U.S. patent application number 17/178412 was filed with the patent office on 2021-06-10 for compositions and methods for treating an inflammatory disease or disorder.
The applicant listed for this patent is YALE UNIVERSITY. Invention is credited to Marcel De Zoete, Richard Flavell, Noah Palm.
Application Number | 20210170014 17/178412 |
Document ID | / |
Family ID | 1000005417584 |
Filed Date | 2021-06-10 |
United States Patent
Application |
20210170014 |
Kind Code |
A1 |
Flavell; Richard ; et
al. |
June 10, 2021 |
COMPOSITIONS AND METHODS FOR TREATING AN INFLAMMATORY DISEASE OR
DISORDER
Abstract
The invention relates to compositions and methods for treating
inflammatory diseases and disorders in a subject in need thereof.
In certain aspects, the invention relates to immunogenic
compositions (e.g., vaccines) to diminish the number or pathogenic
effects of one or more bacteria associated with the development or
progression of an inflammatory disease or disorder.
Inventors: |
Flavell; Richard; (Guilford,
CT) ; Palm; Noah; (New Haven, CT) ; De Zoete;
Marcel; (New Haven, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YALE UNIVERSITY |
New Haven |
CT |
US |
|
|
Family ID: |
1000005417584 |
Appl. No.: |
17/178412 |
Filed: |
February 18, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15507357 |
Feb 28, 2017 |
10925953 |
|
|
PCT/US2015/047400 |
Aug 28, 2015 |
|
|
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17178412 |
|
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62042878 |
Aug 28, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 39/116 20130101; A61K 2039/55544 20130101; A61K 9/0053
20130101; A61K 2039/58 20130101; A61K 2039/577 20130101; A61K
39/0008 20130101; A61P 31/04 20180101; A61K 39/39 20130101; C12Q
1/689 20130101 |
International
Class: |
A61K 39/116 20060101
A61K039/116; A61K 39/00 20060101 A61K039/00; A61P 31/04 20060101
A61P031/04; A61P 29/00 20060101 A61P029/00; A61K 9/00 20060101
A61K009/00; A61K 39/39 20060101 A61K039/39; C12Q 1/689 20060101
C12Q001/689 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND
DEVELOPMENT
[0002] This invention was made with government support under grant
number 2T32AR007107-37 awarded by the National Institutes of Health
(NIH) and grant number W81XWH-11-1-0745 awarded by the Department
of Defense (DoD). The government has certain rights in the
invention.
Claims
1. A method of treating or preventing an inflammatory disease or
disorder associated with a secretory antibody-bound bacteria in the
microbiota of a subject in need thereof, the method comprising
administering to the subject at least one therapy to diminish the
number or pathogenic effects of a bacteria associated with an
inflammatory disease or disorder, wherein the bacteria is a member
of the Erysipelotrichaceae family.
2. The method of claim 1, wherein the member of the
Erysipelotrichaceae family is a species of Allobaculum.
3. The method of claim 1, wherein the at least one therapy is
selected from the group consisting of a vaccine, an antibiotic and
a passive immunotherapy.
4. The method of claim 3, wherein the vaccine comprises a
heat-killed or an inactivated bacterium.
5. The method of claim 3, wherein the vaccine comprises an
adjuvant.
6. The method of claim 3, wherein the vaccine is administered
orally to the subject.
7. The method of claim 5, wherein the adjuvant is selected from the
group consisting of RIBI, KLH peptide, cholera toxin, E. coli
heat-labile toxin, E. coli enterotoxin, salmonella toxin, AB5
toxin, nanoparticle-based adjuvant, calcium phosphate, liposomes,
virosomes, cochleates, eurocine, archaeal lipids, ISCOMS,
microparticles, monophosphoryl lipid (MPL),
N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), Detox, AS04, AS02,
AS01, OM-174, OM-triacyl, oligonucleotides, double-stranded RNA,
pathogen-associated molecular patterns (PAMPs), TLR ligands,
chitosan, a-galactosylceramide, small-molecule immune potentiators
(SMIPs), a cytokine, a chemokine, DC Choi, PLA (polylactic acid)
microparticles, PLG (poly[lactide-co-glycolide]) microparticles,
Poly(DL-lactide-co-glycolide) microparticles, polystyrene (latex)
microparticles, proteosomes, and 3',5'-Cyclic diguanylic acid
(c-di-GMP).
8. The method of claim 5, wherein the adjuvant is selected from the
group consisting of cholera toxin, E. coli heat-labile toxin, E.
coli enterotoxin, or salmonella toxin.
9. An immunogenic composition for treating or preventing an
inflammatory disease or disorder, the composition comprising a
vaccine to diminish the number or pathogenic effects of a bacteria
associated with an inflammatory disease or disorder, wherein the
bacteria is a member of the Erysipelotrichaceae family.
10. The immunogenic composition of claim 9, wherein the member of
the Erysipelotrichaceae family is a species of Allobaculum.
11. The immunogenic composition of claim 9, wherein the vaccine
comprises a heat-killed or an inactivated bacterium.
12. The immunogenic composition of claim 9, wherein the vaccine
comprises an adjuvant.
13. The immunogenic composition of claim 12, wherein the adjuvant
is selected from the group consisting of RIBI, KLH peptide, cholera
toxin, E. coli heat-labile toxin, E. coli enterotoxin, salmonella
toxin, AB5 toxin, nanoparticle-based adjuvant, calcium phosphate,
liposomes, virosomes, cochleates, eurocine, archaeal lipids,
ISCOMS, microparticles, monophosphoryl lipid (MPL),
N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), Detox, AS04, AS02,
AS01, OM-174, OM-triacyl, oligonucleotides, double-stranded RNA,
pathogen-associated molecular patterns (PAMPs), TLR ligands,
chitosan, a-galactosylceramide, small-molecule immune potentiators
(SMIPs), a cytokine, a chemokine, DC Choi, PLA (polylactic acid)
microparticles, PLG (poly[lactide-co-glycolide]) microparticles,
Poly(DL-lactide-co-glycolide) microparticles, polystyrene (latex)
microparticles, proteosomes, and 3',5'-Cyclic diguanylic acid
(c-di-GMP).
14. The method of claim 12, wherein the adjuvant is selected from
the group consisting of cholera toxin, E. coli heat-labile toxin,
E. coli enterotoxin, or salmonella toxin.
15. A method of treating inflammatory bowel disease (IBD) in a
subject, the method comprising administering to the subject
non-colitogenic bacteria, wherein the non-colitogenic bacteria do
not contribute to the development or progression of IBD in the
subject, and wherein the subject has been diagnosed with IBD via
detection of secretory antibody-bound colitogenic bacteria that do
contribute to the development or progression of IBD, wherein the
non-colitogenic bacteria is Akkermansia mucimphila while the
colitogenic bacteria is a member of the Erysipelotrichaceae
family.
16. The method of claim 15, wherein the member of the
Erysipelotrichaceae family is a species of Allobaculum.
17. The method of claim 15, further comprising administering to the
subject at least one therapy to diminish the number or pathogenic
effects of the colitogenic bacteria present in the subject.
18. The method of claim 17, wherein the at least one therapy is
selected from the group consisting of a vaccine, an antibiotic, and
a passive immunotherapy.
19. The method of claim 18, wherein the at least one therapy is a
vaccine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/507,357, filed Feb. 28, 2017, which is a
national phase application filed under 35 U.S.C. .sctn. 371
claiming benefit to International Patent Application No.
PCT/US2015/047400, filed Aug. 28, 2015, which in turn is entitled
to priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional
Patent Application No. 62/042,878, filed Aug. 28, 2014, each of
which is hereby incorporated herein by reference in their
entirety.
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
[0003] The present application hereby incorporates by reference the
entire contents of the text file named
"047162-5181-01US_Sequence_Listing.txt" in ASCII format. The text
file containing the Sequence Listing of the present application was
created on Feb. 17, 2021 and is 635 bytes in size.
BACKGROUND OF THE INVENTION
[0004] The composition of the intestinal microbiota varies
substantially between individuals and is thought to be a key
determinant of host susceptibility to an increasing variety of
diseases (Blumberg and Powrie, 2012, Sci Transl Med 4:137rv7; Chow
et al., 2011, Curr Opin Immunol 23:473-480; Hooper et al., 2012,
Science 336:1268-1273; Littman and Pamer, 2011, Cell Host Microbe
10:311-323; Lozupone et al., 2012, Nature 489:220-230). In
inflammatory bowel disease (IBD), which includes Crohn's disease
and ulcerative colitis, it is believed that the intestinal
microbiota plays a key role in driving inflammatory responses
during disease development and progression (Abraham and Cho, 2009,
New Engl J Med 361: 2066-2078; Gevers et al., 2014, Cell Host
Microbe 15:382-392; Knights et al., 2013, Gut 62:1505-1510). This
is clearly illustrated in mouse models of IBD, where the effects of
the composition of the intestinal microbiota on disease have been
examined in detail (Saleh and Elson, 2011, Immunity 34:293-302).
These studies have revealed that particular bacterial taxa within
the intestinal microbiota can be uniquely potent drivers of
intestinal disease. For example, Prevotellaceae species drive
chronic intestinal inflammation in mice with inflammasome-mediated
dysbiosis and exacerbate chemically-induced colitis (Elinav et al.,
2011, Cell 145:745-757; Scher et al., 2013, eLife 2:e01202), and
Helicobacter species can drive colitis in mice lacking the
immunoregulatory cytokine interleukin-10 (Kullberg et al., 1998,
Infect Immun 66:5157-5166). Thus, individual members of the
intestinal microbiota vary dramatically in their propensity to
induce inflammatory responses and, thereby, influence the
development and progression of intestinal disease (Saleh and Elson,
2011, Immunity 34:293-302).
[0005] As in mice, specific members of the human intestinal
microbiota that impact disease susceptibility and/or severity by
stimulating chronic inflammatory responses may also play central
roles in the etiology of IBD (Packey and Sartor, 2009, Curr Opin
Infect Dis 22:292-301; Round and Mazmanian, 2009, Nat Rev Immunol
9:313-323). However, identifying such potentially disease-driving
members of the intestinal microbiota in humans has remained a major
challenge (Knights et al., 2013, Gut 62:1505-1510; Round et al.,
2009, Nat Rev Immunol 9:313-323).
[0006] IgA is the predominant antibody isotype produced at mucosal
surfaces and is a critical mediator of intestinal immunity (Pabst,
2012, Nat Rev Immunol 12:821-832; Slack et al., 2012, Front Immnol
3:100). Recognition of enteric pathogens by the intestinal immune
system results in the production of high-affinity, T
cell-dependent, pathogen-specific IgA, which is transcytosed into
the intestinal lumen. In the lumen, these antibodies can bind and
`coat` offending pathogens, and provide protection against
infection through neutralization and exclusion. Indigenous members
of the intestinal microbiota also can stimulate IgA production and
can become coated with IgA (Pabst, 2012, Nat Rev Immunol
12:821-832; Slack et al., 2012, Front Immnol 3:100; van der Waaij
et al., 1994, Cytometry 16:270-279). However, as compared to
pathogen-induced IgA, commensal-induced IgA is generally believed
to be of relatively low-affinity and specificity (Pabst, 2012, Nat
Rev Immunol 12:821-832; Slack et al., 2012, Front Immnol 3:100).
Thus, relative levels of bacterial coating with IgA might be
predicted to correlate with the magnitude of the inflammatory
response triggered by a specific intestinal bacterial species.
[0007] Despite considerable effort, the identification of specific
pathobionts responsible for driving the development of disease in
humans has proven difficult due to the complexity and diversity of
the microbiota, as well as the influence of host genetics and
environment on disease susceptibility. In addition, 16S rRNA
based-metagenomic studies comparing the microbiota of diseased and
normal individuals does not distinguish between the distinct
strains of a bacterial species, some of which may contribute to
disease and some of which may not. Since different strains of the
same bacterial species often colonize the same niche and share
nutrient requirements, one can imagine that non-disease-driving
strains might be used to replace or displace disease-driving
strains of the same bacterial species. Replacement of a
disease-driving bacterial strain with a non-disease-driving strain
from the same bacterial species may reduce, reverse or prevent the
development of disease. Thus, non-disease-driving strains
identified based on low IgA coating may act as `surgical
probiotics,` which specifically target disease-driving bacteria
identified based on high IgA coating that are members of the same
species.
[0008] There is a need in the art to identify the specific bacteria
(e.g., genus, species, strain, sub-strain, etc.) in the microbiota
of a subject that can lead to the development or progression of
diseases and disorders in the subject and to develop compositions
and methods to reduce the pathogenic effects of such bacteria.
Furthermore, there is a need in the art to identify bacteria that
can specifically counteract particular disease-driving members of
the microbiota. The present invention addresses this unmet
need.
SUMMARY OF THE INVENTION
[0009] The invention relates to the discovery that secretory
antibodies can be used to detect and identify microbes present in
the microbiota of a subject that influence susceptibility to or
contribute to the development or progression of diseases or
disorders, including inflammatory diseases and disorders.
[0010] In one embodiment, the invention provides a method of
treating or preventing an inflammatory disease or disorder
associated with a secretory antibody-bound bacteria in the
microbiota of a subject in need thereof. In one embodiment, the
method comprises administering to the subject a vaccine to diminish
the number or reduce the pathogenic effects of at least one type of
bacteria associated with an inflammatory disease or disorder.
[0011] In one embodiment, the at least one type of bacteria is
selected from the group consisting of Acidaminococcus spp.,
Actinomyces spp., Akkermansia muciniphila, Allobaculum spp.,
Anaerococcus spp., Anaerostipes spp., Bacteroides spp., Bacteroides
Other, Bacteroides acidifaciens, Bacteroides coprophilus,
Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis,
Barnesiellaceae spp., Bifidobacterium adolescentis, Bifidobacterium
Other, Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia
producta, Blautia Other, Blautia spp., Bulleidia spp.,
Catenibacterium spp., Citrobacter spp., Clostridiaceae spp.,
Clostridiales Other, Clostridiales spp., Clostridium perfringens,
Clostridium spp., Clostridium Other, Collinsella aerofaciens,
Collinsella spp., Collinsella stercoris, Coprococcus catus,
Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp.,
Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other,
Eggerthella lenta, Enterobacteriaceae Other, Enterobacteriaceae
spp., Enterococcus spp., Erysipelotrichaceae spp., Eubacterium
biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium
spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae
spp., Haemophilus parainfluenzae, Haemophilus Other, Helicobacter
spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp.,
Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae,
Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp.,
Leuconostocaceae spp., Megamonas spp., Megasphaera spp.,
Methanobrevibacter spp., Mitsuokella multacida, Mitsuokella spp.,
Mucispirillum schaedleri, Odoribacter spp., Oscillospira spp.,
Parabacteroides distasonis, Parabacteroides spp., Paraprevotella
spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp.,
Pediococcus Other, Peptococcus spp., Peptoniphilus spp.,
Peptostreptococcus anaerobius, Peptostreptococcus Other,
Phascolarctobacterium spp., Prevotella copri, Prevotella spp.,
Prevotella stercorea, Prevotellaceae, Proteus spp., Rikenellaceae
spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other,
Ruminococcaceae spp., Ruminococcus bromii, Ruminococcus gnavus,
Ruminococcus spp., Ruminococcus Other, Ruminococcus torques,
Slackia spp., S24-7 spp., SMB53 spp., Streptococcus anginosus,
Streptococcus luteciae, Streptococcus spp., Streptococcus Other,
Sutterella spp., Turicibacter spp., UC Bulleidia, UC
Enterobacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC
Pediococcus, Varibaculum spp., Veillonella spp., Sutterella,
Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC
Ruminococcaceae, Veillonella parvula, Veillonella spp., Veillonella
dispar, and Weissella.
[0012] In one embodiment, the vaccine comprises an inactivated
bacterium. In one embodiment, the vaccine is administered orally to
the subject. In some embodiments, the vaccine comprises at least
one adjuvant and/or immunomodulator. An adjuvant and/or
immunomodulator refers to a compound that enhances an immune
response when administered together (or successively) with the
immunological composition/vaccine. Examples of suitable adjuvants
and/or immunomodulators include, but are not limited to, complete
or incomplete Freund's adjuvant, RIBI (e.g., muramyl dipeptides,
etc.), KLH peptide, cholera toxin or a portion thereof, salmonella
toxin or a portion thereof, E. coli heat labile enterotoxin or a
portion thereof, E. coli enterotoxin or a portion thereof, AB5
toxins or a portion thereof, mineral salts, aluminum salts (e.g.,
hydroxide, phosphate, Alum, etc.), calcium phosphate, liposomes,
virosomes (unilamellar liposomal vehicles, immunostimulating
reconstituted influenza virosomes [IRIV]), virus-like particles,
cochleates, eurocine (e.g., monoglycerides with fatty acids, etc.),
archaeal lipids, ISCOMS (e.g., immunostimulating complexes,
structured complex of saponins and lipids, etc.), microparticles
(e.g., PLG, etc.), emulsions (e.g., MF59, Montanides, etc.),
monophosphoryl lipid (MPL) or synthetic derivatives,
N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) or a derivative,
Detox (MPL+CWS), AS04 (Alum+MPL), AS02 (oil-in-water
emulsion+MPL+QS21), AS01 (liposomes+MPL+QS21), OM-174 (e.g., Lipid
A derivative, E. coli, etc.), OM-triacyl, oligonucleotides (e.g.,
CpG, etc.), double-stranded RNA (dsRNA), pathogen-associated
molecular patterns (PAMPs), TLR ligands (e.g., flagellin,
monophosphoryl lipid A, etc.), saponins (e.g., Quils, QS-21, etc.),
chitosan, .alpha.-galactosylceramide, small-molecule immune
potentiators (SMIPs) (e.g., imiquimod, resiquimod [R848], etc.), a
cytokine or chemokine (e.g., IL-2, IL-12, GM-CSF, Flt3, etc.), an
accessory molecule (e.g., B7.1, etc.), liposomes (e.g., DNPC/Chol,
etc.), DC Chol (e.g., lipoidal immunomodulators able to
self-organize into liposomes, etc.), nanoparticle-based adjuvants,
PLA (polylactic acid) microparticles, PLG
(poly[lactide-co-glycolide]) microparticles,
Poly(DL-lactide-co-glycolide) microparticles, polystyrene (latex)
microparticles, proteosomes (e.g., hydrophobic, proteinaceous,
nanoparticles comprised of purified N. meningitidis outer membrane
proteins, etc.), and 3',5'-Cyclic diguanylic acid (c-di-GMP). Such
example adjuvants and/or immunomodulators, as well as others, are
understood by those skilled in the art, are readily described in
available literature, and are useful in the compositions and
methods of the invention.
[0013] In one aspect, the present invention provides an
immunological composition for treating or preventing an
inflammatory disease or disorder, comprising a vaccine to diminish
the number or pathogenic effects of at least one type of bacteria
associated with an inflammatory disease or disorder.
[0014] In one embodiment, the at least one type of bacteria is
selected from the group consisting of Acidaminococcus spp.,
Actinomyces spp., Akkermansia muciniphila, Allobaculum spp.,
Anaerococcus spp., Anaerostipes spp., Bacteroides spp., Bacteroides
Other, Bacteroides acidifaciens, Bacteroides coprophilus,
Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis,
Barnesiellaceae spp., Bifidobacterium adolescentis, Bifidobacterium
Other, Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia
producta, Blautia Other, Blautia spp., Bulleidia spp.,
Catenibacterium spp., Citrobacter spp., Clostridiaceae spp.,
Clostridiales Other, Clostridiales spp., Clostridium perfringens,
Clostridium spp., Clostridium Other, Collinsella aerofaciens,
Collinsella spp., Collinsella stercoris, Coprococcus catus,
Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp.,
Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other,
Eggerthella lenta, Enterobacteriaceae Other, Enterobacteriaceae
spp., Enterococcus spp., Erysipelotrichaceae spp., Eubacterium
biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium
spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae
spp., Haemophilus parainfluenzae, Haemophilus Other, Helicobacter
spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp.,
Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae,
Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp.,
Leuconostocaceae spp., Megamonas spp., Megasphaera spp.,
Methanobrevibacter spp., Mitsuokella multacida, Mitsuokella spp.,
Mucispirillum schaedleri, Odoribacter spp., Oscillospira spp.,
Parabacteroides distasonis, Parabacteroides spp., Paraprevotella
spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp.,
Pediococcus Other, Peptococcus spp., Peptoniphilus spp.,
Peptostreptococcus anaerobius, Peptostreptococcus Other,
Phascolarctobacterium spp., Prevotella copri, Prevotella spp.,
Prevotella stercorea, Prevotellaceae, Proteus spp., Rikenellaceae
spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other,
Ruminococcaceae spp., Ruminococcus bromii, Ruminococcus gnavus,
Ruminococcus spp., Ruminococcus Other, Ruminococcus torques,
Slackia spp., S24-7 spp., SMB53 spp., Streptococcus anginosus,
Streptococcus luteciae, Streptococcus spp., Streptococcus Other,
Sutterella spp., Turicibacter spp., UC Bulleidia, UC
Enterobacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC
Pediococcus, Varibaculum spp., Veillonella spp., Sutterella,
Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC
Ruminococcaceae, Veillonella parvula, Veillonella spp., Veillonella
dispar, and Weissella.
[0015] In one embodiment, the vaccine comprises an inactivated
bacterium. In some embodiments, the vaccine comprises at least one
adjuvant and/or immunomodulator. An adjuvant and/or immunomodulator
refers to a compound that enhances an immune response when
administered together (or successively) with the immunological
composition/vaccine. Examples of suitable adjuvants and/or
immunomodulators include, but are not limited to, complete or
incomplete Freund's adjuvant, MI (e.g., muramyl dipeptides, etc.),
KLH peptide, cholera toxin or a portion thereof, salmonella toxin
or a portion thereof, mineral salts, aluminum salts (e.g.,
hydroxide, phosphate, Alum, etc.), calcium phosphate, liposomes,
virosomes (unilamellar liposomal vehicles, immunostimulating
reconstituted influenza virosomes [IRIV]), virus-like particles,
cochleates, eurocine (e.g., monoglycerides with fatty acids, etc.),
archaeal lipids, ISCOMS (e.g., immunostimulating complexes,
structured complex of saponins and lipids, etc.), microparticles
(e.g., PLG, etc.), emulsions (e.g., MF59, Montanides, etc.),
monophosphoryl lipid (MPL) or synthetic derivatives,
N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) or a derivative,
Detox (MPL+CWS), AS04 (Alum+MPL), AS02 (oil-in-water
emulsion+MPL+QS21), AS01 (liposomes+MPL+QS21), OM-174 (e.g., Lipid
A derivative, E. coli, etc.), OM-triacyl, oligonucleotides (e.g.,
CpG, etc.), double-stranded RNA (dsRNA), pathogen-associated
molecular patterns (PAMPs), E. coli heat labile enterotoxin, TLR
ligands (e.g., flagellin, monophosphoryl lipid A, etc.), AB5 toxins
or a portion thereof, saponins (e.g., Quils, QS-21, etc.),
chitosan, .alpha.-galactosylceramide, small-molecule immune
potentiators (SMIPs) (e.g., imiquimod, resiquimod [R848], etc.), a
cytokine or chemokine (e.g., IL-2, IL-12, GM-CSF, Flt3, etc.), an
accessory molecule (e.g., B7.1, etc.), liposomes (e.g., DNPC/Chol,
etc.), DC Chol (e.g., lipoidal immunomodulators able to
self-organize into liposomes, etc.), PLA (polylactic acid)
microparticles, PLG (poly[lactide-co-glycolide]) microparticles,
Poly(DL-lactide-co-glycolide) microparticles, polystyrene (latex)
microparticles, proteosomes (e.g., hydrophobic, proteinaceous,
nanoparticles comprised of purified N. meningitidis outer membrane
proteins, etc.), and 3',5'-Cyclic diguanylic acid (c-di-GMP). Such
example adjuvants and/or immunomodulators, as well as others, are
understood by those skilled in the art, are readily described in
available literature, and are useful in the compositions and
methods of the invention.
[0016] In one aspect, the present invention provides a method of
treating an inflammatory disease or disorder associated with a
secretory antibody-bound bacteria in the microbiota of a subject in
need thereof, comprising administering to the subject at least one
bacterium of a species of bacteria that does not contribute to the
development or progression of disease in the subject. In one
embodiment, the species of bacteria that does not contribute to the
development or progression of disease is a secretory antibody-bound
bacteria of a subject who does not have the inflammatory disease or
disorder.
[0017] In one embodiment, the invention is a method of identifying
a type (e.g., genus, species, strain, sub-strain, etc.) of bacteria
in the microbiota of a subject that contributes to the development
or progression of an inflammatory disease or disorder in the
subject, including the steps of: isolating secretory antibody-bound
bacteria from the subject's biological sample, amplifying bacterial
nucleic acid from secretory antibody-bound bacteria so isolated,
determining the sequences of the amplicons, identifying the type
(e.g., genus, species, strain, sub-strain, etc.) of antibody-bound
bacteria present in the subject's biological sample by identifying
nucleic acid sequences that are indicative of particular types
(e.g., genus, species, strain, sub-strain, etc.) of bacteria. In
some embodiments, strain and sub-strain identification is performed
by at least one of anaerobic culturing of specific bacteria from
feces, colonization of germ-free mice with bacterial isolates, and
whole bacterial genome sequencing. In another embodiment, the
invention is a method of identifying a type (e.g., genus, species,
strain, sub-strain, etc.) of bacteria in the microbiota of a
subject that specifically counteracts the effects of bacteria in
the microbiota of a subject that contributes to the development or
progression of an inflammatory disease or disorder in the subject
(i.e., probiotic), including the steps of: identifying
phylogenetically similar bacteria that display differential
antibody coating, testing the effects of these bacteria in
germ-free mice, and characterizing these bacteria genetically by
whole bacterial genome sequencing. In some embodiments, the
microbiota of the subject is on or near mucosal surface of the
subject selected from the group consisting of the gastrointestinal
tract, the respiratory tract, genitourinary tract and mammary
gland. In some embodiments, the biological sample is at least one
of a fecal sample, a mucus sample, a sputum sample, and a breast
milk sample. In some embodiments, the bacterial nucleic acid is 16S
rRNA. In some embodiments, the secretory antibody is at least one
selected from the group consisting of IgA1, IgA2, and IgM. In some
embodiments, the inflammatory disease or disorder is at least one
inflammatory disease or disorder selected from the group consisting
of inflammatory bowel disease, celiac disease, colitis, irritable
bowel syndrome, intestinal hyperplasia, metabolic syndrome,
obesity, diabetes, rheumatoid arthritis, liver disease, hepatic
steatosis, fatty liver disease, non-alcoholic fatty liver disease
(NAFLD), and non-alcoholic steatohepatitis (NASH). In some
embodiments, the subject is human.
[0018] In another embodiment, the invention is a method of
identifying a first strain of a species of bacteria in the
microbiota of a subject, wherein the first strain of the species of
bacteria does not contribute to the development or progression of
disease in the subject, and wherein the species of bacteria
comprises at least a second strain of bacteria, and wherein the
second strain of the species of bacteria does contribute to the
development or progression of the inflammatory disease or disorder,
including the steps of isolating the first strain of low- or
non-secretory antibody-bound bacteria from the subject's biological
sample, isolating the second strain of secretory antibody-bound
bacteria from the subject's biological sample, amplifying bacterial
nucleic acid from the first strain of low- or non-secretory
antibody-bound bacteria so isolated, amplifying bacterial nucleic
acid from the second strain of secretory antibody-bound bacteria so
isolated, determining the sequences of the amplicons so amplified,
comparing the sequences of the amplicons so amplified from the
first strain to the sequences of the amplicons so amplified from
the second strain, determining that the first strain of bacteria
and the second strain of bacteria are members of the same species
of bacteria, determining that the first strain does not contribute
to the development or progression of the inflammatory disease or
disorder, and determining that the second strain does contribute to
the development or progression of the inflammatory disease or
disorder. In some embodiments, the method also comprises the step
of culturing at least one of the first strain of bacteria and the
second strain of bacteria. In some embodiments, the method also
comprises the step functionally and phylogenetically characterizing
at least one of the first strain of bacteria and the second strain
of bacteria using at least one selected from the group consisting
of colonization of germ-free mice and whole genome sequencing. In
some embodiments, the microbiota of the subject is on or near
mucosal surface of the subject selected from the group consisting
of the gastrointestinal tract, the respiratory tract, genitourinary
tract and mammary gland. In some embodiments, the biological sample
is at least one of a fecal sample, a mucus sample, a sputum sample,
and a breast milk sample. In some embodiments, the bacterial
nucleic acid is 16S rRNA. In some embodiments, the secretory
antibody is at least one selected from the group consisting of
IgA1, IgA2, and IgM. In some embodiments, the inflammatory disease
or disorder is at least one inflammatory disease or disorder
selected from the group consisting of inflammatory bowel disease,
celiac disease, colitis, irritable bowel syndrome, intestinal
hyperplasia, metabolic syndrome, obesity, diabetes, rheumatoid
arthritis, liver disease, hepatic steatosis, fatty liver disease,
non-alcoholic fatty liver disease (NAFLD), and non-alcoholic
steatohepatitis (NASH). In some embodiments, the subject is
human.
[0019] In another embodiment, the invention is a method of treating
an inflammatory disease or disorder of a subject in need thereof,
including the step of administering to the subject at least one
bacterium that is desired, preferred, neutral, beneficial, and/or
under-represented in the subject's microbiota. In some embodiments,
the at least one bacterium is at least one bacterium of a first
strain of a species of bacteria, wherein the first strain of the
species of bacteria does not contribute to the development or
progression of disease in the subject, and wherein the species of
bacteria comprises at least a second strain of bacteria, and
wherein the second strain of the species of bacteria does
contribute to the development or progression of the inflammatory
disease or disorder.
[0020] In another embodiment, the invention is a method of
diagnosing an inflammatory disease or disorder in a subject in need
thereof by identifying a type (e.g., genus, species, strain,
sub-strain, etc.) of bacteria in the microbiota of the subject that
contributes to the development or progression of an inflammatory
disease or disorder, including the steps of: isolating secretory
antibody-bound bacteria from the subject's biological sample,
amplifying bacterial nucleic acid from secretory antibody-bound
bacteria so isolated, determining the sequences of the amplicons so
amplified, and identifying the type (e.g., genus, species, strain,
sub-strain, etc.) of antibody-bound bacteria present in the
subject's biological sample by identifying nucleic acid sequences
that are indicative of particular types (e.g., genus, species,
strain, sub-strain, etc.) of bacteria, wherein when the type (e.g.,
genus, species, strain, sub-strain, etc.) of antibody-bound
bacteria present in the subject's biological sample is a type
(e.g., genus, species, strain, sub-strain, etc.) of bacteria that
contributes to the development or progression of an inflammatory
disease or disorder, the subject is diagnosed with the inflammatory
disease or disorder. In some embodiments, the microbiota of the
subject is on or near mucosal surface of the subject selected from
the group consisting of the gastrointestinal tract, the respiratory
tract, genitourinary tract and mammary gland. In some embodiments,
the biological sample is at least one of a fecal sample, a mucus
sample, a sputum sample, and a breast milk sample. In some
embodiments, the bacterial nucleic acid is 16S rRNA. In some
embodiments, the secretory antibody is at least one selected from
the group consisting of IgA1, IgA2, and IgM. In some embodiments,
the inflammatory disease or disorder is at least one inflammatory
disease or disorder selected from the group consisting of
inflammatory bowel disease, celiac disease, colitis, irritable
bowel syndrome, intestinal hyperplasia, metabolic syndrome,
obesity, diabetes, rheumatoid arthritis, liver disease, hepatic
steatosis, fatty liver disease, non-alcoholic fatty liver disease
(NAFLD), and non-alcoholic steatohepatitis (NASH). In some
embodiments, the subject is human.
[0021] In one embodiment, the invention is a method of treating an
inflammatory disease or disorder associated with a secretory
antibody-bound bacteria in the microbiota of a subject in need
thereof, the method comprising administering to the subject at
least one therapy to diminish the number or pathogenic effects of
at least one type and strain of bacteria that is over-represented
in the microbiota of the subject. In some embodiments, the at least
one therapy is at least one selected from the group consisting of
at least one vaccine, at least one antibiotic, and at least one
passive immunotherapy. In some embodiments, the microbiota of the
subject is on or near mucosal surface of the subject selected from
the group consisting of the gastrointestinal tract, the respiratory
tract, genitourinary tract and mammary gland. In some embodiments,
the biological sample is at least one of a fecal sample, a mucus
sample, a sputum sample, and a breast milk sample. In some
embodiments, the secretory antibody is at least one selected from
the group consisting of IgA1, IgA2, and IgM. In some embodiments,
the inflammatory disease or disorder is at least one inflammatory
disease or disorder selected from the group consisting of
inflammatory bowel disease, celiac disease, colitis, irritable
bowel syndrome, intestinal hyperplasia, metabolic syndrome,
obesity, diabetes, rheumatoid arthritis, liver disease, hepatic
steatosis, fatty liver disease, non-alcoholic fatty liver disease
(NAFLD), and non-alcoholic steatohepatitis (NASH). In some
embodiments, the subject is human. In some embodiments, the therapy
induces an immune response directed against at least one type and
strain of secretory antibody-bound bacteria present in the
microbiota of the subject. In some embodiments, the method further
comprises administering to the subject at least one probiotic to
increase the number of at least one type and strain of bacteria
under-represented in the microbiota of the subject. In some
embodiments, the method further comprises administering to the
subject at least one `surgical probiotic,` which is a preferred,
desired or beneficial bacterial strain that belongs to the same or
related species as a disease-associated strain of bacteria that was
identified based on IgA coating.
[0022] In one aspect, the present invention provides a method of
treating or preventing an inflammatory disease or disorder
associated with a secretory antibody-bound bacteria in the
microbiota of a subject in need thereof, comprising administering
to the subject a composition comprising a vaccine comprising
inactivated Erysipelotrichaceae spp. and an adjuvant. In one
embodiment, the vaccine is administered orally to the subject. In
one embodiment, the adjuvant is cholera toxin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The following detailed description of preferred embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. For the purpose of illustrating the
invention, there are shown in the drawings embodiments which are
presently preferred. It should be understood, however, that the
invention is not limited to the precise arrangements and
instrumentalities of the embodiments shown in the drawings.
[0024] FIG. 1, comprising FIGS. 1A-1D, depicts the results of
experiments demonstrating that IgA-based sorting and 16S sequencing
of fecal bacteria from specific pathogen free (SPF) mice. (FIG. 1A)
Overview of IgA-based cell sorting of fecal bacteria combined with
16S rRNA gene sequencing (IgA-SEQ). (FIG. 1B) Representative
results and a cartoon of cell sorting of IgA+ and IgA- fecal
bacteria from mice. (FIG. 1C) Heatmap depicting IgA Coating Index
(ICI) scores and average relative abundance of bacterial genera in
Total (Presort), IgA+ and IgA- fractions of fecal bacteria from
C57Bl/6 SPF mice (n=17 samples). Relative abundance heatmaps are
depicted on a logarithmic scale. Genera that are highly coated with
IgA (significantly higher relative abundance in the IgA+ fraction
as compared to the IgA- fraction by LEfSe; P<0.05) are marked
with an asterisk. Genera with ICI >10 are labeled in red. UC,
unclassified in the Greengenes reference database. Gen., classified
as a distinct but unnamed genus in the Greengenes reference
database. (FIG. 1D) Relative abundance of significantly coated
bacterial genera in Presort, IgA+ and IgA- fractions. *P<0.05;
***P<0.001 (Wilcoxon rank-sum). Indicated are mean.+-.standard
error of the mean.
[0025] FIG. 2, comprising FIGS. 2A-2E, depicts the results of
experiments demonstrating that IgA coating identifies colitogenic
bacteria in mice with inflammasome-mediated intestinal dysbiosis
(SPF.sup.dys). (FIG. 2A) Average relative abundance of bacterial
genera of greater than 1% abundance in the intestinal microbiota of
SPF and SPF.sup.dys mice. UC Prevotellaceae is marked with an
arrow. SPF.sup.dys mice were co-housed with Asc.sup.-/- mice to
allow for the acquisition of dysbiosis. (FIG. 2B) Dextran Sodium
Sulfate (DSS)-induced colitis in SPF (n=5) and SPF.sup.dys (n=4)
mice. *P<0.05; **P<0.01; ***P<0.001 (one-way ANOVA). (FIG.
2C) IgA coating of fecal bacteria from 10-16 week old SPF (n=8) and
SPF.sup.dys (n=9) mice at the steady state. ***P<0.001 (unpaired
Student's t-test). (FIG. 2D) Heatmap depicting IgA Coating Index
(ICI) scores and average relative abundance of bacterial genera in
Total (Presort), IgA+ and IgA- fractions of fecal bacteria from
10-16 week old SPF.sup.dys mice (n=14 samples) sampled under steady
state conditions. Relative abundance heatmaps are depicted on a
logarithmic scale. Genera that are highly coated with IgA
(significantly higher relative abundance in the IgA+ fraction as
compared to the IgA- fraction by LEfSe; P<0.05) are marked with
an asterisk. Genera with ICI >10 are labeled in red. (FIG. 2E)
Relative abundance of significantly coated genera in Presort, IgA+
and IgA- fractions. *P<0.05; ***P<0.001 (Wilcoxon rank-sum).
Indicated are mean.+-.standard error of the mean. UC, unclassified
in the Greengenes reference database. Gen., classified as a
distinct but unnamed genus in the Greengenes reference
database.
[0026] FIG. 3, comprising FIGS. 3A-3C, depicts the results of
experiments demonstrating the IgA coating of fecal bacteria from
healthy humans and inflammatory bowel disease patients. (FIG. 3A)
IgA coating of fecal bacteria from 20 healthy subjects, 27 Crohn's
disease patients (CD) and 8 Ulcerative colitis patients (UC).
*P<0.05; ***P<0.001 (one-way ANOVA). (FIG. 3B) Venn-diagram
depicting the distribution of highly coated bacterial species in
healthy, UC and CD patients. Bacterial taxa that showed an ICI
score greater than 10 in at least one subject were classified as
highly coated within that group. (FIG. 3C) Heatmap depicting IgA
coating index (ICI) scores for bacterial species that are uniquely
highly coated (ICI >10) in IBD and never highly coated or never
present in healthy controls. Bars to the right of the heatmap
correspond with the color-coding of the Venn-diagram in panel (3B).
Each column represents an individual human subject. UC,
unclassified in the Greengenes reference database. Spp., classified
as a distinct but unnamed species in the Greengenes reference
database.
[0027] FIG. 4, comprising FIGS. 4A-4E, depicts the results of
experiments demonstrating the isolation of personalized
IBD-associated gut microbiota culture collections, assembly of IgA+
and IgA- consortia and colonization of germ-free mice. (FIG. 4A)
Strategy for isolation of personalized IBD-associated gut
microbiota culture collections. (FIG. 4B) Selection of individual
bacterial isolates comprising IgA+ and IgA- consortia and
colonization of germ-free mice. Specific isolates that were
included in the consortia are boxed in green (IgA-) or red (IgA+).
(FIG. 4C) Barplots depicting relative abundance of bacterial taxa
in IgA+ and IgA- consortia pre-gavage (DO) and in the feces of IgA+
and IgA- colonized mice 2 weeks post-colonization. All members of
the pre-gavage consortia were detectable in colonized mice except
Peptinophilus spp. in the IgA- consortium and Streptococcus spp. in
the IgA+ consortium. UC, unclassified in the Greengenes reference
database. Spp., classified as a distinct but unnamed species in the
Greengenes reference database. (FIG. 4D) IgA coating of fecal
bacteria from germ-free mice colonized with IgA+ (n=5) or IgA-
consortia (n=5) on days 7 and 24 post-colonization. Representative
plots are shown. ***P<0.005 (unpaired Student's t-test).
Indicated are mean.+-.standard error of the mean. (FIG. 4E)
Microbiota localization as visualized by 16S rRNA FISH (red) and
DAPI (blue) staining. The mucus layer is demarked by two dotted
lines.
[0028] FIG. 5, comprising FIGS. 5A-5E, depicts the results of
experiments demonstrating that IBD-associated IgA+ bacteria
exacerbate DSS-induced colitis in gnotobiotic mice. (FIG. 5A)
Timeline of colonization and DSS treatment in germ-free mice
colonized with IgA+ and IgA- consortia. (FIG. 5B) Colon length
after DSS. *P<0.05 (unpaired Student's t-test). (FIG. 5C) Gross
pathology of large bowels after DSS. Note the extensive bleeding
and diarrhea in the IgA+ colonized mice. (FIG. 5D) Colon
histopathology scores after DSS. Scores were assigned as follows:
0, Intact colonic architecture. No acute inflammation or epithelial
injury; 1, Focal minimal acute inflammation; 2, Focal mild acute
inflammation; 3, Severe acute inflammation with multiple crypt
abscesses and/or focal ulceration; 4, Severe acute inflammation,
multiple crypt abscesses, epithelial loss and extensive ulceration.
***P<0.0001 (unpaired Student's t-test). (FIG. 5E)
Representative histology pictures from hematoxylin and eosin
stained colons after DSS. Note that IgA+ colonized mice exhibit
extensive inflammation, crypt abscesses, epithelial loss, and
ulceration, while all IgA- colonized mice showed either no
inflammation or minimal/mild focal inflammation. Data are
representative of 3 independent experiments.
[0029] FIG. 6, comprising FIGS. 6A-6J, depicts the results of
experiments analyzing the IgA coating of fecal bacteria from SPF
C57Bl/6 mice. (FIG. 6A) Representative staining of fecal bacteria
from C57Bl/6 SPF (n=5) and Rag2.sup.-/- mice (n=6), which lack
immunoglobulins, with anti-IgA. Data is representative of more than
5 independent experiments. (FIG. 6B) Dot plot of all mice from
(6A). (FIG. 6C) Gating on IgA coated bacteria demonstrates that the
vast majority of IgA+ events fall within the designated FSC and SSC
gate. SSC, side scatter. FSC, forward scatter. Data is
representative of more than 5 independent experiments. (FIG. 6D)
Representative staining of fecal bacteria from healthy humans, and
Crohn's disease and ulcerative colitis patients with anti-IgA. Data
for all patients is shown in FIG. 3A. (FIG. 6E) Purity of IgA+ and
IgA- fractions after MACS and FACS sorting as determined by flow
cytometry (Presort: 17.2%.+-.2.8; IgA+: 88.3.+-.1.8; IgA-:
3.6.+-.0.4). Indicated are mean.+-.standard error of the mean.
(FIG. 6F) IgA concentrations in total, IgA+ and IgA- bacterial
fractions after MACS sorting as determined by whole bacterial-cell
ELISA. Indicated are mean.+-.standard error of the mean. (FIG. 6G)
Average relative abundance of bacterial genera in Presort (Total),
IgA+, IgA-, and mock-sorted (MACS and FACS) samples (n=4 mice).
Depicted are bacteria of >1% abundance. UC, unclassified in the
Greengenes reference database. Gen., classified as a distinct but
unnamed genus in the Greengenes reference database. (FIG. 6H)
Principal Coordinates Analysis of weighted UniFrac distances of
Presort (total fecal bacteria), IgA+, IgA-, and mock-sorted (MACS
and FACS) samples. PC, Principal Coordinate. PERMANOVA comparisons
of weighted UniFrac distances of Presort, IgA+, IgA-, and
mock-sorted samples demonstrated that IgA+ bacteria were distinct
from Presort and IgA- fractions (P<0.05), while IgA- bacteria
were not significantly different from total bacteria (P=0.266).
Mock sorting did not significantly alter the observed phylogenetic
composition of fecal bacteria (Presort versus MACS: P=0.655;
Presort versus FACS: P=0.606). For MACS mock-sorting, samples were
stained with anti-IgA and sorted by MACS before recombining
positive and negative fractions (Mock Sort MACS); for FACS
mock-sorting, Mock Sort MACS samples were sorted by FACS by gating
on total bacteria by FSC/SCC (Mock Sort FACS). (FIG. 6I) Principal
Coordinate Analysis of weighted UniFrac distances of Total
(Presort), IgA+ and IgA- fecal bacteria from SPF mice (n=17
sampling event). PC, Principal Coordinate. (FIG. 6J) LEfSe
comparisons of IgA+ and IgA- bacterial genera from SPF mice. Taxa
that are significantly enriched in the IgA+ fraction are depicted
in red, and taxa that are significantly enriched in the IgA-
fraction are depicted in green. Significance levels for LEfSe were
P<0.05 and Linear Discriminant Analysis (LDA) Score >2. UC,
unclassified by the Greengenes reference database. Gen., classified
as a distinct but unnamed genus by the Greengenes reference
database.
[0030] FIG. 7, comprising FIGS. 7A-7C, depicts the results of
experiments analyzing the IgA coating of intestinal bacteria from
SPF.sup.dys mice, and dysbiotic wild-type and T cell-deficient
mice. (FIG. 7A) Principal Coordinate Analysis of weighted UniFrac
distances of Total (Presort), IgA+ and IgA- fecal bacteria from SPF
(n=17 sampling events) and SPF.sup.dys mice (n=14 sampling events).
PC, Principal Coordinate. (FIG. 7B) LEfSe comparisons of IgA+ and
IgA- bacterial genera from SPF.sup.dys mice. Taxa that are
significantly enriched in the IgA+ fraction are depicted in red,
and taxa that are significantly enriched in the IgA- fraction are
depicted in green. Significance levels for LEfSe were P<0.05 and
Linear Discriminant Analysis (LDA) Score >2. (FIG. 7C) IgA
Coating Index (ICI) scores in dysbiotic WT C57Bl/6 and T
cell-deficient (Tcrb-/-; Tcrd-/-) mice. C57Bl/6 and Tcrb-/-;Tcrd-/-
mice were co-housed with Asc.sup.-/- mice for at least 6 weeks to
allow for the acquisition of dysbiosis. *P<0.05; **P<0.01;
***P<0.001 (Wilcoxon rank-sum).
[0031] FIG. 8 depicts the results of experiments analyzing the IgA
coating of fecal bacteria from healthy humans, Crohn's disease
patients, and patients with ulcerative colitis. Depicted in the
main heatmap (black, blue, yellow) are IgA coating index (ICI)
scores for bacterial species from 20 healthy humans, 27 Crohn's
disease (CD) patients, and 8 patients with ulcerative colitis (UC).
Each column represents an individual human subject. Bacterial taxa
are clustered (complete linkage clustering using Euclidean
distance) based on ICI scores observed in healthy humans. The first
three columns on the left (red, gray, blue) summarize the
statistical comparisons between relative taxonomic abundance in the
IgA+ and IgA- fraction in each patient group. Bacterial taxa with
significantly higher relative abundance in the IgA+ fraction as
compared to the IgA- fraction by LEfSe and Wilcoxon rank-sum are
red; bacterial taxa with significantly lower relative abundance in
the IgA+ fraction as compared to the IgA- fraction are blue; and
bacterial taxa showing no significant difference in abundance in
the IgA+ and IgA- fractions are grey. The fourth and fifth columns
on the left summarize statistical comparisons between ICI scores in
healthy subjects and IBD patients: gray marks no difference between
diseased and control, green marks taxa where ICI scores are
significantly higher in healthy controls than in diseased patients,
and purple marks taxa where ICI scores are significantly lower in
healthy controls than in diseased patients. Significance levels for
LEfSe and Wilcoxon rank-sum were P<0.05 and Linear Discriminant
Analysis Score >2, and P<0.05, respectively. UC, unclassified
in the Greengenes reference database. Spp., classified as a
distinct but unnamed species in the Greengenes reference
database.
[0032] FIG. 9 depicts the results of experiments demonstrating the
relative abundance of bacterial taxa comprising the IgA+ and IgA-
consortia. NS=not significant; *P<0.05 (Wilcoxon rank-sum).
Indicated are mean.+-.standard error of the mean.
[0033] FIG. 10, comprising FIGS. 10A-10J, depicts the results of
experiments demonstrating that IBD-associated IgA+ and IgA-
bacterial consortia do not induce inflammatory responses under
homeostatic conditions in gnotobiotic mice, and differential IgA
coating identifies distinct strains of B. fragilis. (FIG. 10A)
Colonization of germ-free mice with IgA+ and IgA- consortia. (FIG.
10B) Colon length after 14 days of bacterial colonization. ns, not
significant (unpaired Student's t-test). (FIG. 10C) Gross pathology
of large bowels. (FIG. 10D) Colon histopathology scores. Scores
were assigned as follows: 0, Intact colonic architecture. No acute
inflammation or epithelial injury; 1, Focal minimal acute
inflammation; 2, Focal mild acute inflammation; 3, Severe acute
inflammation with multiple crypt abscesses and/or focal ulceration;
4, Severe acute inflammation, multiple crypt abscesses, epithelial
loss and extensive ulceration. ns, not significant (unpaired
Student's t-test). (FIG. 10E) Representative histology pictures
from hematoxylin and eosin stained colons. (FIG. 10F) Selection of
differentially coated isolates/strains of B. fragilis from human
gut microbiota culture collections and monocolonization of
germ-free mice. (FIG. 10G) Similarity profiles of B. fragilis
(IgA-) and B. fragilis (IgA+) draft genomes aligned via progressive
Mauve. The height of the profile represents the level of
conservation. Sections depicted in white are absent from the
comparison strain. (FIG. 10H) Colon length after DSS. **P<0.005
(unpaired Student's t-test). (FIG. 10I) Colon histopathology scores
6 days after DSS. Scores were assigned as follows: 0, Intact
colonic architecture. No acute inflammation or epithelial injury;
1, Focal minimal acute inflammation; 2, Focal mild acute
inflammation; 3, Severe acute inflammation with multiple crypt
abscesses and/or focal ulceration; 4, Severe acute inflammation,
multiple crypt abscesses, epithelial loss and extensive ulceration.
(FIG. 10J) Representative histology pictures from hematoxylin and
eosin stained colons after DSS.
[0034] FIG. 11 depicts the results of experiments demonstrating
that IgA+ bacteria from healthy humans, unlike IgA+ bacteria from
IBD patients, are non-colitogenic. Germ-free mice were colonized
with IgA- bacteria or IgA+ bacteria from IBD patients, or IgA+
bacteria from healthy humans. One week post-colonization, mice were
treated with 2.5% DSS ad libitum and colon length was measured at
day 6 post-DSS. **P<0.01; N.S. not significant. (One way
ANOVA).
[0035] FIG. 12 depicts the results of experiments demonstrating
that IgA+ species from healthy humans can protect against
colitogenic IgA+ species from patients with IBD. Germ-free mice
were colonized with non-colitogenic IgA- bacteria (IgA- (IBD)), or
colitogenic IgA+ bacteria from IBD patients with
(IgA+(IBD)/IgA+(Healthy)) or without (IgA+(IBD)) IgA+ bacteria from
healthy individuals. One week post-colonization, mice were treated
with 2% DSS ad libitum and colon length was measured at day 6
post-DSS. N.S. not significant (One-way ANOVA).
[0036] FIG. 13 depicts the results of experiments demonstrating
that oral immunization protects against bacterial-driven colitis.
Germ-free mice were colonized with a colitogenic microbiota
consisting of nine strains of non-colitogenic bacteria (IgA-
consortium) and one known colitogenic species (Erysipelotrichaceae
sp.) that was previously isolated from a patient with IBD and
identified based on high IgA-coating. After one week of
colonization, mice were immunized with Cholera Toxin (CT) alone or
CT plus heat-killed Erysipelotrichaceae once weekly for 6 weeks by
oral gavage. After 8 weeks, mice were treated with 1.8% DSS to
induce colitis and colon length was measured at day 6 post-DSS.
***P<0.001 (One way ANOVA).
DETAILED DESCRIPTION
[0037] The present invention relates to the discovery that
secretory antibodies can be used to detect and identify microbes
present in the microbiota of a subject that influence
susceptibility to or contribute to the development or progression
of diseases or disorders.
[0038] Further, the present invention relates to methods of
modifying an altered microbiota having secretory antibody-coated
constituents in a subject in need thereof. In some embodiments, the
invention provides compositions and methods for diminishing
constituents of an altered microbiota that are over-represented in
the altered microbiota as compared with a normal microbiota, such
as over-represented secretory antibody-coated constituents, to
restore the subject's microbiota to a normal microbiota. For
example, in certain embodiments, the invention comprises
compositions and methods relating to a vaccine which induces an
immune response against one or more bacteria associated with the
development or progression of a disease or disorder, thereby
reducing the amount of the one or more bacteria in a subject. In
other embodiments, the invention provides compositions and methods
for supplementing constituents of an altered microbiota that are
under-represented in the altered microbiota, as compared with a
normal microbiota, to restore the subject's microbiota to a normal
microbiota. In further embodiments, the invention provides
compositions and methods for both supplementing constituents of an
altered microbiota that are under-represented in the altered
microbiota, as well as diminishing constituents of an altered
microbiota that are over-represented in the altered microbiota, as
compared with a normal microbiota, to restore the subject's
microbiota to a normal microbiota. In further embodiments, the
invention provides a method for identifying a "surgical probiotic,"
which are desired, preferred, neutral or beneficial strains of
bacteria that are phylogenetically similar to disease-associated
strains of the bacteria.
[0039] In certain embodiments, the present invention provides
methods that combine flow cytometry-based microbial cell sorting
and genetic analyses to detect, to isolate and to identify
secretory antibody-coated (e.g., IgA-coated) microbes from the
microbiota of a subject. Because disease-causing members of the
microbiota, including pathobionts, are recognized by the subject's
immune system, their presence triggers an immune response,
including antibody production and secretion. In some embodiments of
the methods described herein, the presence of an immune response
(e.g., antibody production and secretion) in the subject serves as
a marker and a means for isolating and identifying pathobionts, and
putative pathobionts, that are the targets of the subject's immune
response. Thus, the methods described herein can isolate and
identify microbes present in the microbiota of a subject that
influence susceptibility to or contribute to the development or
progression of a disease or disorder. The microbiota of the subject
can be any microbiota present on any mucosal surface of subject
where antibody is secreted, including the gastrointestinal tract,
the respiratory tract, genitourinary tract and mammary gland.
[0040] In various embodiments, the present invention relates to the
isolation and identification of members of the microbiota that
influence the development and progression of a disease or disorder,
such as an inflammatory disease or disorder. Thus, the invention
relates to compositions and methods for detecting and determining
the identity of secretory antibody-coated constituents of a
subject's microbiota to determine whether the secretory
antibody-coated constituents of a subject's microbiota contribute
to an altered microbiota associated with an inflammatory disease or
disorder. In various embodiments, the relative proportions of the
secretory antibody-coated and uncoated constituents of a subject's
microbiota are indicative of an altered microbiota associated with
an inflammatory disease or disorder. In some embodiments, the
detection and identification of secretory antibody-coated
constituents of the microbiota of the subject are used to diagnose
the subject as having, or as at risk of developing, an inflammatory
disease or disorder. Thus, in some embodiments, the altered
microbiota of a subject influences susceptibility to or contributes
to the development or progression of a disease or disorder, such as
an inflammatory disease or disorder. In various embodiments, the
inflammatory diseases and disorders associated with altered
microbiota having secretory antibody-coated constituents include,
but are not limited to, at least one of: inflammatory bowel
disease, celiac disease, colitis, irritable bowel syndrome,
intestinal hyperplasia, metabolic syndrome, obesity, diabetes,
rheumatoid arthritis, liver disease, hepatic steatosis, fatty liver
disease, non-alcoholic fatty liver disease (NAFLD), or
non-alcoholic steatohepatitis (NASH).
[0041] As used throughout herein, constituents of an altered
microbiota that are over-represented in the altered microbiota as
compared with a normal microbiota, include constituents that are
uniquely present in the altered microbiota as compared with a
normal microbiota.
Definitions
[0042] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
[0043] As used herein, each of the following terms has the meaning
associated with it in this section.
[0044] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0045] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%,
even more preferably .+-.1%, and still more preferably .+-.0.1%
from the specified value, as such variations are appropriate to
perform the disclosed methods.
[0046] The term "abnormal" when used in the context of organisms,
tissues, cells or components thereof, refers to those organisms,
tissues, cells or components thereof that differ in at least one
observable or detectable characteristic (e.g., age, treatment, time
of day, etc.) from those organisms, tissues, cells or components
thereof that display the "normal" (expected) respective
characteristic. Characteristics which are normal or expected for
one cell or tissue type, might be abnormal for a different cell or
tissue type.
[0047] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to
deteriorate.
[0048] In contrast, a "disorder" in an animal is a state of health
in which the animal is able to maintain homeostasis, but in which
the animal's state of health is less favorable than it would be in
the absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0049] A disease or disorder is "alleviated" if the severity of a
sign or symptom of the disease or disorder, the frequency with
which such a sign or symptom is experienced by a patient, or both,
is reduced.
[0050] The term "dysbiosis," as used herein, refers to imbalances
in quality, absolute quantity, or relative quantity of members of
the microbiota of a subject, which is sometimes, but not
necessarily, associated with the development or progression of a
disease or disorder.
[0051] The term "microbiota," as used herein, refers to the
population of microorganisms present within or upon a subject. The
microbiota of a subject includes commensal microorganisms found in
the absence of disease and may also include pathobionts and
disease-causing microorganisms found in subjects with or without a
disease or disorder.
[0052] The term "pathobiont," as used herein, refers to potentially
disease-causing members of the microbiota that are present in the
microbiota of a non-diseased or a diseased subject, and which has
the potential to contribute to the development or progression of a
disease or disorder.
[0053] An "effective amount" or "therapeutically effective amount"
of a compound is that amount of a compound which is sufficient to
provide a beneficial effect to the subject to which the compound is
administered.
[0054] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which can be used to communicate the usefulness of a
compound, composition, or method of the invention in a kit. The
instructional material of the kit of the invention can, for
example, be affixed to a container which contains the identified
compound, composition, or method of the invention or be shipped
together with a container which contains the identified compound,
composition, or method of the invention. Alternatively, the
instructional material can be shipped separately from the container
with the intention that the instructional material and the
compound, composition, or method of the invention be used
cooperatively by the recipient.
[0055] The term "microarray" refers broadly to both "DNA
microarrays" and "DNA chip(s)," and encompasses all art-recognized
solid supports, and all art-recognized methods for affixing nucleic
acid molecules thereto or for synthesis of nucleic acids
thereon.
[0056] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to any animal, or cells
thereof whether in vitro or in vivo, amenable to the methods
described herein. In certain non-limiting embodiments, the patient,
subject or individual is, by way of non-limiting examples, a human,
a dog, a cat, a horse, or other domestic mammal.
[0057] A "therapeutic" treatment is a treatment administered to a
subject who exhibits signs or symptoms of pathology, for the
purpose of diminishing or eliminating those signs or symptoms.
[0058] As used herein, "treating a disease or disorder" means
reducing the severity and/or frequency with which a sign or symptom
of the disease or disorder is experienced by a patient. Disease and
disorder are used interchangeably herein.
[0059] The phrase "biological sample" as used herein, is intended
to include any sample comprising a cell, a tissue, feces, or a
bodily fluid in which the presence of a microbe, nucleic acid or
polypeptide is present or can be detected. Samples that are liquid
in nature are referred to herein as "bodily fluids." Biological
samples may be obtained from a patient by a variety of techniques
including, for example, by scraping or swabbing an area of the
subject or by using a needle to obtain bodily fluids. Methods for
collecting various body samples are well known in the art.
[0060] The term "antibody," as used herein, refers to an
immunoglobulin molecule which is able to specifically bind to a
specific epitope on an antigen. Antibodies can be intact
immunoglobulins derived from natural sources or from recombinant
sources and can be immunoreactive portions of intact
immunoglobulins. The antibodies in the present invention may exist
in a variety of forms including, for example, polyclonal
antibodies, monoclonal antibodies, intracellular antibodies
("intrabodies"), Fv, Fab and F(ab)2, as well as single chain
antibodies (scFv), heavy chain antibodies, such as camelid
antibodies, and humanized antibodies (Harlow et al., 1999, Using
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual,
Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad.
Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
[0061] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage as
described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the
antibody, wherein the DNA or amino acid sequence has been obtained
using synthetic DNA or amino acid sequence technology which is
available and well known in the art.
[0062] As used herein, the term "heavy chain antibody" or "heavy
chain antibodies" comprises immunoglobulin molecules derived from
camelid species, either by immunization with a peptide and
subsequent isolation of sera, or by the cloning and expression of
nucleic acid sequences encoding such antibodies. The term "heavy
chain antibody" or "heavy chain antibodies" further encompasses
immunoglobulin molecules isolated from an animal with heavy chain
disease, or prepared by the cloning and expression of VH (variable
heavy chain immunoglobulin) genes from an animal.
[0063] The term "antigen" or "Ag" as used herein is defined as a
molecule that provokes an adaptive immune response. This immune
response may involve either antibody production, or the activation
of specific immunogenically-competent cells, or both. The skilled
artisan will understand that any macromolecule, including virtually
all proteins or peptides, can serve as an antigen. Furthermore,
antigens can be derived from recombinant or genomic DNA or RNA. A
skilled artisan will understand that any DNA or RNA, which
comprises a nucleotide sequences or a partial nucleotide sequence
encoding a protein that elicits an adaptive immune response
therefore encodes an "antigen" as that term is used herein.
Furthermore, one skilled in the art will understand that an antigen
need not be encoded solely by a full length nucleotide sequence of
a gene. It is readily apparent that the present invention includes,
but is not limited to, the use of partial nucleotide sequences of
more than one gene and that these nucleotide sequences are arranged
in various combinations to elicit the desired immune response.
Moreover, a skilled artisan will understand that an antigen need
not be encoded by a "gene" at all. It is readily apparent that an
antigen can be generated synthesized or can be derived from a
biological sample. Such a biological sample can include, but is not
limited to a microbiota sample, tissue sample, a tumor sample, a
cell or a biological fluid.
[0064] The term "adjuvant" as used herein is defined as any
molecule to enhance an antigen-specific adaptive immune
response.
[0065] As used herein, an "immunoassay" refers to any binding assay
that uses an antibody capable of binding specifically to a target
molecule to detect and quantify the target molecule.
[0066] By the term "specifically binds," as used herein with
respect to an antibody, is meant an antibody which recognizes and
binds to a specific antigen, but does not substantially recognize
or bind other molecules in a sample. For example, an antibody that
specifically binds to an antigen from one species may also bind to
that antigen from one or more species. But, such cross-species
reactivity does not itself alter the classification of an antibody
as specific. In another example, an antibody that specifically
binds to an antigen may also bind to different allelic forms of the
antigen. However, such cross reactivity does not itself alter the
classification of an antibody as specific.
[0067] In some instances, the terms "specific binding" or
"specifically binding," can be used in reference to the interaction
of an antibody, a protein, or a peptide with a second chemical
species, to mean that the interaction is dependent upon the
presence of a particular structure (e.g., an antigenic determinant
or epitope) on the chemical species; for example, an antibody
recognizes and binds to a specific protein structure rather than to
proteins generally.
[0068] A "coding region" of a gene consists of the nucleotide
residues of the coding strand of the gene and the nucleotides of
the non-coding strand of the gene which are homologous with or
complementary to, respectively, the coding region of an mRNA
molecule which is produced by transcription of the gene.
[0069] A "coding region" of a mRNA molecule also consists of the
nucleotide residues of the mRNA molecule which are matched with an
anti-codon region of a transfer RNA molecule during translation of
the mRNA molecule or which encode a stop codon. The coding region
may thus include nucleotide residues comprising codons for amino
acid residues which are not present in the mature protein encoded
by the mRNA molecule (e.g., amino acid residues in a protein export
signal sequence).
[0070] "Complementary" as used herein to refer to a nucleic acid,
refers to the broad concept of sequence complementarity between
regions of two nucleic acid strands or between two regions of the
same nucleic acid strand. It is known that an adenine residue of a
first nucleic acid region is capable of forming specific hydrogen
bonds ("base pairing") with a residue of a second nucleic acid
region which is antiparallel to the first region if the residue is
thymine or uracil. Similarly, it is known that a cytosine residue
of a first nucleic acid strand is capable of base pairing with a
residue of a second nucleic acid strand which is antiparallel to
the first strand if the residue is guanine. A first region of a
nucleic acid is complementary to a second region of the same or a
different nucleic acid if, when the two regions are arranged in an
antiparallel fashion, at least one nucleotide residue of the first
region is capable of base pairing with a residue of the second
region. Preferably, the first region comprises a first portion and
the second region comprises a second portion, whereby, when the
first and second portions are arranged in an antiparallel fashion,
at least about 50%, and preferably at least about 75%, at least
about 90%, or at least about 95% of the nucleotide residues of the
first portion are capable of base pairing with nucleotide residues
in the second portion. More preferably, all nucleotide residues of
the first portion are capable of base pairing with nucleotide
residues in the second portion.
[0071] "Isolated" means altered or removed from the natural state.
For example, a nucleic acid or a peptide naturally present in its
normal context in a living animal is not "isolated," but the same
nucleic acid or peptide partially or completely separated from the
coexisting materials of its natural context is "isolated." An
isolated nucleic acid or protein can exist in substantially
purified form, or can exist in a non-native environment such as,
for example, a host cell.
[0072] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0073] The term "polynucleotide" as used herein is defined as a
chain of nucleotides. Furthermore, nucleic acids are polymers of
nucleotides. Thus, nucleic acids and polynucleotides as used herein
are interchangeable. One skilled in the art has the general
knowledge that nucleic acids are polynucleotides, which can be
hydrolyzed into the monomeric "nucleotides." The monomeric
nucleotides can be hydrolyzed into nucleosides. As used herein
polynucleotides include, but are not limited to, all nucleic acid
sequences which are obtained by any means available in the art,
including, without limitation, recombinant means, i.e., the cloning
of nucleic acid sequences from a recombinant library or a cell
genome, using ordinary cloning technology and PCR, and the like,
and by synthetic means.
[0074] As used herein, the terms "peptide," "polypeptide," and
"protein" are used interchangeably, and refer to a compound
comprised of amino acid residues covalently linked by peptide
bonds. A protein or peptide must contain at least two amino acids,
and no limitation is placed on the maximum number of amino acids
that can comprise a protein's or peptide's sequence. Polypeptides
include any peptide or protein comprising two or more amino acids
joined to each other by peptide bonds. As used herein, the term
refers to both short chains, which also commonly are referred to in
the art as peptides, oligopeptides and oligomers, for example, and
to longer chains, which generally are referred to in the art as
proteins, of which there are many types. "Polypeptides" include,
for example, biologically active fragments, substantially
homologous polypeptides, oligopeptides, homodimers, heterodimers,
variants of polypeptides, modified polypeptides, derivatives,
analogs, fusion proteins, among others. The polypeptides include
natural peptides, recombinant peptides, synthetic peptides, or a
combination thereof.
[0075] The term "probiotic" refers to one or more bacteria that can
be administered to a subject to aid in the restoration of a
subject's microbiota by increasing the number of bacteria that are
desired, preferred, neutral, beneficial and/or under-represented in
the subject's microbiota. Similarly, a "surgical probiotic` is a
strain of bacteria that is desired, preferred, neutral, beneficial
and/or under-represented in the subject's microbiota and that is
phylogenetically similar to a disease-associated strain of the
bacteria.
[0076] "Variant" as the term is used herein, is a nucleic acid
sequence or a peptide sequence that differs in sequence from a
reference nucleic acid sequence or peptide sequence respectively,
but retains essential biological properties of the reference
molecule. Changes in the sequence of a nucleic acid variant may not
alter the amino acid sequence of a peptide encoded by the reference
nucleic acid, or may result in amino acid substitutions, additions,
deletions, fusions and truncations. Changes in the sequence of
peptide variants are typically limited or conservative, so that the
sequences of the reference peptide and the variant are closely
similar overall and, in many regions, identical. A variant and
reference peptide can differ in amino acid sequence by one or more
substitutions, additions, deletions in any combination. A variant
of a nucleic acid or peptide can be a naturally occurring such as
an allelic variant, or can be a variant that is not known to occur
naturally. Non-naturally occurring variants of nucleic acids and
peptides may be made by mutagenesis techniques or by direct
synthesis.
[0077] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
Description
[0078] The present invention relates to the discovery that
secretory antibodies, such as IgA1, IgA2 or IgM, can be used to
detect and identify microbes present in the microbiota of a subject
that influence susceptibility to or contribute to the development
or progression of a diseases or disorder, such as an inflammatory
disease or disorder. Thus, in certain aspects the present invention
relates to methods of modifying an altered microbiota population in
a subject in need thereof. For example, in certain embodiments, the
present invention relates to compositions and methods of reducing
the level of one or more bacteria associated with the development
or progression of a disease or disorder. In one embodiment, the
invention provides a vaccine that reduces the amount of one or more
bacteria associated with the development or progression of a
disease or disorder. For example, in certain embodiments, the
vaccine comprises an inactivated bacteria associated with the
development or progression of a disease or disorder, thereby
inducing an immune response against the bacteria.
[0079] In one embodiment, the invention relates to compositions and
methods for detecting, identifying and determining the absolute
number or relative proportions of the secretory antibody-coated and
uncoated constituents of a subject's microbiota, to determine
whether a subject's microbiota is an altered microbiota associated
with a disease or disorder, such as an inflammatory disease or
disorder. The microbiota of the subject can be any microbiota
present on any mucosal surface of subject where antibody is
secreted, including the gastrointestinal tract, the respiratory
tract, genitourinary tract and mammary gland.
Methods of Treatment
[0080] In some embodiments, the invention relates to methods of
modifying an altered microbiota having secretory antibody-coated
constituents in a subject in need thereof.
[0081] In other embodiments, the invention provides compositions
and methods for diminishing constituents of an altered microbiota
that are over-represented in the altered microbiota as compared
with a normal microbiota, such as over-represented secretory
antibody-coated constituents, to restore the subject's microbiota
to a normal microbiota. As used throughout herein, constituents of
an altered microbiota that are over-represented in the altered
microbiota as compared with a normal microbiota, include
constituents that are uniquely present in the altered microbiota as
compared with a normal microbiota.
[0082] In some embodiments, the invention provides compositions and
methods for supplementing constituents of an altered microbiota
that are desired, preferred, neutral, beneficial and/or
under-represented in the altered microbiota, as compared with a
normal microbiota, to restore the subject's microbiota to a normal
microbiota.
[0083] In further embodiments, the invention provides compositions
and methods for both supplementing constituents of an altered
microbiota that are under-represented in the altered microbiota, as
well as diminishing constituents of an altered microbiota that are
over-represented in the altered microbiota, as compared with a
normal microbiota, to restore the subject's microbiota to a normal
microbiota. The microbiota of the subject can be any microbiota
present on any mucosal surface of subject where antibody is
secreted, including the gastrointestinal tract, the respiratory
tract, genitourinary tract and mammary gland.
[0084] In conjunction with the diagnostic methods, the present
invention also provides therapeutic methods for treating an
inflammatory disease or disorder associated with an altered
microbiota including secretory antibody-coated microbes, by
modifying the microbiota to that observed in a healthy subject. In
some embodiments, the methods supplement the numbers of the types
of microbes that are under-represented in the altered microbiota.
In other embodiments, the methods diminish the numbers or
pathogenic effects of the types of microbes, including secretory
antibody-coated microbes that are overrepresented in the altered
microbiota. In a further embodiment, the methods both supplement
the numbers of the types of bacteria that are under-represented in
the altered microbiota, and diminish the numbers of the types of
bacteria that are overrepresented in the altered microbiota. In
various embodiments, the inflammatory diseases and disorders
treatable by the methods of the invention include, but are not
limited to: inflammatory bowel disease, celiac disease, colitis,
irritable bowel syndrome, intestinal hyperplasia, metabolic
syndrome, obesity, diabetes, rheumatoid arthritis, liver disease,
hepatic steatosis, fatty liver disease, non-alcoholic fatty liver
disease (NAFLD), or non-alcoholic steatohepatitis (NASH).
[0085] Vaccine
[0086] In other embodiments, modification of the altered microbiota
having over-represented secretory antibody-coated constituents is
achieved by administering to a subject in need thereof a
therapeutically effective amount of a vaccine to induce an immune
response against the over-represented constituent, wherein the
administered vaccine and ensuing immune response diminishes the
number or pathogenic effects of at least one type (e.g., genus,
species, strain, sub-strain, etc.) of secretory antibody-coated
bacteria that is over-represented in the altered microbiota, as
compared with a normal microbiota. In various embodiments, the at
least one type (e.g., genus, species, strain, sub-strain, etc.) of
bacteria that is diminished using the methods of the invention
includes a Segmented Filamentous Bacteria (SFB) or Helicobacter
flexispira, or a bacteria from at least one family selected from
the group consisting of Lactobacillus, Helicobacter, S24-7,
Erysipelotrichaceae and Prevotellaceae. In some embodiments, the
bacteria from the family Prevotellaceae is a bacteria from the
genera of Paraprevotella or Prevotella. In other embodiments, the
secretory antibody-coated constituent of the subject's microbiota
associated with the development or progression of an inflammatory
disease or disorder in the subject is at least one strain of at
least one bacteria selected from Acidaminococcus spp., Actinomyces
spp., Akkermansia muciniphila, Allobaculum spp., Anaerococcus spp.,
Anaerostipes spp., Bacteroides spp., Bacteroides Other, Bacteroides
acidifaciens, Bacteroides coprophilus, Bacteroides fragilis,
Bacteroides ovatus, Bacteroides uniformis, Barnesiellaceae spp.,
Bifidobacterium adolescentis, Bifidobacterium Other,
Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia
producta, Blautia Other, Blautia spp., Bulleidia spp.,
Catenibacterium spp., Citrobacter spp., Clostridiaceae spp.,
Clostridiales Other, Clostridiales spp., Clostridium perfringens,
Clostridium spp., Clostridium Other, Collinsella aerofaciens,
Collinsella spp., Collinsella stercoris, Coprococcus catus,
Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp.,
Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other,
Eggerthella lenta, Enterobacteriaceae Other, Enterobacteriaceae
spp., Enterococcus spp., Erysipelotrichaceae spp., Eubacterium
biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium
spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae
spp., Haemophilus parainfluenzae, Haemophilus Other, Helicobacter
spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp.,
Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae,
Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp.,
Leuconostocaceae spp., Megamonas spp., Megasphaera spp.,
Methanobrevibacter spp., Mitsuokella multacida, Mitsuokella spp.,
Mucispirillum schaedleri, Odoribacter spp., Oscillospira spp.,
Parabacteroides distasonis, Parabacteroides spp., Paraprevotella
spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp.,
Pediococcus Other, Peptococcus spp., Peptoniphilus spp.,
Peptostreptococcus anaerobius, Peptostreptococcus Other,
Phascolarctobacterium spp., Prevotella copri, Prevotella spp.,
Prevotella stercorea, Prevotellaceae, Proteus spp., Rikenellaceae
spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other,
Ruminococcaceae spp., Ruminococcus bromii, Ruminococcus gnavus,
Ruminococcus spp., Ruminococcus Other, Ruminococcus torques,
Slackia spp., S24-7 spp., SMB53 spp., Streptococcus anginosus,
Streptococcus luteciae, Streptococcus spp., Streptococcus Other,
Sutterella spp., Turicibacter spp., UC Bulleidia, UC
Enterobacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC
Pediococcus, Varibaculum spp., Veillonella spp., Sutterella,
Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC
Ruminococcaceae, Veillonella parvula, Veillonella spp., Veillonella
dispar, and Weissella.
[0087] In the context of the present invention, the term "vaccine"
(also referred to as an immunogenic composition) refers to a
substance that induces immunity upon inoculation into animals. In
some instances, the vaccine of the invention can be used to
inducing immunity to one or more bacteria types of the
over-represented constituent.
[0088] In one embodiment, the vaccine comprises at least one
bacterium. For example, in certain embodiments, the vaccine
comprises an inactivated or killed bacterium. Inactivated or killed
indicates the bacterium has lost the ability to cause disease in
mammals but retains an immunogenic property thereof, particularly
the ability to generate a specific immune response against one or
more antigens of the bacterium. The term inactivated bacterium also
includes non-virulent bacterium. Methods for preparing or selecting
inactivated bacteria are well known in the art. They include
heat-inactivation methods, or chemical inactivation methods.
Inactivation may be carried out by exposing the bacterium to a
chemical agent such as formalin, formaldehyde, paraformaldehyde,
.beta.-propiolactone, ethyleneimine, binary ethyleneimine (BEI),
thimerosal, or derivatives thereof. Alternatively, inactivation may
be carried out by physical treatments such as heat treatment or
sonication. Methods of inactivation are well known to those of
skill in the art. The inactivated pathogen may be concentrated by
conventional concentration techniques, in particular by
ultrafiltration, and/or purified by conventional purification
means, in particular using chromatography techniques including but
not limited to gel-filtration, ultracentrifugation on a sucrose
gradient, or selective precipitations.
[0089] In one embodiment, the vaccine comprises an antigen (e.g., a
peptide or polypeptide), a nucleic acid encoding an antigen (e.g.,
an antigen expression vector), a cell expressing or presenting an
antigen or cellular component. For example, in certain embodiments,
the antigen is an antigen of one or more bacteria associated with
the development or progression of a disease or disorder, thereby
inducing an immune response against the one or more bacteria.
[0090] In one embodiment, the vaccine comprises a mixture that
comprises an additional immunostimulatory agent or nucleic acids
encoding such an agent. Immunostimulatory agents include but are
not limited to an antigen, an immunomodulator, an antigen
presenting cell or an adjuvant. An adjuvant refers to a compound
that enhances the immune response when administered together (or
successively) with the immunological composition. Examples of
suitable adjuvants include cholera toxin, E. coli heat-labile
toxin, E. coli enterotoxin, salmonella toxin, alum,
nanoparticle-based adjuvants, .alpha.-interferon (IFN-.alpha.),
.beta.-interferon (IFN-.beta.), .gamma.-interferon, platelet
derived growth factor (PDGF), TNF.alpha., TNF.beta., GM-CSF,
epidermal growth factor (EGF), cutaneous T cell-attracting
chemokine (CTACK), epithelial thymus-expressed chemokine (TECK),
mucosae-associated epithelial chemokine (MEC), IL-1, IL-2, IL-4,
IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, and CD86.
[0091] Examples of suitable adjuvants and/or immunomodulators
include, but are not limited to, complete or incomplete Freund's
adjuvant, RIBI (e.g., muramyl dipeptides, etc.), KLH peptide,
cholera toxin or a portion thereof, salmonella toxin or a portion
thereof, E. coli heat labile enterotoxin or a portion thereof, E.
coli enterotoxin or a portion thereof, AB5 toxins or a portion
thereof, mineral salts, aluminum salts (e.g., hydroxide, phosphate,
Alum, etc.), calcium phosphate, liposomes, virosomes (unilamellar
liposomal vehicles, immunostimulating reconstituted influenza
virosomes [IRIV]), virus-like particles, cochleates, eurocine
(e.g., monoglycerides with fatty acids, etc.), archaeal lipids,
ISCOMS (e.g., immunostimulating complexes, structured complex of
saponins and lipids, etc.), microparticles (e.g., PLG, etc.),
emulsions (e.g., MF59, Montanides, etc.), monophosphoryl lipid
(MPL) or synthetic derivatives,
N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) or a derivative,
Detox (MPL+CWS), AS04 (Alum+MPL), AS02 (oil-in-water
emulsion+MPL+QS21), AS01 (liposomes+MPL+QS21), OM-174 (e.g., Lipid
A derivative, E. coli, etc.), OM-triacyl, oligonucleotides (e.g.,
CpG, etc.), double-stranded RNA (dsRNA), pathogen-associated
molecular patterns (PAMPs), TLR ligands (e.g., flagellin,
monophosphoryl lipid A, etc.), saponins (e.g., Quils, QS-21, etc.),
chitosan, .alpha.-galactosylceramide, small-molecule immune
potentiators (SMIPs) (e.g., imiquimod, resiquimod [R848], etc.), a
cytokine or chemokine (e.g., IL-2, IL-12, GM-CSF, Flt3, etc.), an
accessory molecule (e.g., B7.1, etc.), liposomes (e.g., DNPC/Chol,
etc.), DC Chol (e.g., lipoidal immunomodulators able to
self-organize into liposomes, etc.), nanoparticle-based adjuvants,
PLA (polylactic acid) microparticles, PLG
(poly[lactide-co-glycolide]) microparticles,
Poly(DL-lactide-co-glycolide) microparticles, polystyrene (latex)
microparticles, proteosomes (e.g., hydrophobic, proteinaceous,
nanoparticles comprised of purified N. meningitidis outer membrane
proteins, etc.), and 3',5'-Cyclic diguanylic acid (c-di-GMP). Such
example adjuvants and/or immunomodulators, as well as others, are
understood by those skilled in the art, are readily described in
available literature, and are useful in the compositions and
methods of the invention.
[0092] Furthermore, a vaccine of this invention may be combined
appropriately with a pharmaceutically acceptable carrier. Examples
of such carriers are sterilized water, physiological saline,
phosphate buffer, culture fluid and such. Furthermore, the vaccine
may contain as necessary, stabilizers, suspensions, preservatives,
surfactants and such. The vaccine is administered systemically or
locally. Vaccine administration may be performed by single
administration or boosted by multiple administrations. In one
embodiment, the pharmaceutical carrier is an adjuvant.
[0093] The pharmaceutical composition can further comprise other
agents for formulation purposes according to the mode of
administration to be used. In cases where pharmaceutical
compositions are injectable pharmaceutical compositions, they are
sterile, pyrogen free and particulate free. An isotonic formulation
is preferably used. Generally, additives for isotonicity can
include sodium chloride, dextrose, mannitol, sorbitol and lactose.
In some cases, isotonic solutions such as phosphate buffered saline
are preferred. Stabilizers include gelatin and albumin. In some
embodiments, a vasoconstriction agent is added to the
formulation.
[0094] The vaccine can further comprise a pharmaceutically
acceptable excipient. The pharmaceutically acceptable excipient can
be functional molecules as vehicles, adjuvants, carriers, or
diluents.
[0095] The vaccine or pharmaceutical composition can be
administered by different routes including orally, parenterally,
sublingually, transdermally, rectally, transmucosally, topically,
via inhalation, via buccal administration, intrapleurally,
intravenous, intraarterial, intraperitoneal, subcutaneous,
intramuscular, intranasal intrathecal, and intraarticular or
combinations thereof.
[0096] Passive Immunotherapy
[0097] In other embodiments, modification of the altered microbiota
having over-represented secretory antibody-coated constituents is
achieved by administering to a subject in need thereof a
therapeutically effective amount of a passive immunotherapy or
passive vaccine, such as by the administration of immunoglobulin
(e.g., IgA) against the over-represented constituent, wherein the
administered passive vaccine and ensuing immune response diminishes
the number or pathogenic effects of at least one type (e.g., genus,
species, strain, sub-strain, etc.) of secretory antibody-coated
bacteria that is over-represented in the altered microbiota, as
compared with a normal microbiota. In some embodiments, the
immunoglobulin is administered orally. Alternatively, the
immunoglobulin can be administered rectally or by enema. In various
embodiments, the at least one type (e.g., genus, species, strain,
sub-strain, etc.) of bacteria that is diminished using the methods
of the invention includes a Segmented Filamentous Bacteria (SFB) or
Helicobacter flexispira, or a bacteria from at least one family
selected from the group consisting of Lactobacillus, Helicobacter,
S24-7, Erysipelotrichaceae and Prevotellaceae. In some embodiments,
the bacteria from the family Prevotellaceae is a bacteria from the
genera of Paraprevotella or Prevotella. In other embodiments, the
secretory antibody-coated constituent of the subject's microbiota
associated with the development or progression of an inflammatory
disease or disorder in the subject is at least one strain of at
least one bacteria selected from Acidaminococcus spp., Actinomyces
spp., Akkermansia muciniphila, Allobaculum spp., Anaerococcus spp.,
Anaerostipes spp., Bacteroides spp., Bacteroides Other, Bacteroides
acidifaciens, Bacteroides coprophilus, Bacteroides fragilis,
Bacteroides ovatus, Bacteroides uniformis, Barnesiellaceae spp.,
Bifidobacterium adolescentis, Bifidobacterium Other,
Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia
producta, Blautia Other, Blautia spp., Bulleidia spp.,
Catenibacterium spp., Citrobacter spp., Clostridiaceae spp.,
Clostridiales Other, Clostridiales spp., Clostridium perfringens,
Clostridium spp., Clostridium Other, Collinsella aerofaciens,
Collinsella spp., Collinsella stercoris, Coprococcus catus,
Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp.,
Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other,
Eggerthella lenta, Enterobacteriaceae Other, Enterobacteriaceae
spp., Enterococcus spp., Erysipelotrichaceae spp., Eubacterium
biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium
spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae
spp., Haemophilus parainfluenzae, Haemophilus Other, Helicobacter
spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp.,
Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae,
Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp.,
Leuconostocaceae spp., Megamonas spp., Megasphaera spp.,
Methanobrevibacter spp., Mitsuokella multacida, Mitsuokella spp.,
Mucispirillum schaedleri, Odoribacter spp., Oscillospira spp.,
Parabacteroides distasonis, Parabacteroides spp., Paraprevotella
spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp.,
Pediococcus Other, Peptococcus spp., Peptoniphilus spp.,
Peptostreptococcus anaerobius, Peptostreptococcus Other,
Phascolarctobacterium spp., Prevotella copri, Prevotella spp.,
Prevotella stercorea, Prevotellaceae, Proteus spp., Rikenellaceae
spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other,
Ruminococcaceae spp., Ruminococcus bromii, Ruminococcus gnavus,
Ruminococcus spp., Ruminococcus Other, Ruminococcus torques,
Slackia spp., S24-7 spp., SMB53 spp., Streptococcus anginosus,
Streptococcus luteciae, Streptococcus spp., Streptococcus Other,
Sutterella spp., Turicibacter spp., UC Bulleidia, UC
Enterobacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC
Pediococcus, Varibaculum spp., Veillonella spp., Sutterella,
Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC
Ruminococcaceae, Veillonella parvula, Veillonella spp., Veillonella
dispar, and Weissella.
[0098] Antibiotics
[0099] In other embodiments, modification of the altered microbiota
having over-represented secretory antibody-coated constituents is
achieved by administering to a subject in need thereof a
therapeutically effective amount of antibiotic composition
comprising an effective amount of at least one antibiotic, or a
combinations of several types of antibiotics, wherein the
administered antibiotic diminishes the number or pathogenic effects
of at least one type (e.g., genus, species, strain, sub-strain,
etc.) of secretory antibody-coated bacteria that is
over-represented in the altered microbiota, as compared with a
normal microbiota. In various embodiments, the at least one type
(e.g., genus, species, strain, sub-strain, etc.) of bacteria that
is diminished using the methods of the invention includes a
Segmented Filamentous Bacteria (SFB) or Helicobacter flexispira, or
a bacteria from at least one family selected from the group
consisting of Lactobacillus, Helicobacter, S24-7,
Erysipelotrichaceae and Prevotellaceae. In some embodiments, the
bacteria from the family Prevotellaceae is a bacteria from the
genera of Paraprevotella or Prevotella. In other embodiments, the
secretory antibody-coated constituent of the subject's microbiota
associated with the development or progression of an inflammatory
disease or disorder in the subject is at least one strain of at
least one bacteria selected from Acidaminococcus spp., Actinomyces
spp., Akkermansia muciniphila, Allobaculum spp., Anaerococcus spp.,
Anaerostipes spp., Bacteroides spp., Bacteroides Other, Bacteroides
acidifaciens, Bacteroides coprophilus, Bacteroides fragilis,
Bacteroides ovatus, Bacteroides uniformis, Barnesiellaceae spp.,
Bifidobacterium adolescentis, Bifidobacterium Other,
Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia
producta, Blautia Other, Blautia spp., Bulleidia spp.,
Catenibacterium spp., Citrobacter spp., Clostridiaceae spp.,
Clostridiales Other, Clostridiales spp., Clostridium perfringens,
Clostridium spp., Clostridium Other, Collinsella aerofaciens,
Collinsella spp., Collinsella stercoris, Coprococcus catus,
Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp.,
Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other,
Eggerthella lenta, Enterobacteriaceae Other, Enterobacteriaceae
spp., Enterococcus spp., Erysipelotrichaceae spp., Eubacterium
biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium
spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae
spp., Haemophilus parainfluenzae, Haemophilus Other, Helicobacter
spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp.,
Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae,
Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp.,
Leuconostocaceae spp., Megamonas spp., Megasphaera spp.,
Methanobrevibacter spp., Mitsuokella multacida, Mitsuokella spp.,
Mucispirillum schaedleri, Odoribacter spp., Oscillospira spp.,
Parabacteroides distasonis, Parabacteroides spp., Paraprevotella
spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp.,
Pediococcus Other, Peptococcus spp., Peptoniphilus spp.,
Peptostreptococcus anaerobius, Peptostreptococcus Other,
Phascolarctobacterium spp., Prevotella copri, Prevotella spp.,
Prevotella stercorea, Prevotellaceae, Proteus spp., Rikenellaceae
spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other,
Ruminococcaceae spp., Ruminococcus bromii, Ruminococcus gnavus,
Ruminococcus spp., Ruminococcus Other, Ruminococcus torques,
Slackia spp., S24-7 spp., SMB53 spp., Streptococcus anginosus,
Streptococcus luteciae, Streptococcus spp., Streptococcus Other,
Sutterella spp., Turicibacter spp., UC Bulleidia, UC
Enterobacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC
Pediococcus, Varibaculum spp., Veillonella spp., Sutterella,
Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC
Ruminococcaceae, Veillonella parvula, Veillonella spp., Veillonella
dispar, and Weissella.
[0100] The type and dosage of the administered antibiotic will vary
widely, depending upon the nature of the inflammatory disease or
disorder, the character of subject's altered microbiota, the
subject's medical history, the frequency of administration, the
manner of administration, and the like. The initial dose may be
larger, followed by smaller maintenance doses. The dose may be
administered as infrequently as weekly or biweekly, or fractionated
into smaller doses and administered daily, semi-weekly, etc., to
maintain an effective dosage level. In various embodiments, the
administered antibiotic is at least one of lipopeptide,
fluoroquinolone, ketolide, cephalosporin, amikacin, gentamicin,
kanamycin, neomycin, netilmicin, paromomycin, streptomycin,
tobramycin, cefacetrile, cefadroxil, cefalexin, cefaloglycin,
cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine,
cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine,
ceftezole, cefaclor, cefamandole, cefmetazole, cefonicid,
cefotetan, cefoxitin, cefprozil, cefuroxime, cefuzonam, cefcapene,
cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime,
cefmenoxime, cefodizime, cefotaxime, cefpimizole, cefpodoxime,
cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime,
ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime
cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome,
cefaclomezine, cefaloram, cefaparole, cefcanel, cefedrolor,
cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium,
cefovecin, cefoxazole, cefrotil, cefsumide, ceftaroline,
ceftioxide, cefuracetime, imipenem, primaxin, doripenem, meropenem,
ertapenem, flumequine, nalidixic acid, oxolinic acid, piromidic
acid pipemidic acid, rosoxacin, ciprofloxacin, enoxacin,
lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin,
rufloxacin, balofloxacin, gatifloxacin, grepafloxacin,
levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin,
temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin,
sitafloxacin, trovafloxacin, prulifloxacin, azithromycin,
erythromycin, clarithromycin, dirithromycin, roxithromycin,
telithromycin, amoxicillin, ampicillin, bacampicillin,
carbenicillin, cloxacillin, dicloxacillin, flucloxacillin,
mezlocillin, nafcillin, oxacillin, penicillin g, penicillin v,
piperacillin, pivampicillin, pivmecillinam, ticarcillin,
sulfamethizole, sulfamethoxazole, sulfisoxazole,
trimethoprim-sulfamethoxazole, demeclocycline, doxycycline,
minocycline, oxytetracycline, tetracycline, linezolid, clindamycin,
metronidazole, vancomycin, vancocin, mycobutin, rifampin,
nitrofurantoin, chloramphenicol, or derivatives thereof.
[0101] Probiotics
[0102] In some embodiments, modification of the altered microbiota
is achieved by administering to a subject in need thereof a
therapeutically effective amount of a probiotic composition
comprising an effective amount of at least one type (e.g., genus,
species, strain, sub-strain, etc.) of bacteria, or a combinations
of several types of bacteria, wherein the administered bacteria
supplements the number of the types of bacteria which are
under-represented in the altered microbiota, as compared with a
normal microbiota. In some embodiments, the probiotic is a surgical
probiotic.
[0103] In one embodiment, the invention is a method of treating an
inflammatory disease or disorder of a subject in need thereof,
including the step of administering to the subject at least one
type (e.g., genus, species, strain, sub-strain, etc.) of bacteria,
or a combinations of several types of bacteria, that is desired,
preferred, neutral, beneficial, and/or under-represented in the
subject's microbiota.
[0104] In some embodiments, the at least one type of bacteria is at
least one bacterium of a first strain of a species of bacteria,
wherein the first strain of the species of bacteria does not
contribute to the development or progression of disease in the
subject, and wherein the species of bacteria comprises at least a
second strain of bacteria, and wherein the second strain of the
species of bacteria does contribute to the development or
progression of the inflammatory disease or disorder.
[0105] In some embodiments, the at least one type of bacteria is at
least one bacterium of a species of bacteria identified from a
healthy subject that does not have the disease. For example, in one
embodiment, the species or strain of bacteria is a secretory
antibody-bound bacteria identified from a healthy subject. As
described herein, administration of secretory antibody-bound
bacteria from a healthy subject can treat or prevent an
inflammatory disease or disorder.
[0106] Bacteria administered according to the methods of the
present invention can comprise live bacteria. One or several
different types of bacteria can be administered concurrently or
sequentially. Such bacteria can be obtained from any source,
including being isolated from a microbiota and grown in culture
using known techniques.
[0107] In certain embodiments, the administered bacteria used in
the methods of the invention further comprise a buffering agent.
Examples of useful buffering agents include sodium bicarbonate,
milk, yogurt, infant formula, and other dairy products.
[0108] Administration of a bacterium can be accomplished by any
method suitable for introducing the organisms into the desired
location. The bacteria can be mixed with a carrier and (for easier
delivery to the digestive tract) applied to a liquid or to food.
The carrier material should be non-toxic to the bacteria as wells
as the subject. Preferably, the carrier contains an ingredient that
promotes viability of the bacteria during storage. The formulation
can include added ingredients to improve palatability, improve
shelf-life, impart nutritional benefits, and the like.
[0109] The dosage of the administered bacteria (e.g., probiotic,
surgical probiotic) will vary widely, depending upon the nature of
the inflammatory disease or disorder, the character of subject's
altered microbiota, the subject's medical history, the frequency of
administration, the manner of administration, the clearance of the
agent from the host, and the like. The initial dose may be larger,
followed by smaller maintenance doses. The dose may be administered
as infrequently as weekly or biweekly, or fractionated into smaller
doses and administered daily, semi-weekly, etc., to maintain an
effective dosage level. It is contemplated that a variety of doses
will be effective to achieve colonization of the gastrointestinal
tract with the desired bacteria. In some embodiments, the dose
ranges from about 10.sup.6 to about 10.sup.10 CFU per
administration. In other embodiments, the dose ranges from about
10.sup.4 to about 10.sup.6 CFU per administration.
[0110] In certain embodiments, the present invention relates to a
method for modifying an altered microbiota comprising administering
to a subject in need of such treatment, an effective amount of at
least one gastric, esophageal, or intestinal bacterium, or
combinations thereof. In a preferred embodiment, the bacteria are
administered orally. Alternatively, bacteria can be administered
rectally or by enema.
[0111] The organisms contemplated for administration to modify the
altered microbiota include any of the bacteria identified herein as
under-represented in an altered microbiota. One of the organisms
contemplated for administration to modify the altered microbiota is
at least one Lactobacillus spp. In certain embodiments, the
bacteria administered in the therapeutic methods of the invention
comprise administration of a combination of organisms.
[0112] While it is possible to administer a bacteria for therapy as
is, it may be preferable to administer it in a pharmaceutical
formulation, e.g., in admixture with a suitable pharmaceutical
excipient, diluent or carrier selected with regard to the intended
route of administration and standard pharmaceutical practice. The
excipient, diluent and/or carrier must be "acceptable" in the sense
of being compatible with the other ingredients of the formulation
and not deleterious to the recipient thereof. Acceptable
excipients, diluents, and carriers for therapeutic use are well
known in the pharmaceutical art, and are described, for example, in
Remington: The Science and Practice of Pharmacy. Lippincott
Williams & Wilkins (A. R. Gennaro edit. 2005). The choice of
pharmaceutical excipient, diluent, and carrier can be selected with
regard to the intended route of administration and standard
pharmaceutical practice.
[0113] Although there are no physical limitations to delivery of
the formulations of the present invention, oral delivery is
preferred for delivery to the digestive tract because of its ease
and convenience, and because oral formulations readily accommodate
additional mixtures, such as milk, yogurt, and infant formula. For
delivery to colon, bacteria can be also administered rectally or by
enema.
[0114] In a further embodiment, modification of the altered
microbiota is achieved by both administering at least one type
(e.g., genus, species, strain, sub-strain, etc.) of bacteria to
supplement the numbers of at least one type (e.g., genus, species,
strain, sub-strain, etc.) of bacteria that is under-represented in
the altered microbiota, and administering at least one antibiotic
to diminish the numbers of at least one type (e.g., genus, species,
strain, sub-strain, etc.) of bacteria that is over-represented in
the altered microbiota.
[0115] Methods of Identifying
[0116] The methods of the invention are useful for detecting,
identifying and determining the absolute number or relative
proportions of secretory antibody-coated and uncoated constituents
of a subject's microbiota, to determine whether a subject's
microbiota is an altered microbiota associated with a disease or
disorder, such as an inflammatory disease or disorder. In some
embodiments, the methods of the invention combine a flow
cytometry-based microbial cell sorting and genetic analyses to
detect, to isolate and to identify secretory antibody-coated
microbes from the microbiota of a subject. Pathobionts, as well as
other disease-causing microbes, present in the microbiota of the of
the subject are recognized by the subject's immune system, which
triggers an immune response, including antibody production and
secretion, directed against the pathobionts, and disease-causing
microbes. Thus, in some embodiments of the methods of the
invention, specifically binding secretory antibodies (e.g., IgA,
IgM) produced by the subject and secreted through the mucosa of the
subject, serve as a marker and a means for isolating and
identifying putative pathobionts, pathobionts, and other
disease-causing bacteria, that are the targets of the subject's
immune response. In various embodiments of the methods of the
invention, the secretory antibody is IgA (i.e., IgA1, IgA2), or
IgM, or any combination thereof. The microbiota of the subject can
be any microbiota present on any mucosal surface of subject where
antibody is secreted, including the gastrointestinal tract, the
respiratory tract, genitourinary tract and mammary gland.
[0117] In various embodiments, the present invention relates to the
isolation and identification of constituents of the microbiota of a
subject that influence the development and progression of a disease
or disorder, such as an inflammatory disease and disorder. In one
embodiment, the invention relates to compositions and methods for
detecting and determining the identity of secretory antibody-coated
constituents of a subject's microbiota to determine whether the
secretory antibody-coated constituents of a subject's microbiota
form an altered microbiota associated with an inflammatory disease
or disorder. In various embodiments, the relative proportions of
the secretory antibody-coated and uncoated constituents of a
subject's microbiota are indicative of an altered microbiota
associated with an inflammatory disease or disorder. In some
embodiments, the detection and identification of secretory
antibody-coated constituents of the microbiota of the subject are
used to diagnose the subject as having, or as at risk of
developing, an inflammatory disease or disorder. In other
embodiments, the detection and identification of secretory
antibody-coated constituents of the microbiota of the subject are
used to diagnose the subject as having, or as at risk of
developing, a recurrence or flare of an inflammatory disease or
disorder. In other embodiments, the detection and identification of
secretory antibody-coated constituents of the microbiota of the
subject are used to diagnose the subject as having, or as likely to
have, remission or an inflammatory disease or disorder. In various
embodiments, the inflammatory diseases and disorders associated
with altered microbiota having secretory antibody-coated
constituents include, but are not limited to, at least one of:
inflammatory bowel disease, celiac disease, colitis, irritable
bowel syndrome, intestinal hyperplasia, metabolic syndrome,
obesity, diabetes, rheumatoid arthritis, liver disease, hepatic
steatosis, fatty liver disease, non-alcoholic fatty liver disease
(NAFLD), or non-alcoholic steatohepatitis (NASH).
[0118] In other various embodiments, the present invention relates
to the isolation and identification of constituents of the
microbiota of a subject that are not associated with the
development and progression of a disease or disorder, such as an
inflammatory disease and disorder. In one embodiment, the invention
relates to compositions and methods for detecting and determining
the identity of constituents of the subject's microbiota that are
not substantially bound by secretory antibodies. In various
embodiments, the relative proportions of the low-, or non-secretory
antibody-coated constituents of a subject's microbiota are
indicative of an altered microbiota associated with an inflammatory
disease or disorder.
[0119] In one embodiment, the invention is a method for determining
the relative proportions of the types of secretory antibody-coated
constituents of a subject's microbiota, to identify constituents of
a subject's microbiota that are, and are not, associated with the
development or progression of an inflammatory disease or disorder.
In some embodiments, the detection of particular types of secretory
antibody-coated constituents of the subject's microbiota is used to
diagnose the subject as having, or as at risk of developing, an
inflammatory disease or disorder. In various embodiments, the
inflammatory disease or disorder associated with secretory
antibody-coated constituents of the subject's microbiota include,
but are not limited to, at least one of: inflammatory bowel
disease, celiac disease, colitis, irritable bowel syndrome,
intestinal hyperplasia, metabolic syndrome, obesity, diabetes,
rheumatoid arthritis, liver disease, hepatic steatosis, fatty liver
disease, non-alcoholic fatty liver disease (NAFLD), and
non-alcoholic steatohepatitis (NASH). In some embodiments, the
secretory antibody-coated constituent of the subject's microbiota
associated with the development or progression of an inflammatory
disease or disorder in the subject is a Segmented Filamentous
Bacteria (SFB) or Helicobacter flexispira. In other embodiments,
the secretory antibody-coated constituent of the subject's
microbiota associated with the development or progression of an
inflammatory disease or disorder in the subject is a bacteria from
a family selected from the group consisting of Lactobacillus,
Helicobacter, S24-7, Erysipelotrichaceae and Prevotellaceae. In
some embodiments, the bacteria from the family Prevotellaceae is a
bacteria from the genera of Paraprevotella or Prevotella. In other
embodiments, the secretory antibody-coated constituent of the
subject's microbiota associated with the development or progression
of an inflammatory disease or disorder in the subject is at least
one strain of at least one bacteria selected from Acidaminococcus
spp., Actinomyces spp., Akkermansia muciniphila, Allobaculum spp.,
Anaerococcus spp., Anaerostipes spp., Bacteroides spp., Bacteroides
Other, Bacteroides acidifaciens, Bacteroides coprophilus,
Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis,
Barnesiellaceae spp., Bifidobacterium adolescentis, Bifidobacterium
Other, Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia
producta, Blautia Other, Blautia spp., Bulleidia spp.,
Catenibacterium spp., Citrobacter spp., Clostridiaceae spp.,
Clostridiales Other, Clostridiales spp., Clostridium perfringens,
Clostridium spp., Clostridium Other, Collinsella aerofaciens,
Collinsella spp., Collinsella stercoris, Coprococcus catus,
Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp.,
Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other,
Eggerthella lenta, Enterobacteriaceae Other, Enterobacteriaceae
spp., Enterococcus spp., Erysipelotrichaceae spp., Eubacterium
biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium
spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae
spp., Haemophilus parainfluenzae, Haemophilus Other, Helicobacter
spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp.,
Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae,
Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp.,
Leuconostocaceae spp., Megamonas spp., Megasphaera spp.,
Methanobrevibacter spp., Mitsuokella multacida, Mitsuokella spp.,
Mucispirillum schaedleri, Odoribacter spp., Oscillospira spp.,
Parabacteroides distasonis, Parabacteroides spp., Paraprevotella
spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp.,
Pediococcus Other, Peptococcus spp., Peptoniphilus spp.,
Peptostreptococcus anaerobius, Peptostreptococcus Other,
Phascolarctobacterium spp., Prevotella copri, Prevotella spp.,
Prevotella stercorea, Prevotellaceae, Proteus spp., Rikenellaceae
spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other,
Ruminococcaceae spp., Ruminococcus bromii, Ruminococcus gnavus,
Ruminococcus spp., Ruminococcus Other, Ruminococcus torques,
Slackia spp., S24-7 spp., SMB53 spp., Streptococcus anginosus,
Streptococcus luteciae, Streptococcus spp., Streptococcus Other,
Sutterella spp., Turicibacter spp., UC Bulleidia, UC
Enterobacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC
Pediococcus, Varibaculum spp., Veillonella spp., Sutterella,
Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC
Ruminococcaceae, Veillonella parvula, Veillonella spp., Veillonella
dispar, and Weissella.
[0120] In some embodiments, the invention is a method of
identifying the type or types of secretory antibody-bound bacteria
present in the microbiota of a subject that contribute to the
development or progression of an inflammatory disease or disorder
in the subject. In other embodiments, the invention is a method of
diagnosing an inflammatory disease or disorder in a subject by
identifying a type or types of secretory antibody-bound bacteria in
the microbiota of the subject that contribute to the development or
progression of an inflammatory disease or disorder.
[0121] In some embodiments, the invention is a method of
identifying the type or types of secretory antibody-bound bacteria
present in the microbiota of a subject that does not contribute to
the development or progression of an inflammatory disease or
disorder in the subject. For example, in one embodiment, the method
comprises identifying the type or types of secretory antibody-bound
bacteria present in a healthy subject not having an inflammatory
disease or disorder. In certain embodiments, the identified type or
types of secretory antibody-bound bacteria present in a healthy
subject may be used to treat a subject having an inflammatory
disease or disorder. In certain embodiments, the identified type or
types of secretory antibody-bound bacteria present in a healthy
subject may be used to prevent the development of an inflammatory
disease or disorder in a subject at risk.
[0122] Specific alterations in a subject's microbiota, including
the presence of secretory antibody-coated constituents, can be
detected using various methods, including without limitation
quantitative PCR or high-throughput sequencing methods which detect
relative proportions of microbial genetic markers in a total
heterogeneous microbial population. In some embodiments, the
microbial genetic marker is a bacterial genetic marker. In
particular embodiments, the bacterial genetic marker is at least
some portion of the 16S rRNA. In some embodiments, the relative
proportion of particular constituent bacterial phyla, classes,
orders, families, genera, and species present in the microbiota of
a subject is determined. In other embodiments, the relative
proportion of secretory antibody-coated and/or uncoated constituent
bacterial phyla, classes, orders, families, genera, and species
present in the microbiota of a subject is determined. In some
embodiments, the relative proportion of particular constituent
bacterial phyla, classes, orders, families, genera, and species
present in the microbiota of a subject is determined and compared
with that of a comparator normal microbiota. In other embodiments,
the relative proportion of secretory antibody-coated and/or
uncoated constituent bacterial phyla, classes, orders, families,
genera, and species present in the microbiota of a subject is
determined and compared with that of a comparator normal
microbiota. In various embodiments, the comparator normal
microbiota is, by way of non-limiting examples, a microbiota of a
subject known to be free of an inflammatory disorder, or a
historical norm, or a typical microbiota of the population of which
the subject is a member.
[0123] In one embodiment, the method of the invention is a
diagnostic assay for diagnosing an inflammatory disease or disorder
associated with an altered microbiota in a subject in need thereof,
by determining the absolute or relative abundance of particular
types of secretory antibody-coated constituents of the subject's
microbiota present in a biological sample derived from the subject.
In some embodiments, the subject is diagnosed as having an
inflammatory disease or disorder when particular types of secretory
antibody-coated bacteria are determined to be present in the
biological sample derived from the subject with increased relative
abundance. In some embodiments, the secretory antibody-coated
bacteria determined to be present in the biological sample derived
from the subject with increased relative abundance is a Segmented
Filamentous Bacteria (SFB) or Helicobacter flexispira. In some
embodiments, the secretory antibody-coated bacteria determined to
be present in the biological sample derived from the subject with
increased relative abundance is a bacteria from a family selected
from the group consisting of Lactobacillus, Helicobacter, S24-7,
Erysipelotrichaceae and Prevotellaceae. In some embodiments, the
bacteria from the family Prevotellaceae is a bacteria from the
genera of Paraprevotella or Prevotella. In other embodiments, the
secretory antibody-coated constituent of the subject's microbiota
associated with the development or progression of an inflammatory
disease or disorder in the subject is at least one strain of at
least one bacteria selected from Acidaminococcus spp., Actinomyces
spp., Akkermansia muciniphila, Allobaculum spp., Anaerococcus spp.,
Anaerostipes spp., Bacteroides spp., Bacteroides Other, Bacteroides
acidifaciens, Bacteroides coprophilus, Bacteroides fragilis,
Bacteroides ovatus, Bacteroides uniformis, Barnesiellaceae spp.,
Bifidobacterium adolescentis, Bifidobacterium Other,
Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia
producta, Blautia Other, Blautia spp., Bulleidia spp.,
Catenibacterium spp., Citrobacter spp., Clostridiaceae spp.,
Clostridiales Other, Clostridiales spp., Clostridium perfringens,
Clostridium spp., Clostridium Other, Collinsella aerofaciens,
Collinsella spp., Collinsella stercoris, Coprococcus catus,
Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp.,
Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other,
Eggerthella lenta, Enterobacteriaceae Other, Enterobacteriaceae
spp., Enterococcus spp., Erysipelotrichaceae spp., Eubacterium
biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium
spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae
spp., Haemophilus parainfluenzae, Haemophilus Other, Helicobacter
spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp.,
Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae,
Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp.,
Leuconostocaceae spp., Megamonas spp., Megasphaera spp.,
Methanobrevibacter spp., Mitsuokella multacida, Mitsuokella spp.,
Mucispirillum schaedleri, Odoribacter spp., Oscillospira spp.,
Parabacteroides distasonis, Parabacteroides spp., Paraprevotella
spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp.,
Pediococcus Other, Peptococcus spp., Peptoniphilus spp.,
Peptostreptococcus anaerobius, Peptostreptococcus Other,
Phascolarctobacterium spp., Prevotella copri, Prevotella spp.,
Prevotella stercorea, Prevotellaceae, Proteus spp., Rikenellaceae
spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other,
Ruminococcaceae spp., Ruminococcus bromii, Ruminococcus gnavus,
Ruminococcus spp., Ruminococcus Other, Ruminococcus torques,
Slackia spp., S24-7 spp., SMB53 spp., Streptococcus anginosus,
Streptococcus luteciae, Streptococcus spp., Streptococcus Other,
Sutterella spp., Turicibacter spp., UC Bulleidia, UC
Enterobacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC
Pediococcus, Varibaculum spp., Veillonella spp., Sutterella,
Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC
Ruminococcaceae, Veillonella parvula, Veillonella spp., Veillonella
dispar, and Weissella.
[0124] The results of the diagnostic assay can be used alone, or in
combination with other information from the subject, or from the
biological sample derived from the subject.
[0125] In the assay methods of the invention, a test biological
sample from a subject is assessed for the absolute or relative
abundance of secretory antibody-coated and uncoated constituents of
the microbiota. The test biological sample can be an in vitro
sample or an in vivo sample. In various embodiments, the subject is
a human subject, and may be of any race, sex and age.
Representative subjects include those who are suspected of having
an altered microbiota associated with an inflammatory disease or
disorder, those who have been diagnosed with an altered microbiota
associated with an inflammatory disease or disorder, those whose
have an altered microbiota associated with an inflammatory disease
or disorder, those who have had an altered microbiota associated
with an inflammatory disease or disorder, those who at risk of a
recurrence of an altered microbiota associated with an inflammatory
disease or disorder, those who at risk of a flare of an altered
microbiota associated with an inflammatory disease or disorder, and
those who are at risk of developing an altered microbiota
associated with an inflammatory disease or disorder.
[0126] In some embodiments, the test sample is prepared from a
biological sample obtained from the subject. In some instances, a
heterogeneous population of microbes will be present in the
biological samples. Enrichment of a microbial population for
microbes (e.g., bacteria) bound by secretory antibody (e.g., IgA,
IgM) may be accomplished using separation technique. For example,
microbes of interest may be enriched by separation the microbes of
interest from the initial population using affinity separation
techniques. Techniques for affinity separation may include magnetic
separation using magnetic beads conjugated with an affinity
reagent, affinity chromatography, "panning" with an affinity
reagent attached to a solid matrix, e.g. plate, or other convenient
technique. Other techniques providing separation include
fluorescence activated cell sorting, which can have varying degrees
of sophistication, such as multiple color channels, low angle and
obtuse light scattering detecting channels, impedance channels,
etc. One example of an affinity reagent useful in the methods of
the invention is an antibody, such as anti-species antibody or
anti-isotype (e.g., anti-IgA, anti-IgM) antibody. The details of
the preparation of such antibodies and their suitability for use as
affinity reagents are well-known to those skilled in the art. In
some embodiments, labeled antibodies are used as affinity reagents.
Conveniently, these antibodies are conjugated with a label for use
in separation. Labels include magnetic beads, which allow for
direct separation; biotin, which can be removed with avidin or
streptavidin bound to a support; fluorochromes, which can be used
with a fluorescence activated cell sorter; or the like, to allow
for ease of separation of the particular cell type.
[0127] In various embodiments, the initial population of microbes
is contacted with one or more affinity reagent(s) and incubated for
a period of time sufficient to permit the affinity reagent to
specifically bind to its target. The microbes in the contacted
population that become labeled by the affinity reagent are selected
for by any convenient affinity separation technique, e.g. as
described elsewhere herein or as known in the art. Compositions
highly enriched for a microbe of interest (e.g., secretory
antibody-bound bacteria) are achieved in this manner. The affinity
enriched microbes will be about 70%, about 75%, about 80%, about
85% about 90%, about 95% or more of the composition. In other
words, the enriched composition can be a substantially pure
composition of the microbes of interest.
[0128] In one embodiment, the test sample is a sample containing at
least a fragment of a bacterial nucleic acid. The term, "fragment,"
as used herein, indicates that the portion of a nucleic acid (e.g.,
DNA, RNA) that is sufficient to identify it as comprising a
bacterial nucleic acid.
[0129] In some embodiments, the test sample is prepared from a
biological sample obtained from the subject. The biological sample
can be a sample from any source which contains a bacterial nucleic
acid (e.g., DNA, RNA), such as a bodily fluid or fecal sample, or a
combination thereof. A biological sample can be obtained by any
suitable method. In some embodiments, a biological sample
containing bacterial DNA is used. In other embodiments, a
biological sample containing bacterial RNA is used. The biological
sample can be used as the test sample; alternatively, the
biological sample can be processed to enhance access to nucleic
acids, or copies of nucleic acids, and the processed biological
sample can then be used as the test sample. For example, in various
embodiments, nucleic acid is prepared from a biological sample, for
use in the methods. Alternatively or in addition, if desired, an
amplification method can be used to amplify nucleic acids
comprising all or a fragment of an RNA or DNA in a biological
sample, for use as the test sample in the assessment of the
presence, absence and proportion of particular types of bacteria
present in the sample.
[0130] In some embodiments, hybridization methods, such as Southern
analysis, Northern analysis, or in situ hybridizations, can be used
(see Current Protocols in Molecular Biology, Ausubel, F. et al.,
eds., John Wiley & Sons, including all supplements). For
example, the presence of nucleic acid from a particular type of
bacteria can be determined by hybridization of nucleic acid to a
nucleic acid probe. A "nucleic acid probe," as used herein, can be
a DNA probe or an RNA probe.
[0131] The nucleic acid probe can be, for example, a full-length
nucleic acid molecule, or a portion thereof, such as an
oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides
in length and sufficient to specifically hybridize under stringent
conditions to appropriate target RNA or DNA. The hybridization
sample is maintained under conditions which are sufficient to allow
specific hybridization of the nucleic acid probe to RNA or DNA.
Specific hybridization can be performed under high stringency
conditions or moderate stringency conditions, as appropriate. In a
preferred embodiment, the hybridization conditions for specific
hybridization are high stringency. More than one nucleic acid probe
can also be used concurrently in this method. Specific
hybridization of any one of the nucleic acid probes is indicative
of the presence of the particular type of bacteria of interest, as
described herein.
[0132] In Northern analysis (see Current Protocols in Molecular
Biology, Ausubel, F. et al., eds., John Wiley & Sons, supra),
the hybridization methods described above are used to identify the
presence of a sequence of interest in an RNA, such as unprocessed,
partially processed or fully processed rRNA. For Northern analysis,
a test sample comprising RNA is prepared from a biological sample
from the subject by appropriate means. Specific hybridization of a
nucleic acid probe, as described above, to RNA from the biological
sample is indicative of the presence of the particular type of
bacteria of interest, as described herein.
[0133] Alternatively, a peptide nucleic acid (PNA) probe can be
used instead of a nucleic acid probe in the hybridization methods
described herein. PNA is a DNA mimic having a peptide-like,
inorganic backbone, such as N-(2-aminoethyl)glycine units, with an
organic base (A, G, C, T or U) attached to the glycine nitrogen via
a methylene carbonyl linker (see, for example, 1994, Nielsen et
al., Bioconjugate Chemistry 5:1). The PNA probe can be designed to
specifically hybridize to a particular bacterial nucleic acid
sequence. Hybridization of the PNA probe to a nucleic acid sequence
is indicative of the presence of the particular type of bacteria of
interest.
[0134] Direct sequence analysis can also be used to detect a
bacterial nucleic acid of interest. A sample comprising DNA or RNA
can be used, and PCR or other appropriate methods can be used to
amplify all or a fragment of the nucleic acid, and/or its flanking
sequences, if desired. The bacterial nucleic acid, or a fragment
thereof, is determined, using standard methods.
[0135] In another embodiment, arrays of oligonucleotide probes that
are complementary to target microbial nucleic acid sequences can be
used to detect and identify microbial nucleic acids. For example,
in one embodiment, an oligonucleotide array can be used.
Oligonucleotide arrays typically comprise a plurality of different
oligonucleotide probes that are coupled to a surface of a substrate
in different known locations. These oligonucleotide arrays, also
known as "Genechips," have been generally described in the art, for
example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO
90/15070 and 92/10092. These arrays can generally be produced using
mechanical synthesis methods or light directed synthesis methods
which incorporate a combination of photolithographic methods and
solid phase oligonucleotide synthesis methods. See Fodor et al.,
Science, 251:767-777 (1991), Pirrung et al., U.S. Pat. No.
5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et
al., PCT Publication No. WO 92/10092 and U.S. Pat. No. 5,424,186.
Techniques for the synthesis of these arrays using mechanical
synthesis methods are described in, e.g., U.S. Pat. No.
5,384,261.
[0136] After an oligonucleotide array is prepared, a nucleic acid
of interest is hybridized with the array and scanned for particular
bacterial nucleic acids. Hybridization and scanning are generally
carried out by methods described herein and also in, e.g.,
Published PCT Application Nos. WO 92/10092 and WO 95/11995, and
U.S. Pat. No. 5,424,186, the entire teachings of which are
incorporated by reference herein. In brief, a target bacterial
nucleic acid sequence is amplified by well-known amplification
techniques, e.g., PCR. Typically, this involves the use of primer
sequences that are complementary to the target sequence. Amplified
target, generally incorporating a label, is then hybridized with
the array under appropriate conditions. Upon completion of
hybridization and washing of the array, the array is scanned to
determine the position on the array to which the target sequence
hybridizes. The hybridization data obtained from the scan is
typically in the form of fluorescence intensities as a function of
location on the array.
[0137] Other methods of nucleic acid analysis can be used to detect
microbial nucleic acids of interest. Representative methods include
direct manual sequencing (1988, Church and Gilbert, Proc. Natl.
Acad. Sci. USA 81:1991-1995; 1977, Sanger et al., Proc. Natl. Acad.
Sci. 74:5463-5467; Beavis et al. U.S. Pat. No. 5,288,644);
automated fluorescent sequencing; single-stranded conformation
polymorphism assays (SSCP); clamped denaturing gel electrophoresis
(CDGE); denaturing gradient gel electrophoresis (DGGE) (1981,
Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236), mobility
shift analysis (1989, Orita et al., Proc. Natl. Acad. Sci. USA
86:2766-2770; 1987, Rosenbaum and Reissner, Biophys. Chem.
265:1275; 1991, Keen et al., Trends Genet. 7:5); restriction enzyme
analysis (1978, Flavell et al., Cell 15:25; 1981, Geever, et al.,
Proc. Natl. Acad. Sci. USA 78:5081); heteroduplex analysis;
chemical mismatch cleavage (CMC) (1985, Cotton et al., Proc. Natl.
Acad. Sci. USA 85:4397-4401); RNase protection assays (1985, Myers,
et al., Science 230:1242); use of polypeptides which recognize
nucleotide mismatches, such as E. coli mutS protein (see, for
example, U.S. Pat. No. 5,459,039); Luminex xMAP.TM. technology;
high-throughput sequencing (HTS) (2011, Gundry and Vijg, Mutat Res,
doi:10.1016/j.mrfmmm.2011.10.001); next-generation sequencing (NGS)
(2009, Voelkerding et al., Clinical Chemistry 55:641-658; 2011, Su
et al., Expert Rev Mol Diagn. 11:333-343; 2011, Ji and Myllykangas,
Biotechnol Genet Eng Rev 27:135-158); ion semiconductor sequencing
(2011, Rusk, Nature Methods doi:10.1038/nmeth.f.330; 2011, Rothberg
et al., Nature 475:348-352) and/or allele-specific PCR, for
example. These and other methods can be used to identify the
presence of one or more microbial nucleic acids of interest, in a
biological sample derived from a subject. In various embodiments of
the invention, the methods of assessing a biological sample for the
presence or absence of a particular nucleic acid sequence, as
described herein, are used to detect, identify or quantify
particular constituents of a subject's microbiota, and to aid in
the diagnosis of an altered microbiota associated with an
inflammatory disease or disorder in a subject in need thereof.
[0138] The probes and primers according to the invention can be
labeled directly or indirectly with a radioactive or nonradioactive
compound, by methods well known to those skilled in the art, in
order to obtain a detectable and/or quantifiable signal; the
labeling of the primers or of the probes according to the invention
is carried out with radioactive elements or with nonradioactive
molecules. Among the radioactive isotopes used, mention may be made
of .sup.32P, .sup.33P, .sup.35S or .sup.3H. The nonradioactive
entities are selected from ligands such as biotin, avidin,
streptavidin or digoxigenin, haptenes, dyes, and luminescent agents
such as radioluminescent, chemoluminescent, bioluminescent,
fluorescent or phosphorescent agents.
[0139] Nucleic acids can be obtained from the biological sample
using known techniques. Nucleic acid herein refers to RNA,
including mRNA, and DNA, including genomic DNA. The nucleic acid
can be double-stranded or single-stranded (i.e., a sense or an
antisense single strand) and can be complementary to a nucleic acid
encoding a polypeptide. The nucleic acid content may also be an RNA
or DNA extraction performed on a fresh or fixed biological
sample.
[0140] Routine methods also can be used to extract DNA from a
biological sample, including, for example, phenol extraction.
Alternatively, genomic DNA can be extracted with kits such as the
QIAamp.TM.. Tissue Kit (Qiagen, Chatsworth, Calif.), the Wizard.TM.
Genomic DNA purification kit (Promega, Madison, Wis.), the Puregene
DNA Isolation System (Gentra Systems, Inc., Minneapolis, Minn.),
and the A.S.A.P..TM. Genomic DNA isolation kit (Boehringer
Mannheim, Indianapolis, Ind.).
[0141] There are many methods known in the art for the detection of
specific nucleic acid sequences and new methods are continually
reported. A great majority of the known specific nucleic acid
detection methods utilize nucleic acid probes in specific
hybridization reactions. Preferably, the detection of hybridization
to the duplex form is a Southern blot technique. In the Southern
blot technique, a nucleic acid sample is separated in an agarose
gel based on size (molecular weight) and affixed to a membrane,
denatured, and exposed to (admixed with) the labeled nucleic acid
probe under hybridizing conditions. If the labeled nucleic acid
probe forms a hybrid with the nucleic acid on the blot, the label
is bound to the membrane.
[0142] In the Southern blot, the nucleic acid probe is preferably
labeled with a tag. That tag can be a radioactive isotope, a
fluorescent dye or the other well-known materials. Another type of
process for the specific detection of nucleic acids of exogenous
organisms in a body sample known in the art are the hybridization
methods as exemplified by U.S. Pat. Nos. 6,159,693 and 6,270,974,
and related patents. To briefly summarize one of those methods, a
nucleic acid probe of at least 10 nucleotides, preferably at least
15 nucleotides, more preferably at least 25 nucleotides, having a
sequence complementary to a desired region of the target nucleic
acid of interest is hybridized in a sample, subjected to
depolymerizing conditions, and the sample is treated with an
ATP/luciferase system, which will luminesce if the nucleic sequence
is present. In quantitative Southern blotting, levels of the target
nucleic acid can be determined.
[0143] A further process for the detection of hybridized nucleic
acid takes advantage of the polymerase chain reaction (PCR). The
PCR process is well known in the art (U.S. Pat. Nos. 4,683,195,
4,683,202, and 4,800,159). To briefly summarize PCR, nucleic acid
primers, complementary to opposite strands of a nucleic acid
amplification target nucleic acid sequence, are permitted to anneal
to the denatured sample. A DNA polymerase (typically heat stable)
extends the DNA duplex from the hybridized primer. The process is
repeated to amplify the nucleic acid target. If the nucleic acid
primers do not hybridize to the sample, then there is no
corresponding amplified PCR product. In this case, the PCR primer
acts as a hybridization probe.
[0144] In PCR, the nucleic acid probe can be labeled with a tag as
discussed before. Most preferably the detection of the duplex is
done using at least one primer directed to the target nucleic acid.
In yet another embodiment of PCR, the detection of the hybridized
duplex comprises electrophoretic gel separation followed by
dye-based visualization.
[0145] DNA amplification procedures by PCR are well known and are
described in U.S. Pat. No. 4,683,202. Briefly, the primers anneal
to the target nucleic acid at sites distinct from one another and
in an opposite orientation. A primer annealed to the target
sequence is extended by the enzymatic action of a heat stable DNA
polymerase. The extension product is then denatured from the target
sequence by heating, and the process is repeated. Successive
cycling of this procedure on both DNA strands provides exponential
amplification of the region flanked by the primers.
[0146] Amplification is then performed using a PCR-type technique,
that is to say the PCR technique or any other related technique.
Two primers, complementary to the target nucleic acid sequence are
then added to the nucleic acid content along with a polymerase, and
the polymerase amplifies the DNA region between the primers.
[0147] The expression "specifically hybridizing in stringent
conditions" refers to a hybridizing step in the process of the
invention where the oligonucleotide sequences selected as probes or
primers are of adequate length and sufficiently unambiguous so as
to minimize the amount of non-specific binding that may occur
during the amplification. The oligonucleotide probes or primers
herein described may be prepared by any suitable methods such as
chemical synthesis methods.
[0148] Hybridization is typically accomplished by annealing the
oligonucleotide probe or primer to the DNA under conditions of
stringency that prevent non-specific binding but permit binding of
this DNA which has a significant level of homology with the probe
or primer.
[0149] Among the conditions of stringency is the melting
temperature (Tm) for the amplification step using the set of
primers, which is in the range of about 55.degree. C. to about
70.degree. C. Preferably, the Tm for the amplification step is in
the range of about 59.degree. C. to about 72.degree. C. Most
preferably, the Tm for the amplification step is about 60.degree.
C.
[0150] Typical hybridization and washing stringency conditions
depend in part on the size (i.e., number of nucleotides in length)
of the DNA or the oligonucleotide probe, the base composition and
monovalent and divalent cation concentrations (Ausubel et al.,
1997, eds Current Protocols in Molecular Biology).
[0151] In a preferred embodiment, the process for determining the
quantitative and qualitative profile according to the present
invention is characterized in that the amplifications are real-time
amplifications performed using a labeled probe, preferably a
labeled hydrolysis-probe, capable of specifically hybridizing in
stringent conditions with a segment of a nucleic acid sequence, or
polymorphic nucleic acid sequence. The labeled probe is capable of
emitting a detectable signal every time each amplification cycle
occurs.
[0152] The real-time amplification, such as real-time PCR, is well
known in the art, and the various known techniques will be employed
in the best way for the implementation of the present process.
These techniques are performed using various categories of probes,
such as hydrolysis probes, hybridization adjacent probes, or
molecular beacons. The techniques employing hydrolysis probes or
molecular beacons are based on the use of a fluorescence
quencher/reporter system, and the hybridization adjacent probes are
based on the use of fluorescence acceptor/donor molecules.
[0153] Hydrolysis probes with a fluorescence quencher/reporter
system are available in the market, and are for example
commercialized by the Applied Biosystems group (USA). Many
fluorescent dyes may be employed, such as FAM dyes
(6-carboxy-fluorescein), or any other dye phosphoramidite
reagents.
[0154] Among the stringent conditions applied for any one of the
hydrolysis-probes of the present invention is the Tm, which is in
the range of about 65.degree. C. to 75.degree. C. Preferably, the
Tm for any one of the hydrolysis-probes of the present invention is
in the range of about 67.degree. C. to about 70.degree. C. Most
preferably, the Tm applied for any one of the hydrolysis-probes of
the present invention is about 67.degree. C.
[0155] In another preferred embodiment, the process for determining
the quantitative and qualitative profile according to the present
invention is characterized in that the amplification products can
be elongated, wherein the elongation products are separated
relative to their length. The signal obtained for the elongation
products is measured, and the quantitative and qualitative profile
of the labeling intensity relative to the elongation product length
is established.
[0156] The elongation step, also called a run-off reaction, allows
one to determine the length of the amplification product. The
length can be determined using conventional techniques, for
example, using gels such as polyacrylamide gels for the separation,
DNA sequencers, and adapted software. Because some mutations
display length heterogeneity, some mutations can be determined by a
change in length of elongation products.
[0157] In one aspect, the invention includes a primer that is
complementary to a target bacterial nucleic acid, and more
particularly the primer includes 12 or more contiguous nucleotides
substantially complementary to the sequence flanking the nucleic
acid sequence of interest. Preferably, a primer featured in the
invention includes a nucleotide sequence sufficiently complementary
to hybridize to a nucleic acid sequence of about 12 to 25
nucleotides. More preferably, the primer differs by no more than 1,
2, or 3 nucleotides from the target flanking nucleotide sequence.
In another aspect, the length of the primer can vary in length,
preferably about 15 to 28 nucleotides in length (e.g., 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length).
[0158] The present invention also pertains to kits useful in the
methods of the invention. Such kits comprise components useful in
any of the methods described herein, including for example,
hybridization probes or primers (e.g., labeled probes or primers),
reagents for detection of labeled molecules, means for
amplification of nucleic acids, means for analyzing a nucleic acid
sequence, and instructional materials. For example, in one
embodiment, the kit comprises components useful for analysis of a
bacterial nucleic acid of interest present in a biological sample
obtained from a subject. In a preferred embodiment of the
invention, the kit comprises components for detecting one or more
of the bacterial nucleic acids of interest present in a biological
sample derived from a subject.
EXPERIMENTAL EXAMPLES
[0159] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0160] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples therefore, specifically point out the
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the
disclosure.
Example 1: Immunoglobulin a Coating Identifies Colitogenic Bacteria
in Inflammatory Bowel Disease
[0161] The results presented herein are based on the application of
flow cytometry-based bacterial cell sorting and 16S sequencing to
characterize taxa-specific coating of the intestinal microbiota
with immunoglobulin A (IgA-SEQ) and show that high IgA-coating
uniquely identifies colitogenic intestinal bacteria in a mouse
model of microbiota-driven colitis (see also Palm et al., 2014,
Cell). IgA-SEQ and extensive anaerobic culturing of fecal bacteria
from IBD patients was used to create personalized
disease-associated gut microbiota culture collections with
pre-defined levels of IgA coating. Using these collections,
intestinal bacteria selected on the basis of high coating with IgA
was found to confer dramatic susceptibility to colitis in germ-free
mice. These studies demonstrate that IgA-coating identifies
inflammatory commensals that preferentially drive intestinal
disease. Targeted elimination of such bacteria may reduce, reverse,
or even prevent disease development.
[0162] The materials and methods used in these experiments are now
described.
[0163] Animals
[0164] Asc-/-, Rag2-/-, and Tcrb-/-;Tcrd-/- mice were bred and
maintained at the Yale School of Medicine and all treatments were
in accordance with Yale Animal Care and Use Committee guidelines.
Mice were strictly maintained under SPF conditions with consistent
monitoring for and exclusion of viral, fungal and bacterial
pathogens, as well as helminths and ectoparasites. Wild type
C57Bl/6 mice were from the National Cancer Institute (NCI), and
germ-free C57Bl/6 mice were purchased from the University of
Michigan and the University of North Carolina germ-free facilities.
Germ-free mice were singly housed, and age and sex matched mice
were used for all studies.
[0165] Inflammasome-Mediated Intestinal Dysbiosis
[0166] Intestinal dysbiosis was induced by co-housing two wild type
C57Bl/6 mice from NCI with two Asc-/- mice for at least 6
weeks.
[0167] DSS Colitis
[0168] SPF and SPF.sup.dys mice were treated with 2% Dextran Sodium
Sulfate (MP Biomedicals) in the drinking water ad libitum for 7
days to induce colitis. Weight was measured daily for 14 days.
Gnotobiotic mice were treated with filter sterilized 2.5% DSS in
the drinking water ad libitum for 6 days before end-point
euthanasia.
[0169] ELISA
[0170] Pre-sort, IgA+ and IgA- fractions (after MACS sorting) were
probed for IgA by ELISA (Coating: MP Biomedicals 55478, Detection:
Sigma B2766).
[0171] Fecal IgA Flow Cytometry and Sorting of IgA+ and IgA-
Bacteria
[0172] Fecal homogenates were stained with PE-conjugated Anti-Mouse
IgA (eBioscience clone mA-6E1) or PE-conjugated Anti-Human IgA
(Miltenyi Biotec clone IS11-8E10) prior to flow cytometric analysis
or MACS and FACS sorting. Fecal pellets collected directly from two
co-housed mice or -100 mg of frozen human fecal material were
placed in Fast Prep Lysing Matrix D tubes containing ceramic beads
(MP Biomedicals) and incubated in 1 mL Phosphate Buffered Saline
(PBS) per 100 mg fecal material on ice for 1 hour. Fecal pellets
were homogenized by bead beating for 5 seconds (Minibeadbeater;
Biospec) and then centrifuged (50.times.g, 15 min, 4.degree. C.) to
remove large particles. Fecal bacteria in the supernatants were
removed (100 .mu.l/sample), washed with 1 mL PBS containing 1%
(w/v) Bovine Serum Albumin (BSA, American Bioanalytical; staining
buffer) and centrifuged for 5 min (8,000.times.g, 4.degree. C.)
before resuspension in 1 mL staining buffer. A sample of this
bacterial suspension (20 .mu.l) was saved as the Pre-sort sample
for 16S sequencing analysis. After an additional wash, bacterial
pellets were resuspended in 100 .mu.l blocking buffer (staining
buffer containing 20% Normal Rat Serum for mouse samples or 20%
Normal Mouse Serum for human samples, both from Jackson
ImmunoResearch), incubated for 20 min on ice, and then stained with
100 .mu.l staining buffer containing PE-conjugated Anti-Mouse IgA
(1:12.5; eBioscience clone mA-6E1) or PE-conjugated Anti-Human IgA
(1:10; Miltenyi Biotec clone IS11-8E10) for 30 minutes on ice.
Samples were then washed 3 times with 1 mL staining buffer before
flow cytometric analysis or cell separation.
[0173] Anti-IgA stained fecal bacteria were incubated in 1 ml
staining buffer containing 50 .mu.l Anti-PE Magnetic Activated Cell
Sorting (MACS) beads (Miltenyi Biotec) (15 min at 4.degree. C.),
washed twice with 1 ml Staining Buffer (10,000.times.g, 5 min,
4.degree. C.), and then sorted by MACS (Possel_s program on an
AutoMACS pro; Miltenyi). After MACS separation, 50 .mu.l of the
negative fraction was collected for 16S sequencing analysis (IgA
negative fraction). The positive fraction was then further purified
via Fluorescence Activated Cell Sorting (FACSAria; BD Biosciences).
For each sample, 2 million IgA-positive bacteria were collected,
pelleted (10,000.times.g, 5 min, 4.degree. C.), and frozen along
with the Pre-sort and IgA-negative samples at -80.degree. C. for
future use.
[0174] 16S rRNA Gene Sequencing, Bacterial Genome Sequencing and
Statistical Analyses
[0175] 16S rRNA sequencing of the V4 region and bacterial genome
sequencing were performed on an Illumina miSeq using barcoded
primers. Microbial diversity and statistical analyses were
performed with QIIME, the Vegan package for R and LEfSe (Caporaso
et al., 2010, Nat Methods 7:335-336; Segata et al., 2011, Genome
Biol 12:R60). All bacterial samples were suspended in 400 .mu.l
staining buffer before adding 250 .mu.l 0.1 mm zirconia/silica
beads (Biospec), 300 .mu.l Lysis buffer (200 mM NaCl, 200 mM Tris,
20 mM EDTA, pH 8), 200 .mu.l20% SDS and 500 .mu.l
phenol:chloroform:isoamylalcohol (25:24:1, pH 7.9; Sigma). Samples
were chilled on ice for 4 min and then homogenized by beat beating
(2 min bead beating, 2 min on ice, 2 min bead beating). After
centrifugation (6000.times.g, 4.degree. C.), the aqueous phase was
transferred to a Phase Lock Gel tube (Light; 5 PRIME), an equal
volume of phenol:chloroform:isoamylalcohol was added, and samples
were mixed by inversion and then centrifuged for 3 min
(16,100.times.g, room temperature). The DNA was then precipitated
by adding 1/10 volume of 3M NaOAc (pH5.5) and 1 volume Isopropanol
to the aqueous phase before incubation at -20.degree. C. overnight.
Precipitated DNA was pelleted (20 min, 16,100.times.g, 4.degree.
C.), washed with 500 .mu.l 100% EtOH (3 min, 16,100.times.g,
4.degree. C.), dried (miVac GeneVac 15 min, no heat, Auto Run
setting), and resuspended in 100 .mu.l TE buffer (pH 7; 50.degree.
C. for 30 min). The DNA was then treated with 35 U/ml RNase A
(Qiagen) before purification (QIAquick PCR purification; Qiagen),
and elution in 40 .mu.l Elution Buffer. The V4 region of 16S
ribosomal RNA was then PCR amplified (28 cycles; primer pair
F515/R806) in triplicate (10 .mu.l purified DNA per reaction;
Phusion polymerase, New England Bioscience) (Caporaso et al., 2012,
ISME J 6:1621-1624; Caporaso et al., 2011, P Natl Acad Sci USA
108(Suppl 1):4516-4522). After amplification, PCR triplicates were
pooled, purified (MinElute, Qiagen), and resuspended in 20 .mu.l
H.sub.2O. PCR products were then quantified with Picogreen
(Invitrogen) and pooled at a final concentration of 10 nM before
sequencing on a miSeq sequencer (Illumina, 2.times.250 bp
paired-end reads, up to 200 samples per sequencing run)
[0176] Paired end reads were assembled with a novel pipeline that
uses PANDA-seq (Masella et al., 2012, BMC Bioinformatics 13:31) and
assigns consensus Q scores to the assembled reads (P.D. and A.L.G.,
manuscript in preparation). Microbial diversity was analyzed with
the Quantitative Insights Into Microbial Ecology (QIIME version
1.7) analysis suite. Reads were demultiplexed and quality filtered
with a Q-score cutoff of 30. The open-reference OTU picking
workflow in QIIME and the Greengenes reference database were used
to cluster the reads into 97% identity Operational Taxonomic Units
(OTUs). The Ribosomal Database Project classifier (RDP) and the May
2013 Greengenes taxonomy were used to assign taxonomy to
representative OTUs (Caporaso et al., 2010, Nat Methods 7:335-336;
Lozupone and Knight, 2005, Appl Environ Microb 71:8228-8235; Wang
et al., 2007, Appl Environ Microb 73:5261-5267). OTUs of less than
0.01% relative abundance, and contaminating OTUs that were also
found after sequencing of 16S amplicons from PCR samples without
template DNA, were filtered from OTU tables. Filtered OTU tables
were rarefied to a depth of 5000 sequences per sample for all
further analyses.
[0177] QIIME and the Vegan package for R (version 2.1-21) were used
for all microbial ecology analyses (beta diversity, PCoA,
PERMANOVA/adonis) (Caporaso et al., 2010, Nat Methods 7:335-336).
The Linear Discriminant Analysis Effect Size (LEfSe) Galaxy module
(http://huttenhower.sph.harvard.edu/galaxy/) was used for
additional statistical analyses (Segata et al., 2011, Genome Biol
12:R60). Wilcoxon rank-sum tests were performed using R or Prism
(Graphpad Software). Taxa that were undetectable in both the IgA+
and IgA- fractions in a given sample were considered not present
and were assigned as missing-values for Wilcoxon rank-sum tests. As
LEfSe cannot handle missing values, these missing-values were
replaced with zeros for all LEfSe analyses. To allow for the
calculation of ICI scores for taxa that were undetectable in the
IgA- fraction but detected in the IgA+ fraction, and which are
therefore highly-coated, zeroes in the negative fraction were
replaced with a relative abundance of 0.0002, which is the limit of
detection (1 sequence in 5000). The results presented for mice are
combined from at least 4 independent experiments.
[0178] Genomic DNA for whole genome bacterial sequencing was
isolated using a QIAmp DNA isolation kit (Qiagen). A Nextera XT kit
(Illumina) was used to prepare barcoded genomic DNA libraries, and
paired-end (2.times.300) sequencing was performed on a MiSeq
(Illumina). Sequences were assembled into contigs with the SPAdes
genome assembler 3.0 (Bankevich et al., 2012, J Comput Biol
19:455-477) on Basespace (Illumina) and aligned with progressive
Mauve (Darling et al., 2004, Genome Res 14:1394-1403).
[0179] Human Fecal Samples
[0180] The human study protocol was approved by the Institutional
Review Board (Protocol No. 10-1047) of the Icahn Medical School at
Mount Sinai, N.Y. The healthy subjects were recruited through the
Mount Sinai Biobank or an advertisement. Fresh fecal samples were
collected at home, stored at -20.degree. C. in an insulated foam
shipper, mailed to Mount Sinai overnight and then stored at
-80.degree. C. for further analysis. A short questionnaire was also
administrated to collect participants' health information. Informed
consent was obtained from all subjects.
[0181] Culturing of Human Fecal Bacteria and Generation of
Personalized Microbiota Culture Collections
[0182] Culture methods were essentially as in Goodman et al.
(Goodman et al., 2011, P Natl Acad Sci USA 108:6252-6257) with
minor modifications. Briefly, serial dilutions of fecal material
from 11 IBD patients were plated on three types of media: CDC
Anaerobe 5% Sheep Blood Agar with or without Kanamycin and
Vancomycin (BD Bioscience), and Gut Microbiota Medium (GMM) Agar.
100 to 200 colonies per patient were picked and cultured
individually in GMM for 5 days to establish Culture
Collections.
[0183] Assembly of IgA+ and IgA- Consortia and Colonization of
Germ-Free Mice
[0184] Members of the IgA+ and IgA- consortia were selected from
our IBD microbiota culture collections based on ICI scores
determined by IgA-SEQ. The criteria for selecting the members of
these consortia were as follows: Strains comprising the IgA+
consortium were selected based on high coating in the patient from
whom they were isolated (ICI >10), and were rarely or never
highly coated in healthy controls; in other words, they were
selected to represent bacteria that are uniquely or preferentially
highly coated in IBD. Strains comprising the IgA- consortium were
selected based on low coating (ICI <1) in the patient from whom
they were isolated and were rarely or never highly coated in IBD
patients or controls. Bacterial strains that met these criteria
were cultured in GMM for 4 days before mixing to form consortia.
Singly housed germ-free C57Bl/6 mice were colonized with 100 ?al of
the appropriate consortium by oral gavage.
[0185] Fluorescence In Situ Hybridization
[0186] 16S rRNA FISH was performed with a universal bacterial probe
(EUB388; 5'G*CTGCCTCCCGTAGGAGT-3'[Cy5]) on sections fixed with
Carnoy's solution to preserve the mucus layer as described
previously (Canny et al., 2006, Method Mol Cell Biol
341:17-35).
[0187] Histology
[0188] Colons were fixed in Bouin's solution and embedded in
paraffin. Sections were stained with hematoxylin and eosin and
scored in a blinded manner by a trained pathologist.
[0189] Monocolonization with B. fragilis Isolates
[0190] Germ-free C57Bl/6 mice were monocolonized with B. fragilis
strains by oral gavage. Mice were treated with 2.5% DSS in the
drinking water starting at 5 days after colonization.
Monocolonization was confirmed by 16S sequencing of feces.
[0191] The results of the experiments are now described.
[0192] IgA-SEQ Identifies Highly IgA Coated Members of the
Intestinal Microbiota
[0193] To measure taxa-specific IgA coating in an unbiased and
comprehensive manner, an approach was devised that combines
antibody-based bacterial cell sorting and 16S ribosomal RNA (rRNA)
gene sequencing to isolate and identify IgA coated bacteria from
fecal material (IgA-SEQ, FIG. 1A). First, fecal bacteria from
specific pathogen free (SPF) mice were stained for IgA and it was
confirmed that only a fraction of intestinal bacteria are
measurably IgA coated, as determined by flow cytometry
(7.4%.+-.2.2; FIG. 6A-FIG. 6C) (Kawamoto et al., 2012, Science
336:485-489; Tsuruta et al., 2009, FEMS Immunol Med Mic 56:185-189;
van der Waaij et al., 1994, Cytometry 16:270-279); importantly,
intestinal bacteria from recombination activating gene 2
(Rag2)-deficient mice, which cannot produce antibodies, showed
minimal staining for IgA (0.5%.+-.0.3). Highly IgA coated (IgA+)
and non-coated (IgA-) bacteria were subsequently isolated using a
combination of magnetic activated cell sorting (MACS) and
fluorescence activated cell sorting (FACS). The specificity and
efficacy of the sorting was confirmed by reanalyzing sorted
fractions via flow cytometry (FIG. 1B and FIG. 6E) and ELISA (FIG.
6F). After 16S rRNA gene sequencing, microbial compositions were
compared and visualized using Principal Coordinates Analysis (PCoA)
of weighted UniFrac distances, which revealed that, rather than
comprising a random sampling of all intestinal bacteria, IgA+
bacteria represent a distinct sub-community within the intestinal
microbiota (P<0.05, PERMANOVA) (FIG. 6G and FIG. 6H).
Importantly, as was observed in other recent studies using FACS to
sort fecal bacteria (Ben-Amor et al., 2005, Appl Environ Microb
71:4679-4689; Maurice et al., 2013, Cell 152:39-50; Peris-Bondia et
al., 2011, PLoS One 6:e22448), sorting itself did not artificially
alter microbial composition (P >0.05, PERMANOVA). These data
demonstrate that IgA coating of the intestinal microbiota is
selective across microbial taxa, and show that IgA coated bacteria
represent a taxonomically distinct subset of intestinal bacteria in
mice.
[0194] To identify which specific bacterial taxa were highly coated
with IgA, the relative abundance of bacterial genera in total, IgA+
and IgA- bacterial fractions isolated from the feces of SPF mice
was examined (FIG. 1C, FIG. 1D, FIG. 6I, and Table 1). To quantify
and compare relative levels of IgA coating between taxa, an IgA
Coating Index (ICI) was calculated for each individual bacterial
taxon as follows: ICI=relative abundance (IgA+)/relative abundance
(IgA-). Taxonomic abundance was then compared using the Wilcoxon
rank-sum test and Linear Discriminant Analysis Effect Size (LEfSe;
(Segata et al., 2011, Genome Biol 12:R60)) to determine which taxa
were enriched in either the IgA+ or IgA- fractions (FIG. 6J and
Table 1). These analyses revealed that only four genera were
significantly enriched in the IgA+ fraction in SPF mice
(`significantly coated`; P<0.05): an unclassified genus of the
family S24-7 from the order Bacteroidales, Lactobacillus, SFB, and
an unclassified Erysipelotrichaceae (FIG. 1C and FIG. 1D). Among
these bacteria, only SFB was significantly enriched in the IgA+
fraction and showed an ICI score greater than 10, which was defined
as `highly coated` (P<0.05; ICI >10). In addition, 22 taxa
were significantly enriched in the IgA- fraction (`low- or
non-coated`; P<0.05), while the remaining taxa were neither
enriched nor depleted by IgA-based separation.
TABLE-US-00001 TABLE 1 Taxa Average ICI score P value Bacteroidales
(S24-7 gen.) 1.995277226 6.75E-07 UC Clostridiales 0.079125427
6.72E-07 Oscillospira 0.10337211 6.72E-07 Clostridiales gen.
0.1022536 6.74E-07 UC Lachnospiraceae 0.329611522 2.04E-06
Bacteroides 0.255545029 8.52E-06 Rikenellaceae gen. 0.292006876
9.21E-06 Lachnospiraceae gen. 0.081503842 2.04E-06 Coprococcus
0.139659611 1.33E-06 Ruminococcus (Lachno) 0.04645315 6.55E-07
Ruminococcus 0.038383995 6.54E-07 Turicibacter 0.637961763 ns
Lactobacillus 5.145068807 1.37E-05 Anaerostipes 0.463344316
0.004148803 Mucispirillum 0.082908163 8.13E-07 Adlercreutzia
0.03927932 6.48E-07 UC Ruminococcaceae 0.133643617 0.006425379
Roseburia 0.144031142 ns Unclassified Bacteria 0.171195652 2.21E-05
Erysipelotrichaceae gen. 1.188981043 ns Bilophila 0.09375 5.48E-07
Dehalobacterium 0.205078125 1.37E-05 Anaeroplasma 0.61875 ns
Ruminococcaceae gen. 0.457417582 0.006426379 Dorea 0.090604027
1.91E-06 Mogibacteriaceae gen. 0.166666667 1.13E-05 Clostridium
0.1875 0.000131466 Sutterella 0.066964286 1.30E-06 Coprobacillus
1.125 ns UC Erysipelotrichaceae 6 0.013299629 Prevotellaceae gen.
0.625 ns SFB 12.375 0.000803781
[0195] Colitogenic Members of the Intestinal Microbiota are Highly
Coated with IgA in Mice with Inflammasome-Mediated Intestinal
Dysbiosis
[0196] Next tested was whether IgA coating would identify
disease-driving members of the intestinal microbiota in the context
of a colitogenic intestinal dysbiosis. It was recently found that
mice lacking components of the inflammasome, which is a critical
mediator of innate immunity, harbor a colitogenic intestinal
microbiota that can be transmitted to wild type SPF mice through
co-housing. In this model, susceptibility to colitis is driven by
Prevotellaceae species (Elinav et al., 2011, Cell 145:745-757).
Therefore, IgA-SEQ was performed on SPF mice that had acquired
inflammasome-mediated intestinal dysbiosis through co-housing with
Asc.sup.-/- mice (SPF.sup.dys). As previously reported, co-housing
with Asc.sup.-/- mice altered the composition of the SPF intestinal
microbiota and strongly increased susceptibility to
chemically-induced colitis (FIG. 2A, FIG. 2B and FIG. 7A). Flow
cytometric analysis of IgA coating of the intestinal microbiota of
SPF.sup.dys mice at the steady state revealed an increase in the
percentage of intestinal bacteria coated with IgA as compared to
SPF mice, which is consistent with the explanation that acquisition
of the colitogenic microbiota altered the pattern and/or extent of
IgA coating (FIG. 2C). Indeed, IgA+ bacteria in SPF.sup.dys mice
were distinct from IgA- bacteria and from IgA+ bacteria in control
SPF mice sampled under identical conditions (FIG. 7A; P<0.05,
PERMANOVA). Although 23 taxa in SPF.sup.dys mice showed significant
expansion as a result of co-housing, only two of these taxa were
highly coated with IgA (Table 2, FIG. 2D, FIG. 2E and FIG. 7B;
P<0.05 LEfSe); remarkably, the most abundant highly IgA coated
taxon was an unclassified genus from the Prevotellaceae family,
which is the defining taxon in inflammasome-mediated intestinal
dysbiosis and the main driver of colitis in this model (Elinav et
al., 2011, Cell 145:745-757). Furthermore, Helicobacter sp.
flexispira, which is also acquired during co-housing, was highly
coated with IgA in SPF.sup.dys mice. As in SPF mice, Lactobacillus
remained coated and SFB remained highly coated in SPF.sup.dys
mice.
TABLE-US-00002 TABLE 2 SPF (Mean SPF-Dys (Mean Taxa Abundance)
Abundance) P value UC Bacteroidales 0 0.002514286 2.33E-07
Odoribacter 0 0.007171429 2.35E-07 Parabacteroides 0 0.003742857
2.35E-07 Bacteroidales gen. 0 0.004457143 2.35E-07 Paraprevotella 0
0.003885714 2.37E-07 Prevotella 0 0.0218 2.37E-07 Desulfovibrio 0
0.0034 2.37E-07 UC Helicobacteraceae 0 0.044285714 2.37E-07 AF12
1.17647E-05 0.001628571 5.51E-07 UC Prevotellaceae 5.88235E-05
0.051957143 9.14E-07 UC Clostridia 0 0.001828571 1.00E-06 Rikenella
0 0.000371429 1.34E-05 Dorea 0.000988235 0.005414286 2.26E-05
Allobaculum 1.17647E-05 0.004542857 3.14E-05 UC Erysipelotrichaceae
5.88235E-05 0.018457143 3.58E-05 Streptococcus 0 0.000257143
4.59E-05 Desulfovibrionaceae gen. 0 0.000271429 0.000144935
Lactobacillus 0.007670588 0.021142857 0.000379843 YS2 gen 0
0.000357143 0.000445906 Dehalobacterium 0.001717647 0.003114286
0.001100491 Coriobacteriaceae gen. 8.23529E-05 0.000528571
0.001875312 Helicobacter sp. flexispira 0 0.000342857 0.003324003
Ruminococcus (Lachno) 0.0124 0.021985714 0.006514397
[0197] Strikingly, all of the bacteria that were found to be highly
coated in SPF.sup.dYs mice (Prevotellaceae, Helicobacter and SFB)
are known to drive intestinal inflammation and disease development
in mouse models of colitis (Elinav et al., 2011, Cell 145:745-757;
Kullberg et al., 1998, Infect Immun 66:5157-5166; Stepankova et
al., 2007, Inflamm Bowel Dis 13:1202-1211). In addition, SFB is a
potent driver of intestinal T helper 17 (Th17) cell responses in
mice and has been shown to exacerbate development of arthritis
(Ivanov et al., 2009, Cell 139:485-498; Wu et al., 2010, Immunity
32:815-827).
[0198] Antigen-specific binding of IgA to the intestinal microbiota
can result from both high affinity, T cell-dependent responses and
lower affinity, T cell-independent responses (Bemark et al., 2012,
Ann NY Acad Sci 1247:97-116). In addition, IgA can bind members of
the intestinal microbiota non-specifically, for example through
glycan-dependent binding to certain Gram-positive bacteria (Mathias
and Corthesy, 2011, Gut Microbes 2:287-293). Importantly, IgA
coating of SFB, UC Prevotellaceae, and Helicobacter sp. flexispira
was found to be significantly reduced in SPF.sup.dys mice lacking T
cells, which shows that high IgA coating detected via IgA-SEQ is
largely the result of high-affinity, antigen-specific, T
cell-dependent antibody responses rather than low-affinity T
cell-independent responses (FIG. 7C). In contrast, coating of
Lactobacillus, which was significantly but not highly coated (ICI
<10), was increased in T cell-deficient mice, demonstrating that
these IgA antibodies resulted from either T cell-independent immune
responses or non-specific binding.
[0199] IgA-SEQ Identifies Highly Coated Members of the Intestinal
Microbiota in IBD Patients
[0200] Interactions between the intestinal microbiota and the
immune system play a critical role in IBD development and
progression in humans; however, the specific bacteria responsible
for these effects have remained elusive (Abraham and Cho, 2009, New
Engl J Med 361: 2066-2078; Knights et al., 2013, Gut 62:1505-1510;
Round and Mazmanian, 2009, Nat Rev Immunol 9:313-323). Since the
data show that IgA coating can identify colitogenic members of the
intestinal microbiota in mice, coating of fecal bacteria from 27
patients with Crohn's disease (CD), 8 patients with ulcerative
colitis (UC) and 20 healthy controls was next examined to identify
such organisms in human disease. Similar to mice with intestinal
dysbiosis, and as previously reported (van der Waaij et al., 2004,
Eur J Gastroen Hepat 16:669-674), the proportion of intestinal
bacteria that are coated with IgA was significantly increased in CD
and UC patients as compared to healthy controls (FIG. 3A and FIG.
6D). As expected, both healthy control subjects and IBD patients
exhibited considerable diversity in their gut microbiota
compositions and patterns of IgA coating (FIG. 8). Individual ICI
scores for bacteria in each subject can be found in Table S3 of
Palm et al. (Palm et al., 2014, Cell, 158(5): 1000-1010) and in
Table 3 of U.S. Provisional Patent Application No. 62,042,878, each
of which is herein incorporated by reference. While many species
were highly coated (ICI >10) in both IBD and control groups, 35
species were uniquely highly coated in patients with IBD (FIG. 3B
and FIG. 3C). For example, Streptococcus luteciae, Haemophilus
parainfluenzae, and Collinsella aerofaciens were detected in both
IBD patients and some healthy controls, but were only highly coated
in IBD. In addition, multiple species that were uniquely present in
IBD were highly coated in at least one patient (e.g., unclassified
Bulleidia, Allobaculum spp., Lactobacillus mucosae, unclassified
Pediococcus, and Weissella spp.). Finally, both CD- and UC-specific
highly IgA coated bacterial species could be observed; for
instance, unclassified Clostridiales, unclassified Ruminococcaceae,
and Blautia spp. were uniquely coated in CD, and Eubacterium
dolichum and Eggerthella lenta were uniquely coated in UC.
[0201] Establishment of a Gnotobiotic Mouse Model to Evaluate the
Effects of IgA+ and IgA- Members of the Human Microbiota on
Intestinal Inflammation
[0202] In order to directly test whether IgA coating marks human
intestinal bacteria that preferentially drive intestinal
inflammation, isolation of representative IgA coated and non-coated
bacteria from human IBD patients was attempted. Personalized gut
microbiota culture collections were assembled from eleven IBD
patients using standard anaerobic culture media and a custom rich
medium designed to recover intestinal bacterial species from humans
(Goodman et al., 2011, P Natl Acad Sci USA 108:6252-6257). First,
100-200 single colonies were selected and cultured per patient
sample, and these isolates were taxonomically classified through
high-throughput 16S sequencing (FIG. 4A). The individual bacterial
isolates in each of the personalized gut microbiota culture
collections were then cross-referenced with the matching data from
the IgA-SEQ studies to classify all isolates based on their level
of IgA coating. Finally, individual isolates from these culture
collections were rationally selected and combined to assemble
representative consortia consisting of either isolates that were
classified as highly coated (IgA+ consortium) or isolates that were
classified as low coated (IgA- consortium) (FIG. 4B; see methods
for criteria used to select the IgA+ and IgA- consortia).
Importantly, the taxa comprising the IgA+ and IgA- consortia would
not have been chosen simply based on traditional evaluations of the
relative abundance of these taxa in healthy versus sick individuals
(FIG. 9).
[0203] To directly test the effects of IgA+ versus IgA- bacteria
from IBD patients on intestinal inflammation, germ-free mice were
colonized with the assembled IgA+ or IgA- consortia. As a first
test of the feasibility of this system, the composition of the
intestinal microbiota in these mice was examined two weeks
post-colonization and it was found that all but two bacteria, one
from each consortium, were able to successfully colonize germ-free
mice (FIG. 4C).
[0204] Next was tested whether the human IgA+ consortium would also
preferentially induce the production of IgA when transplanted into
germ-free mice. At seven days post-colonization, before the
induction of a specific IgA response, fecal bacteria from mice
colonized with the IgA+ or IgA- consortia was found to show
equivalent low levels of IgA coating by flow cytometry (FIG. 4D).
However, as compared to the IgA- consortium, the IgA+ consortium
showed dramatically higher levels of IgA coating by day 24
post-colonization, which demonstrates that the IgA+ consortium had
induced a strong and specific IgA response. These data show that
bacteria that preferentially drive IgA responses in human IBD
patients can also drive strong IgA responses in gnotobiotic
mice.
[0205] The bacteria that were identified as highly coated in
SPF.sup.dys mice are known to colonize normally sterile mucosal
environments, such as the inner mucus layer and intestinal crypts
(Elinav et al., 2011, Cell 145:745-757; Ivanov et al., 2009, Cell
139:485-498). To test whether the bacteria in the human IgA+
consortium also exhibit such characteristics, bacterial 16S rRNA
fluorescence in situ hybridization (FISH) was performed on colons
from IgA+ and IgA- consortia colonized mice. Remarkably, the
presence of many bacteria was observed in the mucus layer of mice
colonized with the IgA+ consortium (FIG. 4E). In contrast, in mice
colonized with the IgA- consortium, the inner mucus layer remained
devoid of any detectable bacteria. Thus, one mechanism by which the
bacteria comprising the IgA+ consortium may selectively induce IgA
responses is through the invasion or colonization of normally
sterile mucosal environments.
[0206] IgA Inducing Members of the Intestinal Microbiota Cultured
from IBD Patients Exacerbate DSS Colitis in Gnotobiotic Mice
[0207] Germ-free mice colonized with the IgA- and IgA+ consortia
for two weeks showed no signs of spontaneous intestinal pathology,
which is consistent with the explanation that these bacterial
strains are not immediately pathogenic in wild-type mice under
healthy conditions. However, after the induction of colitis with
DSS, IgA+ mice exhibited obvious and severe intestinal inflammation
with extensive bleeding throughout the intestine, as well as
significant shortening of the colon, while the intestines of IgA-
mice appeared normal (FIG. 5A-FIG. 5C). Histological examination
revealed that IgA+ mice showed significant cellular infiltration
and extensive loss of tissue integrity in the colon, while IgA-
mice showed minimal visible inflammation (FIG. 5D and FIG. 5E).
These data show that bacteria isolated from IBD patients and chosen
based on high IgA coating selectively drive severe intestinal
inflammation in a mouse model of IBD.
[0208] Bacterial isolates from different patients that were
taxonomically assigned as the same species via 16S sequencing often
showed differential IgA coating. While many factors may contribute
to this phenomenon, the observation was consistent with the
explanation that these isolates represent distinct bacterial
strains of the same species that display divergent behaviors that
lead to differential IgA induction. As a proof-of-principle test of
this scenario, two isolates of B. fragilis from the gut microbiota
culture collections were identified and characterized and showed
either high (ICI=37.8) or low (ICI=0.68) IgA coating (FIG. 10F).
Whole genome sequencing demonstrated that these isolates represent
genetically distinct strains of the same species (i.e., B.
fragilis) (FIG. 10G). Finally, germ-free mice were monocolonized
with these two strains and their effects on the development of
DSS-induced colitis were examined. Mice colonized with the IgA+
strain of B. fragilis were found to exhibit more severe colitis
than the mice colonized with the IgA- strain of B. fragilis, as
measured by colon length and histopathology (FIG. 10H-FIG. 10J).
These data demonstrate that different strains of the same bacterial
species exhibit differential effects on the intestinal immune
system and inflammatory disease and that they can be distinguished
based on IgA coating.
[0209] IgA Coating of Intestinal Microbiota
[0210] Taxa-specific coating of the intestinal microbiota with the
secreted immunoglobulin IgA was examined based on the hypothesis
that levels of IgA coating might distinguish between members of the
microbiota that impact disease susceptibility and/or severity by
stimulating inflammatory responses and the remaining members of the
microbiota. High coating with IgA was found to specifically mark a
select group of known inflammation- and disease-driving intestinal
bacteria in mice with inflammasome-mediated colitogenic dysbiosis.
Bacteria isolated from patients with IBD and selected based on high
IgA coating was found to induce potent IgA responses and
dramatically exacerbate development of DSS-induced colitis in
gnotobiotic mice. Thus, the data demonstrate that high coating with
IgA selectively marks inflammatory, and therefore, potentially
disease-driving commensals in mice and humans.
[0211] Bacterial cell-sorting based on IgA coating and 16S rRNA
gene sequencing were combined in order to examine the intestinal
immune response to the intestinal microbiota in an unbiased and
comprehensive manner. Recently, FACS-based bacterial cell sorting
has been combined with next-generation sequencing by others as a
way to examine: the active human gut microbiota (Peris-Bondia et
al., 2011, PLoS One 6:e22448); responses of the intestinal
microbiota to xenobiotics (Maurice et al., 2013, Cell 152:39-50);
the effect of IgA coating on bacterial gene expression (Cullender
et al., 2013, Cell Host Microbe 14:571-581); and IgA coating of the
healthy intestinal microbiota (D'Auria et al., 2013, Sci Rep
3:3515). This type of approach, which combines taxonomic
information with functional information regarding bacterial
viability, behavior, or other bacterial features, will likely
become increasingly common in future studies of the microbiota and
its interactions with the host. The data clearly illustrate the
utility of this approach as a way to functionally classify
intestinal bacteria based on their interactions with and
recognition by the host immune system.
[0212] To maintain intestinal homeostasis, the mucosal immune
system must selectively recognize and respond to pathogenic species
while simultaneously maintaining tolerance to harmless and
symbiotic members of the intestinal microbiota (Belkaid and Hand,
2014, Cell 157:121-141). Because most innate immune receptors
involved in the detection of bacterial pathogens sense microbial
components present in both pathogens and commensals (e.g.,
lipopolysaccharide), the mechanisms by which the immune system
distinguishes between pathogens and commensals remain largely
unknown. However, one way the immune system is thought to
distinguish between pathogens and commensals is by sensing
pathogen-associated activities or behaviors, such as adherence to
the intestinal epithelium, tissue invasion or destruction, or the
ability to colonize normally sterile mucosal environments, such as
intestinal crypts (Sansonetti, 2011, Mucosal Immunol 4:8-14). The
inflammatory commensals that were identified via IgA-SEQ appear to
exhibit similar activities or behaviors. For example,
Prevotellaceae species invade the mucus layer in the large
intestine and colonize colonic crypts (Elinav et al., 2011, Cell
145:745-757); furthermore, SFB firmly adhere to the epithelium in
the small intestine (Ivanov et al., 2009, Cell 139:485-498).
Finally, members of the human IgA+ consortium were found to be
observed in the colonic mucus layer. Since the invasion of normally
sterile sites proximal to the epithelium would naturally lead to
increased stimulation of the innate immune system and increased
availability of antigen for the induction of specific T cell and
antibody responses, these behaviors may at least partially explain
the propensity of IgA coated bacteria to preferentially induce both
IgA responses and the inflammatory responses that lead to
exacerbated disease susceptibility.
[0213] A variety of specific innate and adaptive immune mechanisms
are known to influence IgA responses to the intestinal microbiota.
For example, the Toll-like receptors have been implicated both
T-dependent and T-independent IgA responses to the gut microbiota
(Tezuka et al., 2007, Nature 448:929-933). Furthermore, specific T
cell subsets, including T helper type 17 cells and regulatory T
cells, as well as gamma-delta T cells have been implicated in
coordinating IgA responses (Cong et al., 2009, P Natl Acad Sci USA
106:19256-19261; Fujihashi et al., 1996, J Exp Med 183:1929-1935;
Hirota et al., 2013, Nat Immunol 14:372-379; Kawamoto et al., 2012,
Science 336:485-489). The specific responses observed by IgA-SEQ
were largely T-dependent; however, T-independent responses also
clearly contributed to IgA responses to specific taxa.
[0214] Since highly IgA coated bacteria are constitutive
inhabitants of the intestine and can drive disease, it appears that
the host's IgA response to these bacteria is insufficient to lead
to bacterial clearance or complete neutralization. Nonetheless, the
IgA response may still reduce the level of inflammation caused by
such bacteria. Indeed, SFB expand in the absence of an effective
IgA response (Kato et al., 2014, Immunol Cell Biol 92:49-56;
Shinkura et al., 2004, Nat Immunol 5:707-712). Furthermore,
bacterial-specific IgA has been shown to minimize intestinal
inflammation through bacterial exclusion (Peterson et al., 2007,
Cell Host Microbe 2:328-339). IgA inducing bacteria may drive even
stronger pathological inflammatory responses in mice that are
unable to mount an efficient IgA response.
[0215] The etiology of IBD involves a combination of genetic,
environmental and microbial factors (Abraham and Cho, 2009, New
Engl J Med 361: 2066-2078; Knights et al., 2013, Gut 62:1505-1510).
Here, the microbial contribution to IBD was focused upon and
attempts were made to identify members of the human gut microbiota
that may preferentially impact IBD susceptibility and/or severity.
One reason why it has been difficult to identify disease-driving
bacteria in humans is that the strategies traditionally used to
identify these bacteria in mice, including co-housing and gut
microbiota transfer cannot be applied to humans (Dantas et al.,
2013, Annu Rev Microbiol 67:459-475). In addition, due to the
diversity of the intestinal microbiota in humans, the specific
bacteria that drive disease may differ dramatically from patient to
patient and, therefore, identifying these bacteria may require an
individualized, rather than population-based, approach (Huttenhower
and Consortium, 2012, Nature 486:207-214; Lozupone et al., 2012,
Nature 489:220-230). High IgA coating was found to be able to
identify colitogenic bacteria from patients with IBD by combining:
(i) a functional classification of the intestinal microbiota based
on the host's individual immune response; (ii) anaerobic culturing
of members of the intestinal microbiota from diseased patients; and
(iii) colonization of germ-free mice with human microbial consortia
selected rationally based on their propensity to induce
inflammation and, therefore, become highly coated with IgA. Using
this approach, a subset of the intestinal microbiota from IBD
patients that is characterized by high coating with IgA was shown
to selectively confer susceptibility to colitis in a mouse model of
IBD. Thus, the data demonstrate that the host's individual IgA
response to the intestinal microbiota can be used as a guide to
identify members of the microbiota that preferentially impact
disease susceptibility and/or severity. The ability to identify
these important bacterial taxa in humans in an individualized
manner represents a first step towards the development of
personalized, microbiota-focused therapies that may reduce,
reverse, or even prevent disease development through targeted
elimination or replacement of disease-driving members of the
intestinal microbiota.
[0216] IgA+ Bacteria from Healthy Humans, Unlike IgA+ Bacteria from
Humans with IBD, are Non-Colitogenic
[0217] As described above, it is shown herein that IgA coating
marks colitogenic bacteria in patients with IBD. However it was
found that healthy humans also harbored similar numbers of highly
IgA coated bacteria. The fact that these bacteria induce high
levels of specific IgA suggests that they intimately interact with
the intestinal immune system, but clearly are not driving
pathological intestinal inflammation since their human hosts remain
healthy. Two major possibilities may explain this observation: 1)
the IgA-coated bacteria in healthy humans and IBD patients are
equally capable of driving intestinal inflammation but host
genetics or environmental factors dictate whether disease will
actually occur; or, 2) the IgA-coated bacteria in healthy humans
are fundamentally different from those found in patients with
IBD.
[0218] To directly test whether IgA+ bacteria from healthy humans
have similar or different effects as compared to IgA+ bacteria from
IBD patents, five bacterial strains, cultured from healthy
subjects, were selected that were highly coated with IgA as based
on IgA-SEQ analysis (ICI score >10) in the healthy subject from
whom they were isolated. These healthy IgA+ strains were next
assembled into a representative bacterial consortium. As an initial
test of whether IgA+ bacteria from healthy humans are inflammatory
or immunoregulatory, germ-free mice were colonized with either the
`IgA+ (healthy)` consortium, the colitogenic `IgA+ (IBD)`
consortium, or the non-colitogenic `IgA-` consortium. Colitis was
then induced by administration of 2.5% DSS in the water. As
described previously, the `IgA+ (IBD)` consortium induced severe
colitis, as measured by colon shortening at day 6, while the `IgA-`
consortium did not induce significant colitis. However, unlike the
`IgA+ (IBD)` consortium, the `IgA+ (healthy)` consortium appeared
to result in mild protection from colitis as compared to the `IgA-`
consortium (FIG. 11). These results suggest that `IgA+ ` bacteria
from healthy individuals are qualitatively different from `IgA+ `
bacteria in IBD and may induce immunoregulatory responses that are
associated with a healthy intestine.
[0219] IgA+ Bacteria from Healthy Humans can Protect Against
Bacterial-Driven Colitis
[0220] The finding that `IgA+ ` bacteria from healthy individuals
are non-colitogenic raises the possibility that these bacteria may
be able to protect against colitis. Therefore, it was next tested
whether IgA+ bacteria from healthy humans could reduce or
neutralize the pathogenic effects of colitogenic IgA+ bacteria from
IBD patients. Germ-free mice were colonized with either the
non-colitogenic `IgA-` consortium, the colitogenic `IgA+ (IBD)`
consortium, or a mixture of the colitogenic `IgA+ (IBD)` consortium
and the `IgA+ (healthy)` consortium. One week after colonization,
colitis was induced by administration of 2% DSS ad libitum in the
drinking water. As described previously, the `IgA+ (IBD)`
consortium induced severe colitis, as measured by colon shortening
at day 6, while the `IgA-` consortium induced only mild colitis.
However, the `IgA+ (healthy)` consortium blocked the colitogenic
effects of the `IgA+ (IBD)` consortium completely (FIG. 12). These
results suggest that `IgA+ ` bacteria from healthy individuals can
protect against colitis driven by bacteria found in IBD patients
and show that IgA-SEQ can be used to identify protective bacteria
in healthy individuals.
[0221] Oral Immunization can Protect Against Colitis Driven by
Bacteria Found in IBD Patients
[0222] It was found that specific individual IgA-inducing members
of the gut microbiota isolated from patients with IBD were potently
colitogenic when transferred into germ-free mice. For instance,
mice colonized with the non-colitogenic `IgA-` community did not
show significant signs of colitis when treated with 2% DSS for 6
days, while the `IgA-` community together with the IBD-associated
IgA-inducing bacterium Erysipelotrichaceae spp. led to potent
intestinal inflammation. The observation that the presence of one
individual inflammatory IgA+ bacterial species can transform a
`neutral` microbiota into a dysbiotic, inflammatory microbiota
suggests that neutralizing or removing this inflammatory bacterial
species from the microbiota should ameliorate colitis. To test this
possibility, groups of germ-free mice were colonized with the
`IgA-` community together with Erysipelotrichaceae spp. (`IgA- plus
Ery`) for one week to mimic the microbiota of IBD patients
containing an individual inflammatory bacterial species amongst a
majority of neutral bacterial species. Next, these mice were orally
immunized with heat-killed Erysipelotrichaceae spp. with cholera
toxin (CT) as an adjuvant. Using such low levels of CT as an
adjuvant does not result in intestinal inflammation due to the
toxin but rather induces a highly potent secretory IgA response to
the heat-killed bacteria with which it is co-administered. After
weekly immunizations for 6 weeks, these mice, along with control
mice that were either non-immunized or mock-immunized with CT
alone, were treated with 1.8% DSS to induce colitis. Interestingly,
while non-immunized and mock-immunized mice showed severe
inflammation, as demonstrated by shortening of the colon, mice
immunized with heat-killed Erysipelotrichaceae spp. showed
significant protection from intestinal pathology (FIG. 13). These
results demonstrate that targeting one or more inflammatory IgA+
bacterial species through vaccination can ameliorate inflammatory
responses in the intestine, and suggest that targeting similar
species in IBD patients may be a feasible approach to treat or cure
their disease.
[0223] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention
may be devised by others skilled in the art without departing from
the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
Sequence CWU 1
1
1118DNAArtificial SequenceChemically Synthesized 1gctgcctccc
gtaggagt 18
* * * * *
References