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Title:
COMPOSITIONS FOR MODULATING GUT MICROFLORA POPULATIONS, ENHANCING DRUG POTENCY AND TREATING CANCER, AND METHODS FOR MAKING AND USING SAME
Document Type and Number:
WIPO Patent Application WO/2021/127235
Kind Code:
A1
Abstract:
Provided are compositions, including products of manufacture and kits, and methods, comprising combinations of microbes, such as non-pathogenic, live bacteria and/or bacterial spores, for the control, amelioration, prevention, and treatment of a disease or condition, for example, a cancer. In alternative embodiment, these non-pathogenic, live bacteria and/or bacterial spores are administered to an individual in need thereof, thereby resulting in a modification or modulation of the individual's gut microfloral population(s). In alternative embodiments, by modulating or modifying the individual's gut microbial population(s) using compositions, products of manufacture and methods as provided herein, the pharmacodynamics of a drug administered to the individual is altered, thereby controlling, ameliorating, preventing and/or treating of that cancer. Combinations of microbes are administered with chemotherapy, radiation therapy, an immune checkpoint inhibitor, a Chimeric Antigen Receptor T-cell therapy or other immunotherapy or cancer treatment.

Inventors:
CULLER STEPHANIE J (US)
HASELBECK ROBERT J (US)
VAN DIEN STEPHEN (US)
SWAMINATHAN ANANDH (US)
SATO HIROKAZU (US)
Application Number:
PCT/US2020/065693
Publication Date:
June 24, 2021
Filing Date:
December 17, 2020
Export Citation:
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Assignee:
PERSEPHONE BIOSCIENCES INC (US)
International Classes:
A61K35/38; A61K35/74; A61K35/741; A61P35/00
Domestic Patent References:
WO2019178542A12019-09-19
WO2016063263A22016-04-28
Other References:
See also references of EP 4076482A4
Attorney, Agent or Firm:
EINHORN, Gregory P. (US)
Download PDF:
Claims:
WHAT IS CLAIMED IS:

1. A method for controlling, ameliorating, preventing or treating a cancer in an individual in need thereof, comprising:

(a) administering or having administered to an individual in need thereof a formulation comprising at least two different species or genera (or types) of non- pathogenic bacteria, wherein each of the non-pathogenic bacteria comprise (or are in the form of) a plurality of non-pathogenic colony forming live bacteria, a plurality of non-pathogenic germinable bacterial spores, or a combination thereof; or,

(b) (i) providing a formulation comprising at least two different species or genera (or types) of non-pathogenic bacteria, wherein each of the non-pathogenic bacteria comprise (or are in the form of) a plurality of non-pathogenic colony forming live bacteria, a plurality of non-pathogenic germinable bacterial spores, or a combination thereof, and

(ii) administering or having administered to an individual in need thereof the formulation; wherein the formulation comprises a or any combination of at least two different species or genera of non-pathogenic, live bacteria, or spore thereof, if the bacteria is spore forming, as described Table 1, 5, 10, 11, or 12, or live biotherapeutic compositions or combinations of bacteria as set forth in Table 15 or 16, and optionally the different species or genera (or types) of non-pathogenic, live bacteria are present in approximately equal amounts, or each of the different species or genera (or types) of non-pathogenic, live bacteria or non-pathogenic germinable bacterial spores represent at least about 1%, 5%, 10%, 20%, 30%, 40%, or 50% or more, or between about 1% and 75%, of the total amount of non-pathogenic, live bacteria and non-pathogenic germinable bacterial spores in the formulation, and optionally only or substantially only non-pathogenic, live bacteria are present in the formulation, or only or substantially only non-pathogenic germinable bacterial spores are present in the formulation, or approximately equal amounts of non-pathogenic, live bacteria and non-pathogenic germinable bacterial spores are present in the formulation.

2. The method of claim 1, further comprising administering or having administered one or any one of: a chemotherapy, a radiation therapy, an immune checkpoint inhibitor, a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or an immunotherapy or a cancer treatment, or a combination thereof, and optionally the chemotherapy, the radiation therapy, the immune checkpoint inhibitor, the Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or the immunotherapy or the cancer treatment, or the combination thereof, is administered before, during (concurrently with) and/or after administration the formulation.

3. The method of claim 1, or claim 2 wherein:

(a) the formulation comprises an inner core surrounded by an outer layer of polymeric material enveloping the inner core, wherein the non-pathogenic bacteria or the non-pathogenic germinable bacterial spores are substantially in the inner core, and optionally the polymeric material comprises a natural polymeric material;

(b) the formulation is formulated or manufactured as or in: a nano-suspension delivery system; an encochleated formulation; or, as a multilayer crystalline, spiral structure with no internal aqueous space;

(c) the formulation is formulated or manufactured as a delayed or gradual enteric release composition or formulation, and optionally the formulation comprises a gastro-resistant coating designed to dissolve at a pH of 7 in the terminal ileum, optionally an active ingredient is coated with an acrylic based resin or equivalent, optionally a poly(meth)acrylate, optionally a methacrylic acid copolymer B, NF, optionally EUDRAGIT S™ (Evonik Industries AG, Essen, Germany), which dissolves at pH 7 or greater, optionally comprises a multimatrix (MMX) formulation, and optionally manufactured as enteric coated to bypass the acid of the stomach and bile of the duodenum.

4. The method of any of the preceding claims, or a method of claims 1 to 3, wherein the plurality of non-pathogenic colony forming live bacteria are substantially dormant colony forming live bacteria, or the plurality of non-pathogenic colony forming live bacteria or the plurality of non-pathogenic germinable bacterial spores are lyophilized, wherein optionally the dormant colony forming live bacteria comprise live vegetative bacterial cells that have been rendered dormant by lyophilization or freeze drying.

5. The method of any of the preceding claims, or a method of claims 1 to

4, wherein the formulation comprises at least about 1 x 104 colony forming units (CFUs), or between about 1 x 101 and 1 x 1013 CFUs, 1 x 102 and 1 x 1010 CFUs, 1 x 102 and 1 x 108 CFUs, 1 x 103 and 1 x 107 CFUs, or 1 x 104 and 1 x 106 CFUs, of non-pathogenic live bacteria and/or non-pathogenic germinable bacterial spores.

6. The method of any of the preceding claims, or a method of claims 1 to

5, wherein the formulation comprises at least one (or any one, several, or all of) non- pathogenic bacteria or spore of the family or genus (or class): Agathobaculum (TaxID: 2048137), Alistipes (TaxID: 239759), Anaeromassilibacillus (TaxID: 1924093), Anaerostipes (TaxID: 207244), Asaccharobacter (TaxID: 553372), Bacteroides (TaxID: 816), Barnesiella (TaxID: 397864), Bifidobacterium (TaxID: 1678), Blautia (TaxID: 572511), Butyricicoccus (TaxID: 580596), Clostridium (TaxID: 1485), Collinsella (TaxID: 102106), Coprococcus (TaxID: 33042), Dorea (TaxID: 189330), Eubacterium (TaxID: 1730), Faecalibacterium (TaxID: 216851), Fusicatenibacter (TaxID: 1407607), Gemmiger (TaxID: 204475), Gordonibacter (TaxID: 644652), Lachnoclostridium (TaxID: 1506553), Methanobrevibacter (TaxID: 2172), Parabacteroides (TaxID: 375288), Romboutsia (TaxID: 1501226), Roseburia (TaxID: 841), Ruminococcus (TaxID: 1263), Erysipelotrichaceae (TaxID: 128827), Coprobacillus (TaxID: 100883), Erysipelatoclostridium sp. SNUG30099 (TaxID: 1982626), Erysipelatoclostridium (TaxID: 1505663), Acetatifactor (TaxID: 1427378), Adlercreutzia (TaxID: 447020), Agathobacter (TaxID: 1766253), Anaerotruncus (TaxID: 244127), Bariatricus (TaxID: 1924081), Butyrivibrio (TaxID: 830), Christensenellaceae (TaxID: 990719), Clostridiales (TaxID: 186802), Dialister (TaxID: 39948), Drancourtella (TaxID: 1903506), Eggerthella (TaxID: 84111), Eisenbergiella (TaxID: 1432051), Enterocloster (TaxID: 2719313), Enterococcus (TaxID: 1350), Intestinibacter (TaxID: 1505657), Lachnospira (TaxID: 28050), Lachnospiraceae (TaxID: 186803), Mediterraneibacter (TaxID: 2316020), Negativibacillus (TaxID: 1980693), Oscillibacter (TaxID: 459786), Phocaeicola (TaxID: 909656), Pseudobutyrivibrio (TaxID: 46205), Pseudoflavonifractor (TaxID: 1017280), Ruminococcaceae (TaxID: 541000), Sellimonas (TaxID:

1769710), Solobacterium (TaxID: 123375), Terrisporobacter (TaxID: 1505652), Tidjanibacter (TaxID: 1929083), Veillonella (TaxID: 29465), or a combination thereof.

7. The method of any of the preceding claims, or a method of claims 1 to

6, wherein the formulation comprises at least one (or any one, several, or all of) non- pathogenic bacteria or spore form thereof as set forth in Tables 1, 5, 10, 11, or 12, or included in the combination of non-pathogenic bacteria and/or spores thereof (or spore derived from) as set forth in Table 15 or Table 16.

8. The method of any of the preceding claims, or a method of claims 1 to

7, wherein the formulation comprises combination of non-pathogenic bacteria and/or spores thereof (or spore derived from) as set forth in Table 15 or Table 16.

9. The method of any of the preceding claims, or a method of claims 1 to

8, wherein the formulation comprises water, sterile water, saline, sterile saline, a pharmaceutically acceptable preservative, a carrier, a buffer, a diluent, an adjuvant or a combination thereof.

10. The method of any of the preceding claims, or a method of claims 1 to

9, wherein the formulation is administered orally or rectally, or is formulated and/or administered as a liquid, a food, a gel, a candy, an ice, a lozenge, a tablet, pill or capsule, or a suppository or as an enema formulation, or the formulation is administered as an or is in a form for intra-rectal or intra-colonic administration.

11. The method of any of the preceding claims, or a method of claims 1 to

10, wherein the formulation is administered to the individual in need thereof in one, two, three, or four or more doses, and wherein the one, two, three, or four or more doses are administered on a daily basis (optionally once a day, bid or tid), every other day, every third day, or about once a week, and optionally the two, three, or four or more doses are administered at least a week apart (or dosages are separated by about a week).

12. The method of any of the preceding claims, or a method of claims 1 to 11, wherein the formulation further comprises an antibiotic, or the method further comprises administration of an antibiotic, and optionally at least one dose of the antibiotic is administered before a first administration of the formulation, optionally at least one dose of the antibiotic is administered one day or two days, or more, before a first administration of the formulation.

13. The method of any of the preceding claims, or a method of claims 1 to

12, wherein the inhibitor of the inhibitory immune checkpoint molecule comprises a protein or polypeptide that binds to an inhibitory immune checkpoint protein, and optionally inhibitor of the inhibitory immune checkpoint protein is an antibody or an antigen binding fragment thereof that specifically binds to the inhibitory immune checkpoint protein.

14. The method of any of the preceding claims, or a method of claims 1 to

13, wherein the inhibitor of the inhibitory immune checkpoint molecule targets a compound or protein comprising: a CTLA4 or CTLA-4 (cytotoxic T-lymphocyte- associated protein 4, also known as CD 152, or cluster of differentiation 152); Programmed cell Death protein 1, also known as PD-1 or CD279; Programmed Death-Ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)); PD-L2; A2AR (adenosine A2A receptor, also known as ADORA2A); B7-H3; B7-H4; BTLA (B- and T-lymphocyte attenuator protein); KIR (Killer-cell Immunoglobulin-like Receptor); IDO (Indoleamine-pyrrole 2,3- di oxygenase); LAG3 (Lymphocyte- Activation Gene 3 protein); TIM-3; VISTA (V- domain Ig suppressor of T cell activation protein); or any combination thereof.

15. The method of any of the preceding claims, or a method of claims 1 to

14, wherein the inhibitor of an inhibitory immune checkpoint molecule comprises: ipilimumab or YERVOY®; pembrolizumab or KEYTRUDA®; nivolumab or OPDIVO®; atezolizumab or TECENTRIQ®; avelumab or BAVENCIO®; durvalumab or IMFINZI®; AMP-224 (Medlmmune), AMP-514 (an anti-programmed cell death 1 (PD-1) monoclonal antibody (mAh) (Medlmmune)), PDR001 (a humanized mAh that targets PD-1), STI-A1110 or STI-A1010 (Sorrento Therapeutics), BMS-936559 (Bristol-Myers Squibb), BMS-986016 (Bristol-Myers Squibb), TSR-042 (Tesaro), JNJ-61610588 (Janssen Research & Development), MSB-0020718C, AUR-012, enoblituzumab (also known as MGA271) (MacroGenics, Inc.), MBG453, LAG525 (Novartis), BMS-986015 (Bristol-Myers Squibb), cemiplimab (or LIBTAYO®) (Regeneron), or any combination thereof.

16. The method of any of the preceding claims, or a method of claims 1 to

15, wherein the inhibitor of the inhibitory immune checkpoint molecule, or the stimulatory immune checkpoint molecule, is administered by: intravenous (IV) injection, intramuscular (IM) injection, intratumoral injection or subcutaneous injection; or, is administered orally or by suppository; or the formulation further comprises at least one immune checkpoint inhibitor.

17. The method of any of the preceding claims, or a method of claims 1 to

16, wherein the cancer is melanoma, advanced melanoma, cutaneous or intraocular melanoma, primary neuroendocrine carcinoma of the skin, breast cancer, a cancer of the head and neck, uterine cancer, rectal and colorectal cancer, a cancer of the head and neck, cancer of the small intestine, a colon cancer, a cancer of the anal region, a stomach cancer, lung cancer, brain cancer, non-small-cell lung cancer, ovarian cancer, angiosarcoma, bone cancer, osteosarcoma, prostate cancer; cancer of the bladder; cancer of the kidney or ureter or renal cell carcinoma, or carcinoma of the renal pelvis; a neoplasm of the central nervous system (CNS) or renal cell carcinoma.

18. The method of any of the preceding claims, or a method of claims 1 to

17, comprising, or further comprising, administering, or having administered, or delivering, a genetically (or recombinantly) engineered cell, wherein optionally the genetically engineered cell is: a microbe or spore derived from a microbe as used in a method of any of the preceding claims, or a method of claims 1 to 17; or, a non-pathogenic bacteria or spore form thereof as set forth in Tables 1, 5, 10, 11, or 12; or, a non-pathogenic bacteria or spore form thereof included in a combination of non-pathogenic bacteria and/or spores thereof (or spore derived from) as set forth in Table 15 or Table 16, and optionally the microbe is genetically engineered to express or secrete a heterologous or overexpress an endogenous immunomodulatory molecule, and optionally the immunomodulatory molecule is an immunomodulatory protein or peptide, and optionally the immunomodulatory molecule is an immunostimulatory molecule, and optionally the microbe is genetically engineered to overexpress a pathway for production of at least one short chain fatty acid (SCFA), and optionally the SCFA comprises butyrate or butyric acid, propionate or acetate, and optionally the microbe is genetically engineered by inserting a heterologous nucleic acid into the microbe, and optionally the heterologous nucleic acid encodes an exogenous membrane protein, and optionally the immunostimulatory molecule, protein or peptide comprises a non-specific immunostimulatory protein, and optionally the non-specific immunostimulatory protein comprises a cytokine, and optionally the cytokine comprises an interferon (optionally an IFN-a2a, IFN-a2b), and interleukin (optionally IL-2, IL-4, IL-7, IL-12), an interferon (IFN), a TNF-a, a granulocyte colony- stimulating factor (G-CSF, also known as filgrastim, lenograstim or Neupogen®), a granulocyte monocyte colony-stimulating factor (GM-CSF, also known as molgramostim, sargramostim, Leukomax®, Mielogen® or Leukine®), or any combination thereof, and optionally the immunostimulatory molecule, protein or peptide comprises a specific immunostimulatory protein or peptide, and optionally the specific immunostimulatory protein or peptide comprises an immunogen that can generate a specific humeral or cellular immune response or an immune response to a cancer antigen, and optionally the genetically engineered cell is a lymphocyte, and optionally the genetically engineered cell expresses a chimeric antigen receptor (CAR), and optionally the lymphocyte is a B cell or a T cell (CAR-T cell), and optionally the lymphocyte is a tumor infiltrating lymphocyte (TIL), and optionally the microbe is genetically engineered to substantially decrease, reduce or eliminate the microbe’s toxicity, and optionally the microbe is genetically engineered to comprise a kill switch so the microbe can be rendered non-vital after administration of an appropriate trigger or signal, and optionally the microbe is genetically engineered to secrete anti inflammatory compositions or have an anti-inflammatory effect, and optionally the genetically engineered cell is administered or delivered before administration of, simultaneously with, and/or after administration or delivery of the formulation.

19. A formulation or a pharmaceutical composition comprising:

(a) a combination of microbes as set forth in Tables 15 and 16;

(b) a combination of microbes as used in any of the preceding claims, or a method any of claims 1 to 18; and/or

(c) at least two different species or genera (or types) of non-pathogenic bacteria, wherein each of the non-pathogenic bacteria comprise (or are in the form of) a plurality of non-pathogenic colony forming live bacteria, a plurality of non- pathogenic germinable non-pathogenic bacterial spores, or a combination thereof, and the formulation comprises at least one (or any one, several, or all of) non-pathogenic bacteria or spore of the family or genus (or class): Agathobaculum (TaxID: 2048137), Alistipes (TaxID: 239759), Anaeromassilibacillus (TaxID: 1924093), Anaerostipes (TaxID: 207244), Asaccharobacter (TaxID: 553372), Bacteroides (TaxID: 816), Bamesiella (TaxID: 397864), Bifidobacterium (TaxID: 1678), Blautia (TaxID: 572511), Butyricicoccus (TaxID: 580596), Clostridium (TaxID: 1485), Collinsella (TaxID: 102106), Coprococcus (TaxID: 33042), Dorea (TaxID: 189330),

Eubacterium (TaxID: 1730), Faecalibacterium (TaxID: 216851), Fusicatenibacter (TaxID: 1407607), Gemmiger (TaxID: 204475), Gordonibacter (TaxID: 644652), Lachnoclostridium (TaxID: 1506553), Methanobrevibacter (TaxID: 2172), Parabacteroides (TaxID: 375288), Romboutsia (TaxID: 1501226), Roseburia (TaxID: 841), Ruminococcus (TaxID: 1263), Erysipelotrichaceae (TaxID: 128827), Coprobacillus (TaxID: 100883), Erysipelatoclostridium sp. SNUG30099 (TaxID: 1982626), Erysipelatoclostridium (TaxID: 1505663), Acetatifactor (TaxID: 1427378), Adlercreutzia (TaxID: 447020), Agathobacter (TaxID: 1766253), Anaerotruncus (TaxID: 244127), Bariatricus (TaxID: 1924081), Butyrivibrio (TaxID: 830), Christensenellaceae (TaxID: 990719), Clostridiales (TaxID: 186802), Dialister (TaxID: 39948), Drancourtella (TaxID: 1903506), Eggerthella (TaxID: 84111), Eisenbergiella (TaxID: 1432051), Enterocloster (TaxID: 2719313), Enterococcus (TaxID: 1350), Intestinibacter (TaxID: 1505657), Lachnospira (TaxID: 28050), Lachnospiraceae (TaxID: 186803), Mediterraneibacter (TaxID: 2316020), Negativibacillus (TaxID: 1980693), Oscillibacter (TaxID: 459786), Phocaeicola (TaxID: 909656), Pseudobutyrivibrio (TaxID: 46205), Pseudoflavonifractor (TaxID: 1017280), Ruminococcaceae (TaxID: 541000), Sellimonas (TaxID: 1769710), Solobacterium (TaxID: 123375), Terrisporobacter (TaxID: 1505652), Tidjanibacter (TaxID: 1929083), Veillonella (TaxID: 29465), or a combination thereof.

20. The formulation or a pharmaceutical composition of claim 19, wherein the formulation comprises at least one (or any one, several, or all of) non-pathogenic bacteria or spore form thereof as set forth in Tables 1, 5, 10, 11, or 12, or included in the combination of non-pathogenic bacteria and/or spores thereof (or spore derived from) as set forth in Table 15 or 16.

21. The formulation or pharmaceutical composition of claim 19 to claim 20, wherein the formulation comprises an inner core surrounded by an outer layer of polymeric material enveloping the inner core, wherein the non-pathogenic bacteria or the non-pathogenic germinable bacterial spores are substantially in the inner core, and optionally the polymeric material comprises a natural polymeric material.

22. The formulation or pharmaceutical composition of any of the preceding claims, or a method of claims 19 to 21, wherein the plurality of non- pathogenic colony forming live bacteria are substantially dormant colony forming live bacteria, or the plurality of non-pathogenic colony forming live bacteria or the plurality of non-pathogenic germinable bacterial spores are lyophilized, wherein optionally the non-pathogenic dormant colony forming live bacteria comprise live vegetative bacterial cells that have been rendered dormant by lyophilization or freeze drying.

23. The formulation or pharmaceutical composition of any of the preceding claims, or a method of claims 19 to 22, wherein the formulation comprises at least 1 x 104 colony forming units (CFUs), or between about 1 x 102 and 1 x 108 CFUs, 1 x 103 and 1 x 107 CFUs, or 1 x 104 and 1 x 106 CFUs, of live non-pathogenic bacteria and/or non-pathogenic germinable bacterial spores.

24. The formulation or pharmaceutical composition of any of the preceding claims, or a method of claims 19 to 23, wherein the formulation or pharmaceutical composition comprises water, saline, a pharmaceutically acceptable preservative, a carrier, a buffer, a diluent, an adjuvant or a combination thereof.

25. The formulation or pharmaceutical composition of any of the preceding claims, or a method of claims 19 to 24, the formulation or pharmaceutical composition is formulated for administration orally or rectally, or is formulated as a liquid, a food, a gel, a geltab, a candy, a lozenge, a tablet, pill or capsule, or a suppository.

26. The formulation or pharmaceutical composition of any of the preceding claims, or a method of claims 19 to 25, wherein the formulation or pharmaceutical composition further comprises: a biofilm disrupting or dissolving agent, an antibiotic, an inhibitor of an inhibitory immune checkpoint molecule and/or a stimulatory immune checkpoint molecule (or any composition for use in checkpoint blockade immunotherapy).

27. The formulation or pharmaceutical composition of claim 27, or a method of claims 19 to 27, wherein the inhibitor of an inhibitory immune checkpoint molecule comprises a protein or polypeptide that binds to an inhibitory immune checkpoint protein, and optionally the inhibitor of the inhibitory immune checkpoint molecule is an antibody or an antigen binding fragment thereof that binds to an inhibitory immune checkpoint protein.

28. The formulation or pharmaceutical composition of claim 27, or a method of claims 19 to 28, wherein the inhibitor of an inhibitory immune checkpoint molecule targets a compound or protein comprising: CTLA4 or CTLA-4 (cytotoxic T- lymphocyte-associated protein 4, also known as CD 152, or cluster of differentiation 152); Programmed cell Death protein 1, also known as PD-1 or CD279; Programmed Death-Ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)); PD-L2; A2AR (adenosine A2A receptor, also known as ADORA2A); B7-H3; B7-H4; BTLA (B- and T-lymphocyte attenuator protein); KIR (Killer-cell Immunoglobulin-like Receptor); IDO (Indoleamine-pyrrole 2,3- di oxygenase); LAG3 (Lymphocyte- Activation Gene 3 protein); TIM-3; VISTA (V- domain Ig suppressor of T cell activation protein) or any combination thereof.

29. The formulation or pharmaceutical composition of claim 27, or a method of claims 19 to 28, wherein the inhibitor of an inhibitory immune checkpoint molecule comprises: ipilimumab or YERVOY®; pembrolizumab or KEYTRUDA®; nivolumab or OPDIVO®; atezolizumab or TECENTRIQ®; avelumab or BAVENCIO®; durvalumab or IMFINZI®; AMP-224 (Medlmmune), AMP-514 (an anti -programmed cell death 1 (PD-1) monoclonal antibody (mAh) (Medlmmune)), PDR001 (a humanized mAh that targets PD-1), STI-A1110 or STI-A1010 (Sorrento Therapeutics), BMS-936559 (Bristol-Myers Squibb), BMS-986016 (Bristol-Myers Squibb), TSR-042 (Tesaro), JNJ-61610588 (Janssen Research & Development), MSB-0020718C, AUR-012, enoblituzumab (also known as MGA271) (MacroGenics, Inc.), MBG453, LAG525 (Novartis), BMS-986015 (Bristol-Myers Squibb), cemiplimab (or LIBTAYO®) (Regeneron), or any combination thereof.

30. The formulation or pharmaceutical composition of claim 27, or a method of claims 19 to 26, wherein the stimulatory immune checkpoint molecule comprises a member of the tumor necrosis factor (TNF) receptor superfamily, optionally CD27, CD40, 0X40, GITR (a glucocorticoid-induced TNFR family Related gene protein) or GDI 37, or comprises a member of the B7-CD28 superfamily, optionally CD28 or Inducible T-cell co-stimulator (ICOS).

31. A kit or product of manufacture comprising a formulation or pharmaceutical composition of any of the preceding claims, or a formulation or pharmaceutical composition of claims 19 to 30, wherein optionally the product of manufacture is an implant.

32. Use of a formulation or pharmaceutical composition of any of the preceding claims, or a formulation or pharmaceutical composition of claims 19 to 30, or a kit or product of manufacture of any of the preceding claims, for controlling, ameliorating, preventing or treating a cancer in an individual in need thereof.

33. Use of a formulation or a pharmaceutical composition of any of the preceding claims, or a formulation or pharmaceutical composition of claims 19 to 30, in the manufacture of a medicament for controlling, ameliorating, preventing or treating a cancer in an individual in need thereof.

34. A formulation or pharmaceutical composition of any of the preceding claims, or a formulation or pharmaceutical composition of claims 19 to 30, or a kit or product of manufacture of any of the preceding claims, for use in controlling, ameliorating, preventing or treating a cancer in an individual in need thereof.

35. The Use, kit, formulation or pharmaceutical composition of any of the preceding claims, wherein the cancer is advanced melanoma, non-small-cell lung cancer or renal cell carcinoma.

Description:
COMPOSITIONS FOR MODULATING GUT MICROFLORA POPULATIONS, ENHANCING DRUG POTENCY AND TREATING CANCER, AND METHODS FOR MAKING AND USING SAME

RELATED APPLICATIONS

This Patent Convention Treaty (PCT) International Application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial No. (USSN) 62/951,673, Dec. 20, 2019. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

TECHNICAL FIELD

This invention generally relates to microbiology, pharmacology and cancer therapies. In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, comprising combinations of microbes, such as non-pathogenic, live bacteria and/or bacterial spores, for the control, amelioration, prevention, and treatment of a disease or condition, for example, a cancer. In alternative embodiment, these non-pathogenic, live bacteria and/or bacterial spores are administered to an individual in need thereof, thereby resulting in a modification or modulation of the individual’s gut microfloral population(s). In alternative embodiments, by modulating or modifying the individual’s gut microbial population(s) using compositions, products of manufacture and methods as provided herein, the pharmacodynamics of a drug administered to the individual is altered, for example, the pharmacodynamics of the drug is enhanced, e.g., the individual’s ability to absorb a drug is modified (e.g., accelerated or slowed, or enhanced), or the dose efficacy of a drug is increased (e.g., resulting in the requirement for a lower dose of drug to provide an intended effect), which can result in lowering the effective toxicity of the drug. For example, in alternative embodiments, the modulating or modifying of the individual’s gut microbial population(s) increases the dose efficacy of a cancer drug, thereby controlling, ameliorating, preventing and/or treating of that cancer. In alternative embodiments, the amount, identity, presence, and/or ratio of gut microbiota in a subject is manipulated to facilitate one or more co-treatments, for example, in alternative embodiments, combinations of microbes as provided herein are administered with a chemotherapy, a radiation therapy, an immune checkpoint inhibitor, a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment.

BACKGROUND

Checkpoint inhibitors are a class of cancer drugs that function by enabling the patient’s own immune system to fight the tumor, a treatment approach known as immunotherapy. These agents bind to and block inhibitory signals to T-cells from either antigen presenting cells or cancer cells, thereby allowing excitatory signals to prevail that result in T cell cancer recognition, activation and proliferation, ultimately leading to cancer rejection and elimination. Examples of T-cell inhibitory signal targets and their corresponding immunotherapy (as immunostimulating) agents include: cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), targeted by, e.g., YERVOY ® /Ipilimumab; the programmed cell death protein 1 (PD-1), targeted by, e.g., KEYTRUDA ® /Pembrolizumab, OPDIVO ® /Nivolumab; and its ligand PD-L1, targeted by, e.g. TECENTRIQ ® /Atezolizumab, BAVENCIO®/Avelumab and IMFINZI ® /Durvalumab; , cemiplimab (or LIBTAYO ® ) (Regeneron). Blockade of these inhibitory signals by checkpoint inhibitor immunotherapies has been shown to be particularly effective against advanced melanoma, non-small cell lung cancer, and renal cell carcinoma, yet more than 50% of cancer patients subjected to checkpoint inhibitor therapies fail to respond to the treatment (Ribas A, Wolchok JD (2018) Science (80- ) 359: 1350-1355).

New findings indicate that the likelihood of response or non-response to checkpoint inhibitors are directly correlated to the state of the gut microbiome and its contribution to immunological function of the gastrointestinal tract (Peled et al.

(2017) J Clin Oncol 15:1650-1659; Iida etal. (2013) Science 342, 967-970; Daillere et al. (2016) Immunity 45:931-943; Vetizou et al. (2015) Science 350:1079-1084; Sivan et al. (2015) Science 350:1084-1089; Routy, B. et al. (2018) Science 359, 91- 97; Gopalakrishnan, V. et al. (2018) Science (80- ). 359, 97-103; Matson, V. et al. (2018) Science (80- ). 359, 104-108).

For example, among melanoma patients undergoing anti -PD-1 immunotherapy, those more likely to respond to the therapy tended to have gut microbiomes enriched in anti-inflammatory gut microbes lik Q Faecalibacterium prausnitzii or Akkermansia muciniphila , while non-responding patients were enriched in microbes more associated with chronic inflammation such as Bacteroides species and those of th e Proteobacteria phylum (Routy, B. etal. (2018) Science 359, 91-97; Gopalakrishnan, V. etal. (2018) Science (80- ). 359, 97-103). It was posited that possession of a more anti-inflammatory gut microbiome better primed T-cells of the responder patients to respond to activation by checkpoint inhibition, while the chronic inflammatory state brought on by the dysbiotic microbiota of non-responders led to T- cell exhaustion and a relative inability to be activated by checkpoint inhibition. Thus, an opportunity arises to improve the likelihood of response to checkpoint inhibitor therapies by modification of the composition of the gut microbiome to include immunomodulatory microbes that might be lacking in some patients.

SUMMARY

In alternative embodiments, provided are methods for controlling, ameliorating, preventing or treating a cancer in an individual in need thereof, comprising:

(a) administering or having administered to an individual in need thereof a formulation comprising at least two different species or genera (or types) of non- pathogenic bacteria, wherein each of the non-pathogenic bacteria comprise (or are in the form of) a plurality of non-pathogenic colony forming live bacteria, a plurality of non-pathogenic germinable bacterial spores, or a combination thereof; or,

(b) (i) providing a formulation comprising at least two different species or genera (or types) of non-pathogenic bacteria, wherein each of the non-pathogenic bacteria comprise (or are in the form of) a plurality of non-pathogenic colony forming live bacteria, a plurality of non-pathogenic germinable bacterial spores, or a combination thereof, and

(ii) administering or having administered to an individual in need thereof the formulation; wherein the formulation comprises a or any combination of at least two different species or genera of non-pathogenic, live bacteria, or spore thereof, if the bacteria is spore forming, as described Table 1, 5, 10, 11, or 12, or live biotherapeutic compositions or combinations of bacteria as set forth in Table 15 or 16, and optionally the different species or genera (or types) of non-pathogenic, live bacteria are present in approximately equal amounts, or each of the different species or genera (or types) of non-pathogenic, live bacteria or non-pathogenic germinable bacterial spores represent at least about 1%, 5%, 10%, 20%, 30%, 40%, or 50% or more, or between about 1% and 75%, of the total amount of non-pathogenic, live bacteria and non-pathogenic germinable bacterial spores in the formulation, and optionally only or substantially only non-pathogenic, live bacteria are present in the formulation, or only or substantially only non-pathogenic germinable bacterial spores are present in the formulation, or approximately equal amounts of non-pathogenic, live bacteria and non-pathogenic germinable bacterial spores are present in the formulation.

In alternative embodiments of methods as provided herein:

- the method further comprises administering or having administered one or any one of: a chemotherapy, a radiation therapy, an immune checkpoint inhibitor, a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or an immunotherapy or a cancer treatment, or a combination thereof, and optionally the chemotherapy, the radiation therapy, the immune checkpoint inhibitor, the Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or the immunotherapy or the cancer treatment, or the combination thereof, is administered before, during (concurrently with) and/or after administration the formulation;

- the formulation comprises an inner core surrounded by an outer layer of polymeric material enveloping the inner core, wherein the non-pathogenic bacteria or the non-pathogenic germinable bacterial spores are substantially in the inner core, and optionally the polymeric material comprises a natural polymeric material;

- the formulation is formulated or manufactured as or in: a nano-suspension delivery system; an encochleated formulation; or, as a multilayer crystalline, spiral structure with no internal aqueous space;

- the formulation is formulated or manufactured as a delayed or gradual enteric release composition or formulation, and optionally the formulation comprises a gastro-resistant coating designed to dissolve at a pH of 7 in the terminal ileum, optionally an active ingredient is coated with an acrylic based resin or equivalent, optionally a poly(meth)acrylate, optionally a methacrylic acid copolymer B, NF, optionally EUDRAGIT S™ (Evonik Industries AG, Essen, Germany), which dissolves at pH 7 or greater, optionally comprises a multimatrix (MMX) formulation, and optionally manufactured as enteric coated to bypass the acid of the stomach and bile of the duodenum; - the plurality of non-pathogenic colony forming live bacteria are substantially dormant colony forming live bacteria, or the plurality of non-pathogenic colony forming live bacteria or the plurality of non-pathogenic germinable bacterial spores are lyophilized, wherein optionally the dormant colony forming live bacteria comprise live vegetative bacterial cells that have been rendered dormant by lyophilization or freeze drying;

- the formulation comprises at least about 1 x 10 4 colony forming units (CFUs), or between about 1 x 10 1 and 1 x 10 13 CFUs, 1 x 10 2 and 1 x 10 10 CFUs, 1 x 10 2 and 1 x 10 8 CFUs, 1 x 10 3 and 1 x 10 7 CFUs, or 1 x 10 4 and 1 x 10 6 CFUs, of non-pathogenic live bacteria and/or non-pathogenic germinable bacterial spores;

- the formulation comprises at least one (or any one, several, or all of) non- pathogenic bacteria or spore of the family or genus (or class): Agathobaculum (TaxID: 2048137), Alistipes (TaxID: 239759), Anaeromassilibacillus (TaxID: 1924093), Anaerostipes (TaxID: 207244), Asaccharobacter (TaxID: 553372), Bacteroides (TaxID: 816), Barnesiella (TaxID: 397864), Bifidobacterium (TaxID: 1678), Blautia (TaxID: 572511), Butyricicoccus (TaxID: 580596), Clostridium (TaxID: 1485), Collinsella (TaxID: 102106), Coprococcus (TaxID: 33042), Dorea (TaxID: 189330), Eubacterium (TaxID: 1730), Faecalibacterium (TaxID: 216851), Fusicatenibacter (TaxID: 1407607), Gemmiger (TaxID: 204475), Gordonibacter (TaxID: 644652), Lachnoclostridium (TaxID: 1506553), Methanobrevibacter (TaxID: 2172), Parabacteroides (TaxID: 375288), Romboutsia (TaxID: 1501226), Roseburia (TaxID: 841), Ruminococcus (TaxID: 1263), Erysipelotrichaceae (TaxID: 128827), Coprobacillus (TaxID: 100883), Erysipelatoclostridium sp. SNUG30099 (TaxID: 1982626), Erysipelatoclostridium (TaxID: 1505663), Acetatifactor (TaxID: 1427378), Adlercreutzia (TaxID: 447020), Agathobacter (TaxID: 1766253), Anaerotruncus (TaxID: 244127), Bariatricus (TaxID: 1924081), Butyrivibrio (TaxID: 830), Christensenellaceae (TaxID: 990719), Clostridiales (TaxID: 186802), Dialister (TaxID: 39948), Drancourtella (TaxID: 1903506), Eggerthella (TaxID: 84111), Eisenbergiella (TaxID: 1432051), Enterocloster (TaxID: 2719313), Enterococcus (TaxID: 1350), Intestinibacter (TaxID: 1505657), Lachnospira (TaxID: 28050), Lachnospiraceae (TaxID: 186803), Mediterraneibacter (TaxID: 2316020), Negativibacillus (TaxID: 1980693), Oscillibacter (TaxID: 459786), Phocaeicola (TaxID: 909656), Pseudobutyrivibrio (TaxID: 46205), Pseudoflavonifractor (TaxID: 1017280), Ruminococcaceae (TaxID: 541000), Sellimonas (TaxID: 1769710), Solobacterium (TaxID: 123375), Terrisporobacter (TaxID: 1505652), Tidjanibacter (TaxID: 1929083), Veillonella (TaxID: 29465), or a combination thereof;

- wherein the formulation comprises at least one (or any one, several, or all of) non-pathogenic bacteria or spore form thereof as set forth in Tables 1, 5, 10, 11, or 12, or included in the combination of non-pathogenic bacteria and/or spores thereof (or spore derived from) as set forth in Table 15 or 16.;

- the formulation comprises combination of non-pathogenic bacteria and/or spores thereof (or spore derived from) as set forth in Tables 15 and 16;

- the formulation comprises water, sterile water, saline, sterile saline, a pharmaceutically acceptable preservative, a carrier, a buffer, a diluent, an adjuvant or a combination thereof;

- the formulation is administered orally or rectally, or is formulated and/or administered as a liquid, a food, a gel, a candy, an ice, a lozenge, a tablet, pill or capsule, or a suppository or as an enema formulation, or the formulation is administered as an or is in a form for intra-rectal or intra-colonic administration;

- the formulation is administered to the individual in need thereof in one, two, three, or four or more doses, and wherein the one, two, three, four or five or more doses are administered on a daily basis (optionally once a day, bid or tid or more), every other day, every third day, or about once a week, and optionally the two, three, or four or more doses are administered at least a week apart (or dosages are separated by about a week);

- the formulation further comprises an antibiotic, or the method further comprises administration of an antibiotic, and optionally at least one dose of the antibiotic is administered before a first administration of the formulation, optionally at least one dose of the antibiotic is administered one day or two days, or more, before a first administration of the formulation;

- the inhibitor of the inhibitory immune checkpoint molecule comprises a protein or polypeptide that binds to an inhibitory immune checkpoint protein, and optionally inhibitor of the inhibitory immune checkpoint protein is an antibody or an antigen binding fragment thereof that specifically binds to the inhibitory immune checkpoint protein;

- the inhibitor of the inhibitory immune checkpoint molecule targets a compound or protein comprising: a CTLA4 or CTLA-4 (cytotoxic T-lymphocyte- associated protein 4, also known as CD 152, or cluster of differentiation 152); Programmed cell Death protein 1, also known as PD-1 or CD279; Programmed Death-Ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)); PD-L2; A2AR (adenosine A 2A receptor, also known as ADORA2A); B7-H3; B7-H4; BTLA (B- and T-lymphocyte attenuator protein); KIR (Killer-cell Immunoglobulin-like Receptor); IDO (Indoleamine-pyrrole 2,3- di oxygenase); LAG3 (Lymphocyte- Activation Gene 3 protein); TIM-3; VISTA (V- domain Ig suppressor of T cell activation protein); or any combination thereof;

- the inhibitor of an inhibitory immune checkpoint molecule comprises: ipilimumab or YERVOY ® ; pembrolizumab or KEYTRUDA ® ; nivolumab or OPDIVO ® ; atezolizumab or TECENTRIQ ® ; avelumab or BAVENCIO ® ; durvalumab or IMFINZI ® ; AMP-224 (Medlmmune), AMP-514 (an anti-programmed cell death 1 (PD-1) monoclonal antibody (mAh) (Medlmmune)), PDR001 (a humanized mAh that targets PD-1), STI-A1110 or STI-A1010 (Sorrento Therapeutics), BMS-936559 (Bristol-Myers Squibb), BMS-986016 (Bristol-Myers Squibb), TSR-042 (Tesaro), JNJ-61610588 (Janssen Research & Development), MSB-0020718C, AUR-012, enoblituzumab (also known as MGA271) (MacroGenics, Inc.), MBG453, LAG525 (Novartis), BMS-986015 (Bristol-Myers Squibb), cemiplimab (or LIBTAYO ® ) (Regeneron), or any combination thereof;

- the inhibitor of the inhibitory immune checkpoint molecule, or the stimulatory immune checkpoint molecule, is administered by: intravenous (IV) injection, intramuscular (IM) injection, intratumoral injection or subcutaneous injection; or, is administered orally or by suppository; or the formulation further comprises at least one immune checkpoint inhibitor;

- the cancer is melanoma, advanced melanoma, cutaneous or intraocular melanoma, primary neuroendocrine carcinoma of the skin, breast cancer, a cancer of the head and neck, uterine cancer, rectal and colorectal cancer, a cancer of the head and neck, cancer of the small intestine, a colon cancer, a cancer of the anal region, a stomach cancer, lung cancer, brain cancer, non-small-cell lung cancer, ovarian cancer, angiosarcoma, bone cancer, osteosarcoma, prostate cancer; cancer of the bladder; cancer of the kidney or ureter or renal cell carcinoma, or carcinoma of the renal pelvis; a neoplasm of the central nervous system (CNS) or renal cell carcinoma; and/or - the method comprises, or further comprises, administering, or having administered, or delivering, a genetically (or recombinantly) engineered cell, wherein optionally the genetically engineered cell is: a microbe or spore derived from a microbe as used in a method of any of the preceding claims, or a method as provided herein; or, a non-pathogenic bacteria or spore form thereof as set forth in Tables 1, 5, 10, 11, or 12, or included in the combination of non-pathogenic bacteria and/or spores thereof (or spore derived from) as set forth in Table 15 or 16, and optionally the microbe is genetically engineered to express or secrete a heterologous or overexpress an endogenous immunomodulatory molecule, and optionally the immunomodulatory molecule is an immunomodulatory protein or peptide, and optionally the immunomodulatory molecule is an immunostimulatory molecule, and optionally the microbe is genetically engineered to overexpress a pathway for production of at least one short chain fatty acid (SCFA), and optionally the SCFA comprises butyrate or butyric acid, propionate or acetate, and optionally the microbe is genetically engineered by inserting a heterologous nucleic acid into the microbe, and optionally the heterologous nucleic acid encodes an exogenous membrane protein, and optionally the immunostimulatory molecule, protein or peptide comprises a non-specific immunostimulatory protein, and optionally the non-specific immunostimulatory protein comprises a cytokine, and optionally the cytokine comprises an interferon (optionally an IFN-a2a, IFN-a2b), and interleukin (optionally IL-2, IL-4, IL-7, IL-12), an interferon (IFN), a TNF-a, a granulocyte colony- stimulating factor (G-CSF, also known as filgrastim, lenograstim or Neupogen®), a granulocyte monocyte colony-stimulating factor (GM-CSF, also known as molgramostim, sargramostim, Leukomax®, Mielogen® or Leukine®), or any combination thereof, and optionally the immunostimulatory molecule, protein or peptide comprises a specific immunostimulatory protein or peptide, and optionally the specific immunostimulatory protein or peptide comprises an immunogen that can generate a specific humeral or cellular immune response or an immune response to a cancer antigen, and optionally the genetically engineered cell is a lymphocyte, and optionally the genetically engineered cell expresses a chimeric antigen receptor (CAR), and optionally the lymphocyte is a B cell or a T cell (CAR-T cell), and optionally the lymphocyte is a tumor infiltrating lymphocyte (TIL), and optionally the microbe is genetically engineered to substantially decrease, reduce or eliminate the microbe’s toxicity, and optionally the microbe is genetically engineered to comprise a kill switch so the microbe can be rendered non-vital after administration of an appropriate trigger or signal, and optionally the microbe is genetically engineered to secrete anti inflammatory compositions or have an anti-inflammatory effect, and optionally the genetically engineered cell is administered or delivered before administration of, simultaneously with, and/or after administration or delivery of the formulation.

In alternative embodiments, provided are formulations or pharmaceutical compositions comprising:

(a) a combination of microbes as set forth in Tables 15 to 16;

(b) a combination of microbes as used in a method as provided herein or as provided herein; and/or

(c) at least two different species or genera (or types) of non-pathogenic bacteria, wherein each of the non-pathogenic bacteria comprise (or are in the form of) a plurality of non-pathogenic colony forming live bacteria, a plurality of non- pathogenic germinable non-pathogenic bacterial spores, or a combination thereof, and the formulation comprises at least one (or any one, several, or all of) non-pathogenic bacteria or spore of the family or genus (or class): Agathobaculum (TaxID: 2048137), Alistipes (TaxID: 239759), Anaeromassilibacillus (TaxID: 1924093), Anaerostipes (TaxID: 207244), Asaccharobacter (TaxID: 553372), Bacteroides (TaxID: 816), Bamesiella (TaxID: 397864), Bifidobacterium (TaxID: 1678), Blautia (TaxID: 572511), Butyricicoccus (TaxID: 580596), Clostridium (TaxID: 1485), Collinsella (TaxID: 102106), Coprococcus (TaxID: 33042), Dorea (TaxID: 189330),

Eubacterium (TaxID: 1730), Faecalibacterium (TaxID: 216851), Fusicatenibacter (TaxID: 1407607), Gemmiger (TaxID: 204475), Gordonibacter (TaxID: 644652), Lachnoclostridium (TaxID: 1506553), Methanobrevibacter (TaxID: 2172), Parabacteroides (TaxID: 375288), Romboutsia (TaxID: 1501226), Roseburia (TaxID: 841), Ruminococcus (TaxID: 1263), Erysipelotrichaceae (TaxID: 128827), Coprobacillus (TaxID: 100883), Erysipelatoclostridium sp. SNUG30099 (TaxID: 1982626), Erysipelatoclostridium (TaxID: 1505663), Acetatifactor (TaxID: 1427378), Adlercreutzia (TaxID: 447020), Agathobacter (TaxID: 1766253), Anaerotruncus (TaxID: 244127), Bariatricus (TaxID: 1924081), Butyrivibrio (TaxID: 830), Christensenellaceae (TaxID: 990719), Clostridiales (TaxID: 186802), Dialister (TaxID: 39948), Drancourtella (TaxID: 1903506), Eggerthella (TaxID: 84111), Eisenbergiella (TaxID: 1432051), Enterocloster (TaxID: 2719313), Enterococcus (TaxID: 1350), Intestinibacter (TaxID: 1505657), Lachnospira (TaxID: 28050), Lachnospiraceae (TaxID: 186803), Mediterraneibacter (TaxID: 2316020), Negativibacillus (TaxID: 1980693), Oscillibacter (TaxID: 459786), Phocaeicola (TaxID: 909656), Pseudobutyrivibrio (TaxID: 46205), Pseudoflavonifractor (TaxID: 1017280), Ruminococcaceae (TaxID: 541000), Sellimonas (TaxID:

1769710), Solobacterium (TaxID: 123375), Terrisporobacter (TaxID: 1505652), Tidjanibacter (TaxID: 1929083), Veillonella (TaxID: 29465), or a combination thereof.

In alternative embodiments, of formulations or pharmaceutical compositions as provided herein, or methods as provided herein:

- the wherein the formulation comprises at least one (or any one, several, or all of) non-pathogenic bacteria or spore form thereof as set forth in Tables 1, 5, 10, 11, or 12, or included in the combination of non-pathogenic bacteria and/or spores thereof (or spore derived from) as set forth in Table 15 or 16.;

- the formulation comprises an inner core surrounded by an outer layer of polymeric material enveloping the inner core, wherein the non-pathogenic bacteria or the non-pathogenic germinable bacterial spores are substantially in the inner core, and optionally the polymeric material comprises a natural polymeric material;

- the plurality of non-pathogenic colony forming live bacteria are substantially dormant colony forming live bacteria, or the plurality of non-pathogenic colony forming live bacteria or the plurality of non-pathogenic germinable bacterial spores are lyophilized, wherein optionally the non-pathogenic dormant colony forming live bacteria comprise live vegetative bacterial cells that have been rendered dormant by lyophilization or freeze drying;

- the formulation comprises at least 1 x 10 4 colony forming units (CFUs), or between about 1 x 10 2 and 1 x 10 8 CFUs, 1 x 10 3 and 1 x 10 7 CFUs, or 1 x 10 4 and 1 x 10 6 CFUs, of live non-pathogenic bacteria and/or non-pathogenic germinable bacterial spores; - the formulation or pharmaceutical composition comprises water, saline, a pharmaceutically acceptable preservative, a carrier, a buffer, a diluent, an adjuvant or a combination thereof;

- the formulation or pharmaceutical composition is formulated for administration orally or rectally, or is formulated as a liquid, a food, a gel, a geltab, a candy, a lozenge, a tablet, pill or capsule, or a suppository;

- the formulation or pharmaceutical composition further comprises: a biofilm disrupting or dissolving agent, an antibiotic, an inhibitor of an inhibitory immune checkpoint molecule and/or a stimulatory immune checkpoint molecule (or any composition for use in checkpoint blockade immunotherapy);

- the inhibitor of an inhibitory immune checkpoint molecule comprises a protein or polypeptide that binds to an inhibitory immune checkpoint protein, and optionally the inhibitor of the inhibitory immune checkpoint molecule is an antibody or an antigen binding fragment thereof that binds to an inhibitory immune checkpoint protein;

- the inhibitor of an inhibitory immune checkpoint molecule targets a compound or protein comprising: CTLA4 or CTLA-4 (cytotoxic T-lymphocyte- associated protein 4, also known as CD 152, or cluster of differentiation 152); Programmed cell Death protein 1, also known as PD-1 or CD279; Programmed Death-Ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)); PD-L2; A2AR (adenosine A 2A receptor, also known as ADORA2A); B7-H3; B7-H4; BTLA (B- and T-lymphocyte attenuator protein); KIR (Killer-cell Immunoglobulin-like Receptor); IDO (Indoleamine-pyrrole 2,3- di oxygenase); LAG3 (Lymphocyte- Activation Gene 3 protein); TIM-3; VISTA (V- domain Ig suppressor of T cell activation protein) or any combination thereof;

- the inhibitor of an inhibitory immune checkpoint molecule comprises: ipilimumab or YERVOY ® ; pembrolizumab or KEYTRUDA ® ; nivolumab or OPDIVO ® ; atezolizumab or TECENTRIQ ® ; avelumab or BAVENCIO ® ; durvalumab or IMFINZI ® ; AMP-224 (Medlmmune), AMP-514 (an anti-programmed cell death 1 (PD-1) monoclonal antibody (mAh) (Medlmmune)), PDR001 (a humanized mAh that targets PD-1), STI-A1110 or STI-A1010 (Sorrento Therapeutics), BMS-936559 (Bristol-Myers Squibb), BMS-986016 (Bristol-Myers Squibb), TSR-042 (Tesaro), TNJ-61610588 (Janssen Research & Development), MSB-0020718C, AUR-012, enoblituzumab (also known as MGA271) (MacroGenics, Inc.), MBG453, LAG525 (Novartis), BMS-986015 (Bristol-Myers Squibb), cemiplimab (orLIBTAYO ® ) (Regeneron), or any combination thereof; and/or

- the stimulatory immune checkpoint molecule comprises a member of the tumor necrosis factor (TNF) receptor superfamily, optionally CD27, CD4Q, 0X40, GITR (a glucocorticoid-induced TNFR family Related gene protein) or CD137, or comprises a member of the B7-CD28 superfamily, optionally CD28 or Inducible T- cell co-stimulator (ICOS).

In alternative embodiments, provided are kits or products of manufacture comprising a formulation or pharmaceutical composition as provided herein, wherein optionally the product of manufacture is an implant.

In alternative embodiments, provided are uses of a formulation or pharmaceutical composition as provided herein, or a kit or product of manufacture as provided herein, for controlling, ameliorating, preventing or treating a cancer in an individual in need thereof.

In alternative embodiments, provided are uses of a formulation or a pharmaceutical composition as provided herein in the manufacture of a medicament for controlling, ameliorating, preventing or treating a cancer in an individual in need thereof.

In alternative embodiments, provided are formulations or pharmaceutical compositions as provided herein, or a kit as provided herein, for use in controlling, ameliorating, preventing or treating a cancer in an individual in need thereof. In alternative embodiments, the cancer is melanoma, advanced melanoma, cutaneous or intraocular melanoma, primary neuroendocrine carcinoma of the skin, breast cancer, a cancer of the head and neck, uterine cancer, rectal and colorectal cancer, a cancer of the head and neck, cancer of the small intestine, a colon cancer, a cancer of the anal region, a stomach cancer, lung cancer, brain cancer, non-small-cell lung cancer, ovarian cancer, angiosarcoma, bone cancer, osteosarcoma, prostate cancer; cancer of the bladder; cancer of the kidney or ureter or renal cell carcinoma, or carcinoma of the renal pelvis; a neoplasm of the central nervous system (CNS) or renal cell carcinoma.

The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS

The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

FIG. 1 graphically summarizes the classification level of least common ancestors for each cluster. Microbial genome assemblies from NCBI RefSeq are classified into operational species units by clustering similar genome assemblies together. The least common ancestor in the NCBI hierarchy for the assemblies in each operational species unit (OSU) cluster is determined. For OSU's containing more than one microbial assembly, the rank of the least common ancestor is displayed. Most OSU's have a least common ancestor at the species or genus level, demonstrating consistency between the assigned OSU's and the pre-existing NCBI taxonomic tree, as described in Example 9, below.

FIG. 2 graphically shows the distribution of OSU cluster sizes. Microbial genome assemblies from NCBI RefSeq are classified into operational species units by clustering similar genome assemblies together. The cluster size distribution is visualized, as described in Example 9, below.

FIG. 3 graphically illustrates a principal component analysis (PC A) of microbiome composition obtained from fecal samples. Whole genome sequencing is performed on fecal samples from subjects with and without cancer as well as in remission. The reads are classified, and abundance of each operational species unit is estimated computationally. PCA is performed on centered-log-ratio transformed abundances, and the first two principal coordinates are plotted for cancer, remission, and control sample cohorts, as described in Example 9, below.

FIG. 4 graphically illustrates a PCA plot showing the relationship between longitudinal samples of the same patient. Whole genome sequencing is performed on fecal samples from subjects with and without cancer as well as in remission. The reads are classified and abundance of each operational species unit is estimated computationally. PCA is performed on centered-log-ratio transformed abundances, and the first two principal coordinates are plotted for cancer and control sample cohorts, with longitudinal samples being connected by arrows. Later samples from the same subject are colored darker, as described in Example 9, below.

FIG. 5 graphically illustrates a volcano plot showing the differential abundance of species in cancer and control cohorts. Whole genome sequencing is performed on fecal samples from subjects with and without cancer and the reads are classified and abundance of each operational species unit is estimated computationally. The fold change difference and statistical significance (inverse p value, Mann Whitney U test) is calculated for abundances between cancer and control sample cohorts. The results are displayed on a volcano plot. Each point is an operational species unit, and the area of each point corresponds to the average abundance of that operational species unit across all samples, as described in Example 9, below.

FIG. 6 shows the distribution of abundances of specific organisms among the different patient samples in each cohort. Whole genome sequencing is performed on fecal samples from subjects with and without cancer and the reads are classified and abundance of each operational species unit is estimated computationally. Operational species units with significant differences between cancer and control are displayed, as described in Example 9, below.

FIG. 7 graphically illustrates the distribution of abundances of additional specific organisms among the different patient samples in each cohort, plotted as in Figure 6.

FIG. 8 graphically illustrates a receiver operating characteristic (ROC) curve of the classifier developed based on stool species distribution. A random forest classifier is trained to classify operational species unit abundances for a sample as corresponding to cancer or control. An ROC curve is generated on 145 cancer samples and 88 control samples using leave-one-out cross validation. No hyperparameter optimization was performed, as described in Example 10, below.

FIG. 9 graphically illustrates correlations of species abundance with immune markers obtained from blood analysis. Immune markers with significant correlations to operational species unit relative abundances are plotted. P values are generated by a linear mixed model fit that model immune marker proportions as being linearly related to the logarithm of OSU abundance, with a random effect accounting for cancer and control groups. For CD3+CD56+, the logarithm of the immune marker proportion is used as the output of the mixed model (a) positive correlations; (b) negative correlations, as described in Example 10, below.

FIG. 10 graphically illustrates the distribution of abundances of specific organisms in complete responders (CR), partial responders (PR), and non-responders (NR). Whole genome sequencing is performed on the initial time point fecal samples from subjects undergoing cancer immunotherapy and the reads are classified and abundance of each operational species unit is estimated computationally. Operational species unit abundances are correlated to response to therapy using a score of 2 for complete response, 1 for partial response, 0 for no response, using the Spearman rank correlation. Operational species unit abundances for several notable OSUs are displayed with the corresponding Spearman p values, as described in Example 10, below.

FIG. 11 graphically illustrates the first two principal components of a PC A of centered-log-ratio transformed microbial species abundance values obtained from fecal samples of FMT -treated mice 7 days post-treatment. Circles and Xs represent samples from mice treated with fecal material from two different non-responder patients. Squares and plusses represent samples from mice treated with fecal material from two different responder patients. The large symbols of each type indicate species composition of the human fecal material used for each transplant.

FIG. 12 graphically illustrates principal components 2 and 3 of a PC A of centered-log-ratio transformed microbial species abundance values obtained from fecal samples of FMT-treated mice 7 days post-treatment. Symbols are as described for Figure 11.

FIG. 13 graphically illustrates principal components 3 and 4 of a PC A of centered-log-ratio transformed microbial species abundance values obtained from fecal samples of FMT-treated mice 7 days post-treatment. Symbols are as described for Figure 11.

FIG. 14 graphically illustrates the first two components of a t-Distributed Stochastic Neighbor Embedding (tSNE) of centered-log-ratio transformed microbial species abundance values obtained from fecal samples of FMT-treated mice 7 days post-treatment. Circles and Xs represent samples from mice treated with fecal material from two different non-responder patients. Squares and plusses represent samples from mice treated with fecal material from two different responder patients. FIG. 15 graphically illustrates the first two components of a tSNE of centered-log-ratio transformed microbial species abundance values obtained from fecal samples of FMT-treated mice 7, 13, and 27 days post-treatment. Shading intensity of the points indicates different donors. Circles, 7 days post-treatment; Xs,

13 days post-treatment; squares, 27 days post-treatment.

FIG. 16 illustrates Table 2, as discussed in Example 7, below.

FIG. 17 illustrates Table 3, as discussed in Example 9, below.

FIG. 18 illustrates Table 4, as discussed in Example 9, below.

FIG. 19 illustrates Table 8, as discussed in Example 10, below.

FIG. 20 illustrates Table 9, as discussed in Example 10, below.

FIG. 21 illustrates the gating strategy used to classify immune cell populations based on metal-labeled peptide markers, and cell counts for a representative sample, as discussed in Example 11, below.

FIG. 22 illustrate Table 13, as discussed in Example 10, below.

FIG. 23 illustrate Table 14, as discussed in Example 10, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, comprising novel combinations of microbes, also called live biotherapeutic compositions such as non-pathogenic, live (optionally dormant) bacteria and/or bacterial spores, e.g., such as the exemplary combinations of microbes as listed in Tables 15 and 16, Example 10. In alternative embodiments, the compositions, products of manufacture, kits and methods as provided herein are used as a therapy (e.g., as a mono-therapy or as a co-therapy, or co-treatment) for the control, amelioration, prevention and/or treatment of a disease or condition, for example, a cancer. In alternative embodiments, the compositions, products of manufacture, kits and/or methods as provided herein are administered to an individual receiving a drug, e.g., a cancer therapy, thereby resulting in a modification or modulation of the patient’s gut microfloral population(s), thus resulting in an enhancement of the drug therapy, for example, lowering the dosage or amount of drug needed for effective therapy, or the frequency with which a drug must be administered to be effective. In alternative embodiments, by modulating or modifying the individual’s gut microbial population(s) using compositions, products of manufacture and methods as provided herein, the pharmacodynamics of a drug administered to the patient is altered, for example, the pharmacodynamics of the drug is enhanced, e.g., the individual’s ability to absorb a drug is modified (e.g., accelerated or slowed, or enhanced), or the dose efficacy of a drug is increased (e.g., resulting in needing a lower dose of drug for an intended effect), or the gut microbes act orthogonally on the drug target (e.g., resulting in the presence of the microbe being essential for the drug to have the intended effect). For example, in alternative embodiments, by modulating or modifying the patient’s gut microbial population(s) using compositions, products of manufacture and methods as provided herein the dose efficacy of a cancer drug is increased, thereby enhancing the control or treatment of that cancer.

In alternative embodiments, the amount, identity, presence, and/or ratio of gut microbiota in a subject is manipulated to facilitate a mono-therapy or one or more co treatments; for example, in alternative embodiments, combinations of microbes as provided herein are administered with (e.g., concurrent with, or before and/or after) a chemotherapy, a radiation therapy, an immune checkpoint inhibitor, a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment.

Described here for the first time are novel combinations of specific microbes, for example, bacteria, including for example microbes found in a human gut or recombinantly engineered or cultured microbes, which can be administered as a mono-therapy or as a co-therapy for, in alternative embodiments, cancer or autoimmune patients, where in alternative embodiments the cancer patients are undergoing immune checkpoint inhibitor treatment, or are undergoing a chemotherapy, a radiation therapy, an immune checkpoint inhibitor, a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment.

As described in the Examples, below, we demonstrated a correlation between these combinations of microbes and the metabolic functions associated with them, and the efficacy of treatment in both human patients and mouse cancer models. In alternative embodiments, administering combinations of microbes as provided herein to cancerous mice improves the fraction of animals that show significant tumor size reduction as compared to mice given the same drug but not having their gut microbiome altered using compositions or methods as provided herein. In alternative embodiments, the chemotherapy, radiation therapy, Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment, for example, the immune checkpoint inhibitors (or inhibitors of an inhibitory immune checkpoint molecule) and/or stimulatory immune checkpoint molecules (or more accurately, stimulatory immune molecules), are administered with (e.g., are administered concurrently or sequentially), or formulated with, the combinations of microbes as provided herein, e.g., administered or formulated with non-pathogenic bacteria and/or non-pathogenic germination-competent bacterial spores as provided herein.

The immune checkpoint inhibitors (also described as an inhibitor of an inhibitory immune checkpoint molecule) can function by interfering with regulatory pathways that naturally exist to prevent T cell proliferation. In the tumor microenvironment these inhibitory pathways are highly active, so T cells are often driven to an ineffective state. Checkpoint inhibitors bind to particular proteins in these regulatory pathways associated with inhibition of T cell activation, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), or programmed cell death ligand 1 (PD-L1), thereby allowing excitatory T cell response to tumor antigens. Thus, in alternative embodiments, an inhibitor of an inhibitory immune checkpoint molecule is a molecule that can directly (or specifically) bind to CTLA-4, PD-1, PD-L1, or other component of the inhibitory immune checkpoint to prevent proper binding to its natural corresponding receptor or ligand.

In alternative embodiments, a stimulatory immune checkpoint molecule - which can also be, or more accurately is, described as a stimulatory immune molecule potentiates excitation and activation of T cells, either by enhancing the action of a checkpoint inhibitor or by an independent mechanism.

In alternative embodiments, provided are therapeutic compositions, including formulations and pharmaceutical compositions, comprising non-pathogenic (optionally dormant) live microbes such as bacteria and/or germination-competent bacterial spores, which can be used for the prevention or treatment of a cancer or the side effects of a cancer therapy, e.g., a drug therapy, or can be used or administered with a chemotherapy, a radiation therapy, an immune checkpoint inhibitor, a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment. In alternative embodiments, therapeutic compositions, formulations or pharmaceutical compositions as provided herein, or used to practice methods as provided herein, comprise colony forming (optionally dormant) live bacteria and/or germinable bacterial spores which can be used in mono- or co-therapies, for example, as an adjuvant to an antineoplastic treatment administered to a cancer patient, or administered with or as a supplement to a chemotherapy, a radiation therapy, an immune checkpoint inhibitor, a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment.

In some embodiments, a therapeutic composition as provided herein acts or is used as a probiotic composition which can be administered with, before and/or after a chemotherapy, a radiation therapy, an immune checkpoint inhibitor, a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment. In alternative embodiments, therapeutic compositions (e.g., the formulations) as provided herein, comprise the bacteria and/or spores and an antineoplastic active agent such as an immune checkpoint inhibitor.

In alternative embodiments, therapeutic compositions, formulations or pharmaceutical compositions as provided herein, or used to practice methods as provided herein, comprise colony forming (optionally dormant) live bacteria and/or germinable bacterial spores for use as a mono-therapy or in combination with (e.g., as a co-therapy) or supplementary to a drug (which can be a small molecule or a protein, e.g., a therapeutic antibody) blocking an immune checkpoint for inducing immuno- stimulation in a cancer patient. The therapeutic composition as provided herein and the drug (e.g., an antibody) can be administered separately or together, or at different time points or at the same time, or can be administered sequentially or concurrently.

In alternative embodiments, therapeutic compositions, formulations or pharmaceutical compositions as provided herein comprise colony forming (optionally dormant) live bacteria and/or germinable bacterial spores which can be used as an adjuvant to an anti -cancer or antineoplastic treatment, for example, an immune checkpoint treatment, administered to a cancer patient. In alternative embodiments, the therapeutic composition comprises the antineoplastic or immune checkpoint active agents. In alternative embodiments, the therapeutic composition, formulations or pharmaceutical compositions as provided herein are administered with or after, or both with and after, administration of the antineoplastic or immune checkpoint active agent. In alternative embodiments, the formulation or pharmaceutical composition further comprises, or is manufactured with, an outer layer of polymeric material (e.g., natural polymeric material) enveloping, or surrounding, a core that comprises the combination of microbes as provided herein.

In alternative embodiments, therapeutic compositions, formulations or pharmaceutical compositions as provided herein, or used to practice methods as provided herein, can comprise a pharmaceutically acceptable carrier, diluent, and/or adjuvant. In other embodiments a pharmaceutically acceptable preservative is present. In yet other embodiments, a pharmaceutically acceptable germinate is present. In still other embodiments the therapeutic composition contains, or further comprises, a prebiotic nutrient at an effective dose of 0.005, 0.05, 0.5 , 5.0 milligrams per kilogram body weight.

In alternative embodiments, therapeutic compositions, formulations or pharmaceutical compositions as provided herein, or used to practice methods as provided herein, are in the form of a tablet, geltab or capsule, e.g., a polymer capsule such as a gelatin or a hydroxypropyl methylcellulose (HPMC, or hypromellose) capsule (e.g., VCAPS PLUS™ (Capsugel, Lonza)). In other embodiments, the therapeutic compositions, formulations or pharmaceutical compositions are in or are manufactured as a food or drink, e.g., an ice, candy, lolly or lozenge, or any liquid, e.g., in a beverage.

In alternative embodiments, therapeutic compositions, formulations or pharmaceutical compositions as provided herein, or used to practice methods as provided herein, comprise at least one bacterial type that is not detectable, of low natural abundance, or not naturally found, in a healthy or normal subject’s (e.g., human) gastrointestinal tract. In alternative embodiments, the gastrointestinal tract refers to the stomach, the small intestine, the large intestine and the rectum, or combinations thereof.

In alternative embodiments, provided are methods of ameliorating, preventing or treating cancer and/or at least one symptom resulting from a cancer therapy or of a condition of the gastrointestinal tract.

In alternative embodiments, by administration of a therapeutic composition, formulation or pharmaceutical composition as provided herein to a subject, or practicing a method as provided herein, the microbiome population or composition of the subject is modulated or altered. In alternative embodiments, the term “microbiome” encompasses the communities of microbes that can live sustainably and/or transiently in and on a subject’s body, e.g., in the gut of a human, including bacteria, viruses and bacterial viruses, archaea, and eukaryotes. In alternative embodiments, the term “microbiome” encompasses the “genetic content” of those communities of microbes, which includes the genomic DNA, RNA (ribosomal-, messenger-, and transfer-RNA), the epigenome, plasmids, and all other types of genetic information.

In alternative embodiments, the term "subject" refers to any animal subject including humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), and household pets (e.g., dogs, cats, and rodents). The subject may be suffering from a disease, e.g., a cancer.

In alternative embodiments, the term “type” or “types” when used in conjunction with “bacteria” or “bacterial” refers to bacteria differentiated at the genus level, the species level, the sub-species level, the strain level, or by any other taxonomic method known in the art.

In alternative embodiments, the phrase “dormant live bacteria” refers to live vegetative bacterial cells that have been rendered dormant by lyophilization or freeze drying. Such dormant live vegetative bacterial cells are capable of resuming growth and reproduction immediately upon resuscitation.

In alternative embodiments, the term “spore” also includes “endospore”, and these terms can refer to any bacterial entity which is in a dormant, non-vegetative and non-reproductive stage, including spores that are resistant to environmental stress such as desiccation, temperature variation, nutrient deprivation, radiation, and chemical disinfectants. In alternative embodiments, “spore germination” refers to the dormant spore beginning active metabolism and developing into a fully functional vegetative bacterial cell capable of reproduction and colony formation. In alternative embodiments, “germinanf ’ is a material, composition, and/or physical-chemical process capable of inducing vegetative growth of a dormant bacterial spore in a host organism or in vitro, either directly or indirectly.

In alternative embodiments, the term “colony forming” refers to a vegetative bacterium that is capable of forming a colony of viable bacteria or a spore that is capable of germinating and forming a colony of viable bacteria.

In alternative embodiments, the term “natural polymeric material” comprises a naturally occurring polymer that is not easily digestible by human enzymes so that it passes through most of the human digestive system essentially intact until it reaches the large or small intestine.

In alternative embodiments, therapeutic compositions, formulations or pharmaceutical compositions as provided herein comprise population(s) of non- pathogenic dormant live bacteria and/or bacterial spores. The dormant live bacteria can be capable of colony formation and, in the case of spores, germination and colony formation. Thus, in alternative embodiments, compositions are useful for altering a subject’s gastrointestinal biome, e.g., by increasing the population of those bacterial types or microorganisms, or are capable of altering the microenvironment of the gastrointestinal biome, e.g., by changing the chemical microenvironment or disrupting or degrading intestinal mucin or biofilm, thereby providing treatment of cancer, gastrointestinal conditions, and symptoms resulting from cancer therapy, ultimately increasing the health of the subject to whom they are administered.

In alternative embodiments, the terms “purify,” purified,” and “purifying” are used interchangeably to describe a population’s known or unknown composition of bacterial type(s), amount of that bacterial type(s), and/or concentration of the bacterial type(s); a purified population does not have any undesired attributes or activities, or if any are present, they can be below an acceptable amount or level. In alternative embodiments, the various populations of bacterial types are purified, and the terms “purified,” “purify,” and “purifying” refer to a population of desired bacteria and/or bacterial spores that have undergone at least one process of purification; for example, a process comprising screening of individual colonies derived from fecal matter for a desired phenotype, such as their effectiveness in enhancing the pharmacodynamics of a drug (such as a cancer drug, e.g., a drug inhibitory to an immune checkpoint), e.g., the individual’s ability to absorb a drug is modified (e.g., accelerated or slowed, or enhanced), or the dose efficacy of a drug is increased (e.g., resulting in needing a lower dose of drug for an intended effect), or the immune system is primed for improved drug efficacy, or a selection or enrichment of the desired bacterial types.

Enrichment can be accomplished by increasing the amount and/or concentration of the bacterial types, such as by culturing in a media that selectively favors the growth of certain types of microbes, by screening pure microbial isolates for the desired genotype, or by a removal or reduction in unwanted bacterial types.

In alternative embodiments, bacteria used to practice compositions and methods provided herein are derived from fecal material donors that are in good health, have microbial biomes associated with good health, and are typically free from antibiotic administration during the collection period and for a period of time prior to the collection period such that no antibiotic remains in the donor’s system. In alternative embodiments, the donor subjects do not suffer from and have no family history of renal cancer, bladder cancer, breast cancer, prostate cancer, lymphoma, leukemia, autoimmune disease. In alternative embodiments, donor subjects are free from irritable bowel disease, irritable bowel syndrome, celiac disease, Crohn’s disease, colorectal cancer, anal cancer, stomach cancer, sarcomas, any other type of cancer, or a family history of these diseases. In alternative embodiments, donor subjects do not have and have no family history of mental illness, such as anxiety disorder, depression, bipolar disorder, autism spectrum disorders, panic disorders, obsessive-compulsive disorder, attention-deficit disorders, eating disorders ( e.g . bulimia, anorexia), mood disorder or schizophrenia. In yet other embodiments the donor subjects have no knowledge or history of food allergies or sensitivities.

In alternative embodiments, the health of fecal matter donors is screened prior to the collection of fecal matter, such as at 1, 2, 3, 4, 8, 16, 20, 24, 28, 32, 36, 40, 44, 48, or 52 weeks pre-collection. In alternative embodiments, fecal matter donors are also screened post-collection, such as at 1, 2, 3, 4, 8, 16, 20, 24, 28, 32, 36, 40, 44, 48, or 52 weeks post-collection. Pre- and post- screening can be conducted daily, weekly, bi-weekly, monthly, or yearly. In alternative embodiments, individuals who do not test positive for pathogenic bacteria and/or viruses (e.g. HIV, hepatitis, polio, adeno- associated virus, pox, coxsackievirus, etc.) pre- and post-collection are considered verified donors.

In alternative embodiments, to purify bacteria and/or bacterial spores, fecal matter is collected from donor subjects and placed in an anaerobic chamber within a short time after elimination, such as no more than 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes or more after elimination. In alternative embodiments, fecal matter is collected from donor subjects are placed in an anaerobic chamber within between about 1 minute and 48 hours, or more, after elimination from the donor.

Bacteria from a sample of the collected fecal matter can be collected in several ways. For example, the sample can be mixed with anoxic nutrient broth, dilutions of the resulting mixture conducted, and bacteria present in the dilutions grown on solid anoxic media. Alternatively, bacteria can be isolated by streaking a sample of the collected material directly on anoxic solid media for growth of isolated colonies. In alternative embodiments, to increase the ease of isolating bacteria from fecal samples mixed with anoxic nutrient broth, the resulting mixture can be shaken, vortexed, blended, filtered, and centrifuged to break up and/or remove large non-bacterial matter.

In alternative embodiments, purification of the isolated bacteria and/or bacterial spores by any means known in the art, for example, contamination by undesirable bacterial types, host cells, and/or elements from the host microbial environment can be eliminated by reiterative streaking to single colonies on solid media until at least two replicate streaks from serial single colonies show only a single colony morphology. Purification can also be accomplished by reiterative serial dilutions to obtain a single cell, for example, by conducting multiple 10-fold serial dilutions to achieve an ultimate dilution of 10 2 , 10 3 ,10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or greater. Any methods known to those of skill in the art can also be applied. Confirmation of the presence of only a single bacterial type can be confirmed in multiple ways such as, gram staining, PCR, DNA sequencing, enzymatic analysis, metabolic profiling/analysis, antigen analysis, and flow cytometry using appropriate distinguishing reagents.

In alternative embodiments, purified population(s) of vegetative bacteria that are incorporated into therapeutic bacterial compositions as provided herein, or used to practice methods as provided herein, are fermented in growth media. Suitable growth media include Nutrient Broth (Thermo Scientific™ Oxoid™), Anaerobe Basal Broth (Thermo Scientific™ Oxoid™), Reinforced Clostridial Medium (Thermo Scientific™ Oxoid™), Schaedler Anaerobic Broth (Thermo Scientific™ Oxoid™), MRS Broth (Millipore-Sigma™), Vegitone Actinomyces Broth (Millipore-Sigma™), Vegitone Infusion Broth (Millipore-Sigma™), Vegitone Casein Soya Broth (Millipore- Sigma™), or one of the following media available from Anaerobe Systems: Brain Heart Infusion Broth (BHI), Campylobacter-Thiogly collate Broth (CAMPY-THIO), Chopped Meat Broth (CM), Chopped Meat Carbohydrate Broth (CMC), Chopped Meat Glucose Broth (CMG), Cycloserine Cefoxitin Mannitol Broth with Taurocholate Lysozyme Cysteine (CCMB-TAL), Oral Treponeme Enrichment Broth (OTEB), MTGE-Anaerobic Enrichment Broth (MTGE), Thioglycollate Broth with Hemin, Vit. K, without indicator, (THIO), Thioglycollate Broth with Hemin, Vit. K, without indicator, (THIO), Lactobacilli-MRS Broth (LMRS), Brucella Broth (BRU-BROTH), Peptone Yeast Extract Broth (PY), PY Glucose (PYG), PY Arabinose, PY Adonitol, PY Arginine, PY Amygdalin, PYG Bile, PY Cellobiose, PY DL-Threonine, PY Dulcitol, PY Erythritol, PY Esculin, PYG Formate/Fumarate for FA/GLCf, PY Fructose, PY Galactose, PYG Gelatin, PY Glycerol, Indole-Nitrate Broth, PY Inositol, PY Inulin, PY Lactate for FA/GLCf, PY Lactose, PY Maltose, PY Mannitol, PY Mannose, PY Melezitose, PY Melibiose, PY Pyruvic Acid, PY Raffmose, PY Rhamnose, PY Ribose, PY Salicin, PY Sorbitol, PY Starch, PY Sucrose, PY Trehalose, PY Xylan, PY Xylose, Reinforced Clostridial Broth (RCB), Yeast Casitone Fatty Acids Broth with Carbohydrates (YCFAC Broth). In alternative embodiments, growth media includes or is supplemented with reducing agents such as L-cysteine, dithiothreitol, sodium thioglycolate, and sodium sulfide. In alternative embodiments, fermentation is conducted in stirred-tank fermentation vessels, performed in either batch or fed-batch mode, with nitrogen sparging to maintain anaerobic conditions. pH is controlled by the addition of concentrated base, such as NH4OH or NaOH. In the case of fed-batch mode, the feed is a primary carbon source for growth of the microorganisms, such as glucose. In alternative embodiments, the post-fermentation broth is collected, and/or the bacteria isolated by ultrafiltration or centrifugation and lyophilized or freeze dried prior to formulation.

In alternative embodiments, purified and isolated vegetative bacterial cells used in therapeutic bacterial compositions as provided herein, or used to practice methods as provided herein, have been made dormant; noting that bacterial spores are already in a dormancy state. Dormancy of the vegetative bacterial cells can be accomplished by, for example, incubating and maintaining the bacteria at temperatures of less than 4°C, freezing and/or lyophilization of the bacteria. Lyophilization can be accomplished according to normal bacterial freeze-drying procedures as used by those of skill in the art, such as those reported by the American Type Culture Collection (ATCC) on the ATCC website (see, e.g., (https://www.atcc.org).

In alternative embodiments, the purified population of dormant live bacteria and/or bacterial spores has undetectable levels of pathogenic activities, such as the ability to cause infection and/or inflammation, toxicity, an autoimmune response, an undesirable metabolic response (e.g. diarrhea), or a neurological response. In alternative embodiments, all of the types of dormant live bacteria or bacterial spores present in a purified population are obtained from fecal material treated as described herein or as otherwise known to those of skill in the art. In other embodiments, one or more of the types of dormant live bacteria or bacterial spores present in a purified population is generated individually in culture and combined with one or more types obtained from fecal material. In alternative embodiments, all of the types of dormant live bacteria or bacterial spores present in a purified population are generated individually in culture. In still other embodiments, one or all of the types of dormant live bacteria and/or bacterial spores present in a purified population are non-naturally occurring or engineered. In yet other embodiments, non- naturally occurring or engineered non-bacterial microorganisms are present, with or without dormant live bacteria and/or bacterial spores.

In alternative embodiments, bacterial compositions used in compositions as provided herein, or to practice methods as provided herein, comprise combinations of different bacteria, e.g., comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bacterial types, or more than 20 bacterial types, or between about 2 and 30 bacterial types.

In alternative embodiments, the bacterial compositions comprise at least about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or more (or between about 10 2 to 10 15 ) microbes, for example, dormant live bacteria and/or bacterial spores. In some embodiments each bacterial type is equally represented in the total number of dormant live bacteria and/or bacterial spores. In other embodiments, at least one bacterial type is represented in a higher amount than the other bacterial type(s) found in the composition.

In alternative embodiments, a population of different bacterial types used in compositions as provided herein, or to practice methods as provided herein, can increase microbe populations found in the subject’s gastrointestinal tract by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%, or between about 5% and 2000%, as compared to the subject’s microbiome gastrointestinal population prior to treatment.

In alternative embodiments, the combination of microbes, e.g., combination of bacterial cells and/or spores, used in compositions as provided herein, or to practice methods as provided herein, are mixed with pharmaceutically acceptable excipients, such as diluents, carriers, adjuvants, binders, fillers, salts, lubricants, glidants, disintegrants, coatings, coloring agents, etc. Examples of such excipients are acacia, alginate, alginic acid, aluminum acetate, benzyl alcohol, butyl paraben, butylated hydroxy toluene, citric acid, calcium carbonate, candelilla wax, croscarmellose sodium, confectioner sugar, colloidal silicone dioxide, cellulose, plain or anhydrous calcium phosphate, camuba wax, corn starch, carboxymethylcellulose calcium, calcium stearate, calcium disodium EDTA, copolyvidone, calcium hydrogen phosphate dihydrate, cetylpyridine chloride, cysteine HCL, crossprovidone, calcium phosphate di or tri basic, dibasic calcium phosphate, disodium hydrogen phosphate, dimethicone, erythrosine sodium, ethyl cellulose, gelatin, glyceryl monooleate, glycerin, glycine, glyceryl monostearate, glyceryl behenate, hydroxy propyl cellulose, hydroxyl propyl methyl cellulose, hypromellose, HPMC phthalate, iron oxides or ferric oxide, iron oxide yellow, iron oxide red or ferric oxide, lactose hydrous or anhydrous or monohydrate or spray dried, magnesium stearate, microcrystalline cellulose, mannitol, methyl cellulose, magnesium carbonate, mineral oil, methacrylic acid copolymer, magnesium oxide, methyl paraben, providone or PVP, PEG, polysorbate 80, propylene glycol, polyethylene oxide, propylene paraben, polaxamer 407 or 188, potassium bicarbonate, potassium sorbate, potato starch, phosphoric acid, polyoxyl40 stearate, sodium starch glycolate, starch pregelatinized, sodium carmellose, sodium lauryl sulfate, starch, silicon dioxide, sodium benzoate, stearic acid, sucrose, sorbic acid, sodium carbonate, saccharin sodium, sodium alginate, silica gel, sorbiton monooleate, sodium stearyl fumarate, sodium chloride, sodium metabisulfite, sodium citrate dihydrate, sodium starch, sodium carboxy methyl cellulose, succinic acid, sodium propionate, titanium dioxide, talc, triacetin, and tri ethyl citrate.

In alternative embodiments, the combinations of microbes, e.g., combination of bacterial cells and/or spores, used in compositions as provided herein, or to practice methods as provided herein, are fabricated as colonic or microflora-triggered delivery systems, as described for example, in Basit et al, J. Drug Targeting, 17:1, 64-71;

Kotla, Int J Nanomedicine. 2016; 11: 1089-1095; Bansai et al, Polim Med. 2014 Apr- Jun;44(2):109-18; or, Shah et al, Expert Opin Drug Deliv. 2011 Jun;8(6):779-96.

In alternative embodiments, combinations of microbes, e.g., combination of bacterial cells and/or spores, used in compositions as provided herein, or to practice methods as provided herein, are encapsulated in at least one polymeric material, e.g., a natural polymeric material, such that there is a core of bacterial cells and/or spores surrounded by a layer of the polymeric material, e.g., a polysaccharide. Examples of suitable polymeric materials are those that have been demonstrated to remain intact through the GI tract until reaching the small or large intestine, where they are degraded by microbial enzymes in the intestines. Exemplary natural polymeric materials can include, but are not restricted to, chitosan, inulin, guar gum, xanthan gum, amylose, alginates, dextran, pectin, khava, and albizia gum (Dafe etal. (2017) Int J Biol Macromol; Kofla et al. (2016) Int J Nanomedicine 11 : 1089-1095).

In alternative embodiments, compositions provided herein are suitable for therapeutic administration to a human or other mammal in need thereof. In alternative embodiments the compositions are produced by a process comprising, e.g.,: (a) obtaining fecal material from a mammalian donor subject, (b) subjecting the fecal material to at least one purification treatment under conditions that produce a single bacterial type population of bacteria and/or bacterial spores, or a combination of bacterial types and/or bacterial spores, (c) optionally combining the purified population with another purified population obtained from the same or different fecal material, from cultured conditions, or from a genetic stock center such as ATCC or DSMZ, (d) if the microbes, e.g., bacterial cells, are not dormant, then treating the purified population(s) under conditions that cause vegetative bacterial cells to become dormant, and (e) placing the dormant bacteria and/or bacterial spores in a vehicle for administration.

In alternative embodiments, formulations and pharmaceutical compositions, and microbes, e.g., bacterial cells and/or spores, used in compositions as provided herein or to practice methods as provided herein, are formulated for oral or gastric administration to a mammalian subject. In particular embodiments, the composition is formulated for oral administration as a solid, semi-solid, gel or liquid form, such as in the form of a pill, tablet, capsule, lozenge, food, extract or beverage. Examples of suitable foods are those that require little mastication, such as yogurt, puddings, gelatins, and ice cream. Examples of extracts include crude and processed pomegranate juice, strawberry, raspberry and blackberry. Examples of suitable beverages include cold beverages, such as juices (pomegranate, raspberry, blackberry, blueberry, cranberry, acai, cloudberry, etc., and combinations thereof) and teas (green, black, etc.) and oaked wine.

In alternative embodiments, formulations and pharmaceutical compositions further comprise, or methods as provided herein further comprise administration of, at least one antibiotic, e.g., a doxycycline, chlortetracycline, tetracycline hydrochloride, oxytetracycline, demeclocycline, methacycline, minocycline, penicillin, amoxycillin, erythromycin, vancomycin, clarithromycin, roxithromycin, azithromycin, spiramycin, oleandomycin, josamycin, kitsamysin, flurithromycin, nalidixic acid, oxolinic acid, norfloxacin, perfloxacin, amifloxacin, ofloxacin, ciprofloxacin, sparfloxacin, levofloxacin, rifabutin, rifampicin, rifapentin, sulfisoxazole, sulfamethoxazole, sulfadiazine, sulfadoxine, sulfasalazine, sulfaphenazole, dapsone, sulfacytidine, linezolid or any combination thereof. In alternative embodiments, the antibiotic or a combination of antibiotics are administered before, during and/ or after administration of formulations and pharmaceutical compositions as provided herein.

Gradual or Delayed Release Formulations

In alternative embodiments, exemplary formulations comprise, contain or are coated by an enteric coating to protect a microbe, e.g., a bacteria, in a formulation and pharmaceutical compositions as provided herein to allow it to pass through the stomach and small intestine (e.g., protect the administered combination of microbes such that a substantial majority of the microbes remain viable), although spores are typically resistant to the stomach and small intestines.

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated with a delayed release composition or formulation, coating or encapsulation. In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are designed or formulated for implantation of living microbes, e.g., bacteria or spores, into the gut, including the intestine and/or the distal small bowel and/or the colon. In this embodiment the living microbes, e.g., bacteria pass the areas of danger, e.g., stomach acid and pancreatic enzymes and bile, and reach the intestine substantially undamaged to be viable and implanted in the GI tract.

In alternative embodiments, a formulation or pharmaceutical preparation, or the combination of microbes contained therein, is liquid, frozen or freeze-dried. In alternative embodiments, e.g., for an encapsulated formulation, all are in powdered form. In alternative embodiments, if a formulation or pharmaceutical preparation as provided herein is in a powdered, lyophilate or freeze-dried form, the powder, lyophilate or freeze-dried form can be in a container such as a bottle, cartridge, packet or packette, or sachet, and the powder, lyophilate or freeze-dried form can be hydrated or reconstituted by a liquid, for example by adding water, saline, juice, milk and the like to the powder, lyophilate or freeze-dried form, for example, the powdered, lyophilate or freeze-dried form can be added to the liquid. In alternative embodiments, a powdered, lyophilate or freeze-dried form as provided herein is in a bottle or container, and the liquid is added to the bottle or container, and this mixture can be consumed by an individual in need thereof. In alternative embodiments, a powdered, lyophilate or freeze-dried form as provided herein is in a cartridge that can be part of a container or bottle, and the powdered, lyophilate or freeze-dried form can be mixed with the liquid, e.g., as described in U.S patent no. 8,590,753. In alternative embodiments, a powdered, lyophilate or freeze-dried form as provided herein can be contained in or can be added to a container or bottle as described e.g., in U.S patent nos. 10,315,815; 10,315,803; 10,281,317; 10,183,116; 9,809,374; 9,345,831; 9,173,999; 7,874,420.

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated for delayed or gradual enteric release using cellulose acetate (CA) and polyethylene glycol (PEG), e.g., as described by Defang et al. (2005) Drug Develop. & Indust. Pharm. 31:677-685, who used CA and PEG with sodium carbonate in a wet granulation production process.

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated for delayed or gradual enteric release using a hydroxypropylmethylcellulose (HPMC), a microcrystalline cellulose (MCC) and magnesium stearate, as described e.g., in Huang et al. (2004) European J. of Pharm. & Biopharm. 58: 607-614).

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated for delayed or gradual enteric release using e.g., a poly(meth)acrylate, e.g. a methacrylic acid copolymer B, a methyl methacrylate and/or a methacrylic acid ester, a polyvinylpyrrolidone (PVP) or a PVP-K90 and a EUDRAGIT ® RL PO™, as described e.g., in Kuksal et al. (2006) AAPS Pharm. 7(1), article 1, El to E9.

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20100239667. In alternative embodiments, the composition comprises a solid inner layer sandwiched between two outer layers. The solid inner layer can comprise the non-pathogenic bacteria and/or spores, and one or more disintegrants and/or exploding agents, or one or more effervescent agents or a mixture. Each outer layer can comprise a substantially water soluble and/or crystalline polymer or a mixture of substantially water soluble and/or crystalline polymers, e.g., a polyglycol. These can be adjusted to achieve delivery of the living components to the intestine.

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20120183612, which describes stable pharmaceutical formulations comprising active agents in a non-swellable diffusion matrix. In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are released from a matrix in a sustained, invariant and, if several active agents are present, independent manner and the matrix is determined with respect to its substantial release characteristics by ethylcellulose and at least one fatty alcohol to deliver bacteria distally.

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated for delayed or gradual enteric release as described in U.S. Pat. No. 6,284,274, which describes a bilayer tablet containing an active agent (e.g., an opiate analgesic), a polyalkylene oxide, a polyvinylpyrrolidone and a lubricant in the first layer and a second osmotic push layer containing polyethylene oxide or carboxy- methylcellulose.

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. No. 20030092724, which describes sustained release dosage forms in which a nonopioid analgesic and opioid analgesic are combined in a sustained release layer and in an immediate release layer, sustained release formulations comprising microcrystalline cellulose, EUDRAGIT RSPO™, CAB-O-SIL™, sodium lauryl sulfate, povidone and magnesium stearate. In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20080299197, describing a multi-layered tablet for a triple combination release of active agents to an environment of use, e.g., in the GI tract. In alternative embodiments, a multi-layered tablet is used, and it can comprise two external drug- containing layers in stacked arrangement with respect to and on opposite sides of an oral dosage form that provides a triple combination release of at least one active agent. In one embodiment the dosage form is an osmotic device, or a gastro-resistant coated core, or a matrix tablet, or a hard capsule. In these alternative embodiments, the external layers may contain biofilm dissolving agents and internal layers can comprise viable/ living bacteria, for example, a formulation comprising at least two different species or genera (or types) of non-pathogenic bacteria as used to practice methods as provided herein.

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated as multiple layer tablet forms, e.g., where a first layer provides an immediate release of a formulation or pharmaceutical preparation as provided herein and a second layer provides a controlled-release of another (or the same) bacteria or drug, or another active agent, e.g., as described e.g., in U.S. Pat. No. 6,514,531 (disclosing a coated trilayer immediate/prolonged release tablet), U.S. Pat. No. 6,087,386 (disclosing a trilayer tablet), U.S. Pat. No. 5,213,807 (disclosing an oral trilayer tablet with a core comprising an active agent and an intermediate coating comprising a substantially impervious/impermeable material to the passage of the first active agent), and U.S. Pat. No. 6,926,907 (disclosing a trilayer tablet that separates a first active agent contained in a film coat from a core comprising a controlled-release second active agent formulated using excipients which control the drug release, the film coat can be an enteric coating configured to delay the release of the active agent until the dosage form reaches an environment where the pH is above four).

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20120064133, which describes a release-retarding matrix material such as: an acrylic polymer, a cellulose, a wax, a fatty acid, shellac, zein, hydrogenated vegetable oil, hydrogenated castor oil, polyvinylpyrrolidine, a vinyl acetate copolymer, a vinyl alcohol copolymer, polyethylene oxide, an acrylic acid and methacrylic acid copolymer, a methyl methacrylate copolymer, an ethoxyethyl methacrylate polymer, a cyanoethyl methacrylate polymer, an aminoalkyl methacrylate copolymer, a poly(acrylic acid), a poly(methacrylic acid), a methacrylic acid alkylamide copolymer, a poly(methyl methacrylate), a poly(methacrylic acid anhydride), a methyl methacrylate polymer, a polymethacrylate, a poly(methyl methacrylate) copolymer, a polyacrylamide, an aminoalkyl methacrylate copolymer, a glycidyl methacrylate copolymer, a methyl cellulose, an ethylcellulose, a carboxymethylcellulose, a hydroxypropylmethylcellulose, a hydroxymethyl cellulose, a hydroxyethyl cellulose, a hydroxypropyl cellulose, a crosslinked sodium carboxymethylcellulose, a crosslinked hydroxypropylcellulose, a natural wax, a synthetic wax, a fatty alcohol, a fatty acid, a fatty acid ester, a fatty acid glyceride, a hydrogenated fat, a hydrocarbon wax, stearic acid, stearyl alcohol, beeswax, glycowax, castor wax, carnauba wax, a polylactic acid, polyglycolic acid, a co polymer of lactic and glycolic acid, carboxymethyl starch, potassium methacrylate/divinylbenzene copolymer, crosslinked polyvinylpyrrolidone, polyvinylalcohols, polyvinylalcohol copolymers, polyethylene glycols, non- crosslinked polyvinylpyrrolidone, polyvinylacetates, polyvinylacetate copolymers or any combination thereof. In alternative embodiments, spherical pellets are prepared using an extrusion/ spheronization technique, of which many are well known in the pharmaceutical art. The pellets can comprise one or more formulations or pharmaceutical preparations as provided herein.

In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20110218216, which describes an extended release pharmaceutical composition for oral administration, and uses a hydrophilic polymer, a hydrophobic material and a hydrophobic polymer or a mixture thereof, with a microenvironment pH modifier. The hydrophobic polymer can be ethylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, methacrylic acid-acrylic acid copolymers or a mixture thereof. The hydrophilic polymer can be polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl cellulose, polyethylene oxide, acrylic acid copolymers or a mixture thereof. The hydrophobic material can be a hydrogenated vegetable oil, hydrogenated castor oil, camauba wax, candellia wax, beeswax, paraffin wax, stearic acid, glyceryl behenate, cetyl alcohol, cetostearyl alcohol or and a mixture thereof. The microenvironment pH modifier can be an inorganic acid, an amino acid, an organic acid or a mixture thereof. Alternatively, the microenvironment pH modifier can be lauric acid, myristic acid, acetic acid, benzoic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid, adipic acid, sebacic acid, fumaric acid, maleic acid; glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, sodium dihydrogen citrate, gluconic acid, a salicylic acid, tosylic acid, mesylic acid or malic acid or a mixture thereof.

In alternative embodiments, therapeutic combinations or formulations, or pharmaceuticals or the pharmaceutical preparations as provided herein, or as used in methods as provided herein, are formulated as a delayed or gradual enteric release composition or formulation, and optionally the formulation comprises a gastro- resistant coating designed to dissolve at a pH of 7 in the terminal ileum, for example, an active ingredient is coated with an acrylic based resin or equivalent, for example, a poly(meth)acrylate, for example a methacrylic acid copolymer B, NF, which dissolves at pH 7 or greater, for example, comprises a multimatrix (MMX) formulation. In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are powders that can be included into a suitable carrier, e.g., such as a liquid, a tablet or a suppository. In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are ‘powders for reconstitution’ as a liquid to be drunk, placed down a naso- duodenal tube or used as an enema for patients to take home and self-administer enemas. In alternative embodiments, compositions and formulations as provided herein, and compositions and formulations used to practice methods as provided herein, are micro-encapsulated, formed into tablets and/or placed into capsules, especially enteric-coated capsules. In alternative embodiments, compositions as provided herein are formulated to be effective in a given mammalian subject in a single administration or over multiple administrations. In some embodiments, a substrate or prebiotic required by the bacterial type in a formulation as provided herein is administered for a period of time in advance of the administration of the combination of microbes, e.g., bacterial compositions, as provided herein. Such administration (e.g., of prebiotics) pre-loads the gastrointestinal tract with the substrates needed by the bacterial types of the composition and increases the potential for the bacterial composition to have adequate resources to perform the required metabolic reactions. In other embodiments, the composition is administered simultaneously with the substrates required by the bacterial types a formulation as provided herein. In still other embodiments the substrate or prebiotic is administered alone. In alternative embodiments, efficacy is measured by an increase in the population of those bacterial types in the subject’s intestinal tract, or an increase in the population of those bacterial types originally found in the subject’s intestinal tract before treatment.

In alternative embodiments, compositions as provided herein comprise, further comprise, or have added to: at least one probiotic or prebiotic, wherein optionally the prebiotic comprises an inulin, lactulose, extracts of artichoke, chicory root, oats, barley, various legumes, garlic, kale, beans or flacks or an herb, wherein optionally the probiotic comprises a cultured or stool-extracted microorganism or bacteria, or a bacterial component, and optionally the bacteria or bacterial component comprises or is derived from a Bacteroidetes, a lurmicutes, & Lactobacilli, a Bifidobacteria, an E. coli, a Streptococcus fecalis and equivalents.

In alternative embodiments, compositions as provided herein comprise, further comprise, or have added to: at least one congealing agent, wherein optionally the congealing agent comprises an arrowroot or a plant starch, a powdered flour, a powdered potato or potato starch, an absorbant polymer, an Absorbable Modified Polymer, and/or a corn flour or a corn starch; or, further comprise an additive selected from one or more of a saline, a media, a defoaming agent, a surfactant agent, a lubricant, an acid neutralizer, a marker, a cell marker, a drug, an antibiotic, a contrast agent, a dispersal agent, a buffer or a buffering agent, a sweetening agent, a debittering agent, a flavoring agent, a pH stabilizer, an acidifying agent, a preservative, a desweetening agent and/or coloring agent, vitamin, mineral and/or dietary supplement, or a prebiotic nutrient; or, further comprise, or have added to: at least one Biofilm Disrupting Compound, wherein optionally the biofilm disrupting compound comprises an enzyme, a deoxyribonuclease (DNase), N-acetylcysteine, an auranofm, an alginate lyase, glycoside hydrolase dispersin B; a Quorum-sensing inhibitor, a ribonucleic acid III inhibiting peptide, Salvadora persica extracts, Competence-stimulating peptide, Patulin and penicillic acid; peptides - cathelicidin- derived peptides, small lytic peptide, PTP-7, nitric oxide, neo-emulsions; ozone, lytic bacteriophages, lactoferrin, xylitol hydrogel, synthetic iron chelators, a statin (optionally lovastatin (optionally MEVACOR™), simvastatin (optionally ZOCOR™), atorvastatin (optionally LIPITOR™), pravastatin (optionally PRAVACHOL™), fluvastain (optionally LESCOL™) or rosuvastatin (optionally CRESTOR™)), cranberry components, curcumin, silver nanoparticles, Acetyl-11- keto-P-boswellic acid (AKBA), barley coffee components, probiotics, sinefungin, S- adenosylmethionine, S-adenosyl-homocysteine, Delisea furanones, N-sulfonyl homoserine lactones or any combination thereof.

In alternative embodiments, compositions as provided herein comprise, further comprise, or have added to: a flavoring or a sweetening agent, an aspartamine, a stevia, monk fruit, a sucralose, a saccharin, a cyclamate, a xylitol, a vanilla, an artificial vanilla or chocolate or strawberry flavor, an artificial chocolate essence, or a mixture or combination thereof.

Products of Manufacture and Kits

Provided are products of manufacture, e.g., implants or pharmaceuticals, and kits, containing components for practicing methods as provided herein, e.g., including a formulation comprising a combination of microbes as provided herein, such as e.g., freshly isolated microbes, cultured microbes, or genetically engineered microbes, or at least two different species or genera (or types) of non-pathogenic bacteria, wherein each of the non-pathogenic bacteria comprise (or are in the form of) a plurality of non-pathogenic colony forming live bacteria, a plurality of non-pathogenic germinable bacterial spores, or a combination thereof, and optionally including instructions for practicing methods as provided herein.

Companion Diagnostics and Patient Biomarkers

Provided are biomarkers indicative of patient response or non-response to a composition or method as provided herein, including e.g., a chemotherapy, a radiation therapy, an immune checkpoint inhibitor (e.g., a checkpoint inhibitor therapy), a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment. These biomarkers may be in the form of microbial species abundance in the gut (or abundance in the colon), microbial gene expression or protein expression, or abundance of a metabolite in a stool sample or a sample of bacteria taken from the gut. Alternatively, the biomarkers may be metabolite concentration, cytokine profile, or protein expression in the blood. These biomarkers are used to develop a diagnostic screen to predict in advance whether a patient will naturally respond to therapy or will require microbial intervention to enable the composition or method as provided herein, e.g., checkpoint inhibitors or CAR-T therapy, to function efficaciously or more efficaciously as compared to their effectiveness in the patient if a composition or method as provided herein had not been administered.

Genetic Modification of Microbial Therapeutics

In alternative embodiments, microbes, e.g., bacteria, used in compositions as provided herein, or used to practice methods as provided herein, are genetically engineered. In alternative embodiments, microbes are genetically engineered to increase their efficacy, e.g., to increase the efficacy of a chemotherapy, a radiation therapy, an immune checkpoint inhibitor (e.g., a checkpoint inhibitor therapy), a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment. In alternative embodiments, one several or all of a combination of microbes as provided herein, or used to practice methods as provided herein, are genetically engineered. In alternative embodiments, microbes are genetically engineered to substantially decrease, reduce or eliminate their toxicity. In alternative embodiments, microbes are genetically engineered to comprise a kill switch so they can be rendered non-vital after administration of an appropriate trigger or signal. In alternative embodiments, microbes are genetically engineered to secrete anti inflammatory compositions or have an anti-inflammatory effect. In alternative embodiments, microbes are genetically engineered to secrete an anti-cancer substance.

Microbes, e.g., bacteria, used in compositions as provided herein, or used to practice methods as provided herein, can be genetically engineered using any method known in the art, e.g., as discussed in the Examples, below. For example, one or more gene sequence(s) and/or gene cassette(s) may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid is used to increase expression of an inserted, e.g., heterologous nucleic acid, e.g., a gene or protein encoding sequence or an inhibitory nucleic acid such as an antisense or siRNA-encoding nucleic acid. The inserted nucleic acid of interest can be inserted into a bacterial chromosome at one or more integration sites. For example, in alternative embodiments, microbes are genetically engineered to comprise one or more gene sequence(s) and/or gene cassette(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. In alternative embodiments, the anti-inflammation and/or gut barrier function enhancer molecule comprises a short-chain fatty acid, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2, GLP-1, IL-10, IL-27, TGF-.beta.l, TGF-.beta.2, N-acylphosphatidylethanolamines (NAPES), elafin (also known as peptidase inhibitor 3 or SKALP), trefoil factor, melatonin, PGD2, kynurenic acid, and kynurenine. A molecule may be primarily anti-inflammatory, e.g., IL-10, or primarily gut barrier function enhancing, e.g., GLP-2. In alternative embodiments, microbes are genetically engineered to comprise one or more gene sequence(s) and/or gene cassette(s) that are inhibitory to the activity of, or substantially or completely inhibit expression of, bacterial virulence factors, toxins, or antibiotic resistance functions.

Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and/or Detailed Description sections.

As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of’, “substantially all of’ or “majority of’ encompass at least about 90%, 95%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition.

The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court.

Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of' may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

EXAMPLES

Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994)

Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

The following Examples describe methods and compositions for practicing embodiments as provided herein, including methods for making and using compositions comprising non-pathogenic bacteria and non-pathogenic germinable bacterial spores used to practice methods as provide herein.

Example 1: Anaerobic Culture Conditions

Preparation of Anaerobic Growth Medium

Exemplary bacterial strains described herein are obligate anaerobes that require anaerobic conditions for culture. Growth media suitable for culture of anaerobic bacteria include reducing agents such as L-cysteine, sodium thioglycolate, and dithiothreitol, for the purpose of scavenging and removing oxygen. Appropriate commercially available anaerobic growth media include but are not limited to Anaerobe Basal Broth (Oxoid/Thermo Scientific), Reinforced Clostridial Medium (Oxoid/Thermo Scientific), Wilkins-Chalgren Anaerobe Broth (Oxoid/Thermo Scientific), Schaedler Anaerobe Broth (Oxoid/Thermo Scientific), and Brain Heart Infusion Broth (Oxoid/Thermo Scientific). Animal free medium for anaerobic culture include but are not limited to Vegitone Actinomyces Broth (Millipore-Sigma), MRS Broth (Millipore-Sigma), Vegitone Infusion Broth (Millipore-Sigma), and Vegitone Casein Soya Broth (Millipore-Sigma).

One liter of Anaerobic growth medium is prepared by combining the manufacturer’s recommended amount in grams of dry growth medium powder with 800 ml Reagent Grade Water (NERL™) along with 1 ml 2.5 mg/ml resazurin (ACROS Organics™) in a 2 liter beaker and stirred on a heated stir plate until dissolved. The volume is adjusted to 1 liter by addition of additional Reagent Grade Water, then the volume is brought to a boil while stirring until the red color imbued by the resazurin becomes colorless, indicating removal of oxygen from the solution. The volume is then removed from the stir plate to cool for 10 minutes on the benchtop before further manipulation.

From the 1 -liter volume, 900 ml is transferred to a 1 liter anaerobic media bottle (Chemglass Life Sciences) and then placed back on the heated stir plate to remove any oxygen introduced in the transfer, as indicated by the color of the added resazurin. The anaerobic media bottle is then stoppered with a butyl rubber bung that is secured by a crimped aluminum collar, and then brought into the anaerobic chamber (Coy Lab Type A Vinyl Anaerobic Chamber, Coy Laboratory Products, Grass Lake, MI). The butyl rubber bung is removed to open the bottle within the anaerobic chamber to equilibrate with the anoxic atmosphere while cooling to ambient temperature. The bottle is resealed with a fresh butyl rubber bung and crimped aluminum collar, brought out of the chamber, then sterilized by autoclaving for 20 minutes followed by slow exhaust.

Alternatively, the 1 -liter volume can be aliquoted into smaller 50 ml volumes in 100 ml serum bottles (Chemglass Life Sciences, Vineland New Jersey). The boiled 1 -liter volume is transferred to a one-liter screwcap bottle, which is placed back on the heated stir plate to drive off any oxygen introduced by the transfer. The bottle cap is then securely tightened, and the bottle is immediately brought into the anaerobic chamber, where the cap is loosened to allow the volume to equilibrate with the anoxic atmosphere and to cool for 1 hour. The volume is then transferred in 50 ml aliquots to 100 ml serum bottles using a serological pipette, then the liquid contents cooled to ambient temperature. The bottles are sealed with butyl rubber bungs and crimped aluminum collars, brought out of the chamber, then sterilized by autoclaving for 20 minutes followed by slow exhaust.

Alternatively, the 1 -liter volume can be aliquoted into smaller 10 ml volumes in sealed Hungate tubes (Chemglass Life Sciences, Vineland New Jersey) as follows. The boiled 1 -liter volume is transferred to a one-liter screwcap bottle, which is placed back on the heated stir plate to drive off any oxygen introduced by the transfer. The bottle cap is then securely tightened, and the bottle is immediately brought into the anaerobic chamber, where the cap is loosened to allow the volume to equilibrate with the anoxic atmosphere and to cool for 1 hour. The volume is then transferred in 10 ml aliquots to fill racked Hungate tubes, then allowed to cool to ambient temperature, followed by securely capping and sealing each tube with screwcaps with butyl rubber septa. The sealed Hungate tube aliquots are removed from the anaerobic chamber and then sterilized by autoclaving for 20 minutes followed by slow exhaust.

Alternatively, the 1 liter volume can be combined with 15 grams Agar (Thermo Scientific™) to make solid media in culture plates as follows: The boiled 1 liter volume is poured into a 1 liter screwcap bottle, followed by replacement on a heated stir plate to remove any oxygen introduced by the transfer as indicated by the colorless resazurin oxygen indicator. The bottle is loosely capped and then autoclaved for 20 minutes followed by slow exhaust. Immediately after autoclaving, the cap of the bottle is tightened prior to bringing the bottle into the anaerobic chamber. Once in the anaerobic chamber, the cap is loosened and the contents cooled for 30 minutes, then 25 ml volumes are poured into culture plates and allowed to cool until solidified. The plates are then allowed to dry in the anaerobic chamber for 24 hours prior to use.

Live Cryostorage of Anaerobic Microbes

Individual microbes of interest are prepared for long-term cryogenic live storage by inoculating a pure colony isolate grown on anaerobic solid medium into a prepared Hungate tube containing liquid anaerobic growth medium previously determined to be optimal for the species. The inoculated Hungate tube is then incubated at 37°C until turbidity evident of exponential growth is observed. The Hungate culture is brought into the anaerobic chamber, and 1 ml is transferred by pipette into a 2 ml screwcap cryotube containing anoxic 1 ml Biobank Buffer (Phosphate Buffered Saline (PBS) plus 2% trehalose plus 10 % dimethyl sulfoxide, filter sterilized and bubbled with nitrogen gas to remove oxygen). The resulting 2 ml volume is thoroughly mixed by pipetting, securely tightened, then placed for long term storage in the gaseous phase of a liquid nitrogen Dewar or in a -80°C freezer.

Microbes in fecal matter can be cryogenically preserved for later revival and new strain discovery as follows. Freshly obtained fecal material is brought into the anaerobic chamber and 1 gram is weighed and mixed in a 15 ml conical tube with a solution consisting of 5 ml Anaerobe Basal Broth (ABB) and 5 ml Biobank Buffer. The tube is tightly capped, and the fecal matter is thoroughly suspended in the solution by vortexing for 20 minutes, followed by incubation upright on ice to allow large particles to settle. One ml aliquots of the fecal suspension are then transferred by pipette to a 2 ml screwcap cryotube, securely tightened, then placed for long-term storage in the gaseous phase of a liquid nitrogen Dewar or in a -80°C freezer. Example 2: Fecal Mater Collection from Patients and Processing

Fecal matter donations are acquired from healthy volunteers as well as individuals exhibiting disease symptoms. Donors can be cancer patients being administered approved therapies or participating in clinical trials testing various cancer treatment regimens. Donors can be healthy volunteers that do not exhibit disease symptoms.

Donors receive a stool sampling kit by mail sent to the contact address provided or by their physician. Stool samples are collected by the subject at home, or with necessary assistance if hospitalized. Stool sampling kits consist of the following: gloves, instructions for stool collection, welcome card, freezer pack, Styrofoam container, plastic bracket and plastic commode to aid in stool collection, Bristol stool chart, FedEx shipping labels, and stickers to seal kit prior to shipping. Subjects receive a freezer pack for chilling the samples and are instructed to place it in their freezer overnight upon receipt of the sampling kit. The stool sampling kit also includes a plastic commode that can be placed safely and securely on a toilet seat, allowing the subject to defecate directly into a plastic container. The subject is instructed to use the commode to capture a stool sample, then seal the sample container with a provided snap-cap lid. Subjects are instructed to wear the gloves provided in the kit before removing the sample container from the toilet. The subject is instructed to seal the plastic container inside a specimen bag and remove gloves. The subject is then instructed to remove the ice pack from their home freezer and place it inside the Styrofoam cooler box along with the bagged and sealed stool sample, and the graded Bristol Stool card (form indicating stool collection date/time and consistency). The subject is instructed to close the lid on the foam container and then close the box, sealing with the packing sticker. The subject is instructed to schedule a FedEx pickup at their home within 24 hours of stool collection or drop it off at the nearest FedEx location kinder these conditions the stool has been demonstrated to remain chilled during shipment for as long as 48 hours.

Once received, the stool sample receptacle is given a unique alphanumeric identifier that is used subsequently for sample tracking. The stool is unpacked from the shipping box in a laboratory setting, homogenized, and divided into enough individual aliquots for all projected analyses prior to freezing and storage at -80°C, as described below. All aliquots also bear an alphanumeric identifier corresponding to the subject donor. Any remaining stool after the aliquots are taken is disposed as biohazardous waste.

Preparation of Fecal Matter Samples for Analysis

Fecal matter received from donors can be processed using any method known in the art, for example, as described in USPN 10,493,111; 10,471,107; 10,286,012; 10,314,863; 9,623,056.

For example, received fecal matter in its receptacle is placed on ice and then brought into the anaerobic chamber. The receptacle is opened and approximately 40 g stool is weighed into a tared specimen cup. 15 ml sterile anoxic PBS is then added, and the mixture is homogenized by a hand-held homogenizer to achieve a smooth consistency.

The homogenized fecal matter is then processed and aliquoted for cryo- preservation for several different analyses as follows:

1) For Genomic and Transcriptomic Analyses: homogenized fecal matter is weighed and then an equal volume to weight amount of RNAlater® (Thermo Fisher Scientific) solution is added. The tube is capped tightly and then vortexed for 20 seconds and then placed on ice. A pipette is used to transfer 1 ml aliquots into 2 ml Eppendorf tubes. Aliquoted samples are frozen on dry ice and then stored at - 80°C.

2) Live Cryopreservation for Fecal Microbiome Transfer (FMT) Experiments in Mice: Homogenized fecal matter is combined with FMT Buffer (Phosphate Buffered Saline plus 1% L-Cysteine plus 2% Trehalose plus 30% glycerol). The tube is then vortexed for 20 seconds and then placed on ice. A pipette is used to transfer 1 ml aliquots into 2 ml cryotubes that are then tightly capped. Aliquoted samples are frozen on dry ice and then stored at -80°C.

3) Live Cryopreservation for Isolation and Discovery of Microbes: Homogenized fecal matter is combined in a conical tube with Anaerobe Basal Broth and Biobank Buffer (Phosphate Buffered Saline plus 2% Trehalose plus 10% dimethyl sulfoxide), tightly capped and vortexed for 20 seconds, then put on ice upright and allowed to settle for 10 minutes. Using a pipette, 1 ml aliquots are added to 2 ml cryotubes, which are then tightly capped. Aliquoted samples are frozen on dry ice and then stored at -80°C. For Genomic and Metabolomic Analyses: Homogenized fecal matter is added to a plastic bag. About 1 cm of the tip end of the bag is cut off with scissors, then aliquots are made by manually squeezing 1 ml of the bag contents into 2 ml Eppendorf tubes. Aliquoted samples are frozen on dry ice and then stored at - 80°C.

Example 3: Isolation and Characterization of Pure Microbial Strains from Fecal Matter

In alternative embodiments, microbes used in compositions as provided herein, or used to practice methods as provided herein, are isolated from fecal matter, and can be used on the form of a pure microbial strain isolated from fecal matter.

Individual bacterial strains can be isolated and cultured from fecal matter material for further study and for assembly of therapeutic biologicals, i.e. for manufacturing combinations of microbes as provided herein. The majority of live bacteria that inhabit fecal matter tend to be obligate anaerobes so care must be taken to perform all culture and isolation work in the anaerobic chamber to prevent their exposure to oxygen, and to use various anaerobic growth media that includes reductant compounds as described in Example 1. Growth media that favor growth of target bacteria can be used to improve the ability to find and isolate them as pure living cultures. Different anaerobic growth media are used to enable growth of different subsets of microbes to improve overall ability to isolate and purify an inclusive number of unique bacterial species from each individual fecal material sample.

To begin a microbial isolation and characterization campaign, one cryotube containing cryogenically preserved fecal matter is removed from storage in the liquid nitrogen Dewar, brought into the anaerobic chamber, and then allowed to thaw gently on ice. The entire 1 ml contents are added to 10 ml of Anaerobe Basal Broth (ABB) or another suitable anaerobic growth medium to establish a 1/10 dilution. Successive 10-fold serial dilutions are then performed in ABB to establish 1/100, 1/1000,

1/10000, 1/100000, 1/1000000 dilutions of the fecal matter. From each of the 1/10000, 1/100000, and 1,1000000 dilutions, four 0.1 ml volumes are removed and then added to and spread over solid anaerobic growth medium of choice. The platings are incubated at 37°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days to allow for a wide variety of bacterial colonies to grow. Platings are made from several liquid dilutions of fecal matter to ensure that there will be ones that have numerous yet non overlapping colonies for efficient colony picking.

Colonies are manually picked from plates using sterile pipette tips. Colonies may also be picked by an automated colony picking machine that is enclosed in an anaerobic chamber. Colonies are picked in multiples of 96 to accommodate subsequent 96-well-based genomic DNA isolation steps and large-scale cryogenic storage steps. The individual picked colonies are then struck on solid anaerobic growth medium of choice to isolate single purified colonies from each picked colony, and then incubated at 37°C for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days to allow for visible colony growth to arise. After visible colonies are evident on the streak, single colonies are picked and then each inoculated into an individual well of a 2 ml 96-well deep well block, each well with 1 ml liquid anaerobic growth medium of choice.

Once all wells of the deep-well block have been inoculated with different picked colonies, the deep well block is covered with an adhesive gas-permeable seal and then incubated at 37°C in an incubator within the anaerobic chamber for 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12 days to allow for liquid growth from each isolated colony.

After turbid growth is apparent in all wells, the gas-permeable seal is removed from the 96-well deep well block and a viable stock representation is made by transferring 0.1 ml culture from each well to the corresponding wells of a second 96- well deep-well block, each well containing 0.4 ml of the same anaerobic growth medium plus 0.5 ml Biobank Buffer (Phosphate Buffered Saline plus 2% Trehalose plus 10% dimethyl sulfoxide. The volumes in each well are thoroughly mixed by pipetting up and down several times, then the deep-well block is sealed with an impermeable foil seal rated for -80°C storage, then stored in a -80°C freezer.

Sequence and computational characterization of isolated fecal bacteria

The remaining 0.9 ml culture in the original 96-well deep-well plate is then used for whole genome sequence determination of the isolated strain as follows: The deep-well block is subjected to centrifugation for 20 minutes at 6000 g to pellet the cells. After centrifugation, 0.8 ml supernatant is carefully removed by pipette, leaving 0.1 ml pellet and medium for gDNA processing. Total genomic DNA is extracted from the cell pellet using the MagAttract PowerMicrobiome DNA/RNA EP kit (Qiagen). Genomic DNA is then prepared for Whole Genome Sequencing analysis using the sparQ DNA Frag & Library Prep kit (Quantabio). Sequencing analysis is conducted on the Illumina platform using paired-end 150 bp reads.

Sequencing data is processed to remove low quality reads and adapter contamination using Trim Galore, a wrapper for cutadapt (https://journal.embnet.org/index.php/embnetjournal/article/ view/200).

The high-quality reads for each isolate are compared against each bacterial or archaeal assembly in NCBI RefSeq using mash

(https://genomebiology.biomedcentral.eom/articles/10.l 186/s 13059-016-0997-x).

This identifies the most similar organism in the RefSeq database to each isolate at the species and strain level. If the distance reported by mash is below 0.01, the isolate is assumed to be the same strain as the reference strain. If the distance is less than 0.04, the isolate is assumed to be of the same species as the reference strain. If the distance is greater than 0.04, the isolate is assumed to be of a potentially novel species; these isolates are handled on a case-by-case basis.

Further analysis is performed on isolates of interest by assembling with SPAdes (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342519/) and using mummer (https://journals.plos.org/ploscompbiol/article7idM0.1371/ journal. pebi.1005944) to align the reference genome and isolate genome against each other.

Complete genomes are generated for organisms of special interest using long- read sequencing. High molecular weight genomic DNA is prepared from organisms of interest using a commercially available kit e.g. Genomic-tip (Qiagen). Library preparation on genomic DNA is performed using the Ligation Sequencing Kit (Oxford Nanopore) and sequencing is performed on a MinlON (Oxford Nanopore). Reads are filtered and trimmed for quality and assembly is performed using the assembler Flye (Kolmogorov et al. (2019) Nature Biotechnology 37:540-546). The resulting assembly is polished using multiple rounds of pilon (Walker et al. (2014) PLOS ONE 9:el 12963) with short reads to correct for errors inherent in long read sequencing. Genes are predicted on the polished genome using prodigal (Hyatt etal. (2010) BMC Bioinformatics 11 : 119) or the NCBI Prokaryotic Gene Annotation Pipeline (Tatusova etal. (2016) Nucleic Acids Research 44(14):6614-24). Results of this analysis on isolates collected so far are provided in Table 1. Table 1: Exemplary bacterial strains isolated from human fecal material that can be used alone to practice methods as provided herein, or in making or using combinations of microbe compositions as provided herein. a Listed are the closest genome/species matches for each strain, determined by the analysis described in the text.

Antibiotic Resistance Characterization of Isolated Strains from Fecal Matter

The complete genome sequence of each organism is screened to ensure it contains no genes or pathogenicity island gene clusters encoding known virulence factors, toxins, or antibiotic resistance functions, using publicly available databases such as DBETH55 (for example, see Chakraborty A, et al. (2012 ) Nucleic Acids Res. 40:615-620) and VFDB56 (Chen L, et al. (2005) Nucleic Acids Res . 33:325-328). Each organism is tested by standard antibiotic sensitivity profile techniques such as broth microdilution susceptibility panels or plate-based methods such as disk diffusion method and antimicrobial gradient method (James H. Jorgensen and Mary Jane Ferraro 2009 Clinical Infectious Diseases 49: 1749-1755). Such tests determine the minimal inhibitory concentration (MIC) of an antibiotic on microbial growth. Antibiotics tested include but are not limited to amoxicillin, amoxicillin/clavulanic acid, carbapenem, methicillin, ampicillin, gentamicin, metronidazole, and neomycin. MIC determinations of novel microbes are compared to published values for both sensitive and resistant related strains to make an assessment on sensitivity (CL SI Guideline M45: Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria. Wayne, PA; 2015) to type strains of related microbes to determine possible relative increases in antibiotic resistance.

Example 4: Isolation and Characterization of Pure Microbial Strains from Endospores Purified from Fecal Matter

In alternative embodiments, microbes used in compositions as provided herein, or used to practice methods as provided herein, are derived from, or are cultured as, pure microbial strains derived from endospores purified or derived from fecal matter.

Individual spore-forming bacterial strains can be preferentially isolated and cultured from endospores purified from fecal matter using a protocol adapted from Kearney et al 2018 ISME J. 12:2403-2416. Purified endospores are spread on solid anaerobic medium plates and allowed to germinate and form colonies that can be further characterized. Vegetative cells in the fecal matter are rendered non-viable during the endospore purification process, and thus any resulting colonies are restricted to spore-forming bacteria. Endospores are purified from fecal matter as follows:

Fecal samples are collected and processed in an anaerobic chamber within 30 minutes of defecation. Samples (5 g) are suspended in 20 mL of 1% sodium hexametaphosphate solution (a flocculant) in order to bring biomass into suspension. The suspension is bump vortexed with glass beads to homogenize and centrifuged at 50 x g for 5 min at room temperature to sediment particulate matter and beads. Quadruplicate 1 mL aliquots of the supernatant liquid is transferred into cryovials and stored at -80°C until processing.

The frozen supernatant liquid samples are thawed at 4°C, centrifuged at 4°C and 10,000 x g for 5 minutes, washed and then resuspended in 1 mL Tris-EDTA pH 7.6. The samples are heated at 65°C for 30 minutes with shaking at 100 rpm and then cooled on ice for 5 minutes. Lysozyme (10 mg/mL) is added to a final concentration of 2 mg/mL and the samples are incubated at 37°C for 30 minutes with shaking at 100 rpm. At 30 minutes, 50 pL Proteinase K (>600 mAU/ml) (Qiagen) is added and the samples incubated for an additional 30 minutes at 37°C. 200 pL 6% SDS, 0.3 N NaOH solution is added to each sample and incubated for 1 hour at room temperature with shaking at 100 rpm. Samples are then centrifuged at 10,000 rpm for 30 minutes. At this step, a pellet containing resistant endospores is visible, and the pellet is washed three times at 10,000 x g with 1 mL chilled sterile ddH20. The pellet containing endospores is stored at -20°C until required.

To germinate and resuscitate spore-forming bacterial colonies from the purified endospores, the endospore pellet is brought into the anaerobic chamber, thawed and then suspended in 1.0 ml reduced ABB. Successive 10-fold serial dilutions of the suspended spores are then performed in ABB to establish 1/10, 1/100, 1/1000, 1/10000, 1/100000, 1/1000000 dilutions of the endospore preparation. From each 10-fold serial dilution, four 0.1 ml volumes are removed and then added to and spread over Reinforced Clostridial Medium Agar (Oxoid), with 0.1% intestinal bile salts (taurocholate, cholate, glycocholate) to stimulate endospore germination. The platings are incubated at 37°C for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days to allow for the endospores to germinate and grow as single colonies. These colonies are then manually picked, individually cultivated, and the subjected to identification by whole genome sequencing analysis as described in Example 3.

Example 5: Stability Testing

In alternative embodiments, microbes used in compositions as provided herein, or used to practice methods as provided herein, comprise or can be derived from any one of family or genus (or class): Agathobaculum (TaxID: 2048137), Alistipes (TaxID: 239759), Anaeromassilibacillus (TaxID: 1924093), Anaerostipes (TaxID: 207244), Asaccharobacter (TaxID: 553372), Bacteroides (TaxID: 816), Bamesiella (TaxID: 397864), Bifidobacterium (TaxID: 1678), Blautia (TaxID: 572511), Butyricicoccus (TaxID: 580596), Clostridium (TaxID: 1485), Collinsella (TaxID: 102106), Coprococcus (TaxID: 33042), Dorea (TaxID: 189330), Eubacterium (TaxID: 1730), Faecalibacterium (TaxID: 216851), Fusicatenibacter (TaxID: 1407607), Gemmiger (TaxID: 204475), Gordonibacter (TaxID: 644652), Lachnoclostridium (TaxID: 1506553), Methanobrevibacter (TaxID: 2172), Parabacteroides (TaxID: 375288), Romboutsia (TaxID: 1501226), Roseburia (TaxID: 841), Ruminococcus (TaxID: 1263), Erysipelotrichaceae (TaxID: 128827), Coprobacillus (TaxID: 100883), Erysipelatoclostridium sp. SNUG30099 (TaxID: 1982626), Erysipelatoclostridium (TaxID: 1505663), Acetatifactor (TaxID: 1427378), Adlercreutzia (TaxID: 447020), Agathobacter (TaxID: 1766253), Anaerotruncus (TaxID: 244127), Bariatricus (TaxID: 1924081), Butyrivibrio (TaxID: 830), Christensenellaceae (TaxID: 990719), Clostridiales (TaxID: 186802), Dialister (TaxID: 39948), Drancourtella (TaxID: 1903506), Eggerthella (TaxID: 84111), Eisenbergiella (TaxID: 1432051), Enterocloster (TaxID: 2719313), Enterococcus (TaxID: 1350), Intestinibacter (TaxID: 1505657), Lachnospira (TaxID: 28050), Lachnospiraceae (TaxID: 186803), Mediterraneibacter (TaxID: 2316020), Negativibacillus (TaxID: 1980693), Oscillibacter (TaxID: 459786), Phocaeicola (TaxID: 909656), Pseudobutyrivibrio (TaxID: 46205), Pseudoflavonifractor (TaxID: 1017280), Ruminococcaceae (TaxID: 541000), Sellimonas (TaxID:

1769710), Solobacterium (TaxID: 123375), Terrisporobacter (TaxID: 1505652), Tidjanibacter (TaxID: 1929083), Veillonella (TaxID: 29465), for any combination thereof.

In alternative embodiments, any microbe used in a composition as provided herein, or used to practice methods as provided herein, for example, including a microbe as listed above, can be stored in a sealed container, e.g., at 25° C or 4° C and the container can be placed in an atmosphere having 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95% relative humidity, or between about 20% and 99% relative humidity. In alternative embodiments, after 1 month, 2 months, 3 months, 6 months,

1 year, 1.5 years, 2 years, 2.5 years or 3 years, at least 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain as measured in colony forming units determined by standard protocols.

Example 6 - In Silico Modeling to Discover Microbe-Microbe Interactions

Microbe-microbe interactions are determined to exploit and manipulate metabolic reactions present in the gut microbiome using compositions and methods as provided herein for, e.g., increasing the efficacy of a chemotherapy, a radiation therapy, an immune checkpoint inhibitor (e.g., a checkpoint inhibitor therapy), a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment.

Genome scale metabolic modeling is used as a tool to explore the diversity of metabolic reactions present in the gut microbiome, interpret the omics data described here in the framework of cellular metabolism, and evaluate inter-species interactions. A set of 773 different organism-specific metabolic models have been created (Magnusdottir et al. Nature Biotechnology 2017, 35(l):85-89) and are used in this work. Models are used individually to predict the metabolic capabilities of each organism and combined to enable multispecies simulations that predict how these organisms interact when supplied with a nutrient mix mimicking the typical Western human diet or variations thereof. Simulations are performed using the COBRA™ package v2.0™ (Schellenberger et al., Nature Protocols 2011, 6: 1290-1307) or updated versions thereof. Commensal relationships among the organisms result when one or more species consume a compound that another species produces and can be detected by an increased maximum predicted growth rate of each species when growing together than when each is grown separately. In the cases where commensalism is not predicted in the live biotherapeutics provided, simulations are used to identify a suitable microbial partner that can be included in the live biotherapeutic product, thus improving the ability of the active microbes to grow in the gut ecosystem. Similarly, simulations are used to identify prebiotic compounds to be supplemented that can be utilized by the active species as a carbon or energy source.

Metabolic models are downloaded from the Thiele lab website (https://wwwen.uni.lu/lcsb/research/mol_systemsj3hysiology/i n_silico_models) for the following organisms: Coprococcus comes , Dorea formicigenerans , Anaerostipes hadrus , Dorea longicatena , Coprococcus eutactus, Ruminococcus lactaris, Coprococcus catus, Fusicatenibacter saccharivorans, Lachnoclostridium sp. SNUG30099, Clostridium sporogenes, Eubacterium ventriosum , Blautia obeum , Erysipelotrichaceae bacterium GAM147, Akkermansia muciniphilia, Faecalibacterium prauznitzii, Ruminococcus torques, Ruminococcus gnavus, Eubacterium hallii, Blautia obeum, and Clostridium scindens. The models are then used for simulations in the COBRA v2.0™ package (Schellenberger et al., Nature Protocols 2011, 6:1290-1307). Cell metabolism is simulated by defining nutrient uptake rates (mmol/gDCW-hr) and optimizing for growth of each organism (hr 1 ). Oxygen uptake rate is set to zero, to simulate anaerobic conditions. Values for each nutrient uptake rate are obtained from (Magnusdottir et al. Nature Biotechnology 2017, 35(l):85-89, Supplemental Table 12), as estimated for atypical Western diet. To simulate the gut ecosystem comprising of multiple bacterial species, each organism model is treated as a separate compartment, with the extracellular space in the gut considered an additional compartment. Nutrients can enter and exit the extracellular space freely, to simulate food uptake and waste excretion. Nutrients can enter and exit each microbial species based on the specific transporters present in the respective model. The objective function to be maximized is defined to be the total biomass of all species; i.e., the sum of all individual growth rates. The minimum growth rate of each species is set at 0.001 hr 1 .

The consortia of gut microbe metabolic models are used as a framework for interpreting genomic, transcriptomic, and metabolomic data obtained from the mouse and human studies. Enriched genes or pathways at the genomic or transcriptomic level are mapped to the source organism model to determine the metabolic functions these represent and how they connect with the rest of metabolism in that organism, as well as in the gut ecosystem. Enrichments also in metabolic intermediates or end products of these pathways provide further evidence for these pathways’ contribution to checkpoint inhibitor function.

Example 7: In Silico Simulation of Relevant Microbial Species

Models were downloaded for the following organisms: Akkermansia muciniphilia, Faecalibacterium prausnitzii, Ruminococcus torques, Ruminococcus gnavus, Ruminococcus lactaris, Eubacterium hallii, Blautia obeum, Anaerostipes hadrus, Dorea formicigenerans, Coprococcus comes, Coprocuccus catus, Erysipelotrichaceae sp., and Clostridium scindens. The models are then used for simulations in the COBRA package v2.0 (Schellenberger et ah, Nature Protocols 2011, 6: 1290-1307). Cell metabolism was simulated by defining nutrient uptake rates (mmol/gDCW-hr) and optimizing for growth rate of each organism (hr 1 ). Oxygen uptake rate was set to zero, to simulate anaerobic conditions.

First, simulations were performed to determine the minimal growth substrate requirements of each organism. Starting with all substrate uptake fluxes open, allowing utilization of any nutrient, simulations were performed as nutrient uptake fluxes are systematically removed. This was continued for each organism until a minimal set of carbon sources remained, the removal of any of which would result in zero predicted growth. Normally, this resulted in a single sugar compound (often glucose) and one or more other nutrients such as amino acids, nucleotides, vitamins, or lipids. These other compounds are considered auxotrophic requirements of the organism. Next, the substrate utilization range of the organism was determined. The uptake flux of the primary growth substrate (generally, a sugar) was set to zero, and growth was evaluated with different carbon sources one at a time. The predicted ability to grow using each carbon source was documented. The ability to co-utilize organic acid carbon sources was also evaluated. These compounds generally cannot be used as a sole growth substrate during anaerobic growth but can be taken up in conjunction with a sugar. Simulations were run with the uptake rate of each compound constrained to a non-zero value, while maintaining the uptake of the primary sugar source. If an increase was observed in the predicted growth rate over the use of the sugar alone, then co-utilization is considered to be feasible.

The capability of each strain to produce various fermentation products was evaluated using the models. Some products were predicted to naturally form during the carbon source simulations above, as fermentation products are needed to balance redox in anaerobic conditions. These products were noted. For other compounds, the model was constrained to make each one by setting the output flux to a non-zero value. If the simulation gave a feasible solution, then the organism was considered capable of making this product.

Table 2 (illustrated as FIG. 16). Simulation of selected organisms with constraint- based modeling. a 1 indicates predicted growth on substrate; 0 indicates predicted no growth b 1 indicates compound is predicted to be used as a supplemental carbon source; 0 indicates it cannot be consumed c 1 indicates that model predicts production of fermentation product is feasible; 0 indicates it is not feasible d Compounds that must be supplied in the growth media are indicated by X

Example 8: Laboratory-Scale Fermentation of Isolated Anaerobic Microorganisms In alternative embodiments, microbes used in compositions as provided herein, or used to practice methods as provided herein, comprise use of isolated anaerobic microorganisms, for example, anaerobic bacteria isolated from a fecal sample, e.g., from a donor.

A laboratory-scale fermentation is performed using a Sartorius BIOSTAT A™ bioreactor with 2-liter (L) vessel, using the growth media described in Example 1. While still in the anaerobic chamber, 1 L media is transferred to a sterile feed bottle, which has two ports with tubing leading blocked by pinch clamps and covered in foil to maintain sterility.

The fermentation vessel is sterilized by autoclaving, then flushed with a continuous purge of sterile nitrogen gas with oxygen catalytically removed. Two inlet ports are fitted with tubing leading to a connector blocked with a pinch clamp, and the sampling port fitted with tubing leading to a syringe. The vessel is also fitted with a dissolved oxygen probe, a pH probe, and a thermowell containing a temperature probe. Once anaerobic conditions are ensured, the media is removed from the anaerobic chamber and connected to one of the inlet ports. The other feed bottle port is connected to sterile nitrogen purge. The pinch clamp is removed, and media transferred into the fermentation vessel by peristaltic pump or just by the nitrogen pressure. Once the transfer is complete, both lines are sealed again by the pinch clamps, the feed bottle removed, and returned to the anaerobic chamber.

A 50 mL seed culture of one or more bacteria from the following genera (any one of which are used to practice compositions or methods as provided herein), Agathobaculum (TaxID: 2048137), Alistipes (TaxID: 239759), Anaeromassilibacillus (TaxID: 1924093), Anaerostipes (TaxID: 207244), Asaccharobacter (TaxID: 553372), Bacteroides (TaxID: 816), Barnesiella (TaxID: 397864), Bifidobacterium (TaxID: 1678), Blautia (TaxID: 572511), Butyricicoccus (TaxID: 580596), Clostridium (TaxID: 1485), Collinsella (TaxID: 102106), Coprococcus (TaxID: 33042), Dorea (TaxID: 189330), Eubacterium (TaxID: 1730), Faecalibacterium (TaxID: 216851), Fusicatenibacter (TaxID: 1407607), Gemmiger (TaxID: 204475), Gordonibacter (TaxID: 644652), Lachnoclostridium (TaxID: 1506553), Methanobrevibacter (TaxID: 2172), Parabacteroides (TaxID: 375288), Romboutsia (TaxID: 1501226), Roseburia (TaxID: 841), Ruminococcus (TaxID: 1263), Erysipelotrichaceae (TaxID: 128827), Coprobacillus (TaxID: 100883), Erysipelatoclostridium sp. SNUG30099 (TaxID: 1982626), Erysipelatoclostridium (TaxID: 1505663), are grown to mid-exponential phase in a sealed culture bottle using the same media composition as above, and are transferred into the feed bottle in the anaerobic chamber. Repeating the above transfer procedure, this time with the culture, the fermenter is inoculated.

5 M ammonium hydroxide is prepared in another feed bottle. One port is connected to sterile nitrogen, and the bottle is purged for 5 minutes to remove all oxygen. The outlet tubing is then blocked by a pinch clamp and attached to the other inlet port in the fermentation vessel. This tubing is then threaded into a peristaltic pump head, and the pinch clamp removed. Using the software built into the Biostat A™ unit, this pump is controlled to maintain pH at 7.0.

During growth of the culture, temperature is maintained at 37°C using a temperature controller and heating blanket on the vessel. Nitrogen purge is set at 0.5 L/min to maintain anaerobic conditions and positive pressure in the vessel, and agitation is set at 500 rpm to keep the culture well mixed. Periodic samples are taken using the syringe attached to the sample port. For each sample, optical density is measured at 600 nm wavelength using a spectrophotometer.

Example 9: Patient Data Collection from Clinical Trials and Machine Learning and Data Analysis on the Same

Eligible patients were selected based on current health condition, cancer status (current or in remission), and treatment program. Prior patient medical history was also collected and analyzed when available. This includes but is not limited to prior cancer history, diabetes, autoimmune disease, neurodegenerative disease, heart disease, metabolic syndrome, digestive disease, psychological disorders, HIV, and allergies. In addition, lifestyle and dietary habits were collected, including diet regimen, exercise routine, alcohol, nicotine, and caffeine intake, medical as well as recreational drug use, recent courses of antibiotics, vitamins, and probiotics. In some cases, information and data collected from wearable devices that monitor but is not limited to heart rate, calories burned, steps walked, blood pressure, biochemical release, time spent exercising and seizures. This data was assembled and used as input to the machine learning algorithms with the goal of determining correlations between patient history, wearable devices and treatment efficacy. In addition, relationships between this data and the results of sample analysis described below were elucidated.

For current cancer patients, tumor size and cancer progression are tracked over time and are classified based on radiographic assessment using the Response Criteria in Solid Tumors version 1.1 (Schwartz et al. Eur. J. Cancer 2016, 62:132-137) criteria. This is based on longitudinal measurements of lesions in cancer tissue, given a strict set of guidelines for lesion selection and measurement techniques. Responders to checkpoint inhibitor treatment are defined as patients that were cured or had stable disease lasting at least 6 months, while non-responders are defined as those whose cancer progressed or was stable for less than 6 months. Each patient provided stool samples using the procedures as outlined in Example 2 and buccal swabs of the oral biome. In some cases, Urine, Blood and plasma samples were also taken by healthcare personnel within 1-2 days of the stool samples. Stool, urine and buccal samples were kept on ice or at 4°C until processed. Whole blood was collected into an EDTA tube. Plasma was isolated from the blood by centrifugation at lOOOxg for 10 minutes, followed by centrifugation at 2000xg for 10 minutes. At least three timepoints were taken for each patient, roughly every 6 to 8 weeks.

Flow Cytometry Analysis of Peripheral Blood

Flow cytometry analysis of peripheral blood can provide a non-invasive immune profile of the patients on study (Showe et al. Cancer Res. 2009 Dec 15; 69(24): 9202-9210). The peripheral blood immuno-profile evaluation was performed on blood samples collected from patients on study. Phenotypic markers of lymphocyte subpopulations and regulatory T cells (Tregs) was evaluated using flow cytometry with populations gated to include CD3, CD4, CD8, CD1 lb, CD14, CD15, CD25, CD45, CD56, HLA-DR and FoxP3- expressing cells using antibodies to each cell type (BD Biosciences). Peripheral blood cells were stained with Live/Dead violet dye (Invitrogen, Carlsbad, CA) to gate on live cells. Data was acquired on an LSR P™ flow cytometer (BD Biosciences) and analyzed with FLOWJO™ software (TreeStar, Ashland, OR).

Peripheral Blood Mononuclear Cell (PBMC) Preparation and CyTOF® Analysis

Peripheral blood mononuclear cells (PBMC’s) are isolated from subject blood using a standard kit and stored in liquid nitrogen at 1 x 10 L 6 cells/mL until use. Prior to storage, PBMC’s may be processed using flow sorting or an antibody spin separation kit to select for a certain purified lymphocyte subpopulation, such as T cells. To characterize the immune profile of the PBMCs, single cell proteomics analysis (CyTOF®) is applied. This work is conducted by the Bioanalytical and Single-Cell Facility at the University of Texas, San Antonio, and entails a comprehensive panel of 29 different immune markers, allowing for deep interrogation of cellular phenotype and function (https://www.fluidigm.com/products/helios). To complement these results, RNA sequencing is applied to the entire population of the PBMCs, sorted populations, and also to single cells. Single cell RNAseq is applied using the method developed by 10X Genomics (https://www.10xgenomics.com/ solutions/single-cell/). Finally, cytokine levels are determined using the Human Cytokine 30-Plex Luminex assay

(https://www.thermofisher.com/order/catalog/product/LHC60 03M).

Reassignment of Microbial Genomes into Operational Species Units

Because of the limitations of the NCBI taxonomy tree, and the necessity of including proprietary microbial genome assemblies into the reference alignment sequence database, it is necessary to generate a new taxonomy of microbes. Previous work (e.g., see Jain et al. (2018) Nature CommunicaGtabletions 9(1):5114) shows that species are a biologically relevant construction, with the average genomic distance (1- average nucleotide identity) between strains of a species being less than 0.04. Using this as an inspiration, all microbial assemblies from the NCBI RefSeq (Pruitt et al. (2006) Nucleic Acids Research 35(suppl_l):D61-D65) were assigned into operational species units (OSUs) based on a clustering in which microbial assemblies within a genomic distance of 0.04 are assigned to the same OSU.

All microbial assemblies belonging to bacteria and archaea were acquired from the NCBI RefSeq database. All pairwise distances were calculated between assemblies using mash (Ondov et al. (2016) Genome Biology 17(1): 132). Clustering is performed using DBSCAN (Ester et al. (1996) KDD-9696:226-231) with an epsilon parameter of 0.04. Identified clusters were denoted as operational species units (OSUs). Proprietary microbial assemblies were seamlessly included in this procedure as well.

For each OSU, an integer cluster label was created, and a new taxonomic ID created that is unique from any existing NCBI taxonomic identification numbers. The least common ancestor of each OSU was calculated using the original NCBI taxonomy IDs of its member assemblies, and each OSU taxonomic ID was inserted into the NCBI tree under its least common ancestor. Each OSU is also named using its most common species and label number (e.g. Bifidobacterium adolescentis COOOl).

In FIG. 1, the ranks of the least common ancestor of each OSU that contains more than one assembly are displayed. Most OSUs are consistent with pre-existing NCBI taxonomy, with a least common ancestor at the species or genus level.

However, for 207 out of 2,112 non-singleton OSUs, the least common ancestor is at the family level or higher. The chart in FIG. 2 demonstrates that the frequency of OSUs decreases as the cluster size increases in a log-log fashion.

The new names, reference sequences, and taxonomy were used to generate a new reference database for the alignment program centrifuge (Kim et al. (2016) Genome Research 26:1721-1729). The centrifuge program classifies sequencing reads from a metagenomic fecal sample to reference sequences and uses an expectation- maximization method to estimate relative abundance of the taxa present in the sample. The estimated relative abundances for each OSU are carried into downstream analyses, such as machine learning or differential abundance analysis.

In addition to the method for re-assigning taxonomy described, pre-built databases that use the Genome Taxonomy Database (GTDB) were directly used for centrifuge classification (Parks et al. (2019) bioRxiv 771964, Meric et al. (2019) bioRxiv 712166).

Whole Genome Sequencing of Patient Fecal Samples

Whole genome sequencing was performed as previously described in Example 3 on a total of 450 fecal samples. Of the 450 samples, 322 samples were from cancer patients, 96 were from control subjects, and 32 were from subjects in remission. The results were classified, and abundance was estimated for each sample using centrifuge, using either a reference database built in-house consisting of operational species units (OSUs) or the publicly available GTDB database (Parks et al. (2019) bioRxiv 771964, Meric et al. (2019) bioRxiv 712166).

The results were analyzed for differential relative abundance of organisms (classified as OSUs) between cancer and control cohorts, as well as correlations between relative abundance of organisms and immune markers, as measured by flow cytometry. Principal component analysis was performed to visualize the structure of the data (Fig. 3 and Fig. 4) and exhibited a partial separation between cancer and control samples. This separation is driven by a specific subset of microbes that have differential abundance between the two cohorts (Figs. 5-7 and Table 3). Microbes were ranked based on the magnitude and significance of this difference. Additionally, machine learning was performed to train a model capable of discriminating between a subject with cancer and a control subject.

Metagenomic sequences are also scanned to identify novel CRISPR sequences using a scoring algorithm such as that described in (Moreno-Mateos et al. (2015) Nat. Met. 12:982-988), and for predicted natural product gene clusters using the antiSMASH routine (Medema et al. (2011) Nuc. Acids Res. 39:W339-W346).

Table 3. illustrated as FIG. 17. Whole genome sequencing was performed on fecal samples from subjects with and without cancer and the reads were classified and abundance of each operational species unit (OSU) was estimated computationally.

The fold change difference and statistical significance (inverse p value, Mann Whitney U test) was calculated for abundances between cancer and control sample cohorts. For OSUs with a mean relative abundance of at least 0.05%, p-values were filtered using an adjusted p-value computed using a two-stage Benjamini-Hochberg procedure. OSUs passing the threshold are reported.

Table 4. illustrated as FIG. 18. Whole genome sequencing was performed on fecal samples from subjects with and without cancer and the reads were classified using the GTDB database and abundance of each species was estimated computationally (Centrifuge). For classified hits with a mean relative abundance of at least 0.005%, The fold change difference and statistical significance (inverse p value, Mann Whitney U test) was calculated for abundances between cancer and control sample cohorts.

Example 10: Data Driven and Machine Learning Approaches for Therapeutic Design Whole genome sequencing and flow cytometry analysis were performed on human fecal and blood samples, respectively, as described in Example 9. A machine learning model was fit to discriminate cancer and control samples, using all fecal data collected to date. The model was validated using leave-one-out cross-validation, and performance evaluated using a receiver operating characteristic curve (Fig. 8 and Table 5). Alternatively, the model developed using the GTDB database was validated using Stratified Group K-Fold Cross Validation (Tables 6 to 7).

Table 5. A random forest classifier was trained to classify operational species unit abundances for a sample as corresponding to cancer or control. An ROC curve was generated on 145 cancer samples and 88 control samples using leave-one-out cross validation. Following validation, the model was trained on all the samples and feature importance values are reported.

0.016501858 0.415683892 Blautia sp. AF19-1 OLB C2906

0.013518985 0.764382216 Erysipelotrichaceae bacterium GAM147 C2844 Parolsenella catena Cl 992 listipes sp. An31A C0840 Slackia piriformis YIT 12062 Cl 942 Pseudoflavonifractor sp. An85 C2787 Enterococcus faecium C4060 Faecalitalea cylindroides C2250 Lactobacillus sanfranciscensis TMW 1.1304 C4264 bsiella sp. AM22-9 C2879 Streptococcus mitis C5322 Streptococcus mitis C3901 Butyricimonas virosa C0441 gathobaculum sp. Marseille-P7918 C3297 Bacteroides intestinalis C0161 Senegalimassilia sp. KGMB04484 Cl 994 naeromassilibacillus sp. Anl72 C2773 naeromassilibacillus sp. Marseille-P4683 C3061 Clostridium sp. Marseille-P 3244 C3177 Rothia mucilaginosa C3456

Candidatus Methanomassiliicoccus intestinalis Issoire-Mxl C4599 naerostipes sp. 494a C2731 Paraeggerthella hongkongensis Cl 982 Lactococcus garvieae C6016 Eubacterium sp. AF19-12LB C2907 Lachnospiraceae bacterium oral taxon 096 C2846 Prevotella intermedia C0255 Bacteroides sp. OM05-12 Cl 216 Propionibacterium freudenreichii C3941 Oxalobacter formigenes C5820 Eubacterium sp. ER2 C2579 listipes indistinctus C0222 Traorella massiliensis C3119 Weissella cibaria C5172 Prevotella pleuritidis C0414 Citrobacter sp. FDAARGOS 156 C5320 [Collinsella] massiliensis Cl 944 lloscardovia omnicolens C0021 0.174241239 Lactobacillus gasseri C3569 0.059945469 [Clostridium] hylemonae C2157 0.1191171 Citrobacter amalonaticus C5318 0.068345197 Bacteroides sp. KCTC 15687 Cl 337 0.006391049 Lactococcus garvieae C4388 0.120223702 Faecalicoccus pleomorphus C2383 0.097753343 Lactobacillus animalis C6895 0.149698537 Anaerostipes rhamnosivorans C3039

0.007497948 Enterobacter bugandensis C5325 0.032643624 Lactobacillus mucosae LM1 C4338 0.065872962 Bacteroides propionicifaciens C0324 0.078372213 Streptococcus sobrinus C6344

0.064034551 Ruminococcaceae bacterium D5 C3161 0.015908673 Ruminococcus albus C3136 0.070235779 Selenomonas noxia C2179 0.102015151 Citrobacter werkmanii C4750 0.106931981 Providencia rettgeri C6875 -0.08278651 Anaerococcus lactolyticus C2159 0.026978526 Ruminococcus sp. FC2018 C2499 0.040473615 Robinsoniella peoriensis C2512

0.153859627 Megasphaera hexanoica C2664 0.005437415 Atlantibacter hermannii C7332

0.050219427 Megasphaera sp. AM44-1BH C2918 0.013360056 Clostridium sp. 12(A) C2475

0.075062059 Eggerthella sinensis Cl 979 0.029503909 Proteus vulgaris C6084 0.020972769 Plautia stall symbiont C4087

0.009219528 Bacteroides graminisolvens CO 392 0.034902834 Providencia rettgeri C4489

0.072959896 Candidatus Ishikawaella capsulata Mpkobe C4922

0.060674729 secondary endosymbiont of Ctenarytaina eucalypti C4438

0.000740595 Shimwellia blattae C4368

0.042068637 Bacteroides reticulotermitis JCM 10512 C0437

0.134402606 Proteus mirabilis C3929

0.085291723 Peptoclostridium sp. AF21-18 C2156

0.071303376 Bacteroidales bacterium KA00251 C0708

0.044896419 Klebsiella sp. P0552 C5864

0.020350527 Cronobacter universalis NCTC 9529 C5126

0.042060758 Lelliottia jeotgali C5960

0.010010498 Pseudomonas balearica DSM 6083 C4912 0 0.069859304 Fusobacterium nucleatum C2036

0 0.098855648 Mitsuokella sp. AF21-1AC C2899

Table 6. A logistic regression classifier was trained to classify samples as corresponding to cancer or control on samples with a mean relative abundance of at least 0.005% using the GTDB database. An ROC curve was generated on 322 cancer samples and 92 control samples using Stratified Group K-Fold Cross Validation (AUC = 0.79). Following validation, the model was trained on all the samples and feature importance values are reported.

Feature Importance (Logistic Regression) Organism Name

0.514417994 Collinsella sp900548935

0.486287437 Clostridium sp900539375

0.381613445 UBA1191 sp900545775

0.310730798 Raoultibacter massiliensis

0.289945387 Christensenella minuta

0.283774901 CAG-145 sp900540145

0.27456207 Bacteroides stercoris

0.26468198 Erysipelatoclostridium sp900544435

0.263480075 Phocaeicola salanitronis

0.250041885 Marvinbryantia sp900066075

0.249755758 Odoribacter sp900544025

0.216103903 UBA738 sp003522945

0.207027879 An200 sp900550095

0.195934646 Mediterraneibacter faecis

0.185692545 CAG-170 sp000436735

0.179847461 Megasphaera elsdenii

0.162281593 Methanosphaera stadtmanae

0.159663737 UMGS1611 sp900553435

0.157611925 CAG-177 sp003538135

0.157485555 UBA6398 sp003150315

0.155329072 CAG-492 sp000434015

0.153100473 Dorea sp000433215

0.151760426 Evtepia sp004556345

0.14588862 UMGS1071 sp900542375

0.145040782 Collinsella sp900554585

0.136236542 Clostridium_Q sp003024715

0.131388743 CAG-460 sp900544625

0.130605804 Blautia_A sp900551715

0.12874627 Niameybacter sp900549765

0.127187848 CAG-45 sp002299665

0.098447454 Mailhella sp900541395

0.092072207 SFFH01 sp900548125

0.080714744 Dorea longicatena

0.079070946 Sutterella wadsworthensis A 0.076582096 Negativibacillus sp000435195

0.073355953 UMGS1590 sp900552455

0.061020643 Coprococcus_A sp900548825

0.059560254 Blautia_A sp900066335

0.058625801 Eubacterium_l sp900557275

0.048160806 Firm-11 sp900540045

0.0465729 Dorea longicatena_B

0.045683691 UMGS1491 sp900554775

0.044846674 UMGS1241 sp900549955

0.044173983 CAG-1427 sp000436075

0.040847644 Alistipes sp900541585

0.040245741 Gemmiger variabilis

0.039602886 CAG-495 sp000432275

0.036058062 Bariatricus comes

0.035781984 Oxalobacter formigenes

0.03030392 Frisingicoccus caecimuris

0.025478979 CAG-314 sp000437915

0.023104086 QALW01 sp003150515

0.021151433 Collinsella sp900554325

0.020407288 CAG-485 sp900541835

0.020130762 CAG-452 sp000434035

0.017010213 Agathobacter sp900546625

0.016426446 UBA5394 sp003150565

0.005947673 Blautia_A obeum_B

0.004390397 Coprobacillus cateniformis

0.002233086 Akkermansia sp004167605

0.00152013 Anaerostipes hadrus_A

-0.001234426 Limosilactobacillus fermentum_A

-0.003827343 CAG-115 sp003531585

-0.008153089 Fusobacterium_B sp900541465

-0.014246241 Prevotella sp900552515

-0.016286555 Collinsella sp900551665

-0.021479219 Anaerotignum lactatifermentans

-0.023122468 UMGS1781 sp900553695

-0.024041329 Odoribacter laneus

-0.034455465 UBA11471 sp000434215

-0.037849311 Prevotellamassilia sp000437675

-0.039128417 Angelakisella sp900547385

-0.039646845 Agathobaculum sp900291975

-0.041056608 Eubacterium_R sp000434995

-0.04266878 Eubacterium_F sp900539115

-0.044059805 Alistipes sp000434235

-0.050522202 UMGS1590 sp900553245

-0.051836169 UMGS1688 sp900554085

-0.057847833 Butyricimonas faecalis

-0.066253286 Akkermansia muciniphila_A

-0.067189759 Coprobacter fastidiosus

-0.067646141 CAG-83 sp900550585 -0.083533993 Prevotella sp900554045

-0.085318406 Intestinimonas butyriciproducens

-0.093860595 Eubacterium_F sp000434115

-0.103834319 Eubacterium_R sp900540305

-0.106144597 Desulfovibrio fairfieldensis

-0.113985815 Lachnospira sp900316325

-0.117390396 Porphyromonas sp000768875

-0.122672447 Acidaminococcus intestini

-0.126358887 CAG-303 sp000437755

-0.127237507 Bacteroides caccae

-0.136509832 Prevotella sp900548745

-0.136915786 Dorea sp000433535

-0.137055372 Ligilactobacillus salivarius

-0.151411951 Blautia_A sp900551465

-0.174551647 CAG-83 sp000431575

-0.182703866 Streptococcus vestibularis

-0.188088114 CAG-302 sp900543825

-0.191528797 Butyricimonas virosa

-0.207519696 Dialister sp900343095

-0.208796646 Streptococcus sp000314795

-0.21979495 QANA01 sp900554725

-0.220254926 Enterococcus_B faecium

-0.249373565 COE1 sp001916965

-0.249871731 Mailhella sp003150275

-0.251086664 Lachnospira eligens

-0.299023203 Catenibacterium sp000437715

-0.303053041 GCA-900066755 sp900066755

-0.30357643 CAG-1031 sp000431215

-0.306860922 UBA1691 sp900544375

-0.318039896 CAG-495 sp001917125

-0.32832744 AM07-15 sp003477405

-0.387480395 Ruthenibacterium sp003149955

-0.441806113 Parabacteroides johnsonii

-0.513157387 Bariatricus massiliensis

Table 7. A logistic regression classifier was trained to classify samples as corresponding to cancer (non-responder) or control on samples with a mean relative abundance of at least 0.005% using the GTDB database. An ROC curve was generated on 43 non-responder samples and 92 control samples using Stratified Group K-Fold Cross Validation (AUC = 0.71). Following validation, the model was trained on all the samples and feature importance values are reported. Feature Importance (Logistic Regression) Organism Name

0.568597444 CAG-170 sp000436735

0.543705645 Coprobacillus cateniformis

0.509426281 Mailhella sp900541395

0.482820632 Blautia_A sp003474435

0.471483202 UMGS1611 sp900553435

0.244782119 UMGS911 sp900557415

0.184527891 CAG-354 sp900553015

0.174892874 Blautia_A massiliensis

0.158545809 Agathobacter sp900317585

0.140717769 Negativibacillus sp000435195

0.127151205 Prevotella sp002251385

0.122995374 Coprococcus_A sp900548825

0.118746116 Alistipes_A indistinctus

0.118323236 UMGS1071 sp900542375

0.115663494 Erysipelatoclostridium sp900544435

0.10338765 Collinsella sp900547285

0.102410987 Prevotella sp900556825

0.094993875 UMGS172 sp900539855

0.06348916 Phocaeicola sp900551445

0.061539232 Agathobacter rectalis

0.056113717 Anaerobutyricum hallii

0.053598211 Blautia_A sp900066335

0.053249701 Anaerostipes hadrus_A

0.045497159 Clostridium sp001916075

0.037406556 Holdemanella sp003458715

0.021590668 Christensenella minuta

0.002218293 Collinsella sp900541725

4.2957E-05 Phascolarctobacterium faecium

-0.004703489 Bacteroides togonis

-0.008809374 Paraprevotella clara

-0.03119867 Holdemania sp900120005

-0.031492474 AM51-8 sp900546435

-0.035434119 Phill sp001940855

-0.038913964 Schaedlerella sp004556565

-0.044020829 Lachnospira sp900552795

-0.047515072 Muricomes sp900604355

-0.052967481 Prevotella buccae

-0.071596115 Longicatena sp003433845

-0.0796651 Desulfovibrio fairfieldensis

-0.100975915 Lachnospira sp003537285

-0.115966192 Butyricimonas faecihominis

-0.172472377 Blautia_A sp900551465

-0.187868969 Anaerotruncus massiliensis

-0.19109635 Anaerofustis stercorihominis

-0.206509093 UMGS1688 sp900544575

-0.210914586 Bifidobacterium dentium

-0.228226067 Bacteroides cutis -0.241407669 F23-B02 sp001916715

-0.247678711 COE1 sp001916965

-0.267182222 Ruminococcus_E bromii_B

-0.286160011 Porphyromonas sp001552775

-0.323514014 UBA1691 sp900544715

-0.335225188 GCA-900066755 sp900066755

-0.340598662 Eubacterium_G sp900548465

-0.35989301 Limosilactobacillus fermentum_A

-0.460367032 Mesosutterella massiliensis

-0.475293296 Escherichia flexneri

-0.542914883 Enterococcus_B faecium

-0.599141069 CAG-521 sp000437635

-0.675358406 Phocaeicola sp000436795

-0.774574761 CAG-83 sp900550585

Flow cytometry was performed on cancer and control blood samples as described in Example 9, and correlations between immune markers and organism abundances in the corresponding stool samples were determined (Fig. 9 and Tables 8 and 9). The organisms were also ranked according to differential abundance between responder and non-responder patients (Fig. 10 and Table 10). In addition, linear discriminant analysis (LDA) effect size method (LEfSe) was used to classify microbes identified using the GTDB database enriched in cancer or control (Table 11). Table 8. illustrated as FIG. 18. Flow cytometry was performed on 38 cancer blood samples and 38 control blood samples, along with corresponding whole genome sequencing and classification. All operational species unit (OSU) abundances were correlated against a suite of immune markers (CD1 lb+, CD14+CD15-, CD 14- CD15+, CD15+CD14-, CD15-CD14+, CD3+, CD3+CD56+, CD3+HLADR+, CD3- CD56+, CD3-HLA-DR+, CD3-HLA-DRlow, CD4+, CD4+HLA-DR+, CD8+,

CD8+HLA-DR+, Foxp3+). Correlations and p values were computed on all the samples, or on a subset of samples consisting of just control samples or just cancer samples. The p values obtained from all the samples were filtered using a two-stage Benjamini-Hochberg procedure and correlated with an adjusted p value below 0.15 are reported.

Table 9. illustrated in FIG. 19. Flow cytometry was performed on 38 cancer blood samples and 38 control blood samples, along with corresponding whole genome sequencing and classification. All operational species units (OSUs) were correlated against the CD3+ and CD3+CD56+ immune markers (as a subset of CD45+) using a Spearman rank correlation. Adjusted p values were computed using a two-stage Benjamini-Hochberg procedure for each immune marker, and correlations with an adjusted p value below 0.2 are retained. The retained correlations were further vetted using a linear mixed model that accounts for a random effect induced by group

(cancer vs. control). The logarithm of the OSU abundance was used as the input to the model. For CD3+CD56+, the logarithm of the immune marker proportion was used as the output of the mixed model. The mixed model p values and coefficients are reported. Table 10. Whole genome sequencing was performed on the initial time point fecal samples from subjects undergoing cancer immunotherapy and the reads were classified and abundance of each operational species unit was estimated computationally. Operational species unit abundances were correlated to response to therapy using a score of 2 for complete response, 1 for partial response, 0 for no response, using the Spearman rank correlation. Correlations with a p value below 0.15 are reported.

0.004824863 0.009065354 0.460919277 barnesiae C0323 0.50270646

- Streptococcus

0.001854242 0.011550331 0.447714196 mutans C3345 0.50270646

- Lactobacillus

0.002592642 0.013008588 0.441044685 fermentum C3433 0.50270646

Bacteroides

- heparinolyticus

0.003900899 0.01697159 0.425648294 C1005 0.50270646

Bacteroides coprosuis DSM

0.011114361 0.020991328 0.412834447 18011 C0203 0.50270646

- Blautia obeum

0.001347612 0.021974899 0.410011686 C2901 0.50270646

- Streptococcus

0.005138808 0.022206972 0.409360874 vestibularis C7338 0.50270646 Streptococcus

0.004109069 0.028915901 0.392598094 thermophilus C3480 0.50270646

Bacteroides eggerthii

0.002625559 0.029553117 0.391177567 C0137 0.50270646

Streptococcus sp.

0.00180933 0.029570968 0.391138132 HSISS2 C4629 0.50270646

Bacteroides

0.006421035 0.045485127 0.361828833 coprocola CO 136 0.702951961

Lachnospira

0.00479161 0.066985005 0.333215846 pectinoschiza C2649 0.884186581

Lactobacillus

0.001357573 0.067614268 0.332494913 paragasseri C5843 0.884186581

Escherichia coli

0.002942156 0.074018907 0.325438908 C3313 0.894382098

Intestinibacter

0.001499661 0.089187848 0.310437419 bartlettii C2141 0.894382098

Lactococcus lactis

0.001315245 0.090931568 0.308841524 C3409 0.894382098

Anaerotignum lactatifermentans

0.000593797 0.093500218 0.306532546 C2790 0.894382098

Bifidobacterium

0.001096895 0.100936329 0.300108932 dentium C0003 0.894382098

Odoribacter

0.001297862 0.101670448 0.2994944 splanchnicus CO 185 0.894382098

Faecalimonas

0.002123253 0.113189533 0.290262241 umbilicata C2244 0.894382098

Faecalibacterium

0.014086171 0.120986249 0.284404931 prausnitzii C2138 0.894382098

Tyzzerella nexilis

0.001420926 0.123671567 0.282452495 C2155 0.894382098

Clostridiales bacterium CCNA10

0.000841219 0.131047516 0.277245997 C2953 0.894382098

Clostridium

0.001049951 0.132465355 0.276270029 disporicum C2479 0.894382098

Gordonibacter

0.000534773 0.1330099 0.275897229 pamelaeae Cl 937 0.894382098

Table 11. Linear discriminant analysis (LDA) effect size method (LEfSe) was used to classify microbes (GTDB database) enriched in cancer or control. Analysis was conducted on 322 cancer samples and 96 control samples. LEfSe first identifies features that are statistically different among various populations using the non- parametric factorial Kruskal-Wallis (KW) sum-rank test; It then performs additional pairwise tests to assess whether these differences are consistent with respect to population subclasses using the unpaired Wilcoxon rank-sum test. Lastly, LEfSe uses LDA to estimate the effect size of each differentially abundant feature. A total of 135 species were enriched in cancer patients and 189 species were enriched in healthy individuals.

Blautia_A

17568 sp900120195 Cancer 2.13760 0.0010911377336

17532 Blautia coccoides Cancer 2.39200 0.000968237855956

17534 Blautia hansenii Cancer 2.61227 0.0216950428348

17535 Blautia hominis Cancer 2.00955 0.0132436138036

17536 Blautia sp000432195 Cancer 2.64642 6.48111804063e-05

38844 Streptococcus mutans Cancer 2.19809 0.000766810025194 21762 Eisenbergiella tayi Cancer 2.09239 0.0257014664011 18508 CAG-273 sp000437855 Cancer 2.19293 0.00149432368338

Escherichia 22144 sp000208585 Cancer 2.24534 0.000447045218029 20468 Coprococcus eutactus Cancer 2.35758 0.00917122961097 17540 Blautia sp003287895 Cancer 2.62746 1.36005693245e-05 17547 Blautia sp900556555 Cancer 2.05367 0.0264114231619 Bacteroides

17148 bouchesdurhonensis Cancer 2.10570 0.00282381576978 Anaerostipes 15906 sp000508985 Cancer 2.01922 0.00114807012388

14115 43-108 sp001915545 Cancer 2.64298 1.11878531695e-06 Ruminococcus_H 36509 sp900549945 Cancer 2.33650 0.00895450765663

Anaerobutyricum 15832 h a 11 i i_ A Cancer 2.23321 0.0225920389673

Ruminococcus_A 36428 sp000432335 Cancer 2.53016 0.00139750682718

Hungatella

25300 sp005845265 Cancer 2.20734 2.41051912452e-07 31012 Oscillibacter welbionis Cancer 2.97773 0.000181598180603 Fusobacterium_B 23244 sp900541465 Cancer 2.05886 0.00968800303448 Enterocloster 21884 aldenensis Cancer 2.53945 6.46879345659e-10 26966 Longicatena innocuum Cancer 2.62342 0.000826173225832 Streptococcus

38939 sp000187445 Cancer 2.54944 0.000251671138383

20690 Cronobacter sakazakii Cancer 2.00001 0.00176581732956

Clostridium_Q

20055 symbiosum Cancer 2.60684 4.89158492686e-09

Agathobacter

15178 sp000434275 Cancer 2.00539 0.0481413837296

21731 Eggerthella lenta Cancer 2.94523 0.006101347123

Streptococcus

38891 parasanguinis_D Cancer 2.42213 2.6511957776e-05

Streptococcus

38889 parasanguinis_B Cancer 2.56043 0.000319467593934

Streptococcus

38888 parasanguinis_A Cancer 2.33233 9.51509478653e-05

Streptococcus

38887 parasanguinis Cancer 2.35117 0.00071108799438

Faecalimonas

22512 sp900556835 Cancer 2.23748 0.012095387025

19869 Citrobacter freundii Cancer 2.07185 0.00906080909617

Flavonifractor

23068 sp000508885 Cancer 2.96730 2.14986779138e-09

33819 Providencia rettgeri_D Cancer 2.20376 0.00955245042142

17543 Blautia sp900541955 Cancer 2.50204 0.0173404800143

32690 Phocaeicola dorei Cancer 3.66861 0.000216406212684

32695 Phocaeicola plebeius Cancer 2.44370 0.00392932331679

32699 Phocaeicola sartorii Cancer 2.18940 0.00108006311504

18772 CAG-83 sp001916855 Cancer 2.01482 0.00380931699685

Bacteroides

17198 sp900557355 Cancer 2.48723 0.0172953376659

Bacteroides

17196 sp900556215 Cancer 2.44481 0.00508686372198

Bacteroides

17191 sp900066265 Cancer 2.18952 4.52735545739e-05

44733 Veillonella atypica Cancer 2.18847 0.0402708440438

Mediterraneibacter

27993 torques Cancer 3.55674 0.0496897855081

Streptococcus

38890 parasanguinis_C Cancer 2.20714 0.000599054128583

Eisenbergiella

21757 sp900539715 Cancer 2.18135 0.00915828126624

17157 Bacteroides faecis Cancer 2.61898 0.000484436396227

Anaerotruncus

15918 colihominis Cancer 2.22604 0.00133142885356

Streptococcus

38951 sp001556435 Cancer 2.92682 0.00217861964326

18579 CAG-45 sp900066395 Cancer 2.49950 0.00574455333834

Blautia_A

17554 sp000433815 Cancer 3.19213 2.23528859735e-06

21497 Dorea scindens Cancer 2.79558 2.09572874651e-05

Limosilactobacillus

26866 fermentum Cancer 2.34117 0.0103155939811 Bacteroides

17205 xylanisolvens Cancer 3.21745 0.00012372735459

Enterocloster

21888 clostridioformis Cancer 3.08276 3.13138339085e-12

21886 Enterocloster bolteae Cancer 2.81644 2.15012098387e-ll

Butyricimonas

18199 faecihominis Cancer 2.14057 4.33439876776e-05

UBA1691

41906 sp900544375 Cancer 3.44725 2.33522633211e-10

25980 Klebsiella variicola Cancer 2.09542 0.0155919157839

Enterocloster

21889 clostridioformis_A Cancer 2.54158 2.41667650124e-07

Ruthenibacterium

36521 lactatiformans Cancer 2.77271 8.80361828788e-05

Lachnospira

26241 sp000436535 Cancer 2.04108 0.0461403001471

Anaerobutyricum

15835 sp900016875 Cancer 2.11887 0.0181255372277

21501 Dorea sp000433535 Cancer 3.18022 8.50501585649e-07

Acutalibacter

15033 sp900543555 Cancer 2.20583 2.07942112135e-05

Bacteroides

17156 faecichinchillae Cancer 2.00310 0.00578699520994

Bacteroides

17150 caecimuris Cancer 2.30080 1.18027718738e-06

UBA9502

44095 sp900538475 Cancer 2.56934 0.000419156172297

32688 Phocaeicola coprocola Cancer 3.05308 0.0168486380976

Succiniclasticum

39618 sp900544275 Cancer 2.14647 0.0262474166286

Bacteroides

17197 sp900556625 Cancer 2.67332 0.0181020544302

18198 Butyricimonas faecalis Cancer 2.54767 2.02297766283e-06

Ruminococcus_B

36434 gnavus Cancer 3.40611 0.00343207293924

Ruminococcus_C

36436 callidus Cancer 2.59013 3.11571237196e-06

37769 Sellimonas intestinalis Cancer 2.90188 0.0010421172501

Acidaminococcus

14650 intestini Cancer 2.84483 5.37086074804e-06

Streptococcus

38929 salivarius Cancer 2.93889 0.0133708816272

Parabacteroides

31909 distasonis Cancer 3.49346 0.00845055659135

Lawsonibacter

26428 sp900066825 Cancer 2.27921 0.00119563602136

15902 Anaerostipes caccae Cancer 2.59029 1.32175359294e-05

22142 Escherichia flexneri Cancer 3.07841 0.000448573849446

Streptococcus

39003 vestibularis Cancer 2.91268 1.23303428895e-06

17204 Bacteroides uniformis Cancer 3.76744 0.00767040878452 Erysipelatoclostridium 22082 ramosum Cancer 3.10882 4.49001877438e-05

17179 Bacteroides rodentium Cancer 2.36982 0.000809140186422 Klebsiella

25979 quasivariicola Cancer 2.50337 0.0209044295048 Streptococcus 38737 anginosus_C Cancer 2.56735 0.0450278679091 19879 Citrobacter youngae Cancer 2.10125 0.0268286601359 Phocaeicola 32689 coprophilus Cancer 2.39012 8.788249368e-06 Prevotella

33237 sp000257925 Cancer 2.05312 0.00100376799716

23067 Flavonifractor plautii Cancer 2.95190 2.03840497779e-09 Escherichia 22140 dysenteriae Cancer 2.72638 0.000178346031215 Enterocloster 21898 sp900541315 Cancer 2.07701 0.0133349033913

Enterocloster 21890 lavalensis Cancer 2.05837 5.58379720897e-08 Bacteroides

17201 thetaiotaomicron Cancer 3.50101 0.0235802219915 Streptococcus 38946 sp000448565 Cancer 2.35186 3.7304387141e-05

Longicatena 26964 caecimuris Cancer 2.71146 0.000140435878

Parabacteroides 31921 sp900155425 Cancer 2.04037 0.000627513743016

21401 Dialister sp900343095 Cancer 2.42134 0.0326132648947

18336 CAG-103 sp900543625 Cancer 2.23674 0.000902041176381

17180 Bacteroides salyersiae Cancer 2.66579 2.13189055395e-05 CAG-1031

18337 sp000431215 Cancer 2.57987 0.000146355127079 21512 Dorea sp900543415 Cancer 2.70565 3.39380545885e-10 32682 Phil 12 sp002633275 Cancer 2.31286 0.00199992224058

Faecalimonas 22509 sp900550975 Cancer 2.27491 0.0151214093109

Eubacterium_G 22186 ventriosum Cancer 2.14863 0.000467276332765 Faecalimonas 22513 umbilicata Cancer 2.77402 0.0436444293568

Parasutterella 32208 sp000980495 Cancer 2.31846 0.0356205438601

32727 Phocaeicola vulgatus Cancer 3.87915 0.0142224019801 18334 CAG-103 sp900317855 Cancer 2.18662 0.0252397542571 Ligilactobacillus 26805 salivarius Cancer 2.50862 0.000565702472295

Bacteroides 17147 acidifaciens Cancer 2.02458 0.000193059075288 Prevotella

33256 sp001275135 Cancer 2.00053 0.0228785615692 Phascolarctobacterium

32637 faecium Cancer 3.07562 0.00859527040104

19917 Clostridioides difficile Cancer 2.14826 0.000396856053418

Blautia_A

17574 sp900547615 Cancer 2.00426 0.00105097878926

18469 CAG-217 sp900547275 Cancer 2.17756 0.0150594036026

18461 CAG-194 sp000432915 Cancer 2.41211 0.0114380924628

Blautia_A

17578 sp900551465 Cancer 2.09782 8.7672215879e-05

Parabacteroides

31913 johnsonii Cancer 2.32757 7.57370993477e-07

Ruminococcus_B

36435 sp900544395 Cancer 2.32731 0.000138031227462

Bacteroides

17188 sp003545565 Cancer 2.09073 0.00116767135794 18649 CAG-492 sp000434335 Cancer 2.02629 0.000100614508133

UBA1691

41907 sp900544715 Cancer 2.86541 7.87326411749e-07

17160 Bacteroides fragilis Cancer 2.62172 0.0152421440534

Parabacteroides

31910 distasonis_A Cancer 2.20437 0.0138650485607

Bacteroides

17186 sp002491635 Cancer 2.21447 0.00117561051251

Bacteroides

17189 sp003865075 Cancer 2.61541 4.67903060213e-07

Coprococcus 20471 sp000433075 Cancer 2.05457 8.85142109464e-08

Bacteroides

17167 intestinalis Cancer 2.99981 0.0304061339071

Bacteroides

17168 intestinalis_A Cancer 2.49123 0.011609802117

Enterocloster

21894 sp001517625 Cancer 2.40734 0.00141864996401

17154 Bacteroides cutis Cancer 2.12726 0.038819131362

36679 SFFH01 sp900542445 Control 2.41059 3.30021813163e-06

Ruminococcus_C

36440 sp000980705 Control 3.20407 1.2103932064e-08

UBA11524

41347 sp000437595 Control 2.03222 0.00829874351407

Collinsella

20338 sp900556415 Control 2.08313 1.11217975236e-08

Erysipelatoclostridium

22089 sp900544435 Control 2.42399 1.1132866617e-06

Erysipelatoclostridium

22087 sp003024675 Control 2.25783 2.64720533759e-10

Collinsella

20324 sp900554905 Control 2.21598 1.04125747668e-08

Erysipelatoclostridium

22085 sp000752095 Control 2.96769 4.21528595137e-ll

Collinsella

20321 sp900554645 Control 2.00425 0.000781585095622 Ruminococcus_D

36447 bicirculans Control 3.39071 6.09724921548e-06

CAG-1427

18401 sp000435675 Control 2.05861 0.000239442448034

Agathobaculum

15198 sp900625105 Control 2.45929 0.00687534588514

17538 Blautia sp001304935 Control 2.76252 0.00021477069236

UMGS1241

44369 sp900549955 Control 2.48651 0.000229158432302

Longicatena

26970 sp900411325 Control 2.04471 0.0253206102387

Agathobaculum

15193 sp003481705 Control 2.72460 2.55177393509e-06

Faecalibacterium

22497 sp900539885 Control 2.45021 0.00114034752317

Agathobaculum

15191 butyriciproducens Control 2.40050 9.30926345453e-05

18588 CAG-460 sp900544625 Control 2.45197 0.00302957091049

Ruminococcus_E

36473 sp003438075 Control 2.36465 0.0116416270466

Holdemanella

25246 sp900551285 Control 2.39373 5.39071908999e-06

Holdemanella

25245 sp900547815 Control 2.13257 0.010514300225

Holdemanella

25244 sp003458715 Control 2.16423 0.000129153396687

CAG-1427

18402 sp000436075 Control 2.02925 0.00989909203149

Collinsella

20131 aerofaciens_G Control 2.50700 9.18497857285e-06

Faecalibacterium

22491 prausnitzii_J Control 2.61316 1.05261675827e-06

17200 Bacteroides stercoris Control 3.05136 0.042749136853

CAG-1427

18416 sp900556585 Control 2.27099 0.0267898981999

UBA1191

41454 sp900545775 Control 2.21447 9.43642971559e-06

Faecalibacterium

22490 prausnitzii_l Control 2.65573 1.19439741311e-05

Collinsella

20339 sp900556445 Control 2.31396 1.12520743428e-05

GCA-900066135

23770 sp900543575 Control 2.13602 2.91176542674e-08

Blautia_A

17575 sp900548245 Control 2.61892 1.33647845585e-07

18784 CAG-83 sp900547745 Control 2.05677 0.000783754622785

Bifidobacterium

17344 adolescentis Control 3.89216 0.0472240909999

25848 KLE1615 sp900066985 Control 2.60806 1.08359063226e-05

Blautia_A

17562 sp900066145 Control 2.06312 0.000228185017914 18450 CAG-180 sp000432435 Contra 3.28508 0.00107407122157

Phocaeicola

32723 sp900553715 Contra 2.61412 0.032610595599

UBA1191

41455 sp900549125 Contra 2.50874 8.12599169877e-05

21661 ER4 sp000765235 Contra 2.34673 0.000662818789913

18331 CAG-103 sp000432375 Contra 2.87783 5.58314397621e-07

Sellimonas

37771 sp002161525 Contra 2.51538 0.0307760188802

18338 CAG-110 sp000434635 Contra 2.74437 6.44929544018e-05

Gemmiger

24117 sp900539695 Contra 2.05341 4.66989314069e-05 24112 Gemmiger formicilis Contra 2.44504 0.00333770203037 33197 Prevotella copri_A Contra 2.65431 0.0283791459871

Gemmiger

24118 sp900540595 Contra 2.16137 1.2620604518e-05

UMGS1071

44359 sp900542375 Contra 2.09882 0.000974900001284

21409 Dialister sp900555245 Contra 2.63436 0.00333770203037

Eubacterium_F

22173 sp003491505 Contra 2.36858 3.51840365919e-05

21500 Dorea sp000433215 Contra 2.33715 1.97114808776e-08

19946 Clostridium saudiense Contra 2.17730 0.0230705246422

Clostridium

19949 sp000435835 Contra 2.05791 0.00616715858917

Blautia_A

17560 sp003478765 Contra 2.33260 2.52783279408e-06

Blautia_A

17563 sp900066165 Contra 2.88048 0.00298338599418

Blautia_A

17566 sp900066355 Contra 2.55805 5.85243145116e-06

UBA11774

41419 sp003507655 Contra 2.48337 0.0450169284476

Blautia_A

17559 sp003477525 Contra 2.26051 0.00340034813393

21493 Dorea longicatena Contra 3.31604 5.28875486139e-09

Blautia_A

17565 sp900066335 Contra 2.75595 9.24085724755e-ll

18785 CAG-83 sp900548615 Contra 2.00739 0.00287952903975

18783 CAG-83 sp900545585 Contra 2.51557 4.66505048711e-06

Bifidobacterium

17413 sp002742445 Contra 2.50849 0.00126433385994

Agathobacter

15188 sp900550845 Contra 2.36342 0.000411600171088

Agathobacter

15183 sp900546625 Contra 2.45178 0.00017678701388

Agathobacter

15181 sp900317585 Contra 2.82123 0.000142395876762

Agathobacter

15186 sp900549895 Contra 2.34044 0.0348666029549

18651 CAG-492 sp900553225 Contra 2.53389 0.000269274517967 Cloacibacillus

19908 porcorum Contra 2.01540 0.0327228894077

Butyrivibrio_A

18241 crossotus Contra 2.37277 0.00126177720823

Butyrivibrio_A

18243 sp900543865 Contra 2.29993 0.000757328088867

Collinsella

20287 sp900551365 Contra 2.10823 5.77069184548e-06

UMGS1375

44382 sp900066615 Contra 2.31480 0.00204908177496

Acetatifactor 14550 sp900066365 Contra 2.28979 0.00350713933387

Barnesiella 17230 intestinihominis Contra 2.45687 0.00109479017124

Blautia_A 17558 sp003474435 Contra 2.11266 1.13585564617e-09

Mediterraneibacter 27982 faecis Contra 3.12051 3.79965372336e-09

UMGS1375

44383 sp900551235 Contra 2.10198 1.99431838413e-06

17549 Blautia_A massiliensis Contra 3.35483 9.0678522457e-06 44304 UCG-010 sp003150115 Contra 2.07626 1.93454349489e-08

Terrisporobacter

40350 sp900557165 Contra 2.27319 0.0201956139629

Blautia_A

17555 sp000436615 Contra 2.77455 1.50755393035e-07

17550 Blautia_A obeum Contra 3.28966 5.87804863778e-05

17551 Blautia_A obeum_B Contra 2.10105 0.00117560721128 18491 CAG-269 sp003525075 Contra 2.94816 1.10382253131e-06 30848 Odoribacter laneus Contra 2.42804 0.02830176635

Blautia_A

17564 sp900066205 Contra 2.57852 3.49087257679e-ll

Adlercreutzia

15043 celatus_A Contra 2.07439 0.0164550857066

Roseburia

36088 inulinivorans Contra 2.43035 0.0265096583225

Collinsella

20185 sp900541475 Contra 2.45893 1.78086972764e-07

Faecalibacterium

22483 prausnitzii_A Contra 2.53717 1.48409829345e-07

14374 AM51-8 sp003478275 Contra 2.04747 0.001001970988

21494 Dorea longicatena_B Contra 3.01526 4.84949138423e-07

18771 CAG-83 sp000435975 Contra 2.58227 0.00441888019065

18673 CAG-533 sp000434495 Contra 2.22485 0.00514615742321

18475 CAG-245 sp000435175 Contra 2.22165 0.0435372051499

15831 Anaerobutyricum hallii Contra 3.09528 0.00014746116909

Anaerobutyricum

15836 sp900554965 Contra 2.70371 0.000916439523968

Faecalibacterium

22482 prausnitzii Contra 3.19093 2.18287972068e-06 Collinsella

20276 sp900550185 Contra 2.08412 7.91309114891e-05

Collinsella

20272 sp900549455 Contra 2.49443 1.41645935 llle-08

Gemmiger

24122 sp900554145 Contra 2.36155 4.05221257543e-06

Veillonella

44754 sp900556785 Contra 2.24720 0.0330231957551

Ruminococcus_E

36477 sp003526955 Contra 3.37778 0.0144137210572

Enterocloster

21892 sp000431375 Contra 2.38993 0.00857107906373

18445 CAG-177 sp003538135 Contra 2.07697 0.000716445637701

40005 TFOl-11 sp001414325 Contra 2.66655 0.000363496777214

Bifidobacterium

17366 catenulatum Contra 2.20765 0.0114038100379

26235 Lachnospira eligens_B Contra 2.56502 0.0154865827583

36087 Roseburia intestinalis Contra 3.09407 0.0279857822864

Fusicatenibacter

23215 saccharivorans Contra 3.44159 2.62500713715e-06

Clostridium

19959 sp900540255 Contra 2.64288 0.000134946507533

Olsenella_E

30930 sp003150175 Contra 2.10495 9.32929729361e-05

18510 CAG-273 sp003507395 Contra 3.10648 7.55800913874e-06

Ruminococcus_C

36438 sp000437175 Contra 2.50387 0.00215829381827

Blautia_A

17579 sp900551715 Contra 2.07825 5.77555977528e-12

18631 CAG-485 sp900541835 Contra 2.21771 0.00687270499319

Clostridium_Q

20052 sp003024715 Contra 2.13420 5.46070638709e-05

18846 CAG-964 sp000435335 Contra 2.16784 0.0427477385672

21907 Enterococcus faecalis Contra 2.55268 0.000973238270192

Barnesiella

17233 sp003150885 Contra 2.22322 0.00884314924177

Bifidobacterium

17404 ruminantium Contra 2.66841 0.00855402261808

Negativibacillus

29933 sp000435195 Contra 2.17199 0.0422015141754

18346 CAG-110 sp003525905 Contra 2.33171 0.00175739825879

Coprococcus_A

20478 sp900548825 Contra 2.31292 1.03016482881e-08

40012 TFOl-11 sp003529475 Contra 2.55554 2.41121115527e-06

18426 CAG-170 sp000432135 Contra 2.41207 0.000193772855899

Eubacterium_R

22237 sp000433975 Contra 2.42086 0.00209914829707

Faecalibacterium

22498 sp900539945 Contra 2.93189 8.29721553513e-07

Faecalibacterium

22499 sp900540455 Contra 2.40470 0.000272278325887 Faecalibacterium

22484 prausnitzii_C Contra 3.14427 3.49956264954e-08

44737 Veillonella dispar_A Contra 2.58590 0.011756073445

EubacteriumJ

22199 ramulus Contra 2.44993 0.000781473771325

Collinsella

20133 aerofaciens_l Contra 2.42591 4.45271353249e-07

Fusicatenibacter

23216 sp900543115 Contra 2.62476 0.00684254308783

18577 CAG-45 sp000438375 Contra 2.02767 0.0318370090133

Ruminococcus_C

36437 sp000433635 Contra 2.14431 8.48212740565e-05

Oscillibacter

30995 sp001916835 Contra 2.10314 0.000287891879703

18843 CAG-95 sp900066375 Contra 2.50108 0.00140687944173

18482 CAG-269 sp000437215 Contra 2.72572 0.028711054205

15903 Anaerostipes hadrus Contra 3.45161 4.07263251646e-05

UMGS743

44517 sp900545085 Contra 2.14080 0.00458547437347

36674 SFEL01 sp004557245 Contra 2.08035 1.95437989749e-05

Anaerostipes

15904 hadrus_A Contra 3.04152 7.21486613455e-09

UMGS1491

44405 sp900554775 Contra 2.24642 0.000330918704025

Ruminococcus_FI

36508 sp003531055 Contra 2.95780 0.000800746402152

15468 Alistipes sp000434235 Contra 2.30490 0.0124196560779

18511 CAG-273 sp003534295 Contra 2.70758 0.0159319228906

20477 Coprococcus_A catus Contra 2.29379 1.01475697641e-06

Collinsella

20167 sp900540895 Contra 2.39447 9.35633056563e-09

Ruminococcus_A

36429 sp000437095 Contra 2.42518 7.23070236119e-05

Coprococcus

20473 sp900066115 Contra 2.25740 1.14391166168e-08

Intestinibacter

25571 sp900540355 Contra 2.27861 0.0497710656561

17226 Bariatricus comes Contra 3.17691 4.0099294784e-10

Roseburia

36096 sp900552665 Contra 2.24323 0.0231106169811

24113 Gemmiger qucibialis Contra 3.16098 2.99842836046e-05

Faecalibacterium

22496 sp003449675 Contra 2.23613 1.90744603181e-07

UBA7182

43535 sp003481535 Contra 2.09576 1.84717329358e-07

Gemmiger

24119 sp900540775 Contra 2.56966 1.80894410074e-07

Ruminococcus_A

36431 sp003011855 Contra 2.66427 5.49451308118e-08

18480 CAG-269 sp000431335 Contra 3.04994 6.96985807634e-06

18484 CAG-269 sp001915995 Contra 2.01969 0.025830997993 18485 CAG-269 sp001916005 Contra 2.10988 3.25915762025e-07

18648 CAG-492 sp000434015 Contra 2.21000 1.30539446398e-07

Lachnospira

26240 sp000436475 Contra 2.55618 0.000628363206244

18679 CAG-536 sp000434355 Contra 2.82212 0.00456621092046

15176 Agathobacter rectalis Contra 3.41921 0.000310178035846

21491 Dorea formicigenerans Contra 2.80870 5.93297276419e-06

Lachnospira

26245 sp003451515 Contra 2.56136 0.0023267608224

18509 CAG-273 sp000438355 Contra 2.76462 0.0305438756183

Collinsella

20345 sp900557455 Contra 2.00151 2.5548792017e-07

Collinsella

20342 sp900556605 Contra 3.12583 9.53488707721e-05

25241 Holdemanella biformis Contra 2.52660 0.00128748752692

Romboutsia

36078 timonensis Contra 2.47361 0.000416035368085

18438 CAG-170 sp900556635 Contra 2.15669 2.45053105973e-05

Megasphaera

28004 sp000417505 Contra 2.70507 0.0488599018113

Faecalibacterium

22488 prausnitzii_G Contra 2.98235 1.81752622445e-05

Faecalibacterium

22489 prausnitzii_H Contra 2.77389 3.98763445095e-06

18433 CAG-170 sp900545925 Contra 2.09412 1.07072435143e-06

Coprococcus

20469 eutactus_A Contra 2.96335 0.00490028391811

Clostridium

19952 sp001916075 Contra 2.47249 9.76605249819e-06

Prevotella

33438 sp900551275 Contra 2.63503 0.0105872123676

Mediterraneibacter

27983 lactaris Contra 2.82901 3.28992544736e-05

Bifidobacterium

17358 bifidum Contra 2.90693 0.00395513293096

22277 Evtepia sp004556345 Contra 2.09867 0.000584164019016

40011 TFOl-11 sp003524945 Contra 2.99299 0.0106460099673

Faecalibacterium

22486 prausnitzii_E Contra 2.31452 6.15296343593e-06

Lachnospira

26247 sp900316325 Contra 2.63101 0.0141817065492

A composite score was then assigned to each organism, accounting for both their correlations to immune markers and fold change between cancer and control cohorts (Tables 12, 13, and 14). The score is defined as the geometric mean of three metrics: fold change between cancer and control samples, CD3+ correlation, and CD3+CD56+ correlation. Table 12. Operational species units (OSUs) with a mean abundance of at least 0.05% with significant differences between cancer and control cohorts for inclusion into the therapeutic. For each OSU, CD3+ and CD3+CD56+ correlations are included in the table as per the linear mixed model analysis or set to zero if the mixed model correlation is negative or if the Spearman correlation was not significant enough to necessitate mixed model analysis. The cancer and control fold change, CD3+ correlation, and CD3+CD56+ correlation for each OSU were converted to percentile scores, and a combined score for each OSU was generated as the geometric mean of each of the three percentiles.

Erysipelotrichaceae bacterium GAM147

1.13356E-08 0.764382216 C2844 0.417881066 0.481640465 99.3759725

Dorea sp. AM58-8

0.000236114 0.432405099 C2913 0.395242652 0.415256323 94.53854775

[Ruminococcus]

0.000111496 0.304525941 torques C2636 0.282433356 0.290799727 81.68743735

4.96202E-05 0.504914016 Blautia obeum C2129 0.441968558 0 75.35264806

Firmicutes bacterium

1.19211E-05 0.565340143 AF12-30 C2644 0.279890636 0 73.10872098

Blautia sp. AF19-

3.1747E-07 0.415683892 10LB C2906 0.3738098 0 71.5962122

Clostridium sp.

0.016231058 0.392581823 AF36-4 C2893 0.39447635 0 71.56015136

Faecalibacterium

3.36506E-05 0.474788291 prausnitzii C2184 0.277732589 0 71.19231957

Ruminococcus sp.

3.45381E-06 0.557690435 OF03-6AA C2904 0.246561859 0 69.34836951

Dorea longicatena

1.9624E-05 0.436129729 C2413 0.268680793 0 69.18884624

Bifidobacterium

- pseudocatenulatum

0.013509112 0.452532206 C0013 0.256499594 0 69.12262094 Bifidobacterium

0.008426878 0.517722756 bifidum C0005 0.239694858 0 68.55170178

- Coprococcus comes

1.77058E-05 0.449283525 C2152 0.245550239 0 67.24353586 Ruminococcus sp. KGMB03662 C2557 0.219878468 0 66.11567283

Clostridium sp. OF10-22XD C2132 0.320929597 0 65.97044298

Faecalibacterium prausnitzii C2138 0.249514696 0 65.6229349

Firmicutes bacterium AF25-13AC C2695 0.257170198 0 65.61067466

Coprococcus catus

C2881 0.35595352 0 65.4138845

Faecalibacterium prausnitzii C2650 0.237566644 0 65.21900583

Gemmiger formicilis C3234 0.27442242 0.283691046 64.00839092

Oscillibacter sp. ER4 C2580 0.362077922 0 63.36416394

Anaerostipes hadrus

C2144 0.224716336 0 63.19608844

Ruminococcus lactaris C2149 0 0.37621326 61.28393922

Eubacterium ventriosum C2128 0.32683527 0 59.96015726

Blautia luti C2436 0.251838688 0 59.70580459

Anaerobutyricum hallii C3263 0.255775803 0 58.66605169

Faecalitalea cylindroides C2250 0 0.322093794 58.53674188

Dorea formicigenerans

C2197 0 0.383267259 56.61344825

Asaccharobacter celatus C1952 0.219961719 0.298085866 55.95335448

Barnesiella intestinihominis

C0275 0.239107807 0 55.71314544

Alistipes putredinis DSM 17216 C0133 0.306064417 0 53.10583588

Dorea longicatena

C2131 0 0 52.8235779

Collinsella aerofaciens Cl 933 0 0 52.30053782

Dorea sp. OM07-5 C2890 0 0 51.58644796

Clostridium sp. AF23-8 C2908 0.272672591 0 51.08317009

Anaerobutyricum hallii C2206 0 0 51.03761433 - [Clostridium] amygdalinum C2887 0 0.37771702 50.67666817

Eubacterium sp. OM08-24 C2896 0.285831465 0 50.55687075

- Romboutsia timonensis C3123 0 0 50.28695213

Faecalibacterium 1 prausnitzii C2651 0 0 50.09574515

- Ruminococcus callidus C2440 0 0 49.51318366

[Eubacterium] rectale 1 C2102 0 0 49.31591789

- Blautia sp. TF11- 31AT C2841 0 0 48.91658119

- Bifidobacterium adolescentis COOOl 0 0 48.71444425

- Subdoligranulum sp. APC924/74 C2870 0 0 48.51061574

Ruminococcus sp. AM42-11 C2945 0 0 48.30505982

- Blautia sp. KGMB01111 C3003 0 0 48.09773941

- Clostridium disporicum C2479 0 0 47.88861616

Bacteroides

- heparinolyticus C1005 0 0 47.25002508

- Firmicutes bacterium TM09-10 C2909 0 0 47.0332788

- Bifidobacterium animalis C0002 0.248008991 0 47.00940403

- [Eubacterium ] eligens C2123 0 0 46.37075

Clostridium sp. AM49-4BH C2934 0 0 46.14564573

Roseburia hominis C2266 0.267204375 0 46.03382224

- Roseburia sp. AM59- 24XD C2936 0 0 45.91832359

- Roseburia inulinivorans C2207 0 0.286753247 45.8451976

- Faecalibacterium sp. AF28-13AC C2810 0 0 45.45680166

Agathobaculum

- butyriciproducens C2850 0 0 44.74642259

- Faecalibacterium prausnitzii C2863 0 0 44.50454531 Anaeromassilibacillus sp. Marseille-P &l 6 C2925 0.353693881 0 44.32727446

Roseburia intestinalis C2158 0.270102529 0 44.05697948

Faecalibacterium prausnitzii C2864 0 0 44.01274212

Firmicutes bacterium AF22-6AC C2933 0 0 43.5096953

Faecalibacterium prausnitzii C2191 0 0 42.73256973

Bacteroides finegoldii C0138 0 0 42.46714319

Lactococcus lactis C3326 0 0 42.1983566

Bacteroides massiliensis C0310 0 0 41.92610156

Clostridium sp. AF20-17LB C2921 0 0 41.65026396

Fusicatenibacter saccharivorans

C2643 0 0 41.37072356

Clostridium sp. AF46-9NS C2891 0 0 41.08735355

Streptococcus thermophilus C3480 0 0 40.80002

[ Clostridium ] spiroforme C2146 0 0 40.50858134

Holdemanella biformis C2160 0 0 40.21288772

Bifidobacterium longum C0000 0 0 39.91278036

Roseburia sp. OM04- 15AA C2892 0.232754614 0 39.82976685

Firmicutes bacterium AF36-3BH C2905 0 0 39.60809076

Clostridium sp. AM18-55 C2845 0 0 38.98423732

Ruminococcus sp. AF31-8BH C2903 0 0 38.66468002

Bacteroides stercoris C0134 0 0 38.00921984

Coprococcus eutactus C2642 0 0 36.98143604

Eisenbergiella tayi C2259 0 0 35.51525914 Eubacterium

- saphenum ATCC 49989 C2183 0 0 35.12919314

- Eubacterium ramulus C2442 0 0 33.91686307

- Bacteroides uniformis C0132 0 0 33.49285783

- [Eubacterium ] siraeum C2135 0 0 33.05783641

- Intestinibacter bartlettii C2141 0 0 32.151682311 Blautia obeum C2901 0 0 30.68817687

- Ruminococcus sp. AF24-32LB C2894 0 0 30.16793778

- Megamonas funiformis C2294 0 0 29.629109

- Akkermansia sp. KLE1605 C1918 0 0 29.06993521

- Bacteroides nordii C0263 0 0 28.48837982

- Blautia wexlerae C2171 0 0 27.88205907

- Clostridium sp. TM06-18 C2922 0 0 27.24815505

Candidatus

Ishikawaella

- capsulata Mpkobe C4922 0 0 26.58329888

- Parabacteroides goldsteinii C0282 0 0 25.88341081

- Alistipes sp. 5CBH24 1 C0283 0 0 25.14347607

- Lachnospira pectinoschiza C2649 0 0 24.35722212

- Clostridium sp. AF34-13 C2653 0 0 23.5166394

Catenibacterium mitsuokai DSM 1 15897 C2204 0 0 22.61124205

- Eubacterium sp. TM06-47 C2917 0 0 21.626875

- Coprococcus eutactus C2140 0 0 20.54367678

- Roseburia faecis C2648 0 0 19.33234001

- Bacteroides faecis C0221 0 0 17.94655471 Bacteroides sp.

0.686897002 0.016216198 OF04-15BH C1226 0 0 16.30552706

Lawsonibacter asaccharolyticus

0.77256713 0.008006904 C2612 0 0.303877071 14.43735499

- Bacteroides fragilis

0.226827239 0.011837149 C0096 0 0 14.24418991

- Odoribacter

0.804445324 0.006154069 splanchnicus CO 185 0 0 0

Table 13 (illustrated as FIG. 22). Microbe rankings were based on classified species results using the GTDB database with a mean abundance of at least 0.005% with significant differences between cancer and control cohorts for inclusion into the therapeutic (inverse p value, Mann Whitney U test). For each classified species hit, CD3+ and CD3+CD56+ correlations are included in the table as per the linear mixed model analysis or set to zero if the mixed model correlation is negative or if the Spearman correlation was not significant enough to necessitate mixed model analysis. The cancer and control fold change, CD3+ correlation, and CD3+CD56+ correlation for each OSU were converted to percentile scores, and a combined score for each species level hit was generated by computing the geometric mean of each of the three percentiles.

Table 14 (illustrated as FIG. 23). Microbe rankings were based on classified species results using the GTDB database with a mean abundance of at least 0.005% with significant differences between cancer and control cohorts for inclusion into the therapeutic (LDA score, LEfSe). For each classified species hit, CD3+ and CD3+CD56+ correlations are included in the table as per the linear mixed model analysis or set to zero if the mixed model correlation is negative or if the Spearman correlation was not significant enough to necessitate mixed model analysis. The cancer and control fold change, CD3+ correlation, and CD3+CD56+ correlation for each OSU were converted to percentile scores, and a combined score for each species level hit was generated by computing the geometric mean of each of the three percentiles.

Machine Learning for Live Biotherapeutic Design

The top 32 scoring organisms from Example 9 (Table 6) is selected for screening in simulated microbial mixes. Each combination of 4 organisms from the 32 (listed in Table 15, below) is evaluated in silico using the trained machine learning model. For the cancer samples in the model, relative species abundances for the four organisms in the putative mix are increased in silico by a certain amount (here 0.5%). This simulates in silico the physical action of adding microbes to the gut microbiome. Classification is then performed using the machine learning model to estimate the probability that each augmented sample is a cancer sample. The hypothesis is that combinations of microbes that make cancer samples appear more like control samples according to the model are better candidates for therapeutic mixes. Each putative mix is scored by its mean predicted cancer probability across all the augmented cancer samples, with lower mean predicted cancer probabilities corresponding to notionally better therapeutic candidates. The top 30 exemplary live biotherapeutic compositions (exemplary microbial combinations) are then validated experimentally as described in Examples 12, and 16 to 22 as described below.

A similar procedure was then followed by selecting each possible combination of 4 organisms from the top 6 listed in Table 13. These combinations are shown in Table 16.

Table 15. List of exemplary live biotherapeutic compositions, i.e., list of exemplary microbial combinations.

Mix Organism Name (Operational Species Unit)

1 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844

2 Bifidobacterium bifidum C0005 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149

3 Bifidobacterium bifidum C0005

Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644

4 Bifidobacterium bifidum C0005

Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904

5 Bifidobacterium bifidum C0005

Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844

6 Bifidobacterium bifidum C0005 Blautia obeum C2129 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifidum C0005 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Blautia obeum C2129 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Blautia obeum C2129 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Blautia obeum C2129 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifiidum C0005 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Bifidobacterium bifiidum C0005 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifiidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifiidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifiidum C0005 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Blautia obeum C2129

Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Bifidobacterium bifiidum C0005 Blautia obeum C2129

Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Bifidobacterium bifiidum C0005 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Bifidobacterium bifiidum C0005 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Blautia obeum C2129

Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Blautia obeum C2129 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifiidum C0005 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifiidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Blautia obeum C2129

Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129

Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Blautia obeum C2129 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129

Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Blautia obeum C2129 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Blautia obeum C2129 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifiidum C0005 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifidum C0005 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Blautia obeum C2129

Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129

Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129

Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Dorea longi catena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Blautia obeum C2129 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Blautia obeum C2129

Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Bifidobacterium bifiidum C0005 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Bifidobacterium bifiidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Bifidobacterium bifiidum C0005 Blautia obeum C2129

Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Dorea longi catena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Coprococcus comes C2152 Dorea sp. AMS 8-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Blautia obeum C2129 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Blautia obeum C2129 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Coprococcus comes C2152 Dorea longicatena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Coprococcus comes C2152 Dorea longi catena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Blautia obeum C2129 Dorea longi catena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Coprococcus comes C2152 Dorea longi catena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Coprococcus comes C2152 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Coprococcus comes C2152 Dorea longi catena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Blautia obeum C2129 Coprococcus comes C2152 Dorea longi catena C2131 Dorea sp. AM58-8 C2913 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM 147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium G AMI 47 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM 147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM 147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Firmi cutes bacterium AF 12-30 C2644 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Firmi cutes bacterium AF 12-30 C2644 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Firmi cutes bacterium AF 12-30 C2644 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Bifidobacterium bifidum C0005 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129

Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Blautia obeum C2129

Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifiidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Bifidobacterium bifiidum C0005 Blautia obeum C2129

Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Bifidobacterium bifidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Bifidobacterium bifidum C0005 Blautia obeum C2129 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF12-30 C2644 Ruminococcus lactaris C2149 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longi catena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longi catena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longi catena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longi catena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus lactaris C2149 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Dorea longi catena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Firmicutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM 147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Blautia obeum C2129 Clostridium sp. AF36-4 C2893 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Coprococcus comes C2152 Erysipelotrichaceae bacterium GAM 147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Fir mi cutes bacterium AF 12-30 C2644 Ruminococcus lactaris C2149 Blautia obeum C2129 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus lactaris C2149 Ruminococcus sp. OF03-6AA C2904 Blautia obeum C2129 Coprococcus comes C2152 Dorea longicatena C2131 Erysipelotrichaceae bacterium GAM 147 C2844 Firmi cutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Erysipelotrichaceae bacterium GAM 147 C2844 Dorea sp. AM58-8 C2913 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Ruminococcus lactaris C2149

Firmicutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Dorea longicatena C2131 Blautia obeum C2129 Coprococcus comes C2152 Bifidobacterium catenulatum COO 14 Blautia sp. AF19-10LB C2906 Ruminococcus sp. OF03-6AA C2904 Dorea longicatena C2131 Blautia obeum C2129 Coprococcus comes C2152 Bifidobacterium catenulatum COO 14 Blautia sp. AF19-10LB C2906 Ruminococcus sp. OF03-6AA C2904 Dorea longicatena C2131

Blautia obeum C2129 Coprococcus comes C2152 Bifidobacterium catenulatum COO 14 Blautia sp. AF19-10LB C2906 Erysipelotrichaceae bacterium GAM 147 C2844 Dorea sp. OM07-5 C2890 Faecalibacterium prausnitzii C2184 Dorea longicatena C2413 Anaerobutyricum hallii C2206 Faecalibacterium prausnitzii C2650 Faecalibacterium prausnitzii C2651 Anaerostipes hadrus C2144 Dorea formicigenerans C2197 [Ruminococcus] torques C2636 Coprococcus catus C2881 Faecalibacterium sp. AF28-13 AC C2810 [Clostridium] amygdalinum C2887 Roseburia inulinivorans C2207 Asaccharobacter celatus Cl 952 Erysipelotrichaceae bacterium GAM 147 C2844 Dorea sp. AM58-8 C2913 Bifidobacterium bifiidum C0005 Clostridium sp. AF36-4 C2893

Dorea longicatena C2131 Firmicutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Coprococcus comes C2152 Bifidobacterium catenulatum COO 14 Blautia sp. AF19-10LB C2906 Dorea formicigenerans C2197 [Ruminococcus] torques C2636 Coprococcus catus C2881 Erysipelotrichaceae bacterium GAM147 C2844 Dorea sp. AM58-8 C2913

Dorea longicatena C2131 Bifidobacterium catenulatum COO 14 Dorea formicigenerans C2197 Coprococcus comes C2152 Coprococcus catus C2881 Erysipelotrichaceae bacterium GAM147 C2844 Dorea sp. AM58-8 C2913

Firmicutes bacterium AF 12-30 C2644 Ruminococcus sp. OF03-6AA C2904 Dorea longicatena C2131 Blautia obeum C2129 Dorea sp. OM07-5 C2890 Coprococcus comes C2152 Dorea longicatena C2413 Faecalibacterium prausnitzii C2650 Blautia sp. AF19-10LB C2906 [Ruminococcus ] torques C2636 Erysipelotrichaceae bacterium GAM 147 C2844 Dorea sp. AM58-8 C2913 Bifidobacterium bifiidum C0005 Clostridium sp. AF36-4 C2893 Ruminococcus lactaris C2149 Erysipelotrichaceae bacterium GAM 147 C2844 Dorea sp. AM58-8 C2913 Ruminococcus lactaris C2149 Dorea formicigenerans C2197 [Clostridium] amygdalinum C2887 Roseburia inulinivorans C2207 Asaccharobacter celatus C1952 Erysipelotrichaceae bacterium GAM147 C2844 Dorea sp. AM58-8 C2913 Bifidobacterium bifidum C0005 Clostridium sp. AF36-4 C2893 Ruminococcus lactaris C2149

Dorea formicigenerans C2197 [Clostridium] amygdalinum C2887 Roseburia inulinivorans C2207 Asaccharobacter celatus C1952 Erysipelotrichaceae bacterium GAM147 C2844 Dorea sp. AM58-8 C2913 Ruminococcus lactaris C2149 Bifidobacterium bifidum C0005 Bifidobacterium catenulatum COO 14 Bifidobacterium pseudocatenulatum COO 13 Blautia luti C2436 Blautia obeum C2129 Blautia obeum C2901 Blautia sp. AF19-10LB C2906 Blautia luti C2436 Blautia obeum C2129 Blautia obeum C2901 Blautia sp. AF19-10LB C2906 Blautia sp. KGMB01111 C3003 Blautia sp. TF11-31AT C2841 Blautia wexlerae C2171 Clostridium sp. AF20-17LB C2921

Clostridium sp. AF23-8 C2908 Clostridium sp. AF34-13 C2653 Clostridium sp. AF36-4 C2893 Clostridium sp. AM18-55 C2845 Clostridium sp. AM49-4BH C2934 Clostridium sp. OF10-22XD C2132 Collinsella aerofaciens C1933 Collinsella bouchesdurhonensis C1956 Collinsella sp. TM05-38 C1984 Coprococcus catus C2881 Coprococcus comes C2152 Coprococcus eutactus C2642 Dorea formicigenerans C2197 Dorea longicatena C2131 Dorea longicatena C2413 Dorea sp. AM58-8 C2913 Dorea sp. OM07-5 C2890 Eubacterium ramulus C2442 Eubacterium ramulus C2852 Eubacterium saphenum ATCC 49989 C2183 Eubacterium ventriosum C2128 Faecalibacterium prausnitzii C2138 Faecalibacterium prausnitzii C2184 Faecalibacterium prausnitzii C2650 Faecalibacterium prausnitzii C2651 Faecalibacterium prausnitzii C2863 Faecalibacterium prausnitzii C2864 Faecalibacterium sp. AF28-13AC C2810 Firmicutes bacterium AF 12-30 C2644

Firmicutes bacterium AF22-6AC C2933 Firmicutes bacterium AF25-13AC C2695 Firmicutes bacterium AM41-11 C2946 Firmicutes bacterium TM09-10 C2909 Roseburia inulinivorans C2207 Roseburia sp. AM59-24XD C2936 Roseburia sp. OM04-15AA C2892 Ruminococcus callidus C2440 Ruminococcus lactaris C2149 Ruminococcus sp. AF31-8BH C2903 Ruminococcus sp. AM42-11 C2945 Ruminococcus sp. KGMB03662 C2557 Ruminococcus sp. OF03-6AA C2904 Flavonifractor plautii C2284 [Clostridium] scindens C2143 [Clostridium] bolteae C2137 Flavonifractor plautii C2284 [Clostridium] scindens C2143 [Clostridium] bolteae C2137 Blautia hansenii C3044 [Clostridium] clostridioforme C2275 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia sp. AF19-10FB C2906 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia sp. AF19-10FB C2906

Firmicutes bacterium AF 12-30 C2644 Dorea longicatena C2131 Coprococcus comes C2152 Blautia obeum C2129 Faecalibacterium prausnitzii C2184 Dorea longicatena C2413

238 Dorea longicatena C2131 Coprococcus comes C2152 Blautia obeum C2129 Faecalibacterium prausnitzii C2184 Dorea longicatena C2413 [Ruminococcus] torques C2636

239 Erysipelotrichaceae bacterium GAM147 C2844 Dorea longicatena C2131

Coprococcus comes C2152 Blautia obeum C2129 Faecalibacterium prausnitzii C2184 Dorea longicatena C2413

240 Erysipelotrichaceae bacterium GAM147 C2844 Ruminococcus sp. OF03-6AA C2904 Blautia sp. AF19-10LB C2906

Firmicutes bacterium AF 12-30 C2644 Dorea longicatena C2131 Coprococcus comes C2152 Blautia obeum C2129 Faecalibacterium prausnitzii C2184 Dorea longicatena C2413 [Ruminococcus] torques C2636

241 Erysipelotrichaceae bacterium GAM147 C2844 Dorea longicatena C2131

Coprococcus comes C2152 Blautia obeum C2129 Faecalibacterium prausnitzii C2184 Dorea longicatena C2413 [Ruminococcus] torques C2636

Table 16. The top 6 scoring organisms using LEfSe from Table 13 have been selected for screening in simulated microbial mixes. All possible combinations of 4 organisms from the top 6 are shown. Blautia_A obeum Dorea longicatena_B Mediterraneibacter faecis CAG-269 sp000431335 Erysipelatoclostridium sp000752095 Blautia_A obeum Dorea longicatena_B Faecalibacterium prausnitzii_C Erysipelatoclostridium sp000752095 Dorea longicatena_B Faecalibacterium prausnitzii_C CAG-269 sp000431335 Erysipelatoclostridium sp000752095 Blautia_A obeum Dorea longicatena_B CAG-269 sp000431335 Erysipelatoclostridium sp000752095 Blautia_A obeum Faecalibacterium prausnitzii_C CAG-269 sp000431335 Erysipelatoclostridium sp000752095 Dorea longicatena_B Mediterraneibacter faecis Faecalibacterium prausnitzii_C Dorea longicatena_B Mediterraneibacter faecis Faecalibacterium prausnitzii_C CAG-269 sp000431335 Erysipelatoclostridium sp000752095 Mediterraneibacter faecis Faecalibacterium prausnitzii_C CAG-269 sp000431335 Blautia_A obeum Dorea longicatena_B Faecalibacterium prausnitzii_C CAG-269 sp000431335 Blautia_A obeum Mediterraneibacter faecis Faecalibacterium prausnitzii_C CAG-269 sp000431335 Erysipelatoclostridium sp000752095 Dorea longicatena_B Mediterraneibacter faecis CAG-269 sp000431335 Erysipelatoclostridium sp000752095 Blautia A obeum Mediterraneibacter faecis CAG-269 sp000431335 Erysipelatoclostridium sp000752095 Blautia_A obeum Dorea longicatena_B Mediterraneibacter faecis

Example 12: Cytokine and Immune Cell Characterization in Patient Blood Samples Cytokine analysis of blood plasma

Plasma was obtained from 1 mL blood by centrifugation at 2000xg for 10 minutes. The plasma fraction was removed from the top and transferred to a clean tube. To remove any residual cells that may have carried over, the plasma was centrifuged again at 2000xg for 10 minutes, and the top layer was transferred to another tube, taking care to not take any red blood that may have settled to the bottom of the tube. Cytokine analysis was performed on 25 selected plasma samples by Eve Technologies (website link) using the 48-plex Luminex assay.

Mann-Whitney test was applied to each cytokine to identify those with significant differential abundance between samples corresponding to checkpoint inhibitor complete responders (CR, N=6) and non-responders (NR, N=8). The remaining 11 samples were from patients identified as partial responders (PR) or stable disease (SD); due to the unclear phenotype, these were not included in the statistical analysis. Compounds with significant concentration differences between CR and NR samples (p<0.05) are listed in Table 17.

Table 17. Average fluorescence values for CR and NR samples exhibiting significant differential abundance.

Compound CR Average NR Average CR/NR ratio P -value

Eotaxin 20.91 36.11 0.58 0.0046

IFNgamma 0.51 4.41 0.12 0.0132

IL.2 0.55 1.77 0.31 0.0141

IL.27 1065.91 2326.60 0.46 0.0337

MIP.la 30.02 44.87 0.67 0.0132

CyTOF analysis ofPBMCs isolated from whole blood

Peripheral blood mononuclear cells (PBMCs) were isolated from approximately 8 mL blood using SepMate™ tubes following the manufacturer’s instmctions. Following isolation, cells were resuspended in lmL PBS + 2% FBS. IOUL of the cell suspension was mixed with lOuL if Trypan Blue Stain 0.4% and applied to a cell counter plate to determine viable cell concentration. The cell suspension was then diluted in 90% PBS + 10% DMSO to achieve a cell density of 1c10 L 7 cells/mL. Cells were then frozen at a controlled rate of 1 °C/min to a final temperature of -150 °C in liquid nitrogen.

Mass cytometry (CyTOF) was performed on 25 selected PBMC samples by the University of Texas Health Center at San Antonio (UTHCSA). A 30 marker antibody panel focused on human immune-oncology relevant markers (Fluidigm) was used to quantify different cell populations. The markers and associated metal labels are given in Table 18. Markers were gated using the strategy shown in FIG. 21 to determine the immune cell types and subtypes. Cell populations were reported either as a percentage of all viable cells and/or of the parent cell type. Table 18 List of antibodies and metal labels used for CyTOF analysis.

Immune Marker Metal

CCR4 158Gd

CCR5 144Nd

CCR7 159Tb

CDlla 142Nd

CD127 176Yb

CD134 [0X40] 150Nd

CD137 [4-1BB] 173Yb

CD 152 [CTLA-4] 161Dy

CD16 148Nd

CD161 164Dy

CD2 151Eu

CD223 [LAG3] 175Lu

CD25 149Sm

CD27 167Er

CD278 [ICOS] 168Er CD279 [PD-1] 155Gd CD28 160Gd CD3 170Er

CD366 [Tim-3] 153Eu

CD4 145Nd

CD44 166Er

CD45 154Sm

CD45RA 169Tm

CD45RO 165Ho

CD49d 141Pr CDS 143Nd

CD57 172Yb

CD69 162Dy

CD7 147Sm

CD8a 146Nd

CD9 171Yb

CD95 [Fas] 152Sm

CXCR3 156Gd

H LA-DR 174Yb

FIG. 21. Gating strategy used to classify immune cell populations based on metal- labeled peptide markers, and cell counts for a representative sample.

Mann-Whitney test was applied to each population type or subtype to identify those with significant differential abundance between samples corresponding to checkpoint inhibitor complete responders (CR, N=6) and non-responders (NR, N=8). The remaining 11 samples were from patients identified as partial responders (PR) or stable disease (SD); due to the unclear phenotype, these were not included in the statistical analysis. Cell populations with significant abundance differences between CR and NR samples (p<0.05) are listed in Table 19.

Table 19 Cell population abundance values, as a percentage either of total live cells or of the parent cell type, as indicated, for CR and NR samples exhibiting significant differential abundance.

CR NR

Cell Population Average Average CR/NR P -value

% of CD3-CD44+CDlla+ in Siglet alive 13.09 31.67 0.41 0.0132

% of B cells in Siglet alive 9.92 4.92 2.02 0.0046

% of CD3-HLADR+CD45RA_low in

Siglet alive 10.44 23.57 0.44 0.0095

% of Monocytes in Siglet alive 10.12 22.29 0.45 0.0183

% of CD3+CD4-CD8+CD45RO_lo CD45RA+ in alive 13.40 8.28 1.62 0.0337

% in B cells in CD45+CD3- 43.37 26.18 1.66 0.0132

Example 12: Gene Expression Analysis of Microbial Treatment in Co-Culture

Live biotherapeutic compositions as provided herein, including the exemplary combinations of microbes 1 to 241, as described in Table 15, Example 10, are evaluated in co-culture for immunomodulatory effects. Live biotherapeutics are co- cultured with human colonic cells (CaCo2) to investigate the effects of the bacteria on the host. Live biotherapeutic compositions are also co-cultured on CaCo2 cells that were stimulated with Interleukin 1 (ILl) to mimic the effect of the bacteria in an inflammatory environment. The effects in both scenarios are evaluated through gene expression analysis either by PCR or by next generation sequencing approaches. Cytokine Production in THP-1 Cells Induced by Live Biotherapeutics

Live biotherapeutic compositions as provided herein, including for example the exemplary combinations of microbes 1 to 241, Table 15, Example 10, and single bacterial strains are evaluated alone and in combination with lipopolysaccharide (LPS) on cytokine production in THP-1 cells, a model cell line for monocytes and macrophages.

THF-1 cells are differentiated into M0 medium for 48 h with 5 ng/mL phorbol-12-myristate-13-acetate (PMA). These cells are subsequently incubated with the live biotherapeutic composition at a final concentration of 10 8 /ml, with or without the addition of LPS at a final concentration of 100 ng/ml. Alternatively, the bacterial cells are centrifuged, and the resulting supernatant is added to the THF-1 cell preparation. The bacteria are then washed off and the cells allowed to incubate under normal growing conditions for 24 h. The cells are then spun down and the resulting supernatant is analyzed for cytokine content using a Luminex 200 analyzer or equivalent method.

Cytokine Production in Immature Dendritic Cells Induced by Live Biotherapeutic Compositions

Live biotherapeutic compositions as provided herein, including the exemplary combinations of microbes 1 to 241, as described in Table 15, Example 10, and single bacterial strains are evaluated alone and in combination with LPS on cytokine production in immature dendritic cells. A monocyte population is isolated from peripheral blood mononuclear cells (PBMCs). The monocyte cells are subsequently differentiated into immature dendritic cells. The immature dendritic cells are plated out at 200,000 cells/well and incubated with the live biotherapeutic composition at a final concentration of 10 7 /ml in RPMI media, with the optional addition of LPS at a final concentration of 100 ng/ml. Alternatively, the bacterial cells are centrifuged, and the resulting supernatant is added to the dendritic cell preparation. The negative control involves incubating the cells with RPMI media alone and positive controls incubating the cells with LPS at a final concentration of 100 ng/ml. The cytokine content of the cells is then analyzed.

Cytokine Production and Analysis in PBMCs

Peripheral blood mononuclear cells (PBMC’s) are isolated from subject blood using a standard kit and stored in liquid nitrogen at 1 x 10 6 cells per mL until use.

Prior to storage, PBMC’s may be processed using flow sorting or an antibody spin separation kit to select for a certain purified lymphocyte subpopulation, such as T cells.

PBMCs are thawed at 37°C and then transferred to a growth medium consisting of RPMI-1640 (Lonza, Switzerland), with 10% heat inactivated FCS added, as well as 0.1% penicillin-streptavidin, 1% L-glutamine, and DNase at 10 mg/mL to inhibit aggregation. Cells are centrifuged at 200 x g for 15 minutes and then counted using trypan blue and spread into 24 well plates at 1 x 10 6 cells per well (1 mL per well) (Kechaou et al. (2013) Applied and Environmental Microbiology 79:1491-1499; Martin et al. (2017) Frontiers in Microbiology 8:1226).

An overnight bacterial culture is inoculated using a pre-stocked isolated bacterial strain. This strain is grown at 37°C for 10 to 20 hours in a YBHI medium with added cellobiose (1 mg/mL), maltose (1 mg/mL) and cysteine (0.5 mg/mL) in an anaerobic chamber filled with 85% nitrogen, 10% carbon dioxide, and 5% hydrogen (Martin et al., 2017). The growth medium may also be Reinforced Clostridial Medium (RCM) (Thermo Fisher, USA), which may also be supplemented with cysteine (0.5 mg/mL) or arginine (1 mg/mL).

At the end of the anaerobic culture, the culture supernatant and bacterial cells alone are saved for co-culture with PBMC’s. Microbial culture supernatant is saved directly after centrifugation at -80°C. Cells are saved by washing with phosphate buffered saline (PBS) and then storing in PBS with 15% glycerol. Bacteria are quantified using phase contrast microscopy and stored at a final concentration of 10 5 or 10 6 cells per mL (Haller et al. (2000) Infection and Immunity 68; Rossi et al.

(2015) Scientific Reports 6:18507) at -80°C . Bacteria may also be pasteurized prior to storage by treatment at 70°C for 30 minutes (Plovier et al. (2017) Nature Medicine 23:107-113).

Prior to co-culture, supernatant is thawed on ice and 200 pL of supernatant is diluted in 1 mL of total volume of PBMC growth medium. Microbial growth medium is used as a negative control. This 1 mL is added to the 1 mL of PBMC in each well, resulting in a 10% final level of microbial culture supernatant in a 2 mL culture containing 1 x 10 6 PBMCs. Each combination of PBMCs and supernatant is performed in duplicate or triplicate.

Prior to co-culture, bacteria are thawed on ice and then washed at 4°C with PBMC growth medium. 1 mL of the bacterial suspension is added to the 1 mL of PBMC culture in each well of the plate, resulting in a final 2 mL culture containing 1 x 10 6 PBMC’s and 1 x 10 5 or 1 x 10 6 (potentially pasteurized) bacteria.

The co-culture of PBMC’s and supernatant or purified bacteria is incubated for 2, 6, 16, 24, or 48 hours at 37°C in 10% carbon dioxide.

After co-culture, the supernatant is harvested and treated with a protease inhibitor (Complete EDTA-Free protease inhibitor, Roche Applied Bioscience) to protect cytokines and stored directly at -80°C for cytokine profiling. The pelleted cells are treated with RNAlater (Thermo Fisher, USA) and saved for RNA sequencing.

Cytokine analysis is performed on saved co-culture supernatant using ELISA or a Luminex system. Cytokines measured may include but are not limited to, IL-10, IL-2, and IFN-gamma.

RNA sequencing is performed on PBMC’s saved in RNAlater post co-culture. Standard pseudo-alignment is performed using Kallisto (Bray et al. (2016) Nature Biotechnology 34:525-527) and differential expression is analyzed using DESeq2 (Love et al. (2014) Genome Biology 15:550) to identify differential expression between different microbes and different PBMC donors.

Statistical analyses are performed to identify microbes that exhibit desired immunomodulatory effects in vitro , which include but are not limited to inducing production of IFN-gamma and lowering expression of genes associated with T cell exhaustion (PD1, CTLA4, VISTA, TIM3, TIGIT, LAG3).

Example 13: Genetic Modification of Therapeutic Microbes

Microbes of interest, including microbes as provided herein, e.g., as listed in Table 1, 5, 10, 11 or 12, including bacteria from all the genuses listed herein, and including the combinations of microbes as provided herein, e.g., the exemplary combinations 1 to 241 as described in Table 15, Example 10, or as identified from the in vivo and ex vivo analyses described in Example 10 and Example 11, are interrogated or investigated to identify mechanisms of action, and the discovered mechanisms are leveraged using a genetic modification or modifications to amplify the microbe’s therapeutic effect.

In alternative embodiments, this is accomplished in two stages. First, complementary bioinformatic and experimental approaches are used to identify the genes within a microbe of interest responsible for its therapeutic effect. Second, synthetic biology techniques are used to engineer over-expression of the identified genes within the original organism of discovery or inserted for overexpression in the genome of a chassis organism. Chassis organisms include any microbe as described herein, including genuses of bacteria as provided herein, and also include bacteria as listed in Tables 1, 3, 4, 5, 6, 7 and/or 8, including Bacillus subtilis , Escherichia coli Nissle , or any microbes listed in the combinations as provided herein in Table 15 or Table 16, or the original organism of interest itself.

In alternative embodiments, microbes as provided herein are genetically modified to increase expression of existing therapeutically effective genes, or to install extra copies of these genes, or to install into a microbe lacking these functions any one of these genes. Methods for genetic engineering/augmenting a microbe of interest, e.g., a gut microbe, to alter expression of existing therapeutically effective genes or to install extra copies of said genes or to install said genes in a microbe lacking these functions are numerous in the art. Techniques applied to gut microbes and related organisms for experimental gene disruption, gene replacement or gene expression modulation include CRISPR-Cas9 genome editing (Bruder et al (2016) Applied and Environmental Microbiology 82:6109-6119), bacterial conjugation (Cuiv et al (2015) Nature Scientific Reports 5:13282; Ronda et al. (2019) Nature Methods 16:167-170), gene replacement mutagenesis by homologous recombination (Cartman et al (2012) Applied Environmental Microbiology 78:4683-4690; Heap et al (2007) Journal of Microbiological Methods 70:452-464), random transposon mutagenesis (Cartman and Minton (2010) Applied Environmental Microbiology 76: 1103-1109), and antisense-based gene expression attenuation (Forsyth et al (2002) Molecular Microbiology 43:1387-1400; Kedar et al (2007) Antimicrobial Agents and Chemotherapy 51:1708-1718.

Genes of interest inserted into microbes as provided herein, or whose expression is increased in microbes as provided herein, can be engineered to immediately follow and be under inducible control by various promotor elements that are functional in gut microbes. Highly inducible and controllable promoter elements are available for bacteria in the gram -negative genus Bacteroides (Lim et al (2017) Cell 169:547-558; Bencivenga-Barry et al (2019) Journal of Bacteriology doi:

10.1128/JB.00544-19). Some of these are responsive to various diet-derived polysaccharides, while those often most useful for use for inducible function determination in animal models such as mouse rely on induction by tetracycline derivatives like anhydrotetracycline at sub -bactericidal levels. Anhydrotetracycline can be employed as an inducer for engineered promoters in gut Clostridia (Dembek et al (2017) Frontiers of Microbiology 8: 1793). Promoters that respond to bile acids are identified in gram-positive gut Clostridium species (Wells and Hyemon (2000) Applied Environmental Microbiology 66: 1107-1113) and in Eubacterium species (Mallonee et al. (1990) Journal of Bacteriology 172:7011-7019. Also, inducible promoters that respond to sugars such as lactose (Banerjee et al (2014) Applied Environmental Microbiology 80-2410-2416) and arabinose (Zhang et al (2015) Biotechnology for Biofuels 8:36) are identified and useful in related Clostridial species. Genes inserted in exemplary recombinant bacterium can be induced under low-oxygen conditions from promoters driven by transcriptions factors such as FNR (fumarate and nitrate reductase) (Oxer et al (1991) Nucleic Acids Research, 19, 11: 2889-2892). Genes of interest inserted in microbes as provided herein can also be engineered to immediately follow and be under constitutive control by various promotor elements that are functional in gut microbes. Constitutive promoter libraries and promoter-RBS (ribosome binding site) pairs have been created for bacteria in the gram -negative genus Bacteroides (Mimee et al (2015) Cell Syst. 1, 62-71) and computational models have been developed from Bacillus subtilis promoter sequences data sets for promoter prediction in Gram-positive bacteria (Coelho et al (2018) Data Br. 19, 264-270).

Engineering of Metabolic Pathways in Therapeutic Microbes

In one embodiment, an organism used to practice embodiments as provided herein is genetically modified to overexpress a pathway for production of any short chain fatty acid (SCFA), including butyrate or butyric acid, propionate and acetate. Butyric acid is naturally produced in many gut microorganisms and is derived from two molecules of acetyl-CoA, a central metabolic intermediate that is ubiquitous in microorganisms. In one embodiment, the native pathway is overexpressed, e.g., as discussed herein. In another embodiment, a heterologous pathway is constructed by introducing one or more genes from a different organism, including all genes derived from different organisms. Condensation of two acetyl-CoA molecules is catalyzed by a ketothiolase (EC:2.3.1.9), such as the atoB gene from Escherichia coli , to produce one molecule of acetoacetyl-CoA (Sato et al. (2007) J. Biosci. Bioengineer. 103:38-

44). Alternative candidates are obtained by Basic Local Alignment Search Tool

(BLAST) search of this sequence (Altschul et al. (1997) Nuc. Acids. Res. 25:3389-

3402), obtaining homologous genes either known or predicted to encode similar enzyme function. Exemplary gene candidates are obtained using the following

GenBank accession numbers. atoB Escherichia coli NP_416728.1 yqeF Escherichia coli NP_417321.2 phaA Cupriavidus necator YP 725941 bktB Cupriavidus necator AAC38322.1 thiA Clostridium acetobutylicum NP_349476.1 thiB Clostridium acetobutylicum NP 149242.1

The second step in the pathway involves reduction of acetoacetyl-CoA to 3- hydroxybutyryl-CoA by a hydroxyacyl-CoA dehydrogenase (EC: 1.1.1.35), such as that encoded by hbd in Clostridium acetobutylicum (Atsumi et al. (2008) Metab. Eng. 10(6):305-311). Similarly, to above, alternate candidates are identified in the literature or by BLAST. Exemplary candidates are as follows. paaH Escherichia coli NP_415913.1 hbd Clostridium acetobutylicum NP 349314.1 hbd Pseudomonas putida KT2440 NC_002947.4

RSP 3970 Rhodobacter sphaeroides 2.4.1 YP 345236.1

The next step is the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA by an enoyl-CoA hydratase, also known as crotonase (EC:42.1.55), such as that encoded by the crt gene of Clostridium acetobutylicum (Kim et al. (2014) Biochem. Biophys. Res. Commun. 451:431-435) or the homologs listed below crt Clostridium acetobutylicum NC 003030.1 echA18 Mycobacterium bovis AF2122/97 NC_002945.4 maoC Escherichia coli NP 415905.1 crt Bacillus thuringiensis NC 005957.1 Next, crotonyl-CoA is reduced to butyryl-CoA through the action of an enoyl- CoA reductase (EGO.1.38 or EGO.1.44), such as that encoded by the bed gene of Clostridium acetobutylicum (Boynton et al. (1996) J. Bacteriol. 178:3015-3024). Activity of this enzyme can be enhanced by expressing bed in conjunction with expression of the C. acetobutylicum etfAB genes, which encode an electron transfer flavoprotein. Several eukaryotic enzymes with this activity have also been identified, such as TER from Euglena gracilis , that upon removal of the mitochondrial targeting leader sequence have demonstrated superior activity in E. coli (Hoffmeister et al. (2005) J. Biol. Chem. 280:4329-4338). Protein sequences for these and other exemplary sequences can be obtained using the following GenBank accession numbers. bed Clostridium acetobutylicum NP 34.9317.1 etfA Clostridium acetobutylicum NP 349315.1 etfB Clostridium acetobutylicum NP 349316.1

TER Euglena gracilis Q5EU90. 1

TDE0597 Treponema denticola NP 97.1211.1

The final step of this pathway is CoA removal from butyryl-CoA to generate butyric acid. Although numerous CoA hydrolases occur in most bacteria, e.g., tesB from E. coli ((Naggert et al. (1991) J. Biol. Chem. 266:11044-11050), it is desirable to recover energy from hydrolysis of the thioester bond in the form of ATP. The sucCD complex of E. coli (EC:6.2.1.5) is one example of this, known to catalyze the conversion of succinyl-CoA and ADP to succinate and ATP (Buck et al. (1985) Biochem. 24:6245-6252). Another example is sucD, succinic semialdehyde dehydrogenase, from Porphyromonas gingivalis (Yim et al. (2011) Nat. Chem. Biol. 7:445-452). Another option, using phosphotransbutylase/ butyrate kinase (EC:2.3.1.19, EC:2.7.2.7), is catalyzed by the gene products of bukl, buk2, and ptb from C. acetobutylicum (Walter et al. (1993) Gene 134: 107-111) or homologs thereof. Finally, an acetyltransferase capable of transferring the CoA group from butyryl-CoA to acetate can be applied (EC:2.8.3.9), such as Cat3 from C. kluyveri (Sohling and Gottschalk (1996) J. Bacteriol. 178:871-880). Protein sequences for these and other exemplary sequences can be obtained using the following GenBank accession numbers. ptb Clostridium acetobutylicum NP 349676 bukl Clostridium acetobutylicum NP 349675 buk2 Clostridium acetobutylicum Q97II1 sucC Escherichia coli NP_415256.1 sucD Escherichia coli AAC73823.1 cat3 Clostridium kluyveri EDK35586.1 tesB Escherichia coli NP 414986

In another embodiment, a microbe used to practice embodiments as provided herein is genetically modified to metabolize bile acids, also referred to as bile salts to indicate the predominant form at neutral pH, that are produced in the liver and present in the gut at about 1 mM concentration. Two such types of bile acid conversion processes are catalyzed by bacteria. The first is deconjugation, which removes either taurine or glycine that is frequently found conjugated to bile acids (Ridlon et al.

(2016) Gut Microbes 7:22-39; Masuda et al. (1981) Microbiol. Immunol. 25:1-11). This is catalyzed by bile salt hydrolase (BSH) enzymes (EC:3.5.1.24), which are widespread in many gut bacteria. Some BSHs have broad substrate specificity, while others are very specific for a particular bile salt. The substrate range of a BSH of interest is determined by assay of purified BSH or crude lysates from the native host, on a panel of glycine and taurine conjugated bile salts (Jones et al. (2008) Proc. Nat. Acad. Sci. USA 105:13580-13585). To enhance the activity and substrate range of bile salt deconjugation in the engineered microbe, native BSHs of interest and/or heterologous genes from other microbes are introduced. Exemplary genes are listed below. Still others are found by GenBank search or BLAST of these sequences to identify homologs. bsh Bifidobacterium longum AF148138.1 bsh Bifidobacterium animalis AY530821.1 bsh Enterococcus faecalis GG688660.1 bsh3 Lactobacillus plantarum ACL98170.1 cbh2 Bacteroides vulgatis RIB33278.1 cbah Clostridium butyricum EEP54620.1

The other type of bile acid metabolism introduced into a microbe used to practice embodiments as provided herein is capable of converting primary to secondary bile acids, which entails removal of the 7-alpha-hydroxy or 7-beta hydroxy group from the primary bile acid; for example, the conversion of cholic acid to deoxycholic acid or chenodeoxycholic acid to lithocholic acid. The archetype pathway for this process is encoded by the bai gene cluster in Clostridium scindens (Coleman et al. (1987) J. Bacteriol. 169:1516-1521; Ridlon et al. (2006) J. Lipid. Res. 47:241-259) and has been well characterized. In addition, a functional C. scindens dihydroxylation was established in Clostridium sporogenes (Funabashi et al. (2019) BioRxiv). The first step is a bile acid-CoA ligase (baiB, EC:6.2.1.7) to activate the molecule for the subsequent reaction steps. Next, an alcohol dehydrogenase (baiA,

EC: 1.1.1.395) oxidizes the 3-hydroxyl to a keto group. An NADEfflavin oxidoreductase then introduces a double bond into the ring by either baiCD (EC:1.3.1.115) or baiH (EC:1.3.1.116), depending on the substrate. The coA is then removed or transferred to another primary bile acid by a CoA transferase (baiF, EC:2.8.3.25). The 7-alpha or 7-beta-hydroxy group is then removed by a dehydratase (baiE or bail, respectively, EC:4.2.1.106) to form a second double bond in a conjugated position to the other one. Enzymes encoded by baiH and baiCD then serve to reduce the double bonds consecutively, and finally the alcohol dehydrogenase reduces the 3-keto back to a hydroxyl. High bile acid dihydroxylation activity has also been observed in Eubacterium sp. strain VPI 12708, Eubacterium sp. strain Y-l 113, Eubacterium sp. strain I- 10, Eubacterium sp. strain M-18, Eubacterium sp. strain TH-82, Clostridium sp. strain TO-931, and Clostridium sp. strain HD- 17. Homologs for some of the bai genes have been identified in these organisms (Doemer et al. (1997) Appl. Environ. Microbiol. 63:1185-1188), and thus represent alternate gene candidates. Homologs of all essential genes for pathway function were also identified in Clostridium hylemonae DSM 15053, Dorea sp. D7, and a novel Firmicutes bacterium (Das et al. (2019) BMC Genomics 20:517).

To introduce the conversion pathway into the genetically modified host, the following C. scindens genes or suitable homologs are expressed: baiA, baiB, baiCD, baiE, baiF, and baiH. In some embodiments, the baiG gene, encoding a transporter, is also expressed. In other embodiments, the bail gene predicted to encode a delta-5- ketoisomerase, is introduced in order to enable dihydroxylation of secondary bile acids requiring this step.

Tryptophan derivatives are produced by many microbes, including gut bacteria, and have been implicated in strengthening the epithelial cell barrier and modulating the expression of pro-inflammatory genes by T cells in the GI tract (Bercik et al. (2011) Gastroenterology 141 :599-609). A gut microbe is engineered to overexpress one or more tryptophan derivatives by either overexpressing native genes or introducing heterologous genes described below.

In one embodiment, a microbe used to practice embodiments as provided herein is engineered to convert tryptophan to indole by introduction of a tryptophanase, such as that encoded by the tnaA gene of E. coli (Li and Young (2013) Microbiology 159:402-410). Other candidates are found by literature search or BLAST of the sequence to find homologs, as exemplified by the following: tna A Escherichia coli NP_415256.1 tnaA Bacteroides thetaiotamicron NP_810405.1 tnaA Vibrio tasmaniensis LGP32 VS RS05915 tnaA Treponema denticola TDE0251

In another embodiment, a microbe used to practice embodiments as provided herein is engineered to convert tryptophan to indoleacetate. This pathway begins with a tryptophan aminotransferase (EC:2.6.1.27) such as that encoded by the Taml gene of Ustilago maydis (Zuther et al. (2008) Mol. Microbiol. 68:152-172), which uses a- ketoglutarate as the amino acceptor and produces indolepyruvate. Although a microbial sequence for this enzyme is not currently in GenBank, activity has been reported in Clostridium sporogenes (O’Neil et al. (1968) Arch. Biochem. Biophys. 127:361-369). Alternatively, a deaminating tryptophan oxidase (EGO.3.10) such as that encoded by the vioA gene of Chromobacterium violaceum (August et al. (2000)

J. Mol. Microbiol. Biotechnol. 2:513-519) uses molecular oxygen to oxidize and deaminate tryptophan to produce indolepyruvate. Alternative candidates include those indicated as follows: vioA Chromobacterium violaceum CV RS16140

WP 133678757 Paludibacterium purpuratum WP 133678757.1

WP_034786442 Janthinobacterium lividum WP_034786442.1

The next gene to be introduced encodes an indolepyruvate decarboxylase (EG4.1.1.74), which produces indole-3 -acetaldehyde from indolepyruvate. An example is the ipdC gene from Enter obacter cloacae (Koga et al. (1991) Mol. Gen. Genet. 226:10-16). Other exemplary genes can be accessed by the GenBank accession numbers listed below: ipdC Enterobacter cloacae WP 013098183.1

CFNIHl RS23020 Citrobacter freundii CFNIHl RS23020 ipdC Rhodopseudomonas palustris CGA009 TX73 RS 15890 ipdC Azospirillum brasilense AMK58 RS11560

Indole-3 -acetaldehyde is then oxidized to indoleacetate by an aldehyde dehydrogenase (EC: 1.2.1.3), such as that encoded by the aldA gene of Pseudomonas syringae (McClerklin et al. (2018) PLoS Pathog. 14:el006811). Numerous aldehyde dehydrogenases exist, though the best candidates are those homologous to this aldA or others with known activity on indole-3 -aldehyde or similar molecules. Exemplary gene candidates can be accessed by the GenBank accession numbers listed below: aldA Pseudomonas syringae PSPT0 0092

CFNIH1_RS23020 Citrobacter freundii CF H1 RS23020

WP 005887684.1 Pseudomonas cor onafaciens WP 005887684.1

SPOG 02634 Schizosaccharomyces cryophilus OY26 SPOG 02634

In another embodiment, a tryptophan decarboxylase (EC:4.1.1.28) is introduced into a microbe used to practice embodiments as provided herein to produce tryptamine. This activity is rare among bacteria, but two such enzymes have recently been identified: CLOSPO 02083 from Clostridium sporogenes and RUMGNA 01526 from Ruminococcus gnavus (Williams et al. (2014) Cell Host Microbe 16:495-503).

In another embodiment, the pathway to produce indolepropionate (IP A) is introduced into the genetically modified microbe. IPA has been implicated in intestinal barrier fortification by engaging the pregnane X receptor (Venkatesh et al. (2014) Immunity 41 :296-310) and is known to be synthesized by a small number of gut bacteria (Elsden et al. (1976) Arch. Microbiol. 107:283-288). However, the pathway for its synthesis is uncharacterized. The genes encoding this pathway have recently been discovered in Clostridium sporogenes , enabling a pathway to be proposed. Indolepyruvate, synthesized as described above, is reduced to indolelactate which is then dehydrated to produce indoleacrylate. Finally, indoleacrylate is reduced to IPA by an acyl-CoA dehydrogenase. These are encoded by the fldH, fldBC, and acdA genes in C. sporogenes , respectively (Dodd et al. (2017) Nature 551:648-652). Homologs of these genes in other microbes are also candidates for expression, found by BLAST of the C. sporogenes genes. In another embodiment, a microbe used to practice embodiments as provided herein is engineered to consume a sugar or polysaccharide, e.g., a cellobiose, which is a reducing sugar consisting of two b -glucose molecules linked by a b (1 4) bond that is recalcitrant to catabolism by most gut microbes. Consumption of cellobiose first requires a specific enzyme II complex (EC:2.7.1.205) of the phosphotransferase system (PTS), such as the celABC operon in E. coli (Keyhani et al. (2000) J. Biol. Chem. 275:33091-33101). When expressed in a heterologous host, this component functions together with the native PTS machinery to import and phosphorylate cellobiose to generate cellobiose-6-phosphate. Alternate candidates for this step are listed below: cel A Enterococcus gilvus WP_10781765.1 celB Enterococcus gilvus WP_010780456.1 celC Enterococcus gilvus WP_010780458.1 cel A Lactococcus lactis subsp. lactis NP_266573.1 celB Lactococcus lactis subsp. lactis NP 266330.1 ptcA Lactococcus lactis subsp. lactis NP_266570.1 celB Bacillus coagulans BF29 RS 14550

A 6-phospho-beta-glucosidase (EC:3.2.1.86) is then required to convert the cellobiose-6P into one molecule of glucose and one molecule of glucose-6-P, both of which are readily used by the host. An example is the 6-phospho-beta-glucosidase from Bacillus coagulans, which has successfully been expressed in E. coli (Zheng et al. (2018) Biotechnology for Biofuels 18:320). Alternate candidates are listed below: cel A Enterococcus gilvus WP_10781765.1 celB Enterococcus gilvus WP_010780456.1 celC Enterococcus gilvus WP_010780458.1 cel A Lactococcus lactis subsp. lactis NP_266573.1 celB Lactococcus lactis subsp. lactis NP 266330.1 ptcA Lactococcus lactis subsp. lactis NP_266570.1 celB Bacillus coagulans BF29 RS 14550

In another embodiment, a microbe used to practice embodiments as provided herein is genetically modified by deleting or reducing expression of genes to eliminate or reduce production of metabolites, such as the polyamines putrescine, spermidine, and cadaverine. These molecules are essential for gastrointestinal mucosal cell growth and function, but excess of these compounds has been linked to gut dysbiosis and poor nutrient absorption (Forget et al. (1997) J. Pediatr. Gastroenterol. Nutr. 24:285- 288). The primary routes for polyamine synthesis in bacteria are decarboxylation of the amino acid’s arginine or ornithine. Ornithine decarboxylase (ODC, EC:4.1.1.17) converts ornithine to putrescine, while arginine decarboxylase (ADC, EC:4.1.1.19) converts arginine to agmatine, which is subsequently converted to putrescine by agmatinase (EC:3.5.3.11). Putrescine can then be converted to other derivatives such as spermidine. Therefore, a reduction in ODC and/or ADC expression will reduce polyamine production in the host microbe. E. coli contains two ODC isomers, encoded by the speC and speF genes, as well as two isomers of ADC encoded by speA and adiA. BLAST searches using these sequences, or other known bacterial ODC and ADC genes, applied to the genome of the organism of interest is used to identify genes encoding these functions in the organism to be genetically modified. One or both of these genes, or homologs thereof, are then deleted from the host genome using tools such as lambda-red mediated recombination (Datsenko and Wanner (2000) Proc. Nat. Acad. Sci. USA 97:6640-6645), CRISPR-Cas9 genome editing (B ruder et al (2016) Appl. Environ. Microbiol. 82:6109-6119), or any other method resulting in the removal of genes or portions of genes from the chromosome. In another embodiment, these methods are used to replace the native promoters of these genes with alternate promoters of different strengths, or to modify the ribosome binding site, resulting in reduced production of the ODC and ADC enzymes. In yet another embodiment, expression is reduced through a gene silencing mechanism such as antisense RNA-based attenuation (Nakashima et al. (2012) Methods Mol. Biol. 815:307-319) or CRISPR interference (Choudhary et al. (2015) Nat. Comm. 6:6267).

Bioinformatic Discovery of Putative Immunomodulatory Proteins and Genetic Modification of Exemplary Therapeutic Microbes

In alternative embodiments, genetically modified microorganisms as provided herein, including microorganisms as listed in Tables 1, 5, 10, 11 or 12, and a bacterium from a combination of microbes as provided herein, e.g., as in Table 15 or Table 16, are engineered to express immunomodulatory, e.g., immunostimulatory, proteins, or to overexpress endogenous immunomodulatory proteins. In alternative embodiments, the immunomodulatory are secreted or are cell surface-expressed or membrane-expressed proteins. Organisms of interest are bioinformatically interrogated for expression of putative immunomodulatory proteins. Based on immune correlation analysis and the differential relative abundance of organisms between cancer and control samples, certain organisms are identified as being missing from the cancer microbiome and potentially immunostimulatory and having anti-cancer properties. These identified organisms can be incorporated into formulations as provided herein, or into combinations of microbes as provided herein; or, the immunomodulatory proteins they express are identified and genetically engineered into organisms as provided herein, e.g., as listed in Tables 1, 5, 10, 11 or 12. In alternative embodiments, an organism as provided herein (as used in a method as provided herein) is genetically modified to overexpress the discovered immunomodulatory protein or proteins. Organisms potentially immunostimulatory and having anti-cancer properties are highlighted in Example 10.

For example, Dorea formicigenerans is one such organism, with strong positive correlations in both cancer and control cohorts to CD3+ and CD3+CD56+ immune cells in peripheral blood. First, a database of proteins is downloaded and clustered by similarity. Predicted proteins are downloaded from the NCBI RefSeq genomic database for a representative set of microbial genome assemblies. All complete genome assemblies for bacteria and archaea are included. For the taxa of special interest, which include Verrucomicrobia, Clostridia , and Coriobacteria, all assemblies of any status are included. Predicted proteins are downloaded from RefSeq and clustered using mmseqs2 (Steinegger and Soding. (2017) Nature Biotechnology 35:1026-1028). The resulting clusters contain proteins with identical or highly similar sequences. For a specific organism of interest, the protein clustering analysis is used to identify genes that are mostly unique to the organism yet ubiquitous across the organism’s pangenome. These genes are likely candidates to mediate the immunomodulatory functions that are specific to the organism of interest. A standard bioinformatic analysis is performed on genes unique to the organism of interest to identify protein domains within each gene as being signal, cytoplasmic, non- cytoplasmic, or transmembrane domains. Because immunomodulatory genes need to interact with immune cells, they are generally secreted proteins (Quevrain et al.

(2016) Gut 65:415-425) or membrane proteins (Plovier et al. (2017) Nature Medicine 23:107-113). Secreted proteins are identified from the analysis using the signal domains, while membrane proteins are identified by the presence of transmembrane domains. Because proteins with several transmembrane domains tend to be transporters, the focus is on proteins with one or two transmembrane domains. Membrane proteins or secreted proteins from the analysis of genes unique to the organism are prioritized for overexpression in genetically modified microorganisms as provided herein.

In alternative embodiments, genetically modified microorganisms as provided herein are engineered to express exogenous membrane proteins or secreted proteins. Genes unique to the organism of interest that are also membrane proteins or secreted proteins are investigated in a bespoke manner using the publicly available BLAST or Pfam search engines. In one embodiment, the organism is genetically modified to express these or homologues of identified membrane proteins. From this analysis, one protein from Dorea formicigenerans , NCBI Reference Sequence WP_1 18145075.1 is a particularly attractive candidate. The protein family for WP_1 18145075.1 contains 28 protein sequences, of which 26 are from Dorea formicigenerans genomes. There are 27 total Dorea formicigenerans assemblies in the database, so 26 out of 27 assemblies contains a version of protein WP_1 18145075.1. When analyzed on BLAST and Pfam, WP_118145075.1 is identified as a pilus-like protein. Pili and related proteins have a known role in interaction with human cells (Lizano et al. (2007) Journal of Bacteriology 189:1426- 1434; Plovier et al. (2017) Nature Medicine 23:107-113; Ottman, N., et al. (2017) PLOS ONE 12(3):e0173004). Genes may also be identified as containing pilus-like structures or other known immunomodulatory structures using public available techniques such as PilFind (Imam et al. (2011) PLOS ONE 6(12):e28919). In alternative embodiments, these pilus-like structures or other known immunomodulatory structures are engineered into genetically modified microorganisms as provided herein.

Other pili-like proteins of interest and corresponding homologs used in genetically engineered organism as provided herein include the highly abundant outer membrane protein of Akkermansia muciniphila , Amuc l 100 and members of the Amuc_1098 — Amuc l 102 gene cluster, have been shown to induce the production of specific cytokines (IL-8, IL-Ib, IL-6, IL-10 and TNF-a) through activation of Toll like receptors (TLR) 2 and TLR4 (Ottman et al (2017) PLoS One 12, e0173004). Similar outer membrane proteins are belived to be responsible for the induction of cytokine IL-10 by commensal gut microbes such as Faecalibacterium prausnitzii A2- 165 and Lactobacillus pkinlarum WCFS1.

In another embodiment, a genetically engineered organism as provided herein is genetically modified to express homologues of bacterial flagellin to induce TLR5 signaling. TLR5 response to flagellin promotes both innate and adaptive immune functions for gut homeostasis (Leifer et al (2014) Immunol. Lett. 162, 3-9). Recently, flagellin been examined for anti-tumor and radioprotective properties and has shown potential in reducing tumor growth and radiation-associated tissue damage (Hajam et al (2017) Exp. Mol. Med. 49, e373-e373). Some flagellin-based anti-tumor vaccines have also successfully entered into human clinical trials. Flagellins (fliC ' ) and homologues of interest include but are not limited to those from Salmonella Typhimurium ( FliCi ), Escherichia coli , Pseudomonas aeruginosa , Listeria monocytogenes , and Serratia marcescens.

Identification of Immunomodulatory Proteins Via Pooled Screening

In alternative embodiments, microbes used in compositions as provided herein, or as used in methods as provided herein, have enhanced immunomodulatory effects, e.g., immune-stimulatory effects, and these microbes can be generated or derived either by selection using assays, as described below, or by inserting or enhancing the microbe’s immunomodulatory effects by genetic engineering, e.g., by inserting a heterologous nucleic acid into the microbe. In alternative embodiments, microbes that can express or overexpress immunomodulatory proteins or peptides are used with (in addition to, are administered with) microbes used in compositions as provided herein, or microbes used to practice methods as provided herein.

Microbial populations are assayed directly for immunomodulatory effects on dendritic cells. Starting with a fecal sample of interest containing an endogenous microbial population or starting with a synthetic microbial population consisting of pooled microbial isolates of interest, the population can be tested against dendritic cells ex vivo.

Purified dendritic cells are generated as described in previous work (Svensson and Wick. (1999) European Journal of Immunology 29(1): 180-188; Svensson et al. (1997) Journal of Immunology 158(9):4229-4236; Yrlid et al. (2001) Infection and Immunity 69(9):5726-5735). Heat-inactivated, incubated for 30 minutes at 70°C, or live bacteria are added at a 50: 1 ratio and incubated for 4 hours at 37°C in IMDM containing 5% FBS. Following incubation, cells are washed 3x in HBSS to remove excess antigen. A portion of the dendritic cells are saved in RNAlater for future RNA sequencing. When activated, dendritic cells express several co-stimulatory molecules that aid in activating T cells. These molecules (CD40, CD80, and CD86) are upregulated alongside the chemokine receptor CCR7 which homes the activated DC to the spleen or local lymph node (Wilson and O'Neill. (2003) Blood 102(5): 1661- 1669; Ohl et al. (2004) Immunity 21(2):279-288). This set of genes can therefore be used to sort mature, activated DCs from immature DCs that do not stimulate T cells effectively. Cells are stained for expression of one or more of CD86, CD40 and CD80, and sorted via Fluorescence Activated Cell Sorting (FACS).

Purified cells are processed as described previously (Abelin et al. (2017) Immunity 46(2):315-326) for HLA-peptide identification. Briefly, purified cells are dissociated in protein lysis buffer containing protease inhibitors and DNAse, and then sonicated. Following sonication, soluble lysates are incubated with SEPHAROSE™ beads linked to W6/32 antibody which are washed with lysis buffer lacking protease inhibitor, and finally washed with DI water. Peptides are then eluted from the HLA complex on EMPORE Cl 8 STAGETIPS™. Purified protein preparations are then subjected to nanoLC-ESI-MS/MS.

Following LC-MS/MS, individual peptides are identified and matched to the reference genomes of the mix of microbes used in the in vitro activation experiment. A list of candidate peptides is generated by combining peptide abundance data with bioinformatics analysis of protein conservation, localization data, and their likelihood to express and localize to the membrane (Marshall et al. (2016) Cell Reports 16(8):2169-2177; Saladi et al. (2018) Journal of Biological Chemistry 293(13):4913- 4927).

Identification and Validation of Microbes that Activate Immune Cell Receptors

In alternative embodiments, microbes used in compositions as provided herein, or as used in methods as provided herein, can activate immune cell receptors (e.g., such as T cell receptors), and these microbes can be generated or derived either by selection using assays, as described below, or by inserting or enhancing the microbe’s immunomodulatory effects by genetic engineering, e.g., by inserting a heterologous nucleic acid into the microbe. In alternative embodiments, microbes that can express or overexpress proteins or peptides that can activate immune cell receptors are used with (in addition to, are administered with) microbes used in compositions as provided herein, or microbes used to practice methods as provided herein.

In alternative embodiments, ex vivo analyses are used to identify microbes that activate immune cell receptors including but not limited to dendritic cell Toll-like receptors (TLR’s).\Briefly, microbes of interest are co-incubated with human dendritic cells as described in the previous section, except that the co-incubation occurs with a pasteurized and washed microbial isolate rather than a microbial population. Dendritic cells are washed post-incubation. As described in Example 11, dendritic cells are saved and analyzed using RNA sequencing to identify gene expression changes relative to control conditions. The control conditions include both no stimulation i.e. microbial media alone, as well as known agonists for different TLR’s. A computational analysis is performed to ascribe the gene expression of dendritic cells in response to each microbe to some amount of activation of each TLR, thus predicting microbe-TLR interactions.

For each predicted microbe-TLR interaction, the pasteurized and washed microbe is co-incubated with TLR reporter cells (HEK-Blue, InvivoGen), and a plate- based colorimetric assay used to measure TLR activation over time. Validated microbes can be further screened as described previously for specific genes that mediate their mechanistic effects.

Amplification of Therapeutic Effect by Overexpression of Immunomodulatory Genes

In alternative embodiments, microbes used in compositions as provided herein, or as used in methods as provided herein, overexpress immunomodulatory genes (e.g., immunostimulatory genes), and these microbes can be generated or derived either by selection using assays, as described below, or by inserting or enhancing the microbe’s immunomodulatory effects by genetic engineering, e.g., by inserting a heterologous nucleic acid into the microbe.

In alternative embodiments, microbes used in compositions as provided herein, or used to practice methods as provided herein, are selected to express, or overexpress, immunostimulatory proteins or peptides, which can be non-specific immunostimulatory proteins such as a cytokine, e.g., a cytokine such as an interferon (e.g., IFN-a2a, IFN-a2b) IL-2, IL-4, IL-7, IL-12, IFNs, TNF-a, granulocyte colony- stimulating factor (G-CSF, also known as filgrastim, lenograstim or Neupogen®) and granulocyte monocyte colony-stimulating factor (GM-CSF, also known as molgramostim, sargramostim, Leukomax®, Mielogen® or Leukine®), or a specific immunostimulatory protein or peptide, e.g., such as an immunogen that can generate a specific humeral or cellular immune response, e.g., an immune response to a cancer antigen. In alternative embodiments, microbes that can express or overexpress immunostimulatory proteins or peptides are used with (in addition to, are administered with) microbes used in compositions as provided herein, or used to practice methods as provided herein.

Genes identified from a bioinformatic or pooled experimental approach as having an immunomodulatory effect are validated using recombinant expression in an engineered chassis organism. In alternative embodiments, the engineered chassis organism is used as a strong modulator (e.g., stimulator) of immune activity as a component of a live biotherapeutic as provided herein (e.g., as a component of a combination of microbes as provided herein, e.g., as a component of an exemplary combination as listed in Table 15 or Table 16), or the engineered chassis organism can be used in addition to a live biotherapeutic as provided herein.

Nucleic acids encoding protein sequences capable of enhancing a microbe’s immunomodulatory effects are synthesized and cloned or inserted into a microbe, e.g., bacterium, used in a combination of microbes as provided herein (as in e.g., Table 15 or Table 16), including for example any bacterium as listed in Table 1, 5, 10, 11, or 12, e.g., such as a B. subtilis. B. subtilis is a generally recognized as safe (GRAS) organism that has extensive tools available for the cloning and expression of synthetically encoded proteins (see e.g., Popp et al. (2017) Scientific Reports 7(1): 15058). Following cloning, colonies containing each different synthetic protein are grown until logarithmic phase. Each culture is pasteurized and washed as described previously. The cultures are validated for immunomodulatory activity relative to a negative control consisting of the unmodified chassis organism and positive control consisting of the unmodified original microbe of interest.

Each overexpressed gene can be validated for immunomodulatory activity using a TLR reporter assay as described previously, or a co-incubation with dendritic cells followed by mass spectrometry or RNA sequencing as described previously. Validated immunomodulatory engineered microbes can be incorporated into the candidate live biotherapeutic and advanced to in vivo screening in animal models. Example 14: Whole Cell Mutagenesis and Selection Procedures for Therapeutic Microbes

In alternative embodiments, microbes as provided herein (including bacteria from all the genuses listed herein), and including the combinations of microbes as provided herein, e.g., the exemplary combinations 1 to 241 as described in Table 15, Example 10, are genetically modified to enhance a cancer treatment, e.g., to enhance or prolong the efficacy of a chemotherapy, a radiation therapy, an immune checkpoint inhibitor, a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment.

Candidate live biotherapeutic strains are randomly mutagenized to generate a microbe with increased level of production of either a protein or metabolite of interest that may impact cancer treatment. When cells are mutagenized, changes occur in the DNA sequence that could result in changes of expression levels of certain genes.

Often these mutations are lethal, but some strains survive and have altered phenotype, including some with increased expression of genes encoding proteins or metabolic pathways identified from patient data (Examples 9 and 10) or in vitro assays (Example 11). Mutagenesis is carried out by an established treatment such as ultraviolet light, N-ethyl-N-nitrosourea, or ethyl methanesulfonate, followed by culturing on non-selective media to obtain viable cells. Mutagen exposure is first tuned by varying the time or intensity of treatment to a small culture, then selecting the conditions which yield approximately 10-20% of the number of viable colonies compared to a non -treated control. These treatment conditions are then applied to a larger culture of cells, and mutagenized colonies obtained are screened for the phenotype of interest, such as increased production of a protein or metabolite of interest. Clones obtained from this screen are then further characterized by whole genome sequencing.

Example 15: Production of Live Biotherapeutics

In alternative embodiments, microbes as provided herein (including bacteria from all the genuses listed herein), and including the combinations of microbes as provided herein, e.g., the exemplary combinations 1 to 241 as described in Table 15, Example 10, comprise anaerobic bacteria, including anaerobic bacteria isolated from a fecal sample, cultured anaerobic bacteria, or a combination thereof. Individual Culture of Anaerobic Microbes for Mouse Studies

Anaerobic microbes of interest are cultured in multiples of 1 -liter volumes in anaerobic media bottles as follows. Microbes in cryostorage are plated and struck on appropriate anaerobic solid medium and then cultured at 37°C to obtain isolated colonies. For each microbe, a single colony is inoculated into a Hungate tube containing 10 ml appropriate anaerobic growth medium and allowed to grow at 37°C until turbid to create a starter culture. For each microbe of interest, multiple 0.9-liter volumes of appropriate liquid anaerobic medium in 1 L anaerobic bottles (as described in Example 1) are inoculated with 2 ml starter culture each using a needle and syringe. The number of 1 -liter cultures for each microbe is dependent on the necessary final amount of live cell mass for formulation into live biotherapeutics for mouse studies. Inoculated bottles are placed upright on a platform shaker at 115 rpm at 37°C for 48 hours or until growth turbidity is evident. Growth density is monitored by taking 1 ml samples during the course of the cultures for optical density measurements at 600 nm. Optical densities of 1.0 to 4.0 can be obtained after 48 hours depending on the microbe cultured. Prior to large scale culture, cell densities are determined empirically for each microbe by dilution plating and colony counting to determine the colony forming units (CFU) per ml at an optical density of 1.0.

Large scale cultures are grown to attain a final live density of 1.0E8 to 1.0E9 CFU/ml, and then the culture bottles are brought into the anaerobic chamber for harvesting of live cell mass. Once in the chamber, the aluminum collars and butyl rubber bungs are removed, and the 1 -liter contents of each culture bottle are poured into two 500 ml centrifuge bottles with rubber gasketed screw caps. After decanting growth medium, the caps of the centrifuge bottles are tightened for an airtight seal, brought out of the anaerobic chamber, then centrifuged for 20 minutes at 6000 g at 4°C. Centrifuged bottles are then brought into the anaerobic chamber, uncapped, and then the supernatants are poured off and discarded. The remaining cell pellets are then combined with 250 ml ice cold Vehicle Buffer (Phosphate Buffered Saline plus lg/L L-cysteine plus 15% glycerol, filter sterilized and made anoxic by bubbling with filtered nitrogen). The cell pellets are carefully resuspended in the Vehicle Buffer on ice; the resuspended volumes of two pellets are combined into one 500 ml bottle, recapped for an air-tight seal, removed from the anaerobic chamber, then centrifuged for 20 minutes at 6000 g at 4°C. After decanting supernatants in the anaerobic chamber, resulting cell pellets are then carefully resuspended once more with 250 ml ice cold Vehicle Buffer in the anaerobic chamber, removed from the anaerobic chamber, then centrifuged for 20 minutes at 6000 g at 4°C. After removal of supernatant in the anaerobic chamber, each pellet is resuspended in 100 ml ice cold Vehicle Buffer to establish a ten-fold concentration of the original culture cell density.

Within the anaerobic chamber, final resuspended cell pellet volumes for an anaerobic microbe of interest are combined and thoroughly mixed in a sterile bottle by gentle stirring on a stir plate on ice. The volume is then dispensed into 25 ml aliquots in 50 ml conical tubes using a seriological pipette, then a stream of sterile filtered gaseous argon is introduced to each tube to displace the headspace and to serve as an oxygen barrier. Each tube is then tightly capped, and the seal is wrapped with several layers of parafilm. The tubes are then racked upright, removed from the anaerobic chamber, and then allowed to slowly freeze at -80°C. A smaller 5 ml aliquot is also made for each preparation and stored as described above. After 18 hours, the 5 ml aliquots for each microbial strain of interest are removed and allowed to thaw standing in ice water within the anaerobic chamber. The thawed volumes are gently mixed by inversion several times, then subjected to dilution plating on appropriate solid anaerobic medium to determine the live cell density in CFU/ml after freezer storage.

Live Biotherapeutic Assembly for Mouse Studies

Live biotherapeutic compositions of anaerobic microbes of interest, including the combinations of microbes as provided herein, e.g., the exemplary combinations 1 to 241 as described in Table 15, Example 10, are assembled in volumes that are pertinent for projected mouse studies. Enough aliquots for each microbe of interest are removed from storage at -80°C and gently thawed in ice water in the anaerobic chamber. The thawed multiple aliquots are combined in a sterile bottle, gently remixed and then placed on ice. The amount of volume of each microbe to add to a mix is adjusted so that the determined live cell densities for each microbe are equivalent, and final total cell densities can be adjusted by further addition of ice-cold vehicle buffer. Once all requisite volumes for each microbe are added together in a larger sterile bottle, the volume is gently mixed by stirring on a stir plate on ice.

Live biotherapeutic volumes are then re-aliquoted in individual volumes that each comprise a projected daily dose of live microbes in anticipated mouse studies. Determined volumes are each dispensed in 15 ml conical tubes up to 10 ml per aliquot. The volume in each tube is overlaid with a stream of sterile filtered argon to displace oxygen, followed by capping. Live biotherapeutic aliquot tubes are racked upright and allowed to slowly freeze at -80°C. After 48 hours, one aliquot for each microbe mix preparation is thawed and dilution plated to validate the final total CFU/ml, optimally at greater than l.OxlO 9 CFU/ml.

Example 16 - Efficacy of Live Biotherapeutics as an Anticancer Monotherapy Microorganisms in Mouse Study

The sets of microbes to be administered are chosen from either Table 15 (1 — 241) or Table 16 (1-15), described in Example 10 or from engineered microbes described in Examples 13 and 14. Each microbe is isolated from healthy donors, as described in Example 3, or the genetically modified derivatives described in Examples 13 and 14. The live biotherapeutic is cultured and assembled as described in Example 15.

After assembly, PBS-C-G is added to each live biotherapeutic to reduce the total cell density of each live biotherapeutic to the desired dosage level, which can be between 1X10 8 /0.2 ml and 1X10 12 /0.2 ml. Live biotherapeutics are aliquoted into eight 5.0 ml volumes into 15 ml conical tubes and stored at -20°C until required. Animals and Tumor Model

BALB/c mice are obtained from Shanghai Lingchang Biotechnology Co., Ltd (Shanghai, China) or Jackson Laboratory. 6-8-week-old female mice are used. For tumor growth experiments, mice are injected subcutaneously with 2.5 x 10 5 CT-26 colon cancer tumor cells (Griswold and Corbett (1975) Cancer 36:2441-2444), MC38 col on tumor cells (Juneja et al. (2017) J. Exp. Med. 214(4):895), LL/2 lung carcinoma cells (Bertram and Janik (1980) Cancer Lett. 11 :63-73), or EMT6 breast cancer cells (Rockwell et al. (1972) J. Nat. Cancer Inst. 49:735-749). Tumor size is measured twice a week until endpoint, and tumor volume determined as length x width x 0.5.

Tumor Cell Preparation

Cryo vials containing CT-26, MC38, LL/2, or EMT6 tumor cells are thawed and cultured according to manufacturer’s protocol (ATCC CRL-2638). On the day of injection cells are washed in serum free media, counted, and resuspended in cold serum free media at a concentration of 250,000 viable cells/100 pi.

Antibiotic Pre-treatment In some studies, mice are treated daily with 200 pL of antibiotic solution via oral gavage for a duration of 1-2 weeks. The antibiotic solution consists of ampicillin (1 mg/mL)(Alfa Aesar J6380706), gentamicin (1 mg/mL)(Acros Organics AC455310050), metronidazole (1 mg/mL)(Acros Organics AC210440050), neomycin (1 mg/mL)(Alfa Aesar AAJ6149922), and vancomycin (0.5 mg/mL) (Alfa Aesar J6279006) via oral gavage. Animals are given at least 48 hours rest period between antibiotic pre-treatment and the treatment phase to allow for antibiotics to go through the system.

Fecal Microbiota Transplantation (FMT)

Fecal Microbiota Transplantation (FMT) of a human gut microbiome into antibiotic treated mice is a method for standardizing microbiome composition. FMT is performed in some experiments with fecal material derived from responders to checkpoint inhibitor therapy (R) or non-responders to checkpoint inhibitor therapy (NR). Not only does this standardize the mice microbiomes, but also conditions them to favor response or non-response, respectively. Following antibiotic pre-treatment, colonization is performed by oral gavage with 200 mΐ of suspension obtained by homogenizing the fecal samples in PBS. Mouse fecal samples are collected 1-2 times during this period, so that the efficacy of the FMT can be evaluated. Following FMT, a rest period of 5-7 days is allowed to pass prior to treatment initiation.

Tumor Challenge and Treatment

After pre-treatment is complete, tumor inoculation is performed. 100 mΐ of the cell suspension is subcutaneously injected into the rear flank of the mouse. During implantation, a new syringe and needle will be used for every mouse inoculated to minimize tumor ulceration. The cells are drawn up into a 1 mL syringe (no needle attached) to 150 pL with the 50 pL nearest to the plunger being air and 100 pL of cell suspension. Once the cells are drawn up the needle is attached (without priming the needle). For implant, skin is lifted using forceps to ensure a subcutaneous injection, and cells are injected. Mice are marked by ear tagging. Animals are randomized the day following tumor inoculation (Study Day 0). Treatment begins the following day (Study Day 1) and continues for 3 weeks or longer and consists of 200 uL microbe mix or vehicle control (PBS-C-G). Total microbial load is between 10 9 and 10 12 colony forming units (cfu) per dose. Doses are administered at a frequency of at least twice per week, and up to twice per day. Tumor size is routinely monitored by means of a caliper. Stool is collected on day 0, and twice per week until the end of the study, normally 8 hours following a treatment dose.

Peripheral Blood Extraction and Processing

Whole blood is taken via cardiac puncture at the end of the experiment, or via tail bleed during the experiment, and collected into an EDTA tube. Plasma is isolated from an aliquot of the whole blood by centrifugation at 1500xg for 10 minutes, taking the supernatant. A second centrifugation is performed to remove any residual blood cells.

Peripheral blood mononuclear cells (PBMCs) are isolated from blood using a standard kit and stored in liquid nitrogen at 1 x 10 6 cells/mL until use. Prior to storage, PBMC’s may be processed using flow sorting or antibody spin separation kit to select for a certain purified lymphocyte subpopulation, such as T cells.

GI Tract Removal and Analysis

After mice are euthanized at the termination of the study, the intact digestive tract of each mouse from stomach to rectum are removed and kept in a 5 ml Eppendorf tube on ice prior to dissection. Forceps are sterilized by soaking in 100% ethanol and then used to remove the intestine length and stretch it on a work surface covered with cellophane. With the use of ethanol-sterilized dissection scissors, 3 cm lengths of the jejunum nearest to the stomach and the ilium nearest to the cecum/large intestine are excised and then each placed with forceps in a 1.5 ml Eppendorf tube and placed on ice. A 2 cm segment of the cecum/ascending colon is then excised, as are 2 cm segments of the transcending colon and the descending colon, and all are placed in 1.5 ml Eppendorf tubes on ice. Dissection instruments are sterilized by dipping in 100% ethanol between each intestine fragment removal. To each tube containing dissected intestinal segments is added 0.5 ml ice cold PBS buffer. A plastic pestle is used to press and massage the intestinal segment in each tube to expel ruminal matter, which is then removed by pipette and placed in a fresh Eppendorf tube. Tubes containing expelled ruminal matter from each intestinal segment are immediately placed on dry ice and then stored for later analyses at -80°C. Remaining intestinal tissues are then rinsed twice by adding and then removing 0.5 ml ice cold PBS.

Rinsed intestinal fragment tissues are then frozen on dry ice and then stored at -80°C for later analysis. Analyses of Dendritic Cell Subsets in Treated Mice

Cell suspensions from mouse spleen and lymph nodes are prepared by digestion with collagenase and DNase for 60 min and subsequently strained through a 70 mm mesh. Colonic and small intestinal lymphocytes are isolated as previously described (Viaud, S. et al. Science 80(342): 971-976 (2013). In brief, cecum, colon and small intestine are digested in PBS containing 5 mM EDTA and 2 mM DTT shaking at 37°C. A plastic pestle is used to press and massage the intestinal segment in each tube to expel ruminal matter, which is then removed by pipette and placed in a fresh Eppendorf tube. Tubes containing expelled ruminal matter from each intestinal segment are immediately placed on dry ice and then stored for later analyses at -80°C. Remaining intestinal tissues are then rinsed twice by adding and then removing 0.5 ml ice cold PBS. Rinsed intestinal fragment tissues are then frozen on dry ice in RNALater (Thermo Fisher Scientific) and then stored at -80°C for later analysis.

After initial digestion colonic and small intestinal tissue pieces are digested in collagenase/Dnase containing RPMI medium for 30 min. Tissue pieces are further strained through a 70 mm mesh. For flow cytometry analyses, cell suspensions are stained with antibodies against the following surface markers: CD1 lc (N418), CD1 lb (Ml/70), Ly6c (HK1.4), MHC class II (M5/114.15.2), CD24 (Ml/69), CD64 (X54- 5/7.1), CD317 (ebio927), CD45 (30-F11), F4/80 (CEA3-1), CD8a (53-6.7). DAPI is used for dead cell exclusion. Antibodies are purchased from eBiosciences, BD Biosciences or BioLegend respectively. Cell populations are gated as follows: small intestine (migratory fraction): CD103+ DC (CD45+ CD1 lc+MHC-II+ CD103+ CD24+), CD1 lb+ CD 103+ (CD45+ CD1 lc+ MHC-II+ CD 103+ CDllb+ CD24+), CD1 lb+ (CD45+ CD1 lc+MHC-II+ CD1 lb+ CD24+), inflammatory DC (CD45+ CD1 lc+ MHC-II+ CD1 lb+ CD64+ Ly6c+), large intestine: CD103+DC (CD45+

CD1 lc+ MHC-II+ CD 103+ CD24+), CD1 lb+ (CD45+ CD1 lc+ MHC-II+ CD1 lb+ CD24+), inflammatory DC (CD45+ CD1 lc+ MHC-II+ CD1 lb+ CD64+ Ly6c+). Whole genome sequencing

Fecal gDNA is extracted for whole genome sequencing (WGS). Experimental methods for DNA extraction and library preparation are performed using protocols modeled after the Human Microbiome Project (Lloyd-Price et al. (2017) Nature 550(7674):61-66) and validated with samples from healthy volunteers. Sequencing is performed by an outside service provider, using a HISEQ-X® (Illumina) with 2xl50bp paired-end reads, providing approximately 4 million reads per sample. Analysis software such as Centrifuge (Kim, D., et al., Centrifuge: rapid and sensitive classification of metagenomic sequences. Genome Res, 2016. 26(12): p. 1721-1729) are used to align sequence reads to reference genomes and obtain species and strain- level identification.

Metabolomics

Metabolites are extracted from fecal material or blood plasma, using methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) to precipitate protein and dissociate small molecules bound to protein or trapped in the precipitated protein matrix, followed by centrifugation to recover chemically diverse metabolites. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods using positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS using negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS using negative ion mode ESI, and one reserved for backup. Samples are placed briefly on a TurboVap® (Zymark) to remove the organic solvent, followed by injection on one of the instruments mentioned above. Compounds are identified by comparison to library entries of purified standards, that contains the retention time/index (RI), mass to charge ratio (m/z), and chromatographic data (including MS/MS spectral data) on all molecules present in the library. Furthermore, biochemical identifications are based on three criteria: retention index within a narrow RI window of the proposed identification, accurate mass match to the library +/- 10 ppm, and the MS/MS forward and reverse scores. MS/MS scores are based on a comparison of the ions present in the experimental spectrum to ions present in the library entry spectrum. While there may be similarities between these molecules based on one of these factors, the use of all three data points can be utilized to distinguish and differentiate biochemicals. Peaks are quantified as area-under-the-curve detector ion counts.

Immunophenotyping Assays

Immune profiling of whole blood is utilized to assess T cell activation in response to microbial treatment. In some experiments, immune phenotyping is also performed on tissue obtained from the GI tract.

For flow cytometry analysis, 1 mL of RBC Lysis Buffer is added to 0.1 mL of whole blood or homogenized tissue and allowed to incubate at room temperature for 10 minutes. Lysis is quenched by adding 10 mL of cold DPBS. Samples are centrifuged at 1500 rpm for 5 minutes at 4°C. The pellet is aspirated and resuspend in another 10 mL of cold DPBS. Samples are recentrifuged at 1500 rpm for 5 minutes at 4°C. Samples are resuspended in 500 pL of FACS buffer and transferred to a 96-well plate. Samples are stained with Fixable Viability ef780™ (eBioscience), CD45-PEcy7 (BioLegend), CD3-BV605™ (BioLegend), CD8-AF700™ (BioLegend), and CD4- AF488™ (BioLegend). Stained samples are run on a BD LSRFortessa™ flow cytometer and analyses are performed with FlowJo™ (Tree Star).

Alternatively, CyTOF® is applied to characterize the immune profile of the PBMCs. This work is conducted by the Bioanalytical and Single-Cell Facility at the University of Texas, San Antonio, and entails a comprehensive panel of 29 different immune markers, allowing for deep interrogation of cellular phenotype and function (https://www.fluidigm.com/products/helios). To complement these results, RNA sequencing is applied to the entire population of the PBMCs, sorted populations, and also to single cells. Single cell RNAseq is applied using the method developed by 10X Genomics (https://www.10xgenomics.com/solutions/single-cell/). Finally, cytokine levels are determined using the Human Cytokine 30-Plex Luminex assay (https://www.thermofisher.com/order/catalog/product/LHC6003M ).

Example 17 - Therapeutic Effect of Microbes on Efficacy of Cancer Immunotherapy

In this study, live biotherapeutics as provided herein, including combinations of microbes as provided herein, are administered in combination with checkpoint inhibitors (anti-CTLA-4, anti-PD-1, or anti-PD-Ll), to demonstrate the ability of these microbes to enhance the tumor reduction that can be achieved with the checkpoint inhibitor.

Microorganisms in Mouse Study

The sets (or combinations) of microbes to be administered are chosen from the list of exemplary bacterial combinations as set forth in Table 15, listing combinations 1 to 241, or Table 16, as described in Example 10, or from the exemplary engineered microbes described in Examples 12 and 13. Each microbe is isolated from healthy donors, as described in Example 3, or the genetically modified derivatives described in Examples 12 and 13. The live biotherapeutic is cultured and assembled as described in Example 14.

After assembly, PBS-C-G is added to each microbe mix to reduce the total cell density of each microbe mix to the desired dosage level, which can be between 1X10 8 /0.2 ml and 1X10 12 /0.2 ml. Live biotherapeutics are aliquoted into eight 5.0 ml volumes into 15 ml conical tubes and stored at -20°C until required.

Animals and Tumor Model

BALB/c mice are obtained from Shanghai Lingchang Biotechnology Co., Ltd (Shanghai, China). 6-8-week-old female mice are used. For tumor growth experiments, mice are injected subcutaneously with 2.5 c 10 5 CT-26 colon cancer tumor cells (Griswold and Corbett (1975) Cancer 36:2441-2444), MC38 colon tumor cells (Juneja et al. (2017) J. Exp. Med. 214(4):895), LL/2 lung carcinoma cells (Bertram and Janik (1980) Cancer Lett. 11 :63-73), or EMT6 breast cancer cells (Rockwell et al. (1972) J. Nat. Cancer Inst. 49:735-749). Tumor size is measured twice a week until endpoint, and tumor volume determined as length x width x 0.5.

Tumor Cell Preparation

Cryo vials containing CT-26, MC38, LL/2, or EMT6 tumor cells are thawed and cultured according to manufacturer’s protocol (ATCC CRL-2638). On the day of injection cells are washed in serum free media, counted, and resuspended in cold serum free media at a concentration of 250,000 viable cells/100 pi.

Antibiotic Pre-treatment

In some studies, mice are treated daily with 200 pL of antibiotic solution via oral gavage for a duration of 1-2 weeks. The antibiotic solution consists of ampicillin (1 mg/mL)(Alfa Aesar J6380706), gentamicin (1 mg/mL)(Acros Organics AC455310050), metronidazole (1 mg/mL)(Acros Organics AC210440050), neomycin (1 mg/mL)(Alfa Aesar AAJ6149922), and vancomycin (0.5 mg/mL) (Alfa Aesar J6279006) via oral gavage. Animals are given at least 48hrs rest period between antibiotic pre-treatment and the treatment phase to allow for antibiotics to go through system.

Fecal Microbiota Transplantation (FMT)

In alternative embodiments, methods as provided herein comprise use of Fecal Microbiota Transplantation (FMT), or elements used to practice FMT, as described e.g., in USPNs 10,493,111; 10,463,702; 10,383,519; 10,369,175; 10,328,107.

FMT of a human gut microbiome into antibiotic treated mice is a method for standardizing microbiome composition. FMT is performed in some experiments with fecal material derived from responders to checkpoint inhibitor therapy (R) or non responders to checkpoint inhibitor therapy (NR). Not only does this standardize the mice microbiomes, but also conditions them to favor response or non-response, respectively. Following antibiotic pre-treatment, colonization is performed by oral gavage with 200 mΐ of suspension obtained by homogenizing the fecal samples in PBS. Mouse fecal samples are collected 1-2 times during this period, so that the efficacy of the FMT can be evaluated. Following FMT, a rest period of 5-7 days can pass prior to treatment initiation.

Tumor Challenge and Treatment

Tumor inoculation is performed immediately following either the FMT or antibiotic dosing. 100 mΐ of the cell suspension is subcutaneously injected into the rear flank of the mouse. During implantation, a new syringe and needle will be used for every mouse inoculated to minimize tumor ulceration. The cells are drawn up into a 1 mL syringe (no needle attached) to 150 pL with the 50 pL nearest to the plunger being air and 100 pL of cell suspension. Once the cells are drawn up the needle is attached (without priming the needle). For implant, skin is lifted using forceps to ensure a subcutaneous injection, and cells are injected. Mice are marked by ear tagging. Animals are randomized once the tumor volume reaches 40 to 60 mm 3 (Study Day 0), or alternatively 80 to 100 mm 3 , or 100 to 120 mm 3 .

Treatment consists of checkpoint inhibitor, alone or in conjunction with the live biotherapeutics described above. Checkpoint inhibitor is injected intraperitoneally the day following randomization (Day 1) with 100 pg anti-PDl mAb (BioXCell), or with 100 pg anti-PD-Ll mAb, or with 100 pg anti-CTLA-4 mAb (BioXCell) in 100 pi PBS. Dosing of the checkpoint inhibitor is continued twice per week for three weeks starting from day 1. Microbe dosing consists of 200 uL microbe mix or vehicle control (PBS-C-G). Total microbial load is between 10 9 and 10 12 colony forming units (cfu) per dose. Doses are administered at a frequency of at least twice per week, and up to twice per day. Tumor size is routinely monitored by means of a caliper. Stool is collected on day 0, and twice per week until the end of the study, normally 8 hours following a treatment dose.

Peripheral Blood Extraction and Processing

Whole blood is taken via cardiac puncture at the end of the experiment, or via tail bleed during the experiment, and collected into an EDTA tube. Plasma is isolated from an aliquot of the whole blood by centrifugation at 1500xg for 10 minutes, taking the supernatant. A second centrifugation is performed to remove any residual blood cells.

Peripheral blood mononuclear cells (PBMCs) are isolated from blood using a standard kit and stored in liquid nitrogen at 1 x 10 6 cells/mL until use. Prior to storage, PBMC’s may be processed using flow sorting or antibody spin separation kit to select for a certain purified lymphocyte subpopulation, such as T cells.

GI Tract Removal and Analysis

After mice are euthanized at the termination of the study, the intact digestive tract of each mouse from stomach to rectum are removed and kept in a 5 ml Eppendorf tube on ice prior to dissection. Forceps are sterilized by soaking in 100% ethanol and then used to remove the intestine length and stretch it on a work surface covered with cellophane. With the use of ethanol-sterilized dissection scissors, 3 cm lengths of the jejunum nearest to the stomach and the ilium nearest to the cecum/large intestine are excised and then each placed with forceps in a 1.5 ml Eppendorf tube and placed on ice. A 2 cm segment of the cecum/ascending colon is then excised, as are 2 cm segments of the transcending colon and the descending colon, and all are placed in 1.5 ml Eppendorf tubes on ice. Dissection instruments are sterilized by dipping in 100% ethanol between each intestine fragment removal. To each tube containing dissected intestinal segments is added 0.5 ml ice cold PBS buffer. A plastic pestle is used to press and massage the intestinal segment in each tube to expel ruminal matter, which is then removed by pipette and placed in a fresh Eppendorf tube. Tubes containing expelled ruminal matter from each intestinal segment are immediately placed on dry ice and then stored for later analyses at -80°C. Remaining intestinal tissues are then rinsed twice by adding and then removing 0.5 ml ice cold PBS.

Rinsed intestinal fragment tissues are then frozen on dry ice and then stored at -80°C for later analysis.

Analyses of Dendritic Cell Subsets in Treated Mice

Cell suspensions from mouse spleen and lymph nodes are prepared by digestion with collagenase and DNase for 60 min and subsequently strained through a 70 mm mesh. Colonic and small intestinal lymphocytes are isolated as previously described (Viaud, S. et al. Science (80- ). 342, 971-976 (2013). In brief, cecum, colon and small intestine are digested in PBS containing 5 mM EDTA and 2 mM DTT shaking at 37°C. A plastic pestle is used to press and massage the intestinal segment in each tube to expel ruminal matter, which is then removed by pipette and placed in a fresh Eppendorf tube. Tubes containing expelled ruminal matter from each intestinal segment are immediately placed on dry ice and then stored for later analyses at -80°C. Remaining intestinal tissues are then rinsed twice by adding and then removing 0.5 ml ice cold PBS. Rinsed intestinal fragment tissues are then frozen on dry ice in RNALater (Thermo Fisher Scientific) and then stored at -80°C for later analysis.

After initial digestion colonic and small intestinal tissue pieces are digested in collagenase/Dnase containing RPMI medium for 30 min. Tissue pieces are further strained through a 70 mm mesh. For flow cytometry analyses, cell suspensions are stained with antibodies against the following surface markers: CD1 lc (N418), CD1 lb (Ml/70), Ly6c (HK1.4), MHC class II (M5/114.15.2), CD24 (Ml/69), CD64 (X54- 5/7.1), CD317 (ebio927), CD45 (30-F11), F4/80 (CEA3-1), CD8a (53-6.7). DAPI is used for dead cell exclusion. Antibodies are purchased from eBiosciences, BD Biosciences or BioLegend respectively. Cell populations are gated as follows: small intestine (migratory fraction): CD103+ DC (CD45+ CD1 lc+MHC-II+ CD103+ CD24+), CD1 lb+ CD 103+ (CD45+ CD1 lc+ MHC-II+ CD 103+ CDllb+ CD24+), CD1 lb+ (CD45+ CD1 lc+MHC-II+ CD1 lb+ CD24+), inflammatory DC (CD45+ CD1 lc+ MHC-II+ CD1 lb+ CD64+ Ly6c+), large intestine: CD103+DC (CD45+

CD1 lc+ MHC-II+ CD 103+ CD24+), CD1 lb+ (CD45+ CD1 lc+ MHC-II+ CD1 lb+ CD24+), inflammatory DC (CD45+ CD1 lc+ MHC-II+ CD1 lb+ CD64+ Ly6c+). Analysis of Fecal and Blood Samples

Whole genome sequencing, metabolomics, and immunophenotyping are performed on samples collected, as described in Example 15.

Example 18: Fecal Composition Analysis of Tumor Bearing Mice Treated with Fecal Microbiota Transplant

Animals and Tumor Model

BALB/c mice were obtained from the Jackson Laboratory and 6-8-week-old female mice were used. For tumor growth experiments, mice were injected subcutaneously with 2.5 xlO 5 CT-26 colon cancer tumor cells (Griswold and Corbett (1975) Cancer 36:2441-2444). Tumor size was measured twice a week until endpoint, and tumor volume determined as length x width x 0.5.

Tumor Cell Preparation Cryo vials containing CT-26 tumor cells were thawed and cultured according to manufacturer’s protocol (ATCC CRL-2638). On the day of injection cells were washed in serum free media, counted, and resuspended in cold serum free media at a concentration of 250,000 viable cells/100 pi. Cells were prepared for injections by withdrawing 100 pL of the cell suspension into a 1 ml syringe. The cell suspension and filled syringes were kept on ice.

Tumor Implantation

Animals were prepared for injection using standard approved anesthesia and shaved prior to injection. 100 mΐ of the cell suspension was subcutaneously injected into the rear flank of the mouse. Mice were marked by ear tagging.

Antibiotics Protocols

Mice were treated daily with 200 pL of antibiotics via oral gavage 1 week before fecal microbiota transplantation (FMT). Mouse fecal samples were collected twice a week. Animals were given a mix of ampicillin (1 mg/mL)(Alfa Aesar J6380706), gentamicin (1 mg/mL)(Acros Organics AC455310050), metronidazole (1 mg/mL)(Acros Organics AC210440050), neomycin (1 mg/mL)(Alfa Aesar AAJ6149922), and vancomycin (0.5 mg/mL)(Alfa Aesar J6279006) via oral gavage.

Fecal Microbiota Transplantation (FMT)

Fecal Microbiota Transplantation (FMT) of a favorable gut microbiome into antibiotic treated mice is a method for standardizing microbiome composition. FMT was performed with fecal material derived from healthy and cancer patients. Colonization was performed by oral gavage with 200 mΐ of suspension obtained by homogenizing the fecal samples in PBS. Efficient colonization was first checked before tumor inoculation. Mouse fecal samples were collected 1-2 times during this period so that the efficacy of the FMT can be evaluated. Following FMT, a rest period of 5-7 days was allowed prior to checkpoint inhibitor and/or microbe dosing. Blood and fecal pellets were collected at different time points during the experiment.

Mice were pre-treated with antibiotics, FMT was performed, and tumors were inoculated. Randomization began at a tumor volume of 50 mm 3 . Tumor size was measured in all animals receiving antibiotic pre-treatment, followed by FMT transfer from cancer patients. Four FMTs (NR(l)-FMT, NR(2)-FMT, R(1)-FMT, R(2)-FMT) were selected for administration to the mice based on donor cancer patient response to therapy. FMTs NR(1)-FMT and NR(2)-FMT are derived from non -responding cancer patients and FMTs R(1)-FMT and R(2)-FMT are from cancer patients that respond to immunotherapy.

Fecal samples were collected from FMT treated mice 7 days post-dosing and whole genome sequencing was performed. Whole genome sequencing was also performed on the human fecal samples from which the FMT material was generated. The sequencing reads were processed and the species abundances for both mouse and human fecal samples were estimated. The abundances were centered-log-ratio transformed and principal component analysis was performed. As illustrated in Fig.

11, the first principal component clearly separates mouse and human samples, showing a strong difference between species. In contrast, in the second, third, and fourth principal components, mice that received their FMT from the same human donor are nearer to each other as illustrated in Figures (12 and 13). t-Distributed Stochastic Neighbor Embedding (tSNE), a machine learning algorithm for visualization was performed (as, see, e.g., (van der Maaten, L. J.P.; Hinton, G.E. (2008) Journal of Machine Learning Research. 9: 2579-2605) on the centered-log-ratio transformed species abundances from the whole genome sequencing with a Euclidean distance metric in order to embed the data in two dimensions. The tSNE was repeated 50 times to explore hyperparameter space for the best objective value for Kullback-Leibler divergence (also called relative entropy). When performed at only the initial timepoint (7 days post-FMT dosing), tSNE shows that mice receiving their FMT from the same human cluster together in the embedded space as illustrated in Fig. 14. When performed on data from multiple time points (7, 13, 27 days post-FMT dosing), tSNE shows that there is some time variation in the microbiomes of mice that have received an FMT, but that this variation is much less than the variation imparted by different FMT donors as illustrated in Fig. 15.

Example 19: Method of Treating a Subject with a Live Exemplary Biotherapeutic

This example describes administration of a live exemplary biotherapeutic as provided herein, including a combination of bacteria as provided herein, e.g., as set forth in Table 15 or 16, Example 10, to an individual in need thereof.

A patient is suffering from cancer. The patient is administered live biotherapeutic compositions, i.e., a formulation or a pharmaceutical composition comprising a combination of microbes (e.g., bacteria) as provided herein, (Table 15 or Table 16, and as described in Example 10) either in monotherapy or in combination with chemotherapy, radiation therapy, a checkpoint inhibitor, a Chimeric Antigen Receptor (CAR) T-cell therapy (CAR-T) or other immunotherapy or cancer treatment, and the patient can be administered the live biotherapeutic for the duration of treatment or for only one or several segments of treatment.

In alternative embodiments, each or one of the microbes used in the bacterial combination is (at least initially) isolated from a healthy donor or donors, as described in Example 3, or is a genetically modified derivative as described in Examples 12 and 13, or is a cultured derivative either.

In alternative embodiments, the patient is administered a live biotherapeutic at a dose of between about 10 5 to 10 15 bacteria, or at a dose of about 10 10 , 10 11 or 10 12 bacteria total or per dose, which can be in a lyophilized form, e.g., or formulated in an enteric coated capsule. In alternative embodiments, the patient takes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or more live biotherapeutic capsules (e.g., by mouth or suppository) once, twice or three times or more per day, and the patient can resume a normal diet after about 1, 2, 4, 8, 12, or 24 or more hours.

In another embodiment, the patient may take the live biotherapeutic capsule(s) by mouth before, during, and/or immediately after a meal.

In another embodiment, the patient is given a course of antibiotics before treatment, e.g., between one to seven days, or between about one to two weeks prior to the first dose of the live biotherapeutic (e.g., as capsule(s)), or three weeks prior, or four weeks prior, or up to 6 months prior to the first dose of live biotherapeutic.

In another embodiment, dosing of the live biotherapeutic, e.g., as capsule(s), is started one to seven days,, or one to two weeks, prior to administration of a first dose of a chemotherapy, a first checkpoint inhibitor dose, start of a CAR-T therapy or any immunotherapy or cancer therapy.

In another embodiment, dosing of the live biotherapeutic capsule(s) is continued 1 month, 6 months, 1 year, or more, or between about one week and 2 years, following termination of the treatment, e.g., checkpoint inhibitor administration, chemotherapy or any immunotherapy.

In alternative embodiments, patient response to the combination therapy is a measure of success and for solid tumors is based on radiographic assessment using the Response Evaluation Criteria in Solid Tumors (RECIST 1.1) criteria (Schwartz, et al. (2016) Eur. J. Cancer. 62: 132-137) at 6 months after treatment initiation, and again after 12 months and 24 months. Patients are classified as complete responders if all target lesions are gone, partial responders if there is at least 30% reduction in the sum of diameters of all target lesions, progressive disease if there is at least 20% increase in the sum of diameters of all target lesions, and stable disease otherwise. For blood cancers, the Response Evaluation Criteria in Lymphoma (RECIL) criteria is used, based on [18F]2-fluoro-2-deoxy-D-glucose positron-emission tomography (FDG- PET) (Younes, A. et al (2017) Ann. Oncol. 28:1436-1447).

Example 20: Method of Treating a Subject with an Exemplary Live Biotherapeutic Based on Stool Biomarkers

This example describes administration of a live exemplary biotherapeutic as provided herein, including a combination of bacteria as provided herein, e.g., as set forth in Table 15 to Table 16, Example 10, to an individual in need thereof.

A patient is suffering from cancer. The patient’s stool is collected and analyzed using the methods described in Example 9. In one embodiment, whole genome sequencing is performed and the presence of microbes that are characteristic of healthy individuals or checkpoint inhibitor responders is evaluated. The complete organism abundance profile is also plotted on the PCA axes shown in Fig. 3. Based on the abundance profiles of healthy individuals, responders and non-responders collected to date, a classifier is developed to predict if any given microbiome composition represents a responder or non-responder. This may be based on the amount of one or more particular organisms present, position in the PCA plot, or other criteria that combines aspects of the whole genome sequence data. This classifier is applied to the patient’s microbiome composition, to predict whether the patient will likely respond to a checkpoint inhibitor treatment applied in a monotherapy.

In another embodiment, metabolomics is performed on the stool or plasma; a classifier is developed based concentrations of one or more metabolites in all patient data collected to date, and the patient is predicted to be a responder or non-responder based on this classification.

If the patient is classified as a non-responder, a live biotherapeutic will be administered to change the microbiome to be more like that of a responder. The patient is administered one of the present live biotherapeutics (Table 15 or Table 16, and as described in Example 10) in combination with a checkpoint inhibitor, radiation therapy, CAR-T or other immunotherapy for the duration of treatment. Each microbe is isolated from healthy donors, as described in Example 3, or the genetically modified derivatives described in Examples 12 and 13.

In alternative embodiments, the patient is administered a live biotherapeutic at a dose of between about 10 5 to 10 15 bacteria, or at a dose of about 10 10 , 10 11 or 10 12 bacteria total or per dose, which can be in a lyophilized form, e.g., formulated in an enteric coated capsule. The patient takes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or more live biotherapeutic capsules by mouth once, twice or three times per day, and resumes a normal diet after 2, 4, 8, 12, or 24 hours.

In another embodiment, the patient takes the capsule by mouth before, during, or immediately after a meal.

In another embodiment, the patient is given a course of antibiotics before treatment, e.g., between one to seven days, or between about one to two weeks prior to the first dose of microbial cocktail, or three weeks prior, or four weeks prior, or up to 6 months prior to the first dose of live biotherapeutic.

In another embodiment, dosing of the live biotherapeutic is started one to two weeks prior to administration of the first checkpoint inhibitor dose or start of CAR-T therapy. In another embodiment, dosing of the live biotherapeutic is continued 1 month, 6 months, 1 year, or more following termination of checkpoint inhibitor administration.

Patient response to the combination therapy is a measure of success and in solid tumors is based on radiographic assessment using the Response Evaluation Criteria in Solid Tumors (RECIST 1.1) criteria (Schwartz, et al. (2016) Eur. J.

Cancer. 62: 132-137) at 6 months after treatment initiation, and again after 12 months and 24 months. Patients are classified as complete responders if all target lesions are gone, partial responders if there is at least 30% reduction in the sum of diameters of all target lesions, progressive disease if there is at least 20% increase in the sum of diameters of all target lesions, and stable disease otherwise. For blood cancers, the Response Evaluation Criteria in Lymphoma (RECIL) criteria is used, based on [18F]2-fluoro-2-deoxy-D-glucose positron-emission tomography (FDG-PET) (Younes, A. et al (2017) Ann. Oncol. 28:1436-1447). Example 21: Diagnosis of Disease and Method of Treating a Subject with an Exemplary Microbial Therapeutic

This example describes administration of a live exemplary biotherapeutic as provided herein, including a combination of bacteria as provided herein, e.g., as set forth in Table 15 to Table 16, Example 10, to an individual in need thereof.

Stool biomarkers based on microbes present in patients that respond to immuno-oncology (IO) therapy that are also lacking in patients that fail to respond to IO biotherapy can be used to predict the composition of live biotherapeutics for use as co-therapies to augment and improve outcomes of IO treatments for cancer. Conversely, the absence of these microbes in stool samples, as well as the presence of others found to associate with non-responding cancer patients, as detected in NGS analysis of stool samples taken from individuals during routine biomedical tests and procedures, can form a diagnostic pattern of biomarkers that can predict the likelihood that said individuals have or will develop cancer. This diagnostic may be based on the amount of one or more organisms present, position in the PCA plot, or other criteria that combines aspects of the whole genome sequence data. Reliability of such diagnostic is determined by the area under the ROC curve, as exemplified in Figure 8. Such a diagnostic method can be used by itself or in combination with other established tests to detect the presence of cancer. The diagnostic method can also detect gut microbial population patterns that can predict likelihood of a patient to develop cancer in the future, thereby redirecting a patient to further diagnoses, appropriate life-style changes, or prophylactic treatments such as the administration of a live biotherapeutic or live biotherapeutics to restore healthy gut microbe populations.

Example 22: Prophylactic application of a live exemplary biotherapeutic to prevent cancer occurrence in healthy individuals or cancer recurrence in patients in remission

This example describes administration of a live exemplary biotherapeutic as provided herein, including a combination of bacteria as provided herein, e.g., as set forth in Table 15 or Table 16, Example 10, to an individual in need thereof to prevent cancer recurrence, or as a prophylactic in healthy individuals or individuals determined to be at risk of acquiring cancer, e.g., because of (wherein the greater than normal risk is determined by) genetic analysis, family history or predisposing factors. An individual with no history of cancer, or alternatively a cancer patient currently in remission, is administered one of the present live biotherapeutics (Table 15 or 16, and as described in Example 10, or genetically modified variants described in Examples 12 and 13), thereby conditioning the microbiome to best enable the individual’s immune system to eliminate tumors before they substantially form.

Specifically, the individual is administered a live biotherapeutic at a dose of between about 10 5 to 10 15 bacteria, or at a dose of about 10 10 , 10 11 or 10 12 bacteria total or per dose, which can be in a lyophilized form, e.g., formulated in an enteric coated capsule. The individual takes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or more live biotherapeutic capsules by mouth once, twice or three times per day, and resumes a normal diet after 2, 4, 8, 12, or 24 hours. In another embodiment, the individual may take the capsule by mouth before, during, or immediately after a meal.

A number of embodiments of the invention have been described.

Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.