGRSGEIGVVTR
PQVFGAKDSPTYKGTQHGQPFPYDTHIPFLLYGWNVKHGAT
TQQTYIVDIAPTVCAMLHIQMPNGCIGTARNMALGN
MDRQVFQTDSRQRWNRFKWTLRVLITIAILLGVVFVAMFAL
EGSPQMPFRHDYRSVVSASEPLLKDNKRAEVYKSFRDFFKEQ
KMHSNYAKVAARQHRFVGHTDNVTQKYIKEWTDPRMGIRS
AWYVNWDKHAYISLKNNLKNLNMVLPEWYFINPKTDRIEAR
IDQRALKLMRRAHIPVLPMLTNNYNSAFRPEAIGRIMRDSTK
RMGMINELVAACKHNGFAGINLDLEELNINDNALLVTLVKDF
ARVFHANGLYVTQAVAPFNEDYDMQELAKYDDYLFLMAYD
EYNAGSQAGPVSSQRWVEKATDWAAKNVPNDKIVLGMAT
YGYNWAQGQGGTTMSFDQTMATALNAGAKVNFNDDTYN
LNFSYQDEDDGTLHQVFFPDAVTTFNIMRFGATYHLAGFGL
WRLGTEDSRIWKYYGKDLSWESAARMPIAKIMQLSGTDDV
NFVGSGEVLNVTSEPHAGRIGIVLDKDNQLIIEERYLSLPATYT
Poly-beta-1,6-N- VQRLGKCKEKQLVLTFDDGPDSRWTPKVLSILKHYKVPAAFF
acetyl-D- MVGLQIEKNIPIVKDVFNQGCTIGNHTFTHHNMIENSDRRSF
39 Q5HKQO
glucosamine_synt AELKLTRMLIESITGQSTILFRAPYNADADPTDHEEIWPMIIAS
hase RRNYLFVGESIDPNDWQQGVTADQIYKRVLDGVHQEYGHIIL
LHDAGGDTREPTVTALPRIIETLQREGYQFISLEKYLGMSRQTL
MPPIKKGKEYYAMQANLSLAELIYHISDFLTALFLVFLVLGFM
RLVFMYVLMIREKRAENRRNYAPIDPLTAPAVSIIVPAYNEEV
NIVRTISNLKEQDYPSLKIYLVDDGSKDNTLQRVREVFENDDK
VVIISKKNGGKASALNYGIAACSTDYIVCVDADTQLYKDAVSK
LMKHFIADKTGKLGAVAGNVKVGNQRNMLTYWQAIEYTTS
QNFDRMAYSNINAITVIPGAIGAFRKDVLEAVGGFTTDTLAE
DCDLTMSINEHGYLIENENYAVAMTEAPESLRQFIKQRIRWC
FGVMQTFWKHRASLFAPSKGGFGMWAMPNMLIFQYIIPTF
SPIADVLMLFGLFSGNASQIFIYYLIFLLVDASVSIMAYIFEHESL
WVLLWIIPQRFFYRWIMYYVLFKSYLKAIKGELQTWGVLKRT
GHVKGAQTIS
39
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MSQINGRISQIIGPVIDVYFDTKGENPEKVLPNIYDALRVKKA
DGQDLIIEVQQQIGEDTVRCVAMDNTDGLQRGLEVVPTGSP
IVMPAGEQIKGRMMNVIGQPIDGMSALQMEGAYPIHREAP
KFEDLSTHKEMLQTGIKVIDLLEPYMKGGKIGLFGGAGVGKT
VLIMELINNIAKGHNGYSVFAGVGERTREGNDLIRDMLESGV
ATP_synthase_su IRYGEKFRKAMDEGKWDLSLVDSEELQKSQATLVYGQMNEP
40 bunit_beta,_sodiu P29707
PGARASVALSGLTVAEEFRDHGGKNGEAADIMFFIDNIFRFT
m_ion_specific QAGSEVSALLGRMPSAVGYQPTLASEMGAMQERITSTKHGS
ITSVQAVYVPADDLTDPAPATTFTHLDATTELSRKITELGIYPA
VDPLGSTSRILDPLIVGKEHYDCAQRVKQLLQKYNELQDIIAIL
GMDELSDDDKLVVNRARRVQRFLSQPFTVAEQFTGVKGVM
VPIEETIKGFNAILNGEVDDLPEQAFLNVGTIEDVKEKAKQLLE
ATKA
MNPIYKIITSILFCVLSINTMAQDLTGHVTSKADDKPIAYATVT
LKENRLYAFTDEKGNYTIKNVPKGKYTVVFSCMGYASQTVVV
MVNAGGATQNVRLAEDNLQLDEVQVVAHRKKDEITTSYTID
RKTLDNQQIMTLSDIAQLLPGGKSVNPSLMNDSKLTLRSGTL
ERGNASFGTAVEVDGIRLSNNAAMGETAGVSTRSVSASNIES
VEVVPGIASVEYGDLTNGVVKVKTRRGSSPFIVEGSINQHTRQ
IALHKGVDLGGNVGLLNFSIEHARSFLDAASPYTAYQRNVLSL
RYMNVFMKKSLPLTLEVGLNGSIGGYNSKADPDRSLDDYNK
VKDNNVGGNIHLGWLLNKRWITNVDLTAAFTYADRLSESYT
NESSNATQPYIHTLTEGYNIAEDYDRNPSANIILGPTGYWYLR
GFNDSKPLNYSLKMKANWSKAFGKFRNRLLVGGEWTSSMN
Cluster:
RGRGTYYADMRYAPSWREYRYDALPSLNNIAIYAEDKLSMD
41 Uncharacterized G6AGX5
VNERQNAELTAGIREDITSIPGSEYGSVGSFSPRMNARYVFRF
protein
GQNSWLNSMTLHAGWGRSVKIPSFQVLYPSPSYRDMLAFA
STSDADNRSYYAYYTYPSMARYNANLKWQRADQWDLGVE
WRTKIADVSLSFFRSKVSNPYMATDVYTPFTYKYTSPAMLQR
SGIAVADRRFSIDPQTGIVTVSDASGVKSPVTLGYEERNTYVT
NTRYVNADALQRYGLEWIVDFKQIKTLRTQVRLDGKYYHYKA
QDETLFADVPVGLNTRQSDGRLYQYVGYYRGGAATTTNYTA
NASASNGSVSGQVDLNATITTHIPKIRLIVALRLESSLYAFSRAT
SSRGYVVSSGNEYFGVPYDDKTENQTVIVYPEYYSTWDAPDV
LIPFAEKLRWAETNDRGLFNDLAQLVVRTNYPYTLNPNRLSA
YWSANLSVTKEIGRHVSVSFYANNFFNTLSQVHSTQTGLETSL
FGSGYVPSFYYGLSLRLKI
[114] In some embodiments, the Prevotella bacteria is a strain of
Prevotella bacteria
free or substantially free of one or more (e.g., 1,2, 3,4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16,
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17, 18, 19, 20, 21, 22, 23, 24, 25 or more) proteins listed in Table 2 and/or
one or more (e.g., 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25 or more) genes
encoding proteins listed in Table 2. In some embodiments, Prevotella bacteria
is free of all of the
proteins listed in Table 2 and/or all of the genes encoding the proteins
listed in Table 2.
Table 2: Other Prevotella proteins
Seq. Name Uniprot ID
Amino Acid Sequence
ID. No.
MERIDISVLMAVYKKDNPAFLRESLESIFSQTVEAAEVVLLED
GPLTDALYDVIKSYEAIYSTLKVVSYPENRGLGKTLNDGLLLCK
UDP-Gal:alpha-D-
YNLVARMDADDICKPNRLEMEYNWLKSHEDYDVIGSWVDE
GIcNAc-
42 Q03084 FTDNKTRVKSIRKVPEAYDEIKNYAQYRCPINHPTAMYRKAA
diphosphoundeca
VLAVGGYLTEYFPEDYFLWLRMLNNGSKFYNIQESLLWFRYS
prenol
EETVAKRGGWAYACDEVRILVRMLKMGYIPFHVFCQSVVIRF
TTRVMPLPIRQRLYNLIRKT
MSQINGRISQIIGPVIDVYFDTKGENPEKVLPKIHDALRVKRA
NGQDLIIEVQQHIGEDTVRCVAMDNTDGLQRNLEVVPTGSP
IVMPAGDQIKGRMMNVIGQPIDGMEALSMEGAYPIHREAP
KFEDLSTHKEMLQTGIKVIDLLEPYMKGGKIGLFGGAGVGKT
VLIMELINNIAKGHNGYSVFAGVGERTREGNDLIRDMLESGV
IRYGEKFRKAMDEGKWDLSLVDQEELQKSQATLVYGQMNE
ATP_synthase_su
43 A1B8P0 PPGARASVALSGLTVAEEFRDHGGKNGEAADIMFFIDNIFRF
bunit beta
_ TQAGSEVSALLGRMPSAVGYQPTLASEMGTMQERITSTKHG
SITSVQAVYVPADDLTDPAPATTFTHLDATTELSRKITELGIYP
AVDPLGSTSRILDPLIVGKDHYECAQRVKQLLQHYNELQDIIAI
LGMDELSDEDKLVVNRARRVQRFLSQPFTVAEQFTGVKGV
MVPIEETIKGFNAILNGEVDDLPEQAFLNVGTIEDVKEKAKRL
LEATK
MPIGNGQKYQLTIINHTEIIMLIDYKKVNIYQDERLILKDVDFQ
AETGEFIYLIGRVGSGKSSLLKTIYGELDIDSEDAEKAVVLDES
Cell_division_ATP- MPNIKRSRIPALRKQMGIIFQDFQLLHDRSVAKNLKFVLQAT
44 binding_protein_F 005779
GWTSKQKIERRIEEVLAQVGMTDKKNKMPSELSGGEQQRIA
tsE IARALLNTPKIIIADEPTGNLDPETAANIVSILKDSCQAGTTVIM
STHNINLIDQFPGKVYRCHEGELHQLTDKKEVSELAEETAPVE
TIDEPEQND
41
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MKRNILLFICLATSILLLFGLNLTTGSVQIPFADILDILCGRFIGKE
SWEYIILENRLPQTLTAILCGASLSVCGLMLQTAFRNPLAGPD
VFGISSGAGLGVALVMLLLGGTVSTSIFTVSGFLAILTAAFVGA
Hemin_transport_
IAVTALILFLSTLVRNSVLLLIVGIMVGYVSSSAVSLLNFFASEEG
45 system_permease Q56992
VKSYMVWGMGNFGAVSMNHIPLFSILCLIGIIASFLLVKPLNIL
_protein_HmuU
LLGPQYAESLGISTRQIRNILLVVVGLLTAITTAFCGPISFIGLAIP
HIARLLFRTENHQILLPGIVLSGAAIALLCNFICYLPGESGIIPLN
AVTPLIGAPIIIYVIIQRR
MKKYYPWVLVALLWFVALLNYMDRQMLSTMQEAMKVDIA
ELNHAEAFGALMAVFLWIYGIVSPFAGIIADRVNRKWLVVGS
IFVWSAVTYLMGYAESFDQLYWLRAFMGISEALYIPAALSLIA
DWHEGKSRSLAIGIHMTGLYVGQAVGGFGATLAAMFSWHA
Hexuronate trans AFHWFGIIGIVYSLVLLLFLKENPKHGQKSVLQGETKPSKNPFR
46 _
034456
porter GLSIVFSTWAFWVILFYFAVPSLPGWATKNWLPTLFANSLDIP
MSSAGPMSTITIAVSSFIGVIMGGVISDRWVQRNLRGRVYTS
AIGLGLTVPALMLLGFGHSLVSVVGAGLCFGIGYGMFDANN
MPILCQFISSKYRSTAYGIMNMTGVFAGAAVTQVLGKWTDG
GNLGNGFAILGGIVVLALVLQLSCLKPTTDNME
MVTKKTTTKKAPVKKTSAKTTKVKEPSHIGLVKNDAYLAPYED
AIRGRHEHALWKMNQLTQNGKLTLSDFANGHNYYGLHQTA
DGWVFREWAPNATEIYLVGDFNGWNEQEAYQCHRIEGTG
NWELTLPHDAMQHGQYYKMRVHWEGGEGERIPAWTQRV
VQDEASKIFSAQVWAPAEPYVWEKKTFKPQTSPLLIYECHIG
MAQDEEKVGTYNEFREKVLPRIIKDGYNAIQIMAIQEHPYYG
SFGYHVSSFFAASSRFGTPEELKALIDEAHKNGIAVIMDIVHSH
1,4-alpha- AVKNEVEGLGNLAGDPNQYFYPGERHEHPAWDSLCFDYGK
47 glucan_branching P9WN45 DEVLHFLLSNCKYWLEEYHFDGFRFDGVTSMLYYSHGLGEAF
_enzyme_GIgB CNYADYFNGHQDDNAICYLTLANCLIHEVNKNAVTIAEEVSG
MPGLAAKFKDGGYGFDYRMAMNIPDYWIKTIKELPDEAWK
PSSIFWEIKNRRSDEKTISYCESHDQALVGDKTIIFRLVDADMY
WHFRKGDETEMTHRGIALHKMIRLATIAAINGGYLNFMGNE
FGHPEWIDFPREGNGWSHKYARRQWNLVDNEELCYHLLGD
FDRKMLEVITSEKKFNETPIQEIWHNDGDQILAFSRGELVFVF
NFSPSHSYSDYGFLVPEGSYNVVLNTDAREFGGFGFADDTVE
HFTNSDPLYEKDHKGWLKLYIPARSAVVLRKK
MKIDIERIKYFLTVGMFMKTEHSSKRRNMLIRQFQKFYLTVKF
48
Cluster: YihY D9RW24 FFVRDHAASTAQLSFSTIMAIVPIASMIFAIANGFGFGQFLEK
family protein QFREMLSAQPEAATWLLKLTQSYLVHAKTGLFIGIGLMIMLY
SVFSLIRTVETTFDNIWQVKDSRPISRIVIDYTALMFLVPISIIILS
42
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GLSIYFYSFVENLNGLRFLGTIASFSLRYLVPWAILTLMFIVLYV
FMPNAKVKITKTVAPAMIASIAMLCLQAVYIHGQIFLTSYNAI
YGSFAALPLFMLWILASWYICLFCAELCYFNQNLEYYECLIDTE
DICHNDLLILCATVLSHICQRFANDQKPQTALQIKTETHIPIRV
MTDILYRLKEVNLISENFSPTSDEVTYTPTHDTNNITVGEMIA
RLESTPASDFALLGFSPKKAWNHDIYDRVGSIREIYLNELKSINI
KELISYSEN
MMKRPSIARVVKVIICLLTPILLSFSGIGDNDIDKKKSTSKEVDD
TLRIVITGDLLLDRGVRQKIDMAGVDALFSPTIDSLFHSSNYVI
ANLECPVTKIRERVFKRFIFRGEPEWLPTLRRHGITHLNLANN
Capsule_biosynth
HSIDQGRNGLLDTQEQIKKAGMIPIGAGKNMEEAAEPVLIST
49 esis_ A protein_Cap P19579
SPRHVWVISSLRLPLENFLYLPQKPCVSQESIDSLIMRVKRLRA
TDKNCYILLILHWGWEHHFRATPQQREDAHKLIDAGADAIV
GHHSHTLQTIETYRGKPIYYGIGNFIFDQRKPMNSRACLVELSI
TAEKCKAKALPIEIKNCTPYLSK
MILLSFDTEEFDVPREHGVDFSLEEGMKVSIEGTNRILDILKAN
NVCATFFCTGNFAELAPEVMERIKNEGHEVACHGVDHWQP
KPEDVFRSKEIIERVTGVKVAGYRQPRMFPVSDEDIEKAGYLY
Peptidoglycan_de
50 B5ZA76 NSSLNPAFIPGRYMHLTTSRTWFMQGKVMQIPASVSPHLRI
acetylase
PLFWLSMHNFPEWFYLRLVRQVLRHDGYFVTYFHPWEFYDL
KSHPEFKMPFIIKNHSGHELEQRLDRFIKAMKADKQEFITYVD
FVNRQKK
MAKNISFTIKYWKQNGPQDQGHFDTHEMKNIPDDTSFLEM
LDILNEELIAAGDEPFVFDHDCREGICGMCSLYINGTPHGKTE
Fumarate_reducta RGATTCQLYMRRFNDGDVITVEPWRSAGFPVIKDCMVDRT
51 se_iron- P0AC47 AFDKIIQAGGYTTIRTGQAQDANAILISKDNADEAMDCATCI
sulfur_subunit GCGACVAACKNGSAMLFVSSKVSQLALLPQGKPEAAKRAKA
MVAKMDEVGFGNCTNTRACEAVCPKNEKIANIARLNREFIK
AKFAD
MSENKLSTNEQAQTADAPVKASYTEYKVIPSQGYCMIVKCRK
GDQTVVLKTLKEEYRERVLLRNALKREFKQCQRLNHSGIVRY
Serine/threonine-
QGLVEVDGYGLCIEEEYVEGRTLQAYLKENHTDDEKIAIINQIA
52 protein_kinase_Pk P9WI71
DALRYAHQQGVIHRNLKPSNVLVTTQGDYVKLIDFSVLSPED
nH VKPTAETTRFMAPEMKDETLTADATADIYSLGTIMKVMGLTL
AYSEVIKRCCAFKRSDRYSNVDELLADLNNEGSSFSMPKIGKG
TVVLGLIIAVVIGIGALLYNYGGALIDQVGKIDVSSVFSSDAETA
PEDTVKVNTAEQSDSLSTEAEAPAIGKLAFMNRMKPALYKDL
43
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DNIFEKNSADKAKLTKAIKTYYRGLIQANDTLDNEQRAEVDRV
FGDYVKQKKAALN
MRKYICLLLFYLFTFLPLSAQQGNDSPLRKLQLAEMAIKNFYV
DSVN EQKLVE DG I RG M LE KLDP HSTYTDAKETKAM NE PLQG
DFEGIGVQFNMIEDTLVVIQPVVNGPSQKVGILAGDRIVSVN
DSTIAGVKMARIDIMKMLRGKKGTKVKLGVVRRGVKGVLTF
VVTRAKI PVHTI NASYM I RP NVGYI RI ESFG M KTH DE F MSAV
Carboxy- DSLKKKGMKTLLLDLQDNGGGYLQSAVQISNEFLKNNDMIV
53 terminal_processi 034666
YTEGRRARRQNFKAIGNGRLQDVKVYVLVNELSASAAEIVTG
ng_protease_CtpA AIQDNDRGTVVGRRTFGKGLVQRPFDLPDGSMIRLTIAHYYT
PSGRCIQKPYTKGDLKDYEMDIEKRFKHGELTNPDSIQFSDSL
KYYTI R KHRVVYGGGG I MPDNFVPLDTTKFTRYHRMLAAKSI I
INAYLKYADANRQALKAQYSSFDAFNKGYVVPQSLLDEIVAE
GKKEKIEPKDAAELKATLPNIALQI KALTARD IWD M NEYF RV
WNTQSDIVNKAVALATGK
MKLTEQRSSMLHGVLLITLFACAAFYIGDMGWVKALSLSPM
VVGIILGMLYANSLRNNLPDTWVPGIAFCGKRVLRFGIILYGF
RLTFQDVVAVGFPAIIVDAIIVSGTILLGVLVGRLLKMDRSIALL
Cluster: TACGSGICGAAAVLGVDGAIRPKPYKTAVAVATVVIFGTLSM
54 Uncharacterized D9R RG3 FLYPI LYRAG I F DLSPDAMG I
FAGSTI H EVAHVVGAG NAMGA
protein AVSNSAIIVKMIRVMMLVPVLLVIAFFVAKNVAERDDEAGGS
RKI NI PWFAI LFLVVIG F NSLN LLPKE LVDF I NTLDTF LLTMAMS
ALGAETSIDKFKKAGFKPFLLAAILWCWLIGGGYCLAKYLVPV
LGVAC
MNKQFLLAALWLSPLGLYAHKANGIGAVTWKNEAPKERMI
RGIDEDKTHQRFTLSGYVKDRNGEPLINATIYDLTTRQGTMT
NAYG H FSLTLG EGQH El RCSYVGYKTLI ETI DLSANQN HDI I LQ
NEAQLDEVVVTTDLNSPLLKTQTGKLSLSQKDIKTEYALLSSPD
VIKTLQRTSGVADGMELASGLYVHGGNGDENLFLLDGTPLY
HTNHSLGLFSSFNADVVKNVDFYKSGFPARYGGRLSSVIDVR
Cluster: Cna TADGDLYKTHGSYRIGLLDGAFHIGGPIRKGKTSYNFGLRRSW
55 protein B-type X6Q2J4
MDLLTRPAFAIMNHKSDNEDKLSMSYFFHDLNFKLTNIFNER
domain protein SRMSLSVYSGEDRLDAKDEWHSNNSSGYNDVDIYVNRFHW
GNFNAALDWNYQFSPKLFANFTAVYTHNRSTVSSSDEWRFT
RPGEKEQLTLTSHGYRSSIDDIGYRAAFDFRPSPRHHIRFGQD
YTYHRFQPQTYNRFDNYQTNSEAKADTIATHSYNKNVAHQL
TFYAEDEMTLNEKWSLNGGVNADVFHISGKTFATLSPRLSM
KFQPTERLSLKASYTLMSQFVHKIANSFLDLPTDYWVPTTARL
HPMRSWQVAAGAYMKPNKHWLLSLEAYYKRSSHILQYSSW
44
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AGLEPPAANWDYMVMEGDGRSYGVELDADYNVSNLTLHG
SYTLSWTQKKFDDFYDGWYYDKFDNRHKLTLTGRWNITKKI
AAFAAWTFRTGNRMTIPTQYIGLPDVPAQEQGGLTFNSSDD
NTLNFAYEKPNNVILPAYHRLDIGFDFHHTTKKGHERIWNLSF
YNAYCHLNSLWVRVKIDSNNQMKIRNIAFIPVIPSFSYTFKF
MSKQVFQTDSRQRWSYFKWTLRVILTILSLLGIVFLAMFALEG
SPQMPFRHDYRNAVTAASPYTKDNKTAKLYKSFRDFFKEKK
MHNNYAKATIKKQRFIGKADSVTQKYFREWDDPRIGVRSA
WYVNWDKHAYISLKNNIKHLNMVLPEWFFINPKTDKVEYRI
DKQALRLMRRTGIPVLPMLTNNYNSDFHPEAIGRIMRDEKK
RMALINEMVRTCRHYGFAGINLDLEELNIQDNDLLVELLKDFS
RVFHANGLYVTQAVAPFNEDYNMQELAKYNDYLFLMAYDE
HNIESQPGAVSSQRWVEKATDWAAKNVPNDKIVLGMATYG
YDWANGEGGTTVSFDQTMAIAQDADAKVKFDDDTYNVNF
SYQNTDDGKIHHVFFTDAATTFNIMRFGAEYHLAGYGLWRL
GTEDKRIWRFYGKDMSWENVARMSVAKLMQLNGTDDVNF
VGSGEVLEVTTEPHPGDISIRIDKDNRLISEEYYRALPSTYTIQR
Poly-beta-1,6-N- LGKCKDKQLVITFDDGPDSRWTPTVLSTLKKYNVPAAFFMVG
acetyl-D- LQMEKNLPLVKQVYEDGHTIGNHTFTHHNMIENSDRRSYAE
56 P75905
glucosamine_synt LKLTRMLIESVTGHSTILFRAPYNADADPTEHEEIWPMIVASR
hase RNYLFVGESIDPNDWEPNVTSDQIYQRVIDGVHHEDGHIILL
HDAGGSSRKPTLDALPRIIETLQHEGYQFISLEQYLGMGKQTL
MPEINKGKAYYAMQTNLWLAEMIYHVSDFLTALFLVFLALG
MMRLIFMYVLMIREKRAENRRNYAPIDAATAPAVSIIVPGYN
EEVNIVRTITTLKQQDYPNLHIYFVDDGSKDHTLERVHEAFDN
DDTVTILAKKNGGKASALNYGIAACRSEYVVCIDADTQLKND
AVSRLMKHFIADTEKRVGAVAGNVKVGNQRNMLTYWQAIE
YTSSQNFDRMAYSNINAITVVPGAIGAFRKEVIEAVGGFTTDT
LAEDCDLTMSINEHGYIIENENYAVALTEAPETLRQFVKQRIR
WCFGVMQAFWKHRSSLFAPSKKGFGLWAMPNMLIFQYIIP
TFSPLADVLMLIGLFTGNALQIFFYYLIFLVIDASVSIMAYIFEGE
RLWVLLWVIPQRFFYRWIMYYVLFKSYLKAIKGELQTWGVLK
RTGHVKG
MAKKRNKARSRHSLQVVTLCISTAMVLMLIGIVVLTGFTSRN
Cell_division_prot LSSYVKENLTITMILQPDMNTEESAALCERIRTLHYINSLNFISK
57 ein FtsX 034876 EQALKDGTKELGANPAEFAGENPFTGEIEVQLKANYANNDSI
_
RNIVQQLRTYRGVSDITYPQSLVESVNQTLGKISLVLLVIAVLLT
IISFSLINNTIRLSIYAHRFSIHTMKLVGGSWSFIRAPFLRRAVLE
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GLVSALLAIAVLGIGICLLYEKEPEITKLLSWDALIITAIVMLAFG
VIIATFCAWLSVNKFLRMKAGDLYKI
MKNIYFLSDAHLGSLAIDHRRTHERRLVRFLDSIKHKAAAVYLL
GDMFDFWNEYKYVVPKGFTRFLGKISELTDMGVEVHFFTGN
UDP-2,3- HDLWTYGYLEKECGVILHRKPITTEIYDKVFYLAHGDGLGDPD
58 diacylglucosamine P44046
PMFRFLRKVFHNRFCQRLLNFFHPWWGMQLGLNWAKRSR
_hydrolase LKRKDGKEVPYLGEDKEYLVQYTKEYMSTHKDIDYYIYGHRHI
ELDLTLSRKARLLILGDWIWQFTYAVFDGEHMFLEEYVEGESK
P
MVGLDVLCYFIHAKGREKECYFERIIYQITCHSRTKCYLCNIMK
YSIIVPVFNRPDEVEELLESLLSQEEKDFEVVIVEDGSQIPCKEV
CDKYADKLDLHYYSKENSGPGQSRNYGAERAKGEYLLILDSD
Poly-beta-1,6-N- VVLPKGYICAVSEELKREPADAFGGPDCAHESFTDTQKAISYS
acetyl-D- MTSFFTTGGIRGGKKKLDKFYPRSFNMGIRRDVYQELGGFSK
59 P75905
glucosamine_synt MRFGEDIDFSIRIFKAGKRCRLFPEAWVWHKRRTDFRKFWK
hase QVYNSGIARINLYKKYPESLKLVHLLPMVFTVGTALLVLMILFG
LFLQLFPIINVFGSVFIMMGLMPLVLYSVIICVDSTMQNNSLNI
GLLSIEAAFIQLTGYGCGFISAWWKRCVCGMDEFAAYEKNFY
K
MKIEKVHAREIMDSRGNPTVEVEVTLENGVMGRASVPSGAS
TGENEALELRDGDKNRFLGKGVLKAVENVNNLIAPALKGDCV
LNQRAIDYKMLELDGTPTKSKLGANAILGVSLAVAQAAAKAL
NIPLYRYIGGANTYVLPVPMMNIINGGAHSDAPIAFQEFMIR
PVGAPSEKEGIRMGAEVFHALAKLLKKRGLSTAVGDEGGFAP
60 Enolase Q8DTS9 KFDGIEDALDSIIQAIKDAGYEPGKDVKIAMDCAASEFAVCED
GKWFYDYRQLKNGMPKDPNGKKLSADEQIAYLEHLITKYPID
SIEDGLDENDWENWVKLTSAIGDRCQLVGDDLFVTNVKFLE
KGIKMGAANSILIKVNQIGSLTETLEAIEMAHRHGYTTVTSHR
SGETEDTTIADIAVATNSGQIKTGSMSRTDRMAKYNQURIEE
ELGACAKYGYAKLK
MKKLFTIAMLLGVTLGIHAQEVYSLQKCRELALQNNRQLKVS
RMTVDVAENTRKAAKTKYLPRVDALAGYQHFSREISLLSDDQ
Outer_membrane KNAFSNLGTNTFGQLGGQIGQNLTSLAQQGILSPQMAQQL
61 _efflux_protein_B Q8G0Y6
GQLFSNVATPLTQVGNNIGQSINDAFRSNTKNVYAGGIVVN
epC QPIYMGGAIKAANDMAAIGEQVAQNNISLKRQLVLYGVDN
AYWLAISLKKKEALAIRYRDLAQKLNEDVKKMIREGVATRAD
GLKVEVAVNTADMQIARIQSGVSLAKMALCELCGLELNGDIP
LSDEGDADLPPTPSTQFDNYTVSSSDTTGLNEARPELRLLQNA
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VDLSIQNTKLIRSLYMPHVLLTAGYSVSNPNLFNGFQKRFTDL
WNIGITVQVPVWNWGENKYKVRASKTATTIAQLEMDDVRK
KIDLEIEQNRLRLKDANKQLATSQKNMAAAEENLRCANVGFK
EGVMTVTEVMAAQTAWQTSRMAIIDAEISVKLAQTGLQKA
LGGL
MKRTFVTKMVKPIEENSLFFMFMLLVGAFTNVSHRNVFGYIE
LIADVYIICFLLSLCQRTIRQGLVIMLSSVIYVVAIIDTCCKTLFDT
PITPTMLLLAQETTGREATEFFLQYLNLKLFFSAADIILFLAFCHI
VMAVKKMKFSTSYLKQPFVAFVLMFTIFVGMALSIYDKVQLY
TVKNLSGLEVAVTNGFAHLYHPVERIVYGLYSNHLIAKQVDG
VIMANQQIKVDSCSFTSPTIVLVIGESANRHHSQLYGYPLPTT
Phosphoethanola
PYQLAMKNGKDSLAVFTNVVSPWNLTSKVFKQIFSLQSVDE
62 mine transferase Q7CPCO
_
KGDWSKYVLFPAVFKKAGYHVSFLSNQFPYGINYTPDWTNN
_CptA
LVGGFFLNHPQLNKQMFDYRNVTIHNYDEDLLNDYKEIISYK
KPQLIIFHLLGQHFQYSLRCKSNMKKFGIKDYKRMDLTDKEK
QTIADYDNATLYNDFVLNKIVEQFRNKDAIIVYLSDHGEDCYG
KDVNMAGRLTEVEQINLKKYHEEFEIPFWIWCSPIYKQRHRKI
FTETLMARNNKFMTDDLPHLLLYLAGIKTKDYCEERNVISPSF
NNNRRRLVLKTIDYDKALYQ
MFKNHPKGLLQAAFSNMGERFGYVIMNAVLALFLCSKFGLS
DETSGLIASLFLAAIYVMSLVGGVIADRTQNYQRTIESGLVVM
ALGYVALSIPVLATPENNSYLLAFTIFALVLIAVGNGLFKGNLQ
AIVGQMYDDFETEAAKVSPERLKWAQGQRDAGFQIFYVFIN
LGALAAPFIAPVLRSWWLGRNGLTYDAALPQLCHKYINGTIG
Dipeptide_and_tri DNLGNLQELATKVGGNSADLASFCPHYLDVFNTGVHYSFIAS
63 peptide_permeas P36837
VVTMLISLIIFMSSKKLFPMPGKKEQIVNVEYTDEEKASMAKEI
e_B KQRMYALFAVLGISVFFWFSFHQNGQSLSFFARDFVNTDSV
APEIWQAVNPFFVISLTPLIMWVFAYFTKKGKPISTPRKIAYG
MGIAGFAYLFLMGFSLVHNYPSAEQFTSLEPAVRATMKAGP
MILILTYFFLTVAELFISPLGLSFVSKVAPKNLQGLCQGLWLGA
TAVGNGFLWIGPLMYNKWSIWTCWLVFAIVCFISMVVMFG
MVKWLERVTKS
MQKKIKIGLLPRVIIAILLGLFLGYYLPDPAVRVFLTFNSIFSQFL
GFMIPLIIIGLVTPAIAGIGKGAGKLLLATVAIAYVDTIVAGGLS
C4- YGTGTWLFPSMIASTGGAIPHIDKATELTPYFTINIPAMVDV
64 dicarboxylate_tra Q9I4F5
MSSLVFSFIAGLGIAYGGLRTMENLFNEFKTVIEKVIEKAIIPLL
nsport_protein_2 PLYIFGVFLSMTHNGQARQVLLVFSQIIIVILVLHVLILIYEFCIA
GAIVKHNPFRLLWNMLPAYLTALGTSSSAATIPVTLKQTVKN
GVSEEVAGFVVPLCATIHLSGSAMKITACALTICMLTDLPHDP
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GLFIYFILMLAIIMVAAPGVPGGAIMAALAPLSSILGFNEEAQA
LMIALYIAMDSFGTACNVTGDGAIALAVNKFFGKKKETSILS
MISVYSIKPQFQRVLTPILELLHRAKVTANQITLWACVLSLVIGI
LFWFAGDVGTWLYLCLPVGLLIRMALNALDGMMARRYNQI
Inner membrane
65 _
P76090
TRKGELLNEVGDVVSDTIIYFPLLKYHPESLYFIVAFIALSIINEYA
_protein_YnbA
GVMGKVLSAERRYDGPMGKSDRAFVLGLYGVVCLFGINLSG
YSVYIFGVIDLLLVLSTWIRIKKTLKVTRNSQTPE
MKLSTILLSIMLGLSSSTMAQQKDVTIKLIETTDVHGSFFPYDF
ITRKPKSGSMARVYTLVEELRKKDGKDNVYLLDNGDILQGQPI
SYYYNYVAPEKTNIAASVLNYMGYDVATVGNHDIETGHKVY
DKWFKELKFPILGANIIDTKTNKPYILPYYTIKKKNGIKVCVIGM
LTPAIPNWLKESIWSGLRFEEMVSCAKRTMAEVKTQEKPDVI
VGLFHSGWDGGIKTPEYDEDASKKVAKEVPGFDIVFFGHDH
2',3'-cyclic- TPHSSIEKNIVGKDVICLDPANNAQRVAIATLTLRPKTVKGKR
66 P08331
nucleotide QYTVTKATGELVDVKELKADDAFIQHFQPEIDAVKAWSDQVI
GRFENTIYSKDSYFGNSAFNDLILNLELEITKADIAFNAPLLFNA
SIKAGPITVADMFNLYKYENNLCTMRLTGKEIRKHLEMSYDL
WCNTMKSPEDHLLLLSSTQNDAQRLGFKNFSFNFDSAAGID
YEVDVTKPDGQKVRILRMSNGEPFDENKWYTVAVNSYRAN
GGGELLTKGAGIPRDSLKSRIIWESPKDQRHYLMEEIKKAGV
MNPQPNHNWKFIPETWTVPAAARDRKLLFGE
MKLSELKTGETGVIVKVSGHGGFRKRIIEMGFIKGKTVEVLLN
APLQDPVKYKIMGYEVSLRHSEADQIEVLSDVKTHSVGNEEE
QEDNQLEMDSTTYDSTDKELTPEKQSDAVRRKNHTINVALV
GNPNCGKTSLFNFASGAHERVGNYSGVTVDAKVGRAEFDGY
VFNLVDLPGTYSLSAYSPEELYVRKQLVDKTPDVVINVIDSSNL
ERNLYLTTQLIDMHIRMVCALNMFDETEQRGDHIDAQKLSEL
FGVPMIPTVFTNGRGVKELFRQIIAVYEGKEDESLQFRHIHIN
HGHEIENGIKEMQEHLKKYPELCHRYSTRYLAIKLLEHDKDVE
67
Fe(2+)_transporte P33650 QLVSPLGDSIEIFNHRDTAAARVKEETGNDSETAIMDAKYGFI
r_FeoB NGALKEANFSTGDKKDTYQTTHVIDHVLTNKYFGFPIFFLVLL
VMFTATFVIGQYPMDWIEAGVGWLGEFISKNMPAGPVKD
MIVDGIIGGVGAVIVFLPQILILYFFISYMEDCGYMSRAAFIMD
RLMHKMGLHGKSFIPLIMGFGCNVPAVMATRTIESRRSRLIT
MLILPLMSCSARLPIYVMITGSFFALKYRSLAMLSLYIIGVLMA
VAMSRLFSAFVVKGEDTPFVMELPPYRFPTWKAIGRHTWEK
GKQYLKKMGGIILVASIIVWALGYFPLPDDPNMDNQARQEQ
SYIGRIGKAVEPVFRPQGFNWKLDVGLLSGMGAKEIVASTM
GVLYSNDGSFSDDNGYSSETGKYSKLHNLITKDVATMHHISY
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EEAEPIATLTAFSFLLFVLLYFPCVATIAAIKGETGSWGWALFA
AGYTTALAWIVSAVVFQVGMLFM
MESFIIEGGHQLSGTIAPQGAKNEALEVICATLLTSEEVIIRNVP
DILDVNNLIKLLQDIGVKVKKLAPNEFSFQADEVNLDYLESSDF
VKKCSSLRGSVLMIGPLLGRFGKATIAKPGGDKIGRRRLDTHF
LGFKNLGAHFGRVEDRDVYEIQADKLVGTYMLLDEASITGTA
NIIMAAVLAEGTTTIYNAACEPYIQQLCKMLNAMGAKISGIAS
UDP-N-
68 P9WJM1
NLITIEGVKELHSADHRILPDMIEVGSFIGIAAMIGDGVRIKDV
acetylglucosamine
SVPNLGLILDTFHRLGVQIIVDNDDLIIPRQDHYVIDSFIDGTIM
TISDAPWPGLTPDLISVLLVVATQAQGSVLFHQKMFESRLFFV
DKLIDMGAQIILCDPHRAVVVGHDNAKKLRAGRMSSPDIRA
GIALLIAALTAQGTSRIDNIVQIDRGYENIEGRLNALGAKIQRA
EVC
MNIAVIFAGGSGLRMHTKSRPKQFLDLNGKPIIIYTLELFDNH
PNIDAIVVACIESWIPFLEKQLRKFEINKVVKIIPGGKSGQESIY
Ribito1-5-
KGLCAAEEYAQSKGVSNEETTVLIHDGVRPLITEETITDNIKKV
69 phosphate_cytidyl Q8RKI9
EEVGSCITCIPATETLIVKQADDALEIPSRADSFIARAPQSFRLI
yltransferase
DIITAHRRSLAEGKADFIDSCTMMSHYGYKLGTIIGPMENIKIT
TPTDFFVLRAMVKVHEDQQIFGL
[115] In some embodiments, the Prevotella bacteria are from a strain of
Prevotella
bacteria comprising one or more of the proteins listed in Table 1 and that is
free or substantially
free of one or more proteins listed in Table 2. In some embodiments, the
Prevotella bacteria are
from a strain of Prevotella bacteria that comprises all of the proteins listed
in Table 1 and/or all
of the genes encoding the proteins listed in Table 1 and that is free of all
of the proteins listed in
Table 2 and/or all of the genes encoding the proteins listed in Table 2.
Pharmaceutical Compositions
[116] In certain embodiments, provided herein are pharmaceutical
compositions
comprising Prevotella bacteria (e.g., a Prevotella bacteria described herein),
e.g., for use in
methods of treating or preventing a TH2-mediated condition (such as atopic
dermatitis, asthma,
and/or an allergy). In some embodiments, the Prevotella bacteria composition
comprises
Prevotella bacteria and/or a combination of bacteria strains described herein
and a
pharmaceutically acceptable carrier. In some embodiments, the Prevotella
bacteria composition
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comprises a single Prevotella bacteria strain described herein and a
pharmaceutically acceptable
carrier.
[117] In some embodiments, the pharmaceutical compositions comprise
Prevotella
bacteria and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of
bacteria strains or species,
e.g., in addition to Prevotella. In some embodiments, the pharmaceutical
compositions comprise
Prevotella bacteria. In some embodiments, the pharmaceutical composition
comprises
lyophilized Prevotella bacteria. In some embodiments, the pharmaceutical
composition
comprises gamma irradiated Prevotella bacteria. In some embodiments, the
pharmaceutical
composition comprises live Prevotella bacteria.
[118] In some embodiments, to quantify the numbers of Prevotella 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,
nanoparticle tracking
analysis (NTA), Coulter counting, or dynamic light scattering (DLS) or a
combination of these
techniques can be used. NTA and the Coulter counter count particles and show
their sizes. DLS
gives the size distribution of particles, but not the concentration. Bacteria
frequently have
diameters of 1-2 um (microns). The full range is 0.2-20 um. Combined results
from Coulter
counting and NTA can reveal the numbers of bacteria in a given sample. Coulter
counting
reveals the numbers of particles with diameters of 0.7-10 um. For most
bacterial samples, the
Coulter counter alone can reveal the number of bacteria. For NTA, a Nanosight
instrument can
be obtained from Malvern Panalytical. For example, the NS300 can visualize and
measure
particles in suspension in the size range 10-2000nm. NTA allows for counting
of the numbers of
particles that are, for example, 50-1000 nm in diameter. DLS reveals the
distribution of particles
of different diameters within an approximate range of 1 nm ¨ 3 um.
[119] In some embodiments, the Prevotella bacteria may be quantified based
on particle
count. For example, total particle content of a Prevotella bacteria can be
measured using NTA.
[120] In some embodiments, the Prevotella bacteria may be quantified based
on total
cell count (TCC) (e.g., determined by Coulter counter).
[121] In some embodiments, the Prevotella bacteria may be quantified using
a plate
count assay (e.g., by creating serial dilutions of the bacteria, allowing them
to grow on a suitable
medium, and then counting the number of colonies).
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[122] In some embodiments, the Prevotella bacteria may be quantified based
on the
amount of protein, lipid, or carbohydrate. For example, total protein content
of a Prevotella
bacteria preparation can be measured using the Bradford assay or the BCA
assay.
[123] In some embodiments, the Prevotella bacteria are isolated away from
one or
more other bacterial components, e.g., of the source culture. In some
embodiments, the
pharmaceutical composition further comprises other bacterial components or
strains.
[124] In certain aspects, provided are pharmaceutical compositions for
administration to
a subject (e.g., human subject). In some embodiments, the pharmaceutical
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.
[125] In some embodiments, the pharmaceutical composition comprises at
least one
carbohydrate.
[126] In some embodiments, the pharmaceutical composition comprises at
least one
lipid. In some embodiments the lipid comprises at least one fatty acid
selected from lauric acid
(12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1),
margaric acid (17:0),
heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic
acid (18:2), linolenic
acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic
acid (20:1),
eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid
(20:5) (EPA),
docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5),
docosahexaenoic
acid (22:6) (DHA), and tetracosanoic acid (24:0).
[127] In some embodiments, the 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.
[128] In some embodiments, the 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,
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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.
[129] In some embodiments, the 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.
[130] 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.
[131] 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.
[132] In some embodiments, the 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.
[133] In some embodiments, the 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.
[134] In some embodiments, the 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 EILB
emulsifier
surfactants.
[135] In some embodiments, the 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 corn starch,
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potato starch, pregelatinized and modified starches thereof, sweeteners,
clays, such as bentonite,
micro-crystalline 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.
[136] In some embodiments, the pharmaceutical 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.
[137] In some embodiments, the pharmaceutical 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.
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Dose Forms
[138] Dose forms comprising Prevotella bacteria are also provided herein,
e.g., for use
in methods to treat or prevent a TH2-mediated condition (such as atopic
dermatitis and/or a food
allergy). A pharmaceutical composition comprising Prevotella 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 Prevotella
bacteria in the solid dose
form can be isolated Prevotella bacteria. Optionally, the Prevotella bacteria
in the solid dose
form can be lyophilized. Optionally, the Prevotella bacteria in the solid dose
form are live.
Optionally, the Prevotella 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).
[139] In certain embodiments, the pharmaceutical composition provided
herein is
prepared as a solid dosage form comprising Prevotella bacteria and a
pharmaceutically
acceptable carrier.
[140] 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 Prevotella bacteria powder (e.g.,
lyophilized
Prevotella bacteria).
[141] 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.
[142] In some embodiments, the solid dosage form comprises a tablet (> 4mm)
(e.g.,
5mm-17mm). For example, the tablet is a 5mm, 6mm, 7mm, 8mm, 9mm, lOmm, llmm,
12mm,
13mm, 14mm, 15mm, 16mm or 17mm 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.
[143] In some embodiments, the solid dosage form comprises a mini-tablet.
The mini-
tablet can be in the size range of 1 mm-4 mm range. E.g., the mini-tablet can
be a 1 mm mini-
tablet, 1.5 mm mini-tablet, 2 mm mini-tablet, 3 mm mini-tablet, or 4 mm mini-
tablet. 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.
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[144] 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 EIPMC (hydroxyl
propyl methyl
cellulose) or gelatin. 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.
[145] The solid dosage form (e.g., tablet or mini-tablet) described herein
can be
enterically coated.
[146] 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 film coating
applications. Cationic Eudragit
E, insoluble at pH? 5, can prevent drug release in saliva.
[147] The solid dose form (e.g., a capsule) can comprise a single layer
coating, e.g., a
non-enteric coating such as EIPMC (hydroxyl propyl methyl cellulose) or
gelatin.
[148] A pharmaceutical composition comprising Prevotella 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,
Prevotella 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
Prevotella bacteria in the
suspension can be isolated Prevotella bacteria. Optionally, the Prevotella
bacteria in the
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suspension can be lyophilized. Optionally, the Prevotella bacteria in the
solid dose form are live.
Optionally, the Prevotella bacteria in the suspension can be gamma irradiated.
Dosa2e
[149] For oral administration to a human subject, the dose of Prevotella
bacteria can be,
e.g., about 2x106- about 2x1016 particles. The dose can be, e.g., about 1x107-
about lx1015, about
1x108- about lx1014, about 1x109- about lx1013, about lx101 - about lx1014, or
about 1x108-
about 1x1012 particles. The dose can be, e.g., about 2x106, about 2x107, about
2x108, about
2x109, about 1x101 , about 2x101 , about 2x10", about 2x1012, about 2x1013,
about 2x1014, or
about 1x1015 particles. The dose can be, e.g., about 2x10'4 particles. The
dose can be, e.g., about
2x10'2 particles. The dose can be, e.g., about 2x101 particles. The dose can
be, e.g., about 1x101
particles. Particle count can be determined, e.g., by NTA.
[150] For oral administration to a human subject, the dose of Prevotella
bacteria can be,
e.g., based on total protein. The dose can be, e.g., about 5 mg to about 900
mg total protein. The
dose can be, e.g., about 20 mg to about 800 mg, about 50 mg to about 700 mg,
about 75 mg to
about 600 mg, about 100 mg to about 500 mg, about 250 mg to about 750 mg, or
about 200 mg
to about 500 mg total protein. The dose can be, e.g., about 10 mg, about 25
mg, about 50 mg,
about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300
mg, about
400 mg, about 500 mg, about 600 mg, or about 750 mg total protein. The dose
can be, e.g., about
mg total protein. Total protein can be determined, e.g., by Bradford assay or
by the BCA
assay.
[151] For administration by injection (e.g., intravenous administration) to
a human
subject, the dose of Prevotella bacteria can be, e.g., about 1x106- about
lx1016 particles. The
dose can be, e.g., about 1x107- about lx1015, about 1x108- about lx1014, about
1x109- about
lx1013, about lx101 - about lx1014, or about 1x108- about lx1012 particles.
The dose can be, e.g.,
about 2x106, about 2x107, about 2x108, about 2x109, about lx101 , about 2x101
, about 2x10",
about 2x1012, about 2x1013, about 2x1014, or about lx1015 particles. The dose
can be, e.g., about
lx1015particles. The dose can be, e.g., about 2x10'4 particles. The dose can
be, e.g., about 2x1013
particles. Particle count can be determined, e.g., by NTA.
[152] For administration by injection (e.g., intravenous administration),
the dose of
Prevotella bacteria can be, e.g., about 5 mg to about 900 mg total protein.
The dose can be, e.g.,
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about 20 mg to about 800 mg, about 50 mg to about 700 mg, about 75 mg to about
600 mg,
about 100 mg to about 500 mg, about 250 mg to about 750 mg, or about 200 mg to
about 500 mg
total protein. The dose can be, e.g., about 10 mg, about 25 mg, about 50 mg,
about 75 mg, about
100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg,
about 500
mg, about 600 mg, or about 750 mg total protein. The dose can be, e.g., about
700 mg total
protein. The dose can be, e.g., about 350 mg total protein. The dose can be,
e.g., about 175 mg
total protein. Total protein can be determined, e.g., by Bradford assay or by
the BCA assay.
[153] In certain embodiments, the 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 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 of the Prevotella
bacteria. In some embodiments, the pharmaceutical composition comprises no
more than 9 x
1011 total cells (e.g., no more than 1 x 1010 total cells, no more than 2 x
1010 total cells, no more
than 3 x 1010 total cells, no more than 4 x 1010 total cells, no more than 5 x
1010 total cells, no
more than 6 x 1010 total cells, no more than 7 x 1010 total cells, no more
than 8 x 1010 total cells,
no more than 9 x 1010 total cells, no more than 1 x 1011 total cells, no more
than 2 x 1011 total
cells, no more than 3 x 1011 total cells, no more than 4 x 1011 total cells,
no more than 5 x 1011
total cells, no more than 6 x 1011 total cells, no more than 7 x 1011 total
cells, no more than 8 x
1011 total cells) of the Prevotella bacteria. In some embodiments, the
pharmaceutical
composition comprises about 6 x 109 total cells of the Prevotella bacteria. In
some embodiments,
the pharmaceutical composition comprises about 1.6 x 1010 total cells of the
Prevotella bacteria.
In some embodiments, the pharmaceutical composition comprises about 8 x 1010
total cells of the
Prevotella bacteria. In some embodiments, the pharmaceutical composition
comprises about 1.6
x 1011 total cells the Prevotella bacteria. In some embodiments, the
pharmaceutical composition
comprises about 3.2 x 1011 total cells the Prevotella bacteria. In some
embodiments, the
pharmaceutical composition comprises about 8 x 1011 total cells of the
Prevotella bacteria. In
some embodiments, the pharmaceutical composition comprises about 1.6 x 1010 to
about 8 x 1011
total cells of the Prevotella bacteria. In some embodiments, the
pharmaceutical composition
comprises about 1.6 x 1010 to about 1.6 x 1011 total cells of the Prevotella
bacteria. In some
embodiments, the pharmaceutical composition comprises about 8 x 1010 to about
8 x 1011 total
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cells of the Prevotella bacteria. In some embodiments, the pharmaceutical
composition
comprises about 1.6 x 1011 to about 8 x 1011 total cells of the Prevotella
bacteria.
[154] In certain embodiments, provided herein are solid dosage forms
comprising the
Prevotella 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 8 x 1010 total cells of the
Prevotella 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 8 x
1010 total cells) is
administered, e.g., once or twice daily to a subject. In some embodiments, 2
capsules (e.g., each
comprising about 8 x 1010 total cells) are administered, e.g., once or twice
daily to a subject. In
some embodiments, 4 capsules (e.g., each comprising about 8 x 1010 total
cells) are administered,
e.g., once or twice daily to a subject. In some embodiments, 10 capsules
(e.g., each comprising
about 8 x 1010 total cells) are administered, e.g., once or twice daily to a
subject. In some
embodiments, the Prevotella bacteria in the capsule are lyophilized (e.g., in
a powder).
[155] In some embodiments, the solid dosage form comprises a capsule. In
some
embodiments, the capsule is an enteric coated tablet. In some embodiments, the
capsule
comprises about 8 x 1010 total cells of the Prevotella bacteria (e.g., total
dose of a capsule or
plurality of capsules). In some embodiments, the capsule comprises about 1.6 x
1011 total cells of
the Prevotella bacteria (e.g., total dose of a capsule or plurality of
capsules). In some
embodiments, the capsule comprises about 3.2 x 1011 total cells of the
Prevotella bacteria (e.g.,
total dose of a capsule or plurality of capsules). In some embodiments, the
capsule comprises
about 8 x 1011 total cells of the Prevotella bacteria (e.g., total dose of a
capsule or plurality of
capsules). In some embodiments, the Prevotella bacteria in the capsule are
lyophilized (e.g., in a
powder).
[156] 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 17mm in diameter. In some embodiments, the tablet
comprises about 8 x
1010 total cells of the Prevotella bacteria (e.g., total dose of a tablet or
plurality of tablets). In
some embodiments, the tablet comprises about 1.6 x 1011 total cells of the
Prevotella bacteria
(e.g., total dose of a tablet or plurality of tablets). In some embodiments,
the tablet comprises
about 3.2 x 1011 total cells of the Prevotella bacteria (e.g., total dose of a
tablet or plurality of
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tablets). In some embodiments, the tablet comprises about 8 x 10" total cells
of the Prevotella
bacteria (e.g., total dose of a tablet or plurality of tablets). In some
embodiments, the Prevotella
bacteria in the tablet are lyophilized.
[157] 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
1 mm to 4mm in diameter. In some embodiments, the mini-tablet (e.g., enteric
coated mini-tablet)
is a 1 mm 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 8
x 1010 total cells of the Prevotella bacteria (e.g., total dose of a plurality
of mini-tablets). In some
embodiments, the solid dosage form comprises mini-tablets that comprise about
1.6 x 10" total
cells of the Prevotella bacteria (e.g., total dose of a plurality of mini-
tablets). In some
embodiments, the solid dosage form comprises mini-tablets that comprise about
3.2 x 10" total
cells of the Prevotella bacteria (e.g., total dose of a plurality of mini-
tablets). In some
embodiments, the solid dosage form comprises mini-tablets that comprise about
8 x 10" total
cells of the Prevotella bacteria (e.g., total dose of a plurality of mini-
tablets). In some
embodiments, the Prevotella bacteria in the mini-tablets are lyophilized. 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., EIPMC
(hydroxyl propyl
methyl cellulose) or 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
hydroxyl propyl methyl cellulose (1-1PMC). In some embodiments, the capsule
comprises gelatin.
In some embodiments, the mini-tablets (e.g., enteric coated mini-tablets) that
comprise about 8 x
10" total cells of the Prevotella bacteria are contained in a capsule(s),
wherein optionally the
capsule comprises EIPMC.
Gamma-irradiation
[158] Powders (e.g., of Prevotella bacteria) can be gamma-irradiated at
17.5 kGy
radiation unit at ambient temperature.
[159] Frozen biomasses (e.g., of Prevotella bacteria) can be gamma-
irradiated at
25 kGy radiation unit in the presence of dry ice.
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Th2-mediated conditions
[160] A TH2 (type 2)-mediated condition that can be treated and/or
prevented by
Prevotella bacteria (e.g., a pharmaceutical composition thereof) includes a
condition that
involves an increase in interleukin (IL)-4, IL-5, IL-13, IL-19, IL-21, IL-31,
IL-33 and/or TSLP
(Thymic Stromal Lymphopoietin) levels (e.g., mRNA or protein levels), e.g.,
during the onset or
course of the disease.
[161] The Prevotella bacteria (e.g., a pharmaceutical composition thereof)
can decrease
interleukin (IL)-4, IL-5, IL-13, IL-19, IL-21, IL-31, IL-33 and/or TSLP
(Thymic Stromal
Lymphopoietin) levels (e.g., mRNA or protein levels), e.g., the Prevotella
bacteria (e.g., a
pharmaceutical composition thereof) causes a decrease as compared to the level
in the absence of
(or prior to) administration of the Prevotella bacteria (e.g., a
pharmaceutical composition
thereof). The pharmaceutical composition can decrease the levels, e.g., by
about 20%, by about
30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, or
by about
90%.
[162] The TH2-mediated condition that can be treated and/or prevented by
Prevotella
bacteria (e.g., a pharmaceutical composition thereof) includes a condition
that involves an
increase in IgGl, IgE, and/or IgA levels (e.g., mRNA or protein levels), e.g.,
during the onset or
course of the disease.
[163] The Prevotella bacteria (e.g., a pharmaceutical composition thereof)
can decrease
IgGl, IgE, and/or IgA levels (e.g., mRNA or protein levels), e.g., the
Prevotella bacteria (e.g., a
pharmaceutical composition thereof) causes a decrease as compared to the level
in the absence of
(or prior to) administration of the Prevotella bacteria (e.g., a
pharmaceutical composition
thereof). The pharmaceutical composition can decrease the levels, e.g., by
about 20%, by about
30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, or
by about
90%.
[164] The TH2-mediated condition can comprise asthma, atopic dermatitis, an
eosinophilic disease, or an allergy (e.g., seasonal allergy, pet allergy, or
food allergy). The
allergy can comprise a food allergy, seasonal allergic rhinitis, or pet
allergy. The food allergy can
comprise a peanut allergy. The food allergy can comprise a cow's milk, hen
egg, fish, crustacean
shellfish, tree nut, wheat, or soy allergy. The food allergy can comprise an
allergy to a food
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antigen. The food antigen can comprise a peanut antigen. The food allergy can
comprise an
allergy to food antigen and the food antigen can comprise a cow's milk, hen
egg, fish, crustacean
shellfish, tree nut, wheat, or soy antigen.
[165] Some of the particular TH2-mediated conditions are further detailed
below.
Allergic Rhinitis
[166] In some embodiments, the TH2-mediated condition includes allergic
rhinitis (i.e.,
hay fever). Allergic rhinitis occurs when an allergen causes inflammation in
the nose. Potential
allergen types include, individually or in combination, pollens, pet hair,
dander, dust mites, mold,
smoke, and perfume. Symptoms of allergic rhinitis can include, individually or
in combination,
runny or stuffy nose; itchy eyes, mouth, throat or skin; sneezing; coughing;
and fatigue.
Asthma
[167] In some embodiments, the TH2-mediated condition includes asthma
(e.g., allergic
asthma). Asthma occurs when airways, for example in the lungs, are inflamed. A
type of asthma
included among the conditions relevant to the disclosures herein is atopic
asthma. Symptoms of
asthma can include, individually or in combination, coughing, wheezing,
shortness of breath, and
chest tightness.
Atopic Dermatitis
[168] In some embodiments, the TH2-mediated condition includes atopic
dermatitis
(i.e., eczema). Atopic dermatitis occurs when the skin is inflamed. It can
occur along with
allergic rhinitis and/or asthma. Its symptoms can include, individually or in
combination, dry
skin, redness, itching, rashes, and sores.
Urticaria
[169] In some embodiments, the TH2-mediated condition includes urticaria
(i.e., hives) .
Urticaria occurs when the skin develops swollen red welts. The condition can
be caused by blood
plasma leaking out of vessels due to histamine, for example as a result of an
allergic reaction.
The symptoms of urticaria can include, individually or in combination, batches
of welts, itching,
and swelling.
Angioedema
[170] In some embodiments, the TH2-mediated condition includes angioedema
(i.e.,
deep tissue swelling). When the swelling is beneath the skin, the condition
otherwise similar to
urticaria can be classified as angioedema. The condition can be caused by
blood plasma leaking
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out of vessels due to histamine, for example as a result of an allergic
reaction. The symptoms of
angioedema can include, individually or in combination, swelling in the eyes,
mouth, hands, feet,
or throat; difficulty with breathing; and stomach cramps.
Food Allergies
[171] In some embodiments, the TH2-mediated condition includes a food
allergy. Food
allergies occur when food (e.g., an allergen thereof) causes an abnormal
immune response.
Typical types of food that can cause a reaction include eggs, milk, peanuts,
tree nuts (e.g.,
walnuts), fish, shellfish, wheat, and soy. In addition, seeds (e.g., sesame,
mustard), fruits, and
rice can also cause food allergies. The symptoms can include, individually or
in combination,
itching in the mouth; hives; swelling of parts of the body such as the face;
trouble breathing;
abdominal discomfort; and dizziness.
Insect-Induced Allergies
[172] In some embodiments, the TH2-mediated condition includes an insect-
induced
allergy. An insect-induced allergy occurs when the immune system reacts to an
insect sting or
bite. Typical types of insect that can cause such a reaction include wasps,
bees, hornets, yellow-
jackets, ants, mosquitoes, bed bugs, fleas, and ticks.
Drug Allergies
[173] In some embodiments, the TH2-mediated condition includes a drug
allergy. Drug
allergies occur as an adverse reaction upon exposure to a drug. Some types of
drugs that can
cause such a reaction include penicillin, sulfonamides, anticonvusants,
aspirin, and
chemotherapy drugs. Drug allergy symptoms can include, individually or in
combination, fever,
itching, hives, rash, swelling, and shortness of breath.
Anaphylaxis
[174] In some embodiments, the TH2-mediated condition includes anaphylaxis.
Anaphylaxis occurs when the reaction to food, insect venom, or a drug is
severe. It can affect the
whole body, and can alter breathing, blood pressure, and heart rate.
Eosinophilia
[175] In some embodiments, the TH2-mediated condition includes eosinophilia
(e.g.,
primary eosinophilia). Eosinophilia occurs when the eosinophil count exceeds
certain thresholds
at a specific site or in the peripheral blood.
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[176] For each of these TH2-mediated conditions, the methods described
herein for
treating or preventing such conditions can result in lessening, stabilization,
or another
improvement in the symptoms of the particular TH2-mediated condition being
treated, which
symptoms include those described herein.
Additional Therapeutic A2ents
[177] In certain aspects, the methods provided herein include the
administration to a
subject of a bacterium and/or a bacterial composition described herein (e.g.,
a Prevotella
bacteria-containing pharmaceutical composition) either alone or in combination
with another
therapeutic, e.g., to treat or prevent a TH2-mediated condition (such as
atopic dermatitis and/or a
food allergy). The pharmaceutical composition comprising Prevotella bacteria
can be
administered, e.g., in combination with an anti-inflammatory agent. The anti-
inflammatory agent
can be an anti-histamine (such as cetirizine, fexofenadine, or
diphenhydramine), epinephrine, a
corticosteroid (oral or topical) (such as betamethasone valerate,
hydrocortisone or prednisone), a
calcineurin inhibitor (such as tacrolimus or pimecrolimus), cyclosporine,
interferon gamma-1 b,
or dupilumab. Topical corticosteroids that can be used include: Alclometasone
dipropionate,
Betamethasone dipropionate, Betamethasone valerate, Clobetasol propionate,
Desonide,
Desoximetasone Fluocinolone acetonide, Fluocinonide, Fluticasone propionate,
Halobetasol
propionate, Hydrocortisone, Hydrocortisone butyrate, Hydrocortisone valerate,
Mometasone
furoate, Triamcinolone acetonide, and Triamcinolone diacetate.
[178] In some embodiments, the Prevotella bacteria-containing
pharmaceutical
composition and the other therapy can be administered to the subject in any
order. In some
embodiments, the Prevotella bacteria-containing pharmaceutical composition and
the other
therapy are administered conjointly.
[179] In some embodiments, the Prevotella bacteria-containing
pharmaceutical
composition is administered to the subject before the additional 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 Prevotella
bacteria-containing
pharmaceutical composition is administered to the subject after the additional
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,
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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
Prevotella
bacteria-containing pharmaceutical composition and the additional 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
Prevotella bacteria-containing pharmaceutical composition 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). In some embodiments, the subject is
administered an
antibiotic after the Prevotella bacteria-containing pharmaceutical composition
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
Prevotella
bacteria-containing pharmaceutical composition and the antibiotic are
administered to the subject
simultaneously or nearly simultaneously (e.g., administrations occur within an
hour of each
other).
[180] In certain embodiments, the subject may undergo surgery. Types of
surgery
include but are not limited to preventative, diagnostic or staging, curative
and palliative surgery.
[181] In some embodiments, the additional therapeutic is an antibiotic.
"Antibiotics"
broadly refers to compounds capable of inhibiting or preventing a bacterial
infection. Antibiotics
can be classified in a number of ways, including their use for specific
infections, their
mechanism of action, their bioavailability, or their spectrum of target
microbe (e.g., Gram-
negative vs. Gram-positive bacteria, aerobic vs. anaerobic bacteria, etc.) and
these may be used
to kill specific bacteria in specific areas of the host ("niches") (Leekha, et
al 2011. General
Principles of Antimicrobial Therapy. Mayo Clin Proc. 86(2): 156-167). In
certain embodiments,
antibiotics can be used to selectively target bacteria of a specific niche. In
some embodiments,
antibiotics are administered after the bacterial treatment. In some
embodiments, antibiotics are
administered after the bacterial treatment to remove the engraftment.
[182] 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., 0-lactams), the cell membrane (e.g., daptomycin), or
bacterial DNA (e.g.,
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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.
[183] 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.
[184] 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.
[185] 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.
[186] Carbacephems include, but are not limited to, Loracarbef.
Carbacephems are
believed to inhibit bacterial cell wall synthesis.
[187] Carbapenems include, but are not limited to, Ertapenem, Doripenem,
Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal for both Gram-
positive and
Gram-negative bacteria as broad-spectrum antibiotics. Carbapenems are believed
to inhibit
bacterial cell wall synthesis.
[188] 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,
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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 (MRSA). Cephalosporins are believed to inhibit
bacterial cell
wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial
cell walls.
[189] Glycopeptides include, but are not limited to, Teicoplanin,
Vancomycin, and
Telavancin. Glycopeptides are effective, e.g., against aerobic and anaerobic
Gram-positive
bacteria including MRSA and Clostridium difficile. Glycopeptides are believed
to inhibit
bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan
layer of bacterial cell
walls.
[190] 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.
[191] 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.
[192] 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.
[193] 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.
[194] Nitrofurans include, but are not limited to, Furazolidone and
Nitrofurantoin.
[195] Oxazolidonones include, but are not limited to, Linezolid, Posizolid,
Radezolid,
and Torezolid. Oxazolidonones are believed to be protein synthesis inhibitors.
[196] Penicillins include, but are not limited to, Amoxicillin, Ampicillin,
Azlocillin,
Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, 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,
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Borrelia, and Treponema. Penicillins are believed to inhibit bacterial cell
wall synthesis by
disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
[197] Penicillin combinations include, but are not limited to,
Amoxicillin/clavulanate,
Ampicillin/sulbactam, Piperacillin/tazobactam, and Ticarcillin/clavulanate.
[198] 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.
[199] Quinolones and Fluoroquinolone include, but are not limited to,
Ciprofloxacin,
Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin,
Moxifloxacin, Nalidixic
acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, and
Temafloxacin.
Quinolones/Fluoroquinolone are effective, e.g., against Streptococcus and
Neisseria.
Quinolones/Fluoroquinolone are believed to inhibit the bacterial DNA gyrase or
topoisomerase
IV, thereby inhibiting DNA replication and transcription.
[200] 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.
[201] 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.
[202] Anti-mycobacterial compounds include, but are not limited to,
Clofazimine,
Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid,
Pyrazinamide,
Rifampicin, Rifabutin, Rifapentine, and Streptomycin.
[203] 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,
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furazolidone, fusidic acid, Na fusidate, gramicidin, imipenem, indolicidin,
josamycin, magainan
II, metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacin B-
JH1140, 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.
[204] In some embodiments, the additional therapy comprises administering a
therapeutic bacteria (e.g., Prevotella bacteria) and/or a therapeutic
combination of bacteria to the
subject so a healthy microbiome can be reconstituted in the subject. In some
embodiments the
therapeutic bacteria is a probiotic bacteria.
[205] Some of the particular additional therapies are further detailed
below.
Allergic Rhinitis
[206] In some embodiments, the TH2-mediated condition includes allergic
rhinitis, and
the additional therapy includes an agent that can be used to treat allergic
rhinitis. Such agents
include, individually or in combination, corticosteroids (e.g., prednisone,
methylprednisolone,
triamcinolone acetonide, betamethasone), antihistamines (e.g., acrivastine,
alimemazine,
antazoline, astemizole, azelastine, bepotastine, bilastine, bromazine,
brompheniramine,
carbinoxamine, cetirizine, chlorcyclizine, chloropyramine, chlorphenamine,
clemastine,
cyclizine, cyproheptadine, desloratadine, dexbrompheniramine,
dexchlorpheniramine,
dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebastine,
fexofenadine,
hydroxyzine, ketotifen, levocabastine, levocetirizine, loratadine, meclizine,
mepyramine,
mizolastine, olopatadine, orphenadrine, pheniramine, promethazine,
quifenadine, rupatadine,
terfenadine, tripelennamine, triprolidine), mast cell stabilizers (e.g.,
cromolyn), decongestants
(e.g., beclomethasone, budesonide, ciclesonide, dexamethasone, ephedrine,
flunisolide,
fluticasone, fluticasone furoate, fluticasone propionate, levomethamphetamine,
mometasone,
naphazoline, oxymetazoline, phenylephrine, phenylpropanolamine, prednisolone,
propylhexedrine, pseudoephedrine, synephrine, tetryzoline, tixocortol,
tramazoline,
triamcinolone, triamcinoloneacetonide, xylometazoline), leukatriene receptor
antagonists (e.g.,
montelukast, zafirlukast, zileuton, MR-886, meclofenamate sodium), and
immunotherapeutic
agents (e.g., containing a form of the allergen itself, containing a form of
an antibody such as
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omalizumab). These agents can also be used for other types of allergies in
general, which
allergies may occur as the sole type of allergy or may occur in combination
with other types of
allergies.
Asthma
[207] In some embodiments, the TH2-mediated condition includes asthma
(e.g., allergic
asthma), and the additional therapy includes an agent that can be used to
treat asthma. Such
agents include, individually or in combination, the corticosteroids,
decongestants (some of which
are also corticosteroids), mast cell stabilizers, leukotriene modifiers, and
immunotherapeutic
agents provided herein for the treatment of allergic rhinitis. In addition,
for asthma, the agents
also include, individually or in combination, beta-agonists (e.g., salmeterol,
formoterol,
albuterol, levalbuterol), certain corticosterone & beta-agonist combinations
(e.g., fluticasone-
salmeterol, budesonide-formoterol, formoterol-mometasone), additional
immunotherapeutic
agents (e.g., mepolizumab, dupilumab, resulizumab, and benralizumab), and
certain other drugs
(e.g., theophylline, ipratropium). These agents can be provided via the use of
an inhaler, as an
injection (e.g., for antibody forms), or as a pill (e.g., as a sublingual
tablet for allergen
immunotherapy).
Atopic Dermatitis
[208] In some embodiments, the TH2-mediated condition includes atopic
dermatitis, and
the additional therapy includes an agent that can be used to treat atopic
dermatitis. Such agents
include, individually or in combination, corticosteroids (e.g., prednisone)
and immunotherapeutic
agents (e.g., dupilumab) as provided herein for the treatment of allergic
rhinitis or asthma. In
addition, for atopic dermatitis, the agents also include calcineurin
inhibitors (e.g., tacrolimus,
pimecrolimus), certain supplements (e.g., vitamin D), immunosuppressants
(e.g., ciclosporin,
methotrexate, interferon gamma-lb, mycophenolate mofetil, azathioprine), and
other drugs (e.g.,
crisaborole).
Urticaria
[209] In some embodiments, the TH2-mediated condition includes urticaria,
and the
additional therapy includes an agent that can be used to treat urticaria. Such
agents include,
individually or in combination, antihistamines (e.g., diphenhydramine,
loratadine, fexofenadine,
cetirizine, desloratadine), corticosteroids (e.g., prednisone), leukatriene
receptor antagonists, and
immunotherapeutic agents (e.g., omalizumab) as provided herein for the
treatment of allergic
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rhinitis or asthma. In addition, for urticaria, the agents can also include
other antihistamines (e.g.,
ranitidine, cimetidine, famotidine), immunosuppressants (e.g., ciclosporin,
tacrolimus, sirolimus,
mycophenolate), anti-inflammatory medications (e.g., dapsone, sulfasalazine,
hydroxychloroquine), corticosteroids (e.g., cortisone), and hormones (e.g.,
epinephrine).
Angioedema
[210] In some embodiments, the TH2-mediated condition includes angioedema,
and the
additional therapy includes an agent that can be used to treat angioedema.
Such agents include,
individually or in combination, antihistamines (e.g., diphenhydramine,
loratadine, fexofenadine,
cetirizine, desloratadine), corticosteroids (e.g., prednisone), leukatriene
receptor antagonists, and
immunotherapeutic agents (e.g., omalizumab) as provided herein for the
treatment of allergic
rhinitis or asthma. In addition, for angioedema, the agents can also include
other antihistamines
(e.g., ranitidine, cimetidine, famotidine), immunosuppressants (e.g.,
ciclosporin, tacrolimus,
sirolimus, mycophenolate), anti-inflammatory medications (e.g., dapsone,
sulfasalazine,
hydroxychloroquine), corticosteroids (e.g., cortisone), and hormones (e.g.,
epinephrine).
Food Allergies
[211] In some embodiments, the TH2-mediated condition includes a food
allergy, and
the additional therapy includes an agent that can be used to treat food
allergies. Such agents
include, individually or in combination, epinephrine, antihistamines,
glucocorticoid steroids,
immunotherapeutic agents (e.g., containing a form of the allergen itself,
containing a form of an
antibody such as omalizumab).
Insect-Induced Allergies
[212] In some embodiments, the TH2-mediated condition includes an insect-
induced
allergy, and the additional therapy includes an agent that can be used to
treat insect-induced
allergies. Such agents include antihistamines.
Drug Allergies
[213] In some embodiments, the TH2-mediated condition includes a drug
allergy, and
the additional therapy includes an agent that can be used to treat drug
allergies. Such agents
include, individually or in combination, antihistamines (e.g.,
diphenhydramine) and
corticosteroids.
Anaphylaxis
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[214] In some embodiments, the TH2-mediated condition includes anaphylaxis,
and the
additional therapy includes an agent that can be used to treat anaphylaxis.
Such agents include
the same agents that can be used to treat allergies due to food, insect venom,
or drugs can be
used to treat anaphylaxis (e.g., epinephrine).
Eosinophilia
[215] In some embodiments, the TH2-mediated condition includes eosinophilia
(e.g.,
primary eosinophilia), and the additional therapy includes an agent that can
be used to treat
eosinophilia. Such agents include corticosteroids (e.g., prednisone).
[216] For each of these TH2-mediated conditions, in some embodiments, the
disclosed
pharmaceutical compositions can be administered in addition to one or more of
these provided
additional therapies. For example, the pharmaceutical compositions and an
agent of the
additional therapy can be co-administered.
[217] Additional agents that can be useful in downregulating pathways
involving TH2
cell activation include mepolizumab, lebrikizumab, tralokinumab, GSK3772847,
RG6149/AMG282, ANB020, 00000459, BI 671800, AZD1981, A51517499, YM-341619,
AS1810722, SB010, resiquimod, imiquimod, and CYT003.
Administration
[218] In certain aspects, provided herein is a method of delivering a
Prevotella bacteria-
containing pharmaceutical composition described herein to a subject. In some
embodiments of
the methods provided herein, the composition is administered in conjunction
with the
administration of an additional therapeutic (e.g., such as an anti-
inflammatory agent). In some
embodiments, the Prevotella bacteria are co-formulated in a pharmaceutical
composition with
the additional therapeutic. In some embodiments, the Prevotella bacteria
(e.g., a pharmaceutical
composition thereof) are co-administered with the additional therapeutic. In
some embodiments,
the additional therapeutic is administered to the subject before
administration of the Prevotella
bacteria (e.g., a pharmaceutical composition thereof) (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 Prevotella bacteria (e.g., a pharmaceutical
composition thereof) (e.g.,
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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
is used to deliver both the Prevotella bacteria (e.g., a pharmaceutical
composition thereof) and
the additional therapeutic. In some embodiments, different modes of delivery
are used to
administer the Prevotella bacteria (e.g., a pharmaceutical composition
thereof) and the additional
therapeutic. For example, in some embodiments, the Prevotella bacteria (e.g.,
a pharmaceutical
composition thereof) are administered orally while the additional therapeutic
is administered via
injection (e.g., an intravenous and/or intramuscular injection).
[219] In certain embodiments, the pharmaceutical compositions and dosage
forms
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 pharmaceutical compositions and
dosage forms
described herein.
[220] 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. The dose
of the
pharmaceutical 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 condition,
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.
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[221] In some embodiments, the dose administered to a subject is sufficient
to prevent
the condition, delay its onset, or slow or stop its progression or prevent a
relapse of the condition.
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.
[222] Suitable doses and dosage regimens can be determined by conventional
range-
finding techniques known to those of ordinary skill in the art. Generally,
treatment is initiated
with smaller dosages, which are less than the optimum dose of the compound.
Thereafter, the
dosage is increased by small increments until the optimum effect under the
circumstances is
reached. An effective dosage and treatment protocol can be determined by
routine and
conventional means, starting e.g., with a low dose in laboratory animals and
then increasing the
dosage while monitoring the effects, and systematically varying the dosage
regimen as well.
Animal studies are commonly used to determine the maximal tolerable dose
("MTD") of
bioactive agent per kilogram weight. Those skilled in the art regularly
extrapolate doses for
efficacy, while avoiding toxicity, in other species, including humans.
[223] In accordance with the above, in therapeutic applications, 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. Generally, the dose should be sufficient to result in slowing
progression, and preferably
causing regression, of the condition.
[224] Separate administrations can include any number of two or more
administrations
(e.g., doses), 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. In some
embodiments, the
doses may be separated by 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 or 1, 2, 3, or 4 weeks.
Accordingly, the methods
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provided herein include methods of providing to the subject one or more
administrations of a
bacterium, 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, including,
but not limited to,
subject's anti-bacterium antibody titer, the overall health of the subject
and/or the weight of the
subject.
[225] 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 or from the gut. 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 or from the gut;
for example, the time period can be more than the time period for a subject to
clear the bacteria
from normal tissue or from the gut, 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.
[226] In some embodiments, the delivery of an additional therapeutic in
combination
with the Prevotella bacteria (e.g., a pharmaceutical composition thereof)
described herein
reduces the adverse effects and/or improves the efficacy of the additional
therapeutic.
[227] 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 (e.g., subject), 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
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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.
[228] The toxicity of an additional therapy is the level of adverse
effects experienced by
the subject during and following treatment. Adverse events associated with
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, heartburn,
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,
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rapid heartbeat, 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.
[229] In some embodiments, the administration of the Prevotella bacteria
(e.g., a
pharmaceutical composition thereof) treats the condition.
[230] As described herein, the condition to be treated can comprise a TH2-
mediated
condition, such as atopic dermatitis, and/or a food allergy.
EXAMPLES
Example 1: Effects of Prevotella histicola in FITC-driven contact
hypersensitivity model.
[231] Exposure of skin to an antigen can, over time, lead to an allergic
response to that
antigen, for example, in the form of a skin allergy (e.g. dermatitis or atopic
dermatitis or eczema)
and/or in the form of a food allergy (see e.g. Han et al. The atopic march:
current insights into
skin barrier dysfunction and epithelial cell-derived cytokines. Immunol. Rev.
2017.
July;278(1):116-130. Doi: 10.1111/imr.12546; Kawasaki et al. Skin inflammation
exacerbates
food allergy symptoms in epicutaneously sensitized mice. Allergy. 2018.
June;73(6):1313-1321.
Doi: 10.1111/a11.13404; Martel et al. Translational animal models of atopic
dermatitis for
preclinical studies. Yale J. Biol. Med. 2017. Sept;90(3):389-402; Jin et al.
Animal models of
atopic dermatitis. J. Invest. Dermatol. 2009. Jan: 129(1):31-40. Doi
10.1038/jid.2008.106).
[232] The effects of Prevotella histicola Strain B 50329 were studied in a
fluorescein
isothiocyanate (FITC)-driven contact hypersensitivity model (See e.g. Li et
al. T-helper type-2
contact hypersensitivity of Balb/c mice aggravated by dibutyl phthalate via
long-term dermal
exposure. Feb. 3, 2014. https://doi.org/10.1371/journal.pone.0087887; Imai et
al. Effects of
phthalate esters on the sensitization phase of contact hypersensitivity
induced by fluorescein
isothiocyanate. Clin. Exp. Allergy. 2006 Nov; 36(11): 1462-8; Dearman et al.
Role of CD4+ T
helper 2-type cells in cutaneous inflammatory responses induced by fluorescein
isothiocynate.
Immunology. 2000 Dec; 101(4):442-451. Doi: 10.1046/j.1365-2567.2000.01126.x).
[233] Prevotella histicola strain B 50329 was administered by oral gavage,
and the
effects of Prevotella histicola on inflammation were analyzed using various
read-outs.
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[234] The studies were conducted using microbes reconstituted from powder
(e.g. in a
lyophilized form). Sucrose was used as the vehicle for reconstitution.
[235] Every day of gavage, one vial of the Prevotella histicola strain B
50329 and one
vial of anaerobic sucrose was collected from a refrigerator. Anaerobic sucrose
was used to
prepare the proper bacterial dilutions into sterile tubes. For instance, 800u1
of sucrose was added
to 80mg of Prevotella histicola Strain B 50329. In order to ensure proper
resuspension, vials of
resuspended powder were vortexed so that no powder was stuck to the vial. The
mice were
gavaged with resuspended powder at the amount of 100 IA per mouse immediately
after
vortexing (before the powder settles down) and the remaining powder mixture
was discarded (a
freshly prepared dilution must be used every time). Negative control mice were
orally gavaged
with 100u1 of vehicle per day.
FITC-driven hypersensitivity Study Protocol:
[236] Mice were purchased from Taconic and allowed to acclimate to the
vivarium for
at least 1 week prior to the start of the experiment. Mice were housed at 5
animals (or fewer) per
cage, with each cage constituting a different treatment group.
[237] On day 0, mice were anesthetized with isoflurane (one at a time) and
their backs
were shaved.
[238] On day 1, a solution of 0.5 % FITC (w/v) was dissolved in adjuvant
(dibutyl
phthalate (DBP)) and acetone (1:1). To prepare the 0.5 % FITC, 250mg FITC was
dissolved in
25m1 acetone. Once completely dissolved, 25m1 of DBP was added and mixed by
vortexing.
[239] On days 1 and 2, mice were sensitized on the back by applying IA of
the 0.5 %
FITC solution with a pipette. Anaerobic sucrose served as the negative
control. Dexamethasone
served as the positive control (Dexamethasone stock solution was prepared by
resuspending
25mg of dexamethasone (Sigma) in 1.6 ml of 96% ethanol).
[240] On days 1-6, mice were orally gavaged with vehicle (negative control,
group 1) or
Prevotella histicola strain B 50329 (group 3), or injected intraperitoneally
(i.p.) with
Dexamethasone (positive control, group 2) according to following study design:
Group Sensitization Challenge Test substance p.o.
0.5% FITC 0.5% (treatment days 1-6)
in 400 [IL FITC in
20u1
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1 (n=5) Day 1, 2 Day 6 Vehicle (anerobic sucrose)
2 (n=5) Day 1, 2 Day 6 Dexamethasone (lmg/kg)
3 (n=5) Day 1, 2 Day 6 Prevotella histicola strain B
50329 DS (10mg)
DS = Drug substance (freeze-dried)
[241] In addition to the daily gavage (groups 1 and 3) and i.p. injection
(group 2), the
mice were FITC-challenged on day 6 as follows: On day 6, each mouse was
anesthetized with
isoflurane and a baseline left ear measurement was obtained using calipers,
and then 20 IA of
0.5% FITC solution was applied on the left ear (20111 0.5% FITC (w/v)
DBP:acetone (1:1) ("ear
challenge" or "FITC challenge").
[242] On day 7, an 8-hour post ear challenge measurement was obtained using
calipers.
Mice were euthanized and ears were collected for downstream mRNA analyses
using TaqMan
RNA-to-CT 1-Step Kit (Applied Biosystems, ThermoFisher Scientific Catalog#
4392653)
according to manufacturer's instructions.
[243] Results are shown in Figures 1A and 1B.
[244] Oral administration of Prevotella histicola Strain B 50329 (labeled
as "Prevotella
histicola" in the Figures 1A-1B) significantly reduced ear swelling at 8h post-
FITC ear challenge
(Figure 1A).
[245] Compared to the vehicle group, Prevotella histicola Strain B 50329
treatment
reduced gene expression for IL-4, IL-5, IL-33, IL-17a, and TSLP in the ears
(Figure 1B).
Example 2: Effects of Prevotella histicola in FITC-driven contact
hypersensitivity model.
[246] The effect of Prevotella histicola Strain B 50329 in a FITC-driven
contact
hypersensitivity model was tested against two other strains, Strain 1 and
Strain 2, where Strain 1
is a strain of a Prevotella species other than histicola, and Strain 2 is
Strain 2 is a strain of a
different genus than Prevotella.
[247] As described in Example 1, on day 0, mice were anesthetized with
isoflurane and
their backs were shaved. On days 1 and 2, mice were sensitized on the back by
applying 400 IA
of 0.5 % FITC solution with a pipette. On days 1-6, mice were orally gavaged
with vehicle
(sucrose) or the tested bacterial strain, or injected intraperitoneally (i.p.)
with Dexamethaose
(positive control).
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[248] As described in Example 1, on day 6, each mouse was anesthetized with
isoflurane and a baseline left ear measurement was obtained using calipers.
Then the mice were
FITC-challenged with 20[11 of 0.5% FITC solution applied to the left ear.
[249] On day 7, just eight (8) hours following challenge, mice were
euthanized for a
post-challenge ear measurement using calipers.
[250] Results are shown in Figure 2. Oral administration of Prevotella
histicola Strain
B 50329 (labeled as "Prevotella histicola" in Figure 2) significantly reduced
ear swelling at 8
hours post-FITC challenge, while two other bacterial strains (Strain 1 and
Strain 2) did not
(Figure 2). The difference in ear swelling between Prevotella histicola Strain
B 50329 and
Strain 1 (a strain of another species of Prevotella) was not statistically
significant, while the
difference in ear swelling between Prevotella histicola Strain B 50329 and
Strain 2 (strain of
another genus than Prevotella) was statistically significant.
Example 3: Effects of Prevotella histicola in a MC903 model of dermatitis-
associated food
allergy to e22 white protein Ovalbumin (OVA).
[251] A mouse model of dermatitis-associated food allergy was used to
assess the
efficacy of bacterial strains in modulating an allergic reaction.
[252] MC903, a Vitamin D3 analog, induces inflammation and has been used in
a
model of atopic dermatitis (See e.g. Hussain et al. Basophil-derived IL-4
promotes epicutaneous
antigen sensitization concomitant with the development of food allergy. 2017.
American
Academy of Allergy, Asthma & Immunol. http://dx. doi. org/10.1016/j . j
aci.2017. 02. 035;
Moosbrugger-Martinz et al. A mouse model for atopic dermatitis using topical
application of
Vitamin D3 or of its analog MC903. Methods Mol. Biol. 2017;1559:91-106; Li et
al. Topical
vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in
mouse
keratinocytes and trigger an atopic dermatitis. PNAS. Aug. 1, 2006. Vol.
103(31): 11736-
11741)).
[253] MC903 can be used to cutaneously sensitize mice to an antigen of
interest,
resulting in skin sensitization typified by TSLP and IL-4 production (See e.g.
Noti et al.
Exposure to food allergens through inflamed skin promotes intestinal food
allergy via the TSLP-
basophil axis. J. Allergy Clin. Immunol. 2014 May; 133(5): 1390-1399.e6). The
study design for
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the MC903 model of dermatitis-associated food allergy is shown in Figure 3A.
Allergens to be
tested (e.g. OVA) may be obtained from vendors such as Invivogen or Sigma.
Materials and Methods
[254] Female BALB/c mice (6-8 weeks old) were purchased from Taconic Farms.
Animals were housed in specific pathogen¨free conditions in a vivarium (5 mice
or fewer than 5
mice per cage), and all experiments were performed under Institutional Animal
Care and Use
Committee (IACUC) approved protocols and guidelines. Mice were allowed to
acclimate in the
vivarium for one week prior to the start of the experiment. PicoLab Rodent
Diet 20 is provided
and autoclaved water via sipper bottle, given ad libitum and checked daily.
[255] To induce a mouse model mimicking egg food allergy, mice were exposed
to
MC903 (Tocris) and 100 lig ovalbumin (OVA) daily for 14 consecutive days (days
1-14). 45[IM
of MC903 in 20 [IL of 100% ethanol was applied to one ear by pipette and, once
the ear was
dried, 100[Ig of OVA in PBS was applied. 20 [IL ethanol was applied to the
ears of the vehicle
control group.
[256] For the daily oral gavage treatments (days 1-17), negative control
mice received
vehicle (frozen sucrose) and positive control mice received Tofacitinib
(20mg/kg in 100u1/mouse
in 0.5% methylcellulose (p.o.); Tocris). One group of mice received 10mg
Prevotella histicola
Strain B 50329 daily (3.13E+09 TCC/dose), while other groups received 10mg of
either Strain A
(1.23E+10 TCC/dose) or Strain B (7.83E+09 TCC/dose) (bacterial strains that
were not
Prevotella histicola Strain B 50329¨Strain A was a strain of another genus
than Prevotella, and
Strain B was another strain of Prevotella histicola). On day 15, mice were
challenged by oral
gavage (p.o., also referred to as intragastric (i.g.)) with 50 mg of OVA.
[257] On day17.5, mice were orally challenged a second time with 50 mg of
OVA. Ear
inflammation was measured using calipers following the second challenge.
[258] Ear inflammation on Day 18 is shown in Figure 3B (12 hours after the
second
OVA challenge). Treatment with Prevotella histicola Strain B 50329 (labeled as
"P. histicola" in
Figures 3B-3D) significantly reduced ear inflammation compared to the negative
control group.
[259] On Day 18, 12 hours after the second OVA challenge, mice were
euthanized.
Blood (for serum) and tissues (e.g. spleen, draining cervical lymph nodes,
mesenteric lymph
nodes and ears) were collected for ex vivo processing.
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[260] Serum was analyzed by ELISA for anti-OVA IgG1 , anti-OVA IgE using
the
Chondrex, Inc. Mouse Anti-OVA IgG1 Antibody Assay Kit (Catalog# 3013) and
Mouse Anti-
OVA IgE Antibody Assay Kit (Catalog# 3010). Results are shown in Figure 3C.
[261] Ears were homogenized for cytokine detection. Spleen, mesenteric
lymph nodes
(mLN), and cervical lymph nodes (cLN) were analyzed for cytokine expression
following in
vitro PMA stimulation and supernatant collection. Single cell suspensions of
tissues were
prepared, counted, and plated at 200,000 cells/well and restimulated with
PMA/Ionomycin
(eBiosciences Cell Stimulation cocktail Catalog# 00-4975) for 48 hours per
manufacturer's
instructions. Supernatants were collected and used for downstream multiplex
ELISAs to
determine cytokine levels using Meso Scale Discovery (MSD) kits (Catalog# K1
5068L-2). Also,
ears were dissociated in 250u1 T-PER buffer (Thermo Scientific Catalog# 78510)
containing Halt
Protease (Thermo Scientific Catalog# 78444) and protein concentrations
quantified using the
BCA kit (Thermo Scientific Catalog #23227). 10Oug of protein was used to
quantify cytokine
levels using MSD kits (Catalog# K15068L-2). Results are shown in Figure 3D.
Example 4: Effects of Prevotella histicola in MC903 model of type 2 (Th2)
immune response
in OVA and food allergy to complete peanut protein (CPE).
[262] To induce a mouse model mimicking egg food allergy and peanut
allergy, mice
were sensitized to food allergens as described in Example 3 and as depicted in
Figure 4A.
[263] MC903 was prepared in Et0H and applied daily as shown (days 1 thru
14).
[264] Some mice were exposed to MC903 and 10Oug OVA in PBS daily for 14
consecutive days. Other mice were exposed to MC903 and 10Oug complete peanut
protein (CPE)
daily for 14 consecutive days. In addition to the application of antigen (e.g.
OVA or CPE), mice
were gavaged with sucrose vehicle (negative control), Tofacitinib (positive
control), bacterial
Strain A (10mg/dose) or Prevotella histicola Strain B 50329 (10mg/dose).
Strain A is a strain of
another genus than Prevotella.
[265] On day 15, mice were challenged by oral gavage with either 50mg of
OVA or
50mg peanut powder.
[266] On day17.5, mice were challenged a second time by oral gavage with
either 50mg
of OVA or 50mg complete peanut powder (CPE).
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[267] On Day 18, 12 hours after the final antigen challenge, ear thickness
was
measured. Mice were euthanized and serum and tissues were collected as
described in Example
3.
[268] As shown in Figure 4B, Prevotella histicola Strain B 50329 (labeled
"P.
histicola" in Figures 4B-4D) significantly reduced ear inflammation compared
to the vehicle
control groups in both the OVA and peanut allergy (CPE) groups. Antibody
analyses (anti-OVA
IgG1 and anti-OVA IgE) were conducted as described in Example 3 and results
shown in Figure
4C. Cytokine analyses were conducted as described in Example 3 and are shown
in Figure 4D.
Ear RNA was measured as described in Example 1 and results are shown in Figure
4E.
Example 5: MC903-driven atonic dermatitis and anaphylaxis.
[269] To induce a mouse model mimicking egg food allergy, peanut allergy,
and/or
other allergen of interest, mice are sensitized to food allergens as described
in Example 3 and
depicted in Figure 5.
[270] MC903 is prepared in Et0H and applied daily as shown (days 1 thru
14).
[271] Some mice are exposed to MC903 and 10Oug CPE in PBS daily for up to
30
consecutive days. In addition to the application of the model antigen (e.g.
CPE), mice are
gavaged with sucrose vehicle (negative control), Tofacitinib (positive
control; 100111 orally,
daily), bacterial Strain A (10mg/dose) or Prevotella histicola Strain B 50329
(10mg/dose), or
200 g/mouse anti-IL-4 (i.p. days 3, 6, 9 and 12; BioXcell) (days 1-17 or as
long as days 1-30).
[272] On day 30, mice are challenged with 500ug CPE i.p. and observed for
anaphylaxis. Thirty minutes following CPE challenge, body temperature is
measured using a
rectal thermometer.
[273] On Day 31, mice are sacrificed and ear inflammation is measured.
Spleen, ear,
and lymph tissue cytokines are analyzed using methods described in Examples 1
and 3.
Example 6: Tape-stripping peanut allergy model.
[274] As described above, there are various animal models for studying food
allergy.
Some of these models induce sensitization of allergen (e.g. antigen) through
skin barrier defects
(see e.g. Ohsaki et al. Maternal IgG immune complexes induce food allergen-
specific tolerance
in offspring. J Exp Med. 2018. 215:91-113; Kanagaratham et al. Experimental
models for
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studying food allergy. Cell Mol Gastroenterol Hepatol. 2018. 6(3):356-369.el.
Doi:
10.1016/j.jcmgh.2018.05.010). In some models, skin barrier defects are induced
by repeated
tape-stripping of the skin after fur shaving, and the allergen is applied to
the resultantly damaged
skin. This "epicutaneous sensitization" to the allergen is followed, at some
timepoint, by
intragastric challenge to the allergen. For example, mice can be sensitized on
the ear or back,
using tape-stripping and exposure to peanut antigen on either days 1, 2, and 3
(ear sensitization),
or days 0, 3, and 6 (back sensitization), as shown in Figure 6A. For the ear
sensitization groups,
mice are tape-stripped only on day 0, while the back sensitization groups are
tape-stripped on
days 0, 3, and 6. Mice may be challenged on day 21 and, shortly thereafter,
observed and/or
sacrificed for various read-outs including, but not limited to, measuring
serum peanut-specific
IgE, IgGl, and IgA, as well as total IgE, IgGl, and IgA, body temperature,
anaphylaxis scoring,
diarrhea scoring, and/or ex vivo restimulation of mesenteric lymph node (MLN)
cells to assess
various cytokines (including IL-4, IL-5, and TSLP). Alternatively, serum may
be collected at day
35 (see Figure 6B), and in other instances, a study may last as long as 65
days (see Figure 6C).
Mice may be scored for anaphylaxis and diarrhea as shown in Table 3 and Table
4, respectively.
Table 3: Anaphylaxis Scoring
Score Criteria
0 No symptoms
1 Scratching/rubbing around nose and mouth
2 Puffiness around eyes and mouth; diarrhea; pilar erecti; reduced
activity with
increased respiratory rate
3 Wheezing/labored respiration; cyanosis around mouth and tail.
4 No activity after prodding or tremor and convulsions
Table 4: Diarrhea Scoring
Score Description
0 No changes
1 Soft but well formed
2 Soft; non-formed
3 One episode of liquid diarrhea
4 At least two episodes of liquid diarrhea
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[275] Female BALB/c mice were purchased from Taconic Farms and all
experiments
performed under Institutional Animal Care and Use Committee (IACUC) approved
protocols
and guidelines.
[276] Mice were allowed to acclimate in the vivarium for one week prior to
the start of
the experiment. All mice are housed at 5 animals per cage or less, in
individually ventilated
cages with standard bedding and enrichment. PicoLab Rodent Diet 20 was
provided and
autoclaved water via sipper bottle was given ad libitum and checked daily.
Experimental Design
[277] Animal groups were separated as follows (5 mice/group):
[278] 1) Naive mice (no sensitization, no peanut challenge);
[279] 2) Naive mice (no sensitization, peanut challenge on day 21);
[280] 3) Ear sensitization (sensitized days 1, 2, and 3) (no challenge);
[281] 4) Ear sensitization (sensitized days 1, 2, and 3) (peanut challenge
on day 21);
[282] 5) Ear sensitization (sensitized days 1, 2, and 3) (Tofacitinib
20mg/kg) (peanut
challenge on day 21);
[283] 6) Ear sensitization (sensitized days 1, 2, and 3) (P. histicola
10mg) (peanut
challenge day 21);
[284] 7) Back sensitization (sensitized days 0, 3, and 6) (no challenge);
[285] 8) Back sensitization (sensitized days 0, 3, and 6) (peanut challenge
on day 21);
[286] 9) Back sensitization (sensitized days 0, 3, and 6) (Tofacitinib
20mg/kg) (peanut
challenge on day 21); and
[287] 10) Back sensitization (sensitized days 0, 3, and 6) (Prevotella
histi cola Strain B
50329 10mg) (peanut challenge on day 21).
[288] On Day 0, mice were anesthetized with isoflurane, one by one. For the
ear
sensitization groups, the ears of each mouse were repeatedly tape-stripped
(approximately 7-8
times) with cellophane tape. For the back sensitization groups, the backs were
shaved and tape-
stripped with cellophane tape (approximately 7-8 times).
[289] For the ear sensitization groups, mice were tape-stripped on day 0.
On days 1, 2,
and 3, 25 IA of the peanut protein solution (4mg/m1 peanut protein in PBS) was
applied to both
ears with a cotton swab.
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[290] For the back sensitization groups, on days 0, 3, and 6, mice were
tape-stripped
and 100[Ig of peanut protein applied. The sensitized area was covered with a
new waterproof
bandage each time.
[291] For the Prevotella histicola Strain B 50329 groups, mice were orally
gavaged
with 10mg bacteria on days 1 through 21.
[292] On Day 21, mice from groups 2, 4, 5, 6, 8, and 9 were challenged with
a gavage
of 100mg peanut protein. Mice from all groups were cheek bled one hour
following peanut
protein challenge and blood collected for total IgE, peanut-specific IgE,
total IgG1 , peanut-
specific IgGl, total IgA, and peanut-specific IgA ELISAs (Chondrex, per
manufacturer's
instructions).
[293] Inflammation results are shown in Figure 6D. For both the ear groups
and the
back groups, Prevotella histicola Strain B 50329 (labeled "P. histicola" in
Figures 6D-6I)
decreased inflammation.
[294] Total and peanut-specific serum IgE, and IgG1 levels from the day of
challenge
are shown in Figures 6E and 6F, respectively.
[295] Total and peanut-specific serum IgE, IgGl, and IgA levels from 2
weeks post-
challenge are shown in Figures 6G, 6H, and 61, respectively.
Example 7: FITC-CHS model.
[296] Prevotella histicola Strain B 50329 was tested in a FITC-CHS model as
shown in
Figure 7A. The dose tested was 4.69E+09 TCC.
[297] The effects of Prevotella histicola Strain B 50329 on TH2 cytokines
(IL-13, IL-4,
IL-5, IL-31, and IL-33) were examined at day 7. As shown in Figure 7B,
Prevotella histicola
Strain B 50329 (labeled as "Prevotella histicola" in Figures 7B-7C)
significantly lowered levels
of IL-13, IL-5, and IL-31 in gut draining mesenteric lymph nodes. Cytokine
levels were
determined by MSD, with the experiment performed as described in Example 3. As
shown in
Figure 7C, Prevotella histicola Strain B 50329 treatment significantly lowered
levels of IL-13 in
ear draining cervical lymph nodes. The experiment was performed as described
in Example 3.
[298] This example demonstrates that Prevotella histicola Strain B 50329
treatment
significantly lowered TH2 cytokine secretion in the PMA/ionomycin ex vivo re
stimulated cells
from gut draining mesenteric lymph nodes and the ear draining cervical lymph
nodes.
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Example 8: MC903 driven Atopic Dermatitis.
[299] Prevotella histicola Strain B 50329 was tested in an MC903 driven
atopic
dermatitis model as shown in Figure 8A.
[300] For these experiments, mice were purchased from Taconic Labs and
allowed to
acclimate in the vivarium for 1 week prior to start of experiment. Mice were
housed 5 animals
per cage, in individually ventilated cages with standard bedding and
enrichment. Standard Purina
rodent diet (5001) and autoclaved water was provided ad libitum and checked
daily.
[301] On Day 1, mice were anesthetized, and a baseline ear measurement was
taken.
While anesthetized, ears were then sensitized with 45 nIVI calcipitriol
(MC903) by pipetting a
total of 20uL of solution onto both dorsal and ventral sides of the ear. Ears
were sensitized daily
for 14 days.
[302] Mice were orally gavaged daily with Prevotella histicola Strain B
50329 at a dose
of 4.69E+09 TCC, or positive control (tofacitinib), QD, from Day 1 through Day
14.
[303] Ear measurements were taken throughout the study to track changes in
thickness
over time and day 14 to determine change in ear thickness from baseline.
[304] Ex vivo experiments were performed on day 17.
[305] MC903 preparation protocol: 10mg calcipitriol powder was dissolved in
10 mL
99% ethanol to create 1 mg/mL stock. Stock was diluted to 0.01856 mg/mL in 99%
ethanol. The
diluted solution was aliquoted into daily use, light protective tubes at -20
C.
[306] Positive control ¨ Tofacitinib preparation: Tofacitinib was prepared
in 0.5% (w/v)
methylcellulose. Purified water was brought to a boil on a stir/heat plate. An
appropriate amount
of powder methylcellulose was weighed out. The boiling water was stirred and
powder
methylcellulose was added. Stirring was continued until powder was
incorporated into the water.
The mixture was allowed to cool overnight in 4 C while stirring on stir plate.
50 mg stock of
Tofacitinib was resuspended in 0.5% MC (mixture formed a suspension).
Tofacitinib was
administered p.o., 100 uL per mouse.
Results:
[307] As shown in Figure 8B, Prevotella histicola Strain B 50329 (labeled
as
"Prevotella histicola" in Figures 8B-8E) treatment resulted in reduced ear
inflammation in
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MC903 driven atopic dermatitis, as determined by ear measurements over time
(left panel) and
ear inflammation on day 14 (right panel).
[308] The effects of of Prevotella histicola Strain B 50329 on TH2 cytokine
transcript
levels (Tslp, 115, 1131, 114, Cc119, and Ccr4) in ear tissue were examined at
day 17. As shown in
Figure 8C, Prevotella histicola Strain B 50329 treatment significantly lowered
levels of 115,
1131, and Ccr4 transcripts in ear tissue, as determined by qPCR.
[309] The effects of Prevotella histicola Strain B 50329 on mast cell
related gene
transcript levels (Mcpt 1) in the jejunum were examined at day 17. The results
in Figure 8D
demonstrate that Prevotella histicola Strain B 50329 significantly reduced
Mcpatranscript levels
in the jejunum of mice in the MC903-driven atopic dermatitis model, as
determined by qPCR
[310] The effects of Prevotella histicola Strain B 50329 on IL-10 levels in
mesenteric
lymph nodes and spleen were examined at day 17. As shown in Figure 8E,
Prevotella histicola
Strain B 50329 treatment resulted in significantly increased IL-10 levels in
mesenteric lymph
nodes and spleen mice upon ex vivo re-stimulation with PMA/Ionomycin in tissue
isolated from
the MC903-driven atopic dermatitis model, as determined by MSD. The experiment
was
performed as described in Example 3.
[311] The results in this example demonstrate that ear inflammation in
MC903-driven
atopic dermatitis was significantly reduced with Prevotella histicola Strain B
50329 treatment
compared to vehicle treated mice.
[312] Prevotella histicola Strain B 50329 treatment showed decreases in TH2
cytokine
transcripts in ear tissue and a mast cell related transcript (Mcptl) in the
jejunum.
[313] Prevotella histicola Strain B 50329 treatment showed increased levels
of IL-10 in
gut draining mesenteric lymph nodes and spleen cells upon ex vivo re-
stimulation with
PMA/Ionomycin.
Incorporation by Reference
[314] All publications patent applications mentioned herein are hereby
incorporated by
reference in their entirety as if each individual publication or patent
application was specifically
and individually indicated to be incorporated by reference. In case of
conflict, the present
application, including any definitions herein, will control.
87
CA 03143994 2021-12-16
WO 2020/257390
PCT/US2020/038335
Equivalents
[315] 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.
88
