Intestinal microbiota and GVHD

Abstract

To provide intestinal microbiota and GVHD.SOLUTION: The present disclosure describes compositions and methods for increasing the abundance of commensal bacteria belonging to the order Clostridiales, including Blautia, Ruminococcus, Clostridium, Eubacterium, Holdemania and Dorea species, that are associated with reduced lethal GVHD and improved overall survival following bone marrow or hematopoietic stem cell transplant. The present disclosure, therefore, provides methods for reducing the likelihood, incidence or severity of GVHD by (1) avoiding the loss of endogenous beneficial species through antibiotic selection, and (2) by administering a therapeutically effective amount of a composition comprising one or more Clostridiales associated with reduced GVHD to individuals who may lack those strains or have lost those strains from their intestinal microbiota.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Description of Research and Development Funded by the Federal Government This invention was made with government support under grant numbers R01 HL069929, R01-AI080455, R01-AI100288, R01-AI101406, P01-CA023766, and P01-CA023766 from the National Institutes of Health, and contract HHSN272200900059C from the U.S. National Institute of Allergy and Infectious Disease. The government has certain rights in this invention.

[0002] This invention generally relates to graft-versus-host disease (GVHD). More specifically, this invention reports on the role of the gut flora as a predictor of GVHD severity / mortality and strategies for reducing GVHD-related morbidity.

Background Art

[0003] Despite continued improvements in the prognosis of patients receiving allogeneic bone marrow transplantation (allo BMT), GVHD remains a major cause of death in this patient population. 1 Modern immunosuppressive strategies are only partially effective in preventing GVHD and at the same time increase the risk of infections and disease recurrence. Therefore, strategies that reduce GVHD while leaving immune function intact could potentially improve the prognosis. One such strategy is to target the complex community of microorganisms that live in our intestinal tract, collectively known as the gut microbiota.

[0004] The relationship between the microbiota and GVHD has long been suspected but is still not fully understood. Mice transplanted in a germ-free state or 2 mice receiving intestinal decontamination antibiotics 3 develop mild GVHD. Clinical studies initially suggested the benefits of near-complete bacterial decontamination, but 4、5, indicating no obvious benefit later 6~8 , this approach was discontinued in the early 1990s 9 . Partial bowel decontamination is still practiced, but little is known about the optimal antibiotics. One study found that the addition of metronidazole to ciprofloxacin resulted in a significant reduction in acute GVHD, suggesting that anaerobic bacteria may contribute to the etiology of GVHD 10 .

[0005] However, more recent studies have shown that this approach may not be ideal. Administration of metronidazole during allo BMT has been associated with an increase in vancomycin-resistant enterococci in the gut, which precedes enterococcal bacteremia in some patients 11 . Other studies have found that obligate anaerobic bacteria in the gut, particularly certain Clostridium species, are important mediators of gut homeostasis and prevent inflammation by increasing regulatory T cells in the gut 12 .

[0006] In recent years, it has been reported that an increase in bacterial diversity at engraftment is associated with improved overall survival and transplant-related mortality after allo BMT 13 . However, the study populations were heterogeneous and specifically included 45% of patients who received T-cell-depleted allografts. Recipients of this type of transplant have a much lower risk of developing GVHD. Probably due to patient shortage and heterogeneity, it was impossible to determine the subcategories of non-relapse mortality associated with low diversity, including GVHD, infections, and organ failure.

[0007] Therefore, there is a need for treatments that take advantage of the relationship between the gut microbiota and GVHD.

Summary of the Invention

Means for Solving the Problems

[0008] The present disclosure is based on the observation that graft-versus-host disease correlates with large changes in the gut microbiota that occur during bone marrow and / or hematopoietic stem cell transplantation, suggesting that commensal bacteria can be predictors and regulators of GVHD risk and severity.

[0009] Accordingly, the present disclosure relates to methods and compositions for preventing loss of or restoring the mammalian bacterial gastrointestinal microbiota in a subject during bone marrow or hematopoietic stem cell transplantation to prevent, reduce the severity of, or treat GVHD. The present disclosure encompasses several approaches or combinations thereof for first preventing loss of relevant bacteria and then restoring bacteria and supporting the indigenous population or re-colonized bacteria in a subject having a persistent loss of protective bacteria. The approach includes

[0010] (1) selection of antibiotics having lower activity against obligate anaerobic bacteria as a method of preserving and preventing loss of indigenous protective bacteria, and

[0011] (2) providing prebiotics that support the growth of the indigenous population or re-colonized beneficial bacteria, and

[0012] (3) providing probiotics, i.e., administering to the subject a therapeutically effective amount of a therapeutic composition comprising one or more beneficial bacteria for re-colonization of the gastrointestinal tract, when the beneficial bacteria have already been lost.

[0013] In one aspect, the present disclosure relates to a method for restoring gastrointestinal bacteria lost as a result of exposure to, for example, an antibiotic having high activity against anaerobic bacteria, the method comprising administering to a subject in need of such treatment an effective amount of at least one bacterium from the order Clostridiales, or a combination thereof. In certain embodiments, the bacteria are administered orally. Alternatively, the bacteria can be administered rectally, for example, by enema.

[0014] In related aspects, the present disclosure relates to compositions for reducing graft-versus-host disease (GVHD) and GVHD-related mortality. It is based on the observation that there are changes in the gut microbiota that correlate with GVHD-related mortality. In particular, the presence of certain bacterial species that include organisms that ferment xylose, raffinose, cellobiose, or melibiose is particularly effective in reducing GVHD-related mortality.

[0015] In one aspect, the present disclosure relates to a method of reducing the risk of developing graft-versus-host disease (GVHD) and / or treating GVHD in a subject undergoing a bone marrow transplant or a hematopoietic stem cell transplant, the method comprising administering to the subject a therapeutically effective amount of a therapeutic composition comprising one or more bacteria from the Clostridiales, the composition

[0016] (i) promoting the growth or activity of one or more bacterial taxa that are overwhelmingly low in the subject's microbiota either before or after transplantation, or

[0017] (ii) suppressing the growth or activity of one or more bacterial taxa that are overwhelmingly abundant in the subject's microbiota.

[0018] In another aspect, the invention relates to a method of reducing the likelihood, incidence, or severity of GVHD in a subject, the method comprising administering to the subject a composition comprising at least one Clostridiales. In some embodiments, the organism is GenBank It contains 16Sr DNA having the nucleotide sequence of X94966, a nucleotide sequence selected from SEQ ID NOs: 1, 3, 4, 5, 7, 8, 9, 12, and 15, or a sequence about 98% to 100% identical to any of these sequences (about 99 to 100% in some embodiments, about 99.5 to 100% in other embodiments). In some embodiments, the therapeutic composition contains bacteria selected from the genera Blautia, Lachnospira, Eubacterium, Holdemania, and Clostridium. In some embodiments, the bacteria are selected from the group consisting of Lachnospira obum, Clostridium hasawaii, Eubacterium desmolans, Dorea longicatena, Lachnospira lactaris (Blautia producta), Eubacterium contortum, Lachnospira faecis, Holdemania filiformis, Clostridium sordellii, and combinations or mixtures thereof.

[0019] In some embodiments, the Blautia species is Blautia producta.

[0020] Accordingly, in related aspects, the present invention relates to a therapeutic composition containing Clostridium species. In some embodiments, the organism contains 16Sr DNA having the nucleotide sequence of X94966, a nucleotide sequence selected from SEQ ID NOs: 1, 3, 4, 5, 7, 8, 9, 12, and 15, or a sequence about 98% to 100% identical to any of these sequences (about 99 to 100% in some embodiments, about 99.5 to 100% in other embodiments). In some embodiments, the therapeutic composition contains bacteria selected from the genera Blautia, Lachnospira, Eubacterium, Holdemania, and Clostridium. In some embodiments, the bacteria are selected from the group consisting of Lachnospira obum, Clostridium hasawaii, Eubacterium desmolans, Dorea longicatena, Lachnospira lactaris (Blautia producta), Eubacterium contortum, Lachnospira faecis, Holdemania filiformis, Clostridium sordellii, and combinations or mixtures thereof.

[0021] In another related embodiment, the present invention relates to a method for reducing the likelihood of or preventing GVHD, wherein a composition comprising at least one Clostridiales is administered to a subject about 1 week to about 2 weeks prior to allo BMT, in some embodiments about 1 day to about 2 weeks prior to allo BMT, and in some embodiments about 7 to 10 days prior to allo BMT.

[0022] A method for reducing the risk, incidence, or severity of graft-versus-host disease (GVHD) in a subject undergoing bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), the method comprising administering to the subject a therapeutically effective amount of oral vancomycin or ampicillin when the subject has been treated for neutropenic fever by intravenous administration of an antibiotic selected from the group consisting of metronidazole, piperacillin-tazobactam (pip-tazo), and imipenem.

[0023] A method for reducing the risk of developing graft-versus-host disease (GVHD) in a subject after bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), the method comprising determining the abundance of Akkermansia muciniphila in a sample of fecal material from the subject, and administering to the subject a therapeutically effective amount of an antibiotic selected from ampicillin and oral vancomycin when the abundance of Akkermansia muciniphila exceeds 1% to 10%, wherein administration of the antibiotic reduces the abundance of Akkermansia muciniphila and reduces or eliminates the risk of GVHD. In some embodiments, an abundance of Akkermansia muciniphila in the sample exceeding 2% indicates a risk of developing GVHD. The abundance of Akkermansia muciniphila is determined before transplantation, after antibiotic treatment for neutropenic fever associated with transplantation, or both. In certain embodiments, for example, the following items are provided. (Item 1) A therapeutic composition for the prevention and / or treatment of graft-versus-host disease (GVHD) after bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), comprising one or more purified populations of bacteria of the order Clostridiales. (Item 2) The therapeutic composition according to item 1, wherein the bacteria comprise 16S rDNA having a nucleotide sequence of one of SEQ ID NOs: 1, 3, 4, 5, 7, 8, 9, 12, and 15 or a nucleotide sequence approximately 98% to 100% identical to the sequence. (Item 3) The therapeutic composition according to item 1, wherein the bacteria are selected from the genus Blautia, the genus Lachnoclostridium, the genus Eubacterium, the genus Holdemania, and the genus Clostridium, or Blautia-like species. (Item 4) The therapeutic composition according to item 3, wherein the bacteria are selected from the group consisting of Lachnoclostridium ovalum, Clostridium haselwaei, Eubacterium desmolans, Dorea longicatena, Lachnoclostridium lactaris (Blautia producta), Eubacterium contorum, Lachnoclostridium faecis, Holdemania filiformis, Clostridium sordellii, and combinations or mixtures thereof. (Item 5) The therapeutic composition according to items 1 to 4, wherein the bacteria in the composition are live bacteria, frozen bacteria, germinable spores, or combinations thereof. (Item 6) The bacteria are present in a single dose of 10 4 ~10 10 CFU, the therapeutic composition according to items 1 to 5. (Item 7) The bacteria are present in a single dose of 10 5 ~10 9 CFU, the therapeutic composition according to items 1 to 5. (Item 8) The bacteria are present in a single dose of 10 6 ~10 8 CFU, the therapeutic composition according to items 1 to 5. (Item 9) The therapeutic composition according to any one of Items 3 to 5, wherein the bacterium ferments an oligosaccharide selected from xylose, raffinose, cellobiose, or melizitose. (Item 10) The therapeutic composition according to any one of Items 1 to 9, formulated for oral administration. (Item 11) The therapeutic composition according to any one of Items 1 to 9, formulated for colorectal administration. (Item 12) A method for reducing the risk of developing graft-versus-host disease (GVHD) and / or treating GVHD in a subject undergoing bone marrow or hematopoietic stem cell transplantation, the method comprising administering to the subject a therapeutically effective amount of a therapeutic composition comprising one or more bacteria from the order Clostridiales. (Item 13) The method according to Item 12, wherein the bacterium is selected from the genus Blautia, the genus Lachnoclostridium, the genus Eubacterium, the genus Holdemanella, and the genus Clostridium, or a Blautia-like species. (Item 14) The method according to Item 12, wherein the bacterium is selected from the group consisting of Lachnoclostridium ovalum, Clostridium haswaei, Eubacterium desmolans, Dorea longicatena, Lachnoclostridium lactaris (Blautia producta), Eubacterium contortum, Lachnoclostridium faecis, Holdemanella filiformis, Clostridium sordellii, and combinations or mixtures thereof. (Item 15) The composition is (i) promoting the growth or activity of one or more bacterial taxa that are overwhelmingly few in the microbiota of the subject, either before or after transplantation, or (ii) suppressing the growth or activity of one or more bacterial taxa that are overwhelmingly numerous in the microbiota of the subject, the method according to Items 12 to 14. (Item 16) The method according to Item 12 or 15, wherein the method comprises administering to the subject the therapeutic composition according to any one of Items 1 to 9. (Item 17) The method according to item 12, wherein the composition is administered to the subject about 1 day to about 2 weeks after interruption of the treatment of the subject with an antibiotic having high activity against anaerobic bacteria. (Item 18) The method according to item 12, wherein the composition is administered to the subject about 7 to 10 days before allo-BMT or allo-HSCT. (Item 19) The method according to item 12, wherein the composition is administered to the subject about 1 day to about 1 week before allo-BMT or allo-HSCT. (Item 20) A therapeutic composition comprising one or more bacteria from the order Clostridiales for use in the prevention, reduction of risk, and / or treatment of GVHD in an individual undergoing allo-BMT or allo-HSCT. (Item 21) The therapeutic composition according to item 20, wherein the therapeutic composition is selected from the genus Blautia, the genus Lachnoclostridium, the genus Eubacterium, the genus Holdemania, and the genus Clostridium, or Blautia-like species. (Item 22) The therapeutic composition according to item 20, wherein the therapeutic composition is selected from the group consisting of Lachnoclostridium ovalum, Clostridium haswaei, Eubacterium desmolans, Dorea longicatena, Lachnoclostridium lactaris (Blautia producta), Eubacterium contortum, Lachnoclostridium faecis, Holdemania filiformis, Clostridium sordellii, and combinations or mixtures thereof. (Item 23) A nutritional supplement containing a sugar fermented by Clostridium species to support the growth of said species for the treatment of GVHD. (Item 24) The nutritional supplement according to item 23, wherein the sugar is xylose, raffinose, cellobiose, melibiose, or a combination or mixture thereof. (Item 25) A method for reducing the risk of developing graft-versus-host disease (GVHD) in a subject after bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), comprising: (a) determining the abundance of Akkermansia muciniphila in a sample of fecal material from the subject; (b) administering to the subject a therapeutically effective amount of an antibiotic selected from ampicillin and oral vancomycin when the abundance of Akkermansia muciniphila exceeds 1% to 10%; wherein administration of the antibiotic reduces the abundance of Akkermansia muciniphila and reduces or eliminates the risk of GVHD. (Item 26) The method according to item 25, wherein the abundance of Akkermansia muciniphila in the sample exceeds 2%. (Item 27) The method according to item 25, wherein the abundance of Akkermansia muciniphila is determined before transplantation, after antibiotic treatment for neutropenic fever associated with transplantation, or both. (Item 28) A method for screening a subject for the risk of developing GVHD after bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), comprising determining the abundance of Clostridium spp. in a sample of fecal material from the subject, wherein a low abundance of the Clostridium spp. in the sample indicates an increased risk of GVHD. (Item 29) The method according to item 28, wherein the abundance of the Clostridium spp. is from 0.5% to less than 0.01%. (Item 30) The method according to item 28, wherein the abundance of the Clostridium spp. is from 0.25% to less than 0.02%. (Item 31) The method according to item 28, wherein the abundance of the Clostridium spp. is less than 0.05%. (Item 32) The method according to item 28, wherein the Clostridium comprises 16S rDNA having a nucleotide sequence of one of SEQ ID NOs: 1 to 16 or a nucleotide sequence that is about 98% to 100% identical to the sequence. (Item 33) The method according to item 28, wherein the bacterium is Blautia or a Blautia-like species. (Item 34) The method according to item 28, wherein the bacterium is selected from the group consisting of Blautia producta, [Lachnospira] obeum, Clostridium haswaei, Eubacterium desmolans, Dorea longicatena, Lachnospira lactaris (Blautia producta), Eubacterium contortum, Lachnospira faecis, Holdemania filiformis, Clostridium sordellii, and combinations or mixtures thereof. (Item 35) A method for reducing the risk of developing graft-versus-host disease (GVHD) in a subject undergoing bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), the method comprising administering a nutritional supplement to the subject to support the growth of Clostridium species, the supplement comprising a sugar fermented by the species. (Item 36) The method according to item 35, wherein the sugar is xylose, raffinose, cellobiose, melibiose, or a combination or mixture thereof. (Item 37) The method according to item 35, wherein the nutritional supplement is administered before or after transplantation. (Item 38) The method according to item 35, further comprising administering to the subject a therapeutically effective amount of the composition according to items 1 to 9. (Item 39) A method for reducing the risk, incidence, or severity of graft-versus-host disease (GVHD) in a subject undergoing bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), the method comprising administering to the subject a therapeutically effective amount of oral vancomycin or ampicillin when the subject has been treated for neutropenic fever by intravenous administration of an antibiotic selected from the group consisting of metronidazole, piperacillin-tazobactam (pip-tazo), and imipenem. (Item 40) A method for reducing the risk, incidence, or severity of graft-versus-host disease (GVHD) in a subject undergoing bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), the method comprising administering to the subject an antibiotic with reduced activity against anaerobic bacteria selected from the group consisting of intravenous vancomycin, ceftriaxone, cefotaxime, cefepime, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, levofloxacin, and atovaquone. Brief Description of the Drawings

[0024]

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Mode for Carrying Out the Invention

[0025] All patents, publications, applications, and other references cited herein are hereby incorporated by reference in their entirety into this application.

[0026] In practicing the present invention, many conventional techniques in molecular biology, microbiology, and bacteriology are used, and those techniques are within the scope of the art. The content of references, including standard protocols widely known and relied upon by those skilled in the art, is hereby incorporated by reference herein as part of the present disclosure, including the instructions of the manufacturers.

[0027] Regarding technical terms, the terms used in this specification are intended to be construed according to their standard meanings as known to those skilled in the art. For convenience, the definitions of some terms are provided herein.

[0028] As used herein, "patient" or "subject" refers to a mammal, including humans and domestic animals.

[0029] The terms "gut microbiota", "gut flora", and "gastrointestinal microbiota" are used interchangeably to refer to the bacteria in the digestive tract.

[0030] The term "probiotics" refers to substantially pure bacteria (i.e., a single isolate), or a mixture of desired bacteria, and may include any additional components that can be administered to a mammal to restore the microbiota. Such compositions are also referred to herein as "bacterial inoculants".

[0031] The term "prebiotics" refers to an agent that increases the number and / or activity of one or more desired bacteria. Non-limiting examples of prebiotics useful in the methods of the present invention include saccharides such as xylose, raffinose, cellobiose, and melibiose.

[0032] "Therapeutically effective amount" means that the amount of a bacterial inoculant or compound (e.g., a narrow-spectrum antibiotic or antibacterial agent) is sufficient to be effective in treating a disorder or condition when administered to a subject for treating such disorder or condition.

[0033] "Blautia", "Blautia-related", or "Blautia-like species" are gram-stained positive, non-motile, obligate anaerobic bacteria found in the feces of humans and other mammals (Liu et al., 2008). Blautia species include, for example, Blautia producta (ATCC Rincluding 27340-DSM2950, American Type Culture Collection, Manassas, VA). The Blautia-like species include those having a 16S rDNA sequence that is 98% to 100% sequence identical (in some embodiments, 99.5 to 100% identical) to the 16S rDNA of Blautia producta (GenBank X94966). In Table 1 below, several Blautia-related species are shown by name (NCBI name) including their respective 16S rDNA sequences.

[0034] Although not a member of the Clostridiales order, Holdemania filiformis is a bacterium associated with less GVHD and is thus intended to be encompassed by the present disclosure as a potential therapy.

[0035] Antibiotics vary considerably in the strength of their activity against anaerobic symbionts and are herein expressly stated to have either high or low activity against anaerobic bacteria. Antibiotics having high activity against anaerobic bacteria include metronidazole, piperacillin-tazobactam (pip-taxo or P / T), and imipenem. Antibiotics having low activity against anaerobic bacteria include aztreonam, ceftazidime / cefpime, intravenous vancomycin, levofloxacin, ciprofloxacin, cefazolin, atovaquone, and tmp-smx.

[0036] The relevant taxonomic characteristics of the related strains of the organism can be confirmed by the results obtained from 16S rDNA sequence analysis and the Analytical Profile Index (API®) bacterial identification system, as well as other conventional methods used in the art for bacterial identification.

[0037] For patients suffering from hematological malignancies such as leukemia, lymphoma, and other related cancers, allogeneic blood and marrow transplantation (allo BMT) or hematopoietic stem cell transplantation (HSCT) is a very important therapy that can bring about a cure when chemotherapy alone cannot. Every year, more than 25,000 patients worldwide receive all BMT. The main risk of bone marrow / hematopoietic stem cell transplantation still remains graft-versus-host disease (GVHD), which results from the donor immune system recognizing the recipient's organs as foreign and causing life-threatening inflammation. Developing strategies to reduce GVHD while leaving overall immune function intact will bring great benefits to patients.

[0038] In the past, the use of broad-spectrum antibiotics in allo-HSCT recipients was thought to be protective against GVHD. Combinations of broad-spectrum antibiotics were administered for the purpose of complete gut decontamination, which was associated with a reduction in GVHD in mouse models (36) and some (37, 38), but not in all clinical studies (39 - 41). Similarly, the addition of metronidazole to ciprofloxacin resulted in a reduction in GVHD in a small randomized study (42), supporting the hypothesis that gut bacteria contribute to GVHD pathophysiology.

[0039] However, a series of recent studies have described a different association in which allo-HSCT recipients suffering from more severe microbiota damage are more likely to develop severe GVHD (12, 14, 16, 43). Microbiota damage has been observed in several ways, including an increase in commensal Enterococcus species (12), a loss of overall diversity (14), a decrease in commensalism from the genus Blautia, which are members of the Clostridiales order (16), and more recently, low levels of indole, a byproduct of tryptophan metabolism produced by gut bacteria that can be quantified in urine in the form of 3-indoxyl sulfate (43). Consistent with these reports, in the present study, the inventors show that the use of antibiotics with a broader spectrum of higher activity, such as imipenem, results in increased microbiota damage (particularly loss of the Clostridiales order) and increased GVHD severity.

[0040] A complete explanation for the apparent discrepancy between earlier and more recent studies has not yet been fully elucidated, but one potential contributor could be the increase in antibiotic-resistant bacteria, including resistant Enterococcus, which can successfully achieve difficult gut decontamination. An increase in the frequency of colonization with resistant organisms has been observed over time in allo-HSCT recipients (44). A recent study found that gut decontamination ended unsuccessfully in nearly half of the patients who attempted it. Interestingly, patients who achieved decontamination had a much lower incidence of acute GVHD compared to those who did not (38).

[0041] In the present study, the inventors show that different antibiotics used to treat neutropenic fever have different effects on the gut microbiota composition in both patients and mouse models. The inventors also identified several important changes brought about by imipenem treatment in mice with GVHD, including the severity of GVHD in the colon, inflammatory changes, and disruption of the protective colonic mucus barrier.

[0042] One promising approach to reducing the risk, incidence, and severity of GVHD is to target the complex community of microorganisms that exist in the human gut, collectively referred to as the gut microbiota. The relationship between the microbiota and GVHD has been suspected for years but remains incompletely understood. Gut decontamination with antibiotics is practiced in only some, not all, facilities, and there is no consensus on the ideal choice of antibiotic coverage.

[0043] Disclosed herein are results showing that the abundance of bacteria belonging to the Clostridiales taxon, including Blautia, which are generally commensal organisms found in the human gut, predicts protection from life-threatening GVHD in all transplant patients. Furthermore, in a mouse model, introduction of mouse-derived Blautia species reduces GVHD severity. Without wishing to be bound by theory, it is thought to occur by inducing regulatory T cells through the production of short-chain fatty acid metabolites (SCFAs).

[0044] Additional studies characterizing the natural history of Clostridiales and Blautia abundance in all BMT / HSCT recipients showed that the overwhelming majority of patients start with an endogenous population of these organisms in abundance, but many lose them dramatically during transplantation. Interestingly, the loss of Blautia strongly correlates with a decrease in oral nutrient intake in both humans and mice.

[0045] In one embodiment, a nutritional intervention strategy to support Clostridiales / Blautia abundance after all BMT / HSCT provides one way to mitigate GVHD. In a mouse model, it has been shown that these nutritional approaches can successfully prevent the loss of Clostridiales / Blautia and reduce the severity of GVHD. These results identify the microbiota as a powerful therapeutic target that can be employed to significantly reduce GVHD.

[0046] The relationship between the gut microbiota composition and graft-versus-host disease (GVHD) after allogeneic BMT or HSCT is not well understood. Intestinal bacteria have conventionally been thought to contribute to GVHD, but recent animal studies in non-transplant settings have identified a population of obligate anaerobic gut commensals with anti-inflammatory properties.

[0047] In one study, the fecal bacterial composition of 64 patients was evaluated 12 days after BMT. An increase in bacterial diversity was associated with a decrease in GVHD-related mortality. Furthermore, having an increased number of bacteria belonging to the genus Blautia was associated with a decrease in GVHD lethality in this cohort and was confirmed in another independent cohort of 51 patients. Blautia abundance was also associated with improved overall survival. By assessing Blautia abundance against clinical factors, it was found that loss of Blautia was associated with two clinical factors: 1) treatment with antibiotics that suppress anaerobic bacteria, and 2) receiving total parenteral nutrition (TPN) for a longer duration. An increase in the abundance of symbiotic bacteria belonging to the genus Blautia is associated with a decrease in lethal GVHD and an improvement in overall survival.

[0048] In another study, 283 patients were retrospectively examined for neutropenic fever after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Administration of antibiotics with increased activity against anaerobic bacteria, including piperacillin-tazobactam (pip / tazo) or imipenem-cilastatin (imipenem), was found to be associated with an increase in GVHD-related mortality compared to administration of antibiotics with decreased activity against anaerobic bacteria, such as aztreonam or cefepime. Stool microbiota composition analysis showed that pip / tazo and imipenem administration were associated with a more profound loss of members of the Clostridiales bacteria order. Experiments in a mouse model showed similar flora changes by these antibiotics. Furthermore, modeling antibiotic treatment in mice with GVHD reproduced a worsened mortality with imipenem compared to aztreonam.

[0049] The present disclosure describes methods for preventing or treating graft-versus-host disease (GVHD) by preventing loss or restoring a population of certain Clostridiales in the gut to reduce the likelihood, incidence, or severity of GVHD.

[0050] In a first embodiment, the present disclosure includes methods for reducing the risk, incidence, or severity of graft-versus-host disease (GVHD) in a subject undergoing a bone marrow transplant (BMT) or hematopoietic stem cell transplant (HSCT) by taking means to prevent loss of beneficial activity, for example, by avoiding the use of antibiotics that are harmful to anaerobic bacteria, if possible. In this embodiment, the method includes selecting an antibiotic having reduced activity against anaerobic bacteria selected from the group consisting of intravenous vancomycin, ceftriaxone, cefotaxime, cefepime, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, levofloxacin, and atovaquone, and administering it to a subject in need thereof.

[0051] In some embodiments, restoration of the microbiota is achieved by administering to a subject in need thereof a therapeutically effective amount of a probiotic composition comprising an effective amount of at least one bacterial strain, or a combination of several strains, from Clostridiales taxa, where the composition (i) stimulates the growth and / or activity of bacteria that are protective against GVHD, and / or (ii) suppresses the growth and / or activity of bacteria that are in overwhelming majority in GVHD. Support for protective bacteria can be provided in the form of nutritional supplements or prebiotics, and in some cases, sugars fermented by beneficial bacteria. Also contemplated by the present disclosure is suppressing bacteria in overwhelming majority, such as Akkermansia, by administering an antibiotic that prevents excision of those organisms and "pushing out" from beneficial Clostridium species.

[0052] In one embodiment, a method for reducing the likelihood or severity of GVHD comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising one or more bacterial species from the Clostridiales taxon, such as Blautia species / isolate shown to reduce GVHD.

[0053] The bacterial strains administered according to the methods of the present disclosure can include live bacteria. One or several different bacterial inoculants can be administered simultaneously or sequentially (including administering at different times). Such bacteria can be isolated from the microbiota and grown in a medium using known techniques.

[0054] Administration of the bacterial composition can be achieved by any method known in the art that is likely to introduce the organism to the desired location. In one embodiment, the bacteria are administered orally. Alternatively, the bacteria can be administered rectally, for example, by enema.

[0055] The dosage of the bacterial inoculant or compound of the present invention will be apparent to those skilled in the art. Various dosages are effective in achieving gut colonization with the desired bacterial inoculant, for example, 10 6 、10 7 、10 8 、10 9 、and 10 10 CFU can be administered as a single dose. Low doses, for example, 10 4 and 10 5 CFU can also be effective. Subsequent inoculation is contemplated if necessary.

[0056] Organisms contemplated for administration to restore the gastrointestinal microbiota include those shown in Table 1 below.

[0057] Identification of Beneficial Species Species for use in the methods described herein are Blautia producta (ATCC RIt may also include 27340-DSM2950, American Type Culture Collection, Manassas, VA), or those indicated by an asterisk in Table 1 below. New Blautia isolates are identified annually, and in some cases, isolates of other genera have been reclassified as Blautia. That is, for example, Blautia-like or Blautia-related species may include Luminococcus obeum, Luminococcus faecis, Luminococcus lactaris, etc. (see Table 1 below).

[0058] To analyze the association with GVHD-related death (or any other prognosis), a script was utilized that used the Cox proportional hazards test to calculate the association between the log-transformed abundance of each bacterium and the time to the event of interest for the prognosis. This has the advantage of taking into account the time elapsed to the event for each patient. Another major advantage is that the results of the Cox proportional hazards test can be easily adjusted to account for the influence of other potential confounding clinical factors. Here, the inventors performed univariate analysis, as well as multivariate analysis adjusted for the type of transplantation (umbilical cord blood, peripheral blood, or bone marrow) and the intensity of the preconditioning.

[0059] The data were analyzed at the operational taxonomic unit (OTU) level. The nucleotide sequence information of each specific 16S rRNA was blasted against the NCBI 16S database to identify the name.

[0060] Generally, Blautia and Blautia-related species (including species from the genera Luminococcus, Lactococcus, Anaerostipes, Holdemanella, and others) are gram-stained positive, non-motile bacteria that are obligate anaerobes found in the feces of humans and other mammals (Liu et al., 2008). Bacteria shown to have an association with GVHD are shown in Table 1, along with those associated with a lower risk or incidence of GVHD indicated by an asterisk, regardless of whether they are beneficial or harmful.

[0061]

Table 1-1

Table 1-2

Table 1-3

[0062] Brachyspira and Brachyspira-like species, particularly Brachyspira producta or 16S rRNA sequences (GenBank X94966) that are exactly identical to those of any one of SEQ ID NOs: 1, 3, 4, 5, 7, 8, 9, 12, and 15 (e.g., 98% - 100% sequence identity, or in some embodiments, 99.5 - 100% identity) are suitable for use in the disclosed methods.

[0063] The abundance of bacteria from the Clostridiales taxon, including Brachyspira, has also been shown to be predictive of a decrease in GVHD-related mortality in some patients. Interestingly, some of the 17 Clostridium isolates tested (see Narushima et al. Characterization of the 17 strains of regulatory T cell-inducing human-derived Clostridia. Gut Microbes 5:3, 333 - 339 2014, which is incorporated herein by reference for characterization) are very closely related species and may therefore be useful in practicing the methods of the present invention.

[0064] In some embodiments, a method for determining whether a Brachyspira or Brachyspira-like species or isolate is suitable for use in the present invention includes determining the percent identity of the 16S rRNA of a species / isolate having a 16S rRNA sequence of Brachyspira producta (GenBank X94966) or any one of SEQ ID NOs: 1 - 16, and methods for doing so are well known in the art.

[0065] In some embodiments, the Brachia species for use in the present invention include those Brachia and Brachia-like species that ferment certain sugars, such as xylose, raffinose, cellobiose, and melibiose. The supply of nutritional supplements containing these sugars may be suitable for administration to a subject as a prebiotic strategy to reduce GVHD.

[0066] Administration of Brachia / Brachia-related species One or more different bacterial inoculants can be administered simultaneously or sequentially (including administering at different times). The bacterial strains administered according to the methods of the present invention can include live bacteria, frozen bacteria, bacterial spores, or combinations thereof. Such bacteria can be isolated from a suitable microbiota source or obtained from a cell bank (such as the American Type Culture Collection / ATCC) and grown in a medium using known techniques.

[0067] In practicing the methods of the present invention, delivery of Brachia species to a subject for the purpose of reducing the likelihood, incidence, severity, or otherwise preventing or treating GVHD may be achieved using any oral delivery system suitable for administering live microorganisms to an individual in need thereof, such as described in U.S. Published Application No. 2014 / 0112985. Such a delivery system may include a probiotic agent containing at least one live Brachia microorganism that has been shown to correlate with a reduction in GVHD, optionally at least one additional agent, such as a motility agent, and a delivery vehicle, where the oral delivery vehicle releases the probiotic agent into the distal small intestine of the individual. In one embodiment, the Brachia isolate is administered in combination with a sugar known to ferment.

[0068] In other embodiments, oral delivery is achieved via a vehicle selected from the group consisting of pills, tablets, caplets, capsules, soft gels, and coated probiotic granules that release the probiotic agent in the distal small intestine. The present invention further provides oral delivery in a dosage form in which a probiotic agent is present and is selected from immediate release, delayed release, sustained release that is released in the distal small intestine, and targeted release that is targeted to be released in the distal small intestine.

Example

[0069] Method for patient selection and specimen collection In one study, the subjects retrospectively analyzed for the effect of antibiotics on clinical outcomes consisted of 283 adult patients who received all-HSCT at Memorial Sloan Kettering Cancer Center (MSKCC) from 1994 to 2013. Patients who received conventional grafts (non-T cell depleted) were included in this study, and patients who received ex vivo T cell depleted grafts or peri-transplant alemtuzumab were excluded. Stool specimens were collected and stored weekly during the transplant hospitalization. This study was approved by the Institutional Review Board of MSKCC. All study patients provided written informed consent for biospecimen collection and analysis.

[0070] GVHD was clinically diagnosed and pathologically confirmed by biopsy whenever possible and classified according to historical consensus criteria as described previously (see Rowlings PA, Przepiorka D, Klein JP, et al. IBMTR Severity Index for grading acute graft-versus host disease: retrospective comparison with Glucksberg grade. Br J Haematol. 1997). These criteria were applied to GVHD with truly acute characteristics occurring after day 100. Cases of GVHD were further classified by treatment with or without systemic steroids (prednisone or methylprednisolone, at least 0.5 mg / kg per day). The cause of death was determined using a standard algorithm that prioritized the prognosis in the following order: 1) recurrence of the primary disease, 2) transplant failure, 3) GVHD, 4) infection, and 5) organ failure. Thus, in patients without recurrence of the disease or transplant failure, those who were receiving treatment for GVHD at the time of death were considered to have died of GVHD-related death, including those who died of inflammation. The disease risk was determined according to the ASBMT RFI2014 disease classification. The pretreatment intensity was assigned based on a previously established practical definition.

[0071] Sample storage and DNA extraction Stool samples from patients were stored at 4°C for less than 24 hours before freezing at -80°C. Ileum and colon samples from mice were frozen at -80°C. DNA was extracted using one of two methods that yielded similar results.

[0072] For each fecal sample, DNA was extracted using the phenol-chloroform extraction technique (see Ubeda, C., Y. Taur, R. R. Jenq, M. J. Equinda, T. Son, M. Samstein, A. Viale, N. D. Socci, M. R. M. van den Brink, M. Kamboj, and E. G. Pamer. 2010. Intestinal domination by Vancomycin-resistant Enterococcus precedes bloodstream invasion in humans. J. Clin. Invest) or using the Power Soil DNA isolation kit (MO BIO Laboratories).

[0073] 16S analysis of samples For each fecal sample, DNA was extracted and purified. Samples were analyzed using the 454 GS FLX Titanium platform to sequence the V1-V3 regions of the bacterial 16S rRNA gene or alternatively analyzed using the Illumina MiSeq platform to sequence the V4-V5 regions of the 16S rRNA gene. Sequence data were compiled and processed using mothur version 1.34 and QIIME version 1.8.0, and screened and filtered for quality. Operational taxonomic units (OTUs) were classified to the species level using the variants of the Greengenes reference database. Principal component analysis was performed in R software based on the weighted and normalized Unifrac distance matrix of OUT abundances. Data from this study are deposited in the NCBI Sequence Read Archive (url: ncbi.nlm.nih.bov / sra).

[0074] Phylogenetic abundance comparisons were performed using linear discriminant analysis (LDA) effect size (LEfSe) analysis 27 with a logarithmic LDA cut-off of 2.0 to identify biomarkers of GVHD-related death.

[0075] Antibodies and flow cytometry All antibodies were obtained from BD Biosciences-Pharmingen. For cell analysis of surface markers, cells were stained in PBS with 0.5% BSA (PBS / BSA) for 20 minutes at 4°C after Fc block, washed, and resuspended in DAPI in PBS / BSA. After cell surface staining, intracellular staining was performed with an eBioscience kit following the manufacturer's instructions. Dead cells were excluded with the LIVE / DEAD Fixable Dead Cell Stain Kit (Invitrogen). All flow cytometry was performed on an LSR II (BD Biosciences) and analyzed with FlowJo (TreeStar Software).

[0076] Antibiotic Classification Antibiotics used during transplantation hospitalization were divided into those with significant activity against anaerobic bacteria (pip / tazo, ticarcillin clavulanic acid, imipenem, meropenem, metronidazole, oral vancomycin, and clindamycin), and those with decreased activity (intravenous vancomycin, ceftriaxone, ceftazidime, cefepime, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, levofloxacin, atovaquone) (19).

[0077] Transplant Practice As per the institutional practice of the inventors, patients receiving ciprofloxacin prophylaxis and those receiving more intensive pretreatment than a non-myeloablative regimen also received intravenous vancomycin prophylaxis starting on day 2 until day 7 (59). Antibiotic prophylaxis for Pneumocystis jirovecii (trimethoprim sulfamethoxazole, inhaled pentamadine, or atovaquone) was given at the discretion of the transplant physician.

[0078] Analysis of Specimens For each fecal sample, DNA was purified using a phenol-chloroform extraction technique with mechanical disruption (bead beating) based on a previously described protocol (60). Samples were analyzed using the 454 GS FLX Titanium platform to sequence the V1-V3 region of the bacterial 16S rRNA gene or alternatively analyzed using the Illumina MiSeq platform to sequence the V4-V5 region of the 16S rRNA gene. Sequence data were compiled and processed using mothur version 1.34 (61) and screened and filtered for quality (62). Operational taxonomic units (OTUs) were classified to the species level (63) using the variants of the Greengenes reference database (64). Principal component analysis was performed in R software based on the weighted and normalized Unifrac (65) distance matrix of OTU abundances. Data from this study are deposited in the NCBI Sequence Read Archive (see url: www.ncbi.nlm.nih.gov / sra).

[0079] RNA Sequencing and Analysis Mice were sacrificed 16 days after receiving a total of 3 antibiotic treatments. The distal colon was removed, pooled (n = 4, in both the aztreonam and imipenem treatment groups), and then RNA isolation was performed using Trizol LS. RNA was prepared using RiboMinus (Life Technologies). The library was sequenced using the Ion Proton System (Life Technologies). The aligned RNA was analyzed for fold change. Gene expression differences were analyzed in imipenem-treated mice versus aztreonam-treated mice.

[0080] Metagenomic Shotgun Sequencing and Analysis Paired-end raw reads from shotgun sequencing were trimmed using Trimmomatic 0.32(69) with a maximum of two mismatches, a minimum of 30 terminal base scores, and the Illumina TruSeq adapter sequences. The remaining trimmed reads were taxonomically assigned using Kraken(70). Briefly, the trimmed and filtered reads were taxonomically classified by k-mer similarity to bacterial, viral, and fungal k-mer profiles generated from the NCBI genome and chromosome collections (assessed November 12, 2014). Unclassified reads were further queried with BLAST (nt database, March 24, 2015), and non-bacterial reads were discarded. Functional analysis was performed on the quality-filtered reads using HUMAnN v0.99(71) to determine the abundance and pathways of genes within a given metagenomic community. To identify those functional classifications that differed between aztreonam-treated and imipenem-treated subject samples, the inventors used LEfSe(21), a powerful tool for specifically identifying informative biomarkers such as genes, pathways, or organisms among microbial populations.

[0081] Recipients were sacrificed on day 21, and 10-mm segments of colon were carefully harvested along with fecal contents and immersed overnight in an anhydrous methanol-Carnoy's fixative (60% dry methanol, 30% chloroform, and 10% acetic acid)(72). The tissues were then washed in methanol, embedded in paraffin, and then 5-μm sections were placed on glass slides. The slides were deparaffinized, stained by the standard periodic acid Schiff reaction method, and analyzed by light microscopy(73).

[0082] Immunostaining and fluorescence in situ hybridization (FISH) of colon tissue Formalin-fixed colon from the recipient was stained with anti-mouse CD3 antibody A0452 (DAKO), pSTAT3 antibody 9135 (Cell Signaling), CD11b antibody ab133357 (Abcam), and B220 antibody 550286 (BD Pharmingen) in comparison with isotype controls. Immunofluorescence secondary staining was performed with AF488 for pSTAT3 and B220, and with AF594 for CD3 and CD11b. Pieces of colon with fecal material were fixed in Carnoy's and bacterial FISH (EUB338) (35) and immunostaining were performed with MUC2C3 antiserum and DNA by Hoechst 34580 (Life technologies) as described previously (74, 75).

[0083] Assessment of mouse and bone marrow transplantation and graft-versus-host disease. Female C57BL / 6, C57BL / 6 / Ly5.1, and 129S1 / SvlmJ mice were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). Mice used in the experiments were 6 - 9 weeks old. Mice were treated with an intestinal decontamination antibiotic cocktail (ampicillin and vancomycin) to mimic the microbiota that develops in allo BMT patients. The mice were then exposed to a myeloablative single dose of total body irradiation (TBI, 11 Gy from a 137Cs source as split doses with 3-hour intervals between administrations), and then bone marrow and purified splenic T cells from fully MHC-mismatched B10.BR mice (H2k to H2b) were transplanted by intravenous injection. Donor mice were euthanized by asphyxiation using carbon dioxide, and the spleen, femurs, and tibias were aseptically removed. Donor BM was obtained by flushing the tibias and femurs with cold tissue culture medium. To be able to reproducibly induce GVHD by co-injection of T cell-depleted BM and donor splenic T cells in experimental mice, donor BM was incubated with 2.5 μg of anti-Thy-1.2 per 106 BM cells for 30 minutes at 4°C, followed by 10 6T cell depletion (TCD) was performed by incubating at 37°C for 40 minutes with 10 μL of low-tox-M rabbit complement per BM cell. Splenic T cells were purified with anti-CD5 MACS beads (Miltenyi). BM cells (5×10 6 ) and splenic T cells (1×106 per recipient) were transplanted by tail vein injection.

[0084] Isolation of Blautia isolates from mouse or human feces Collect all stool specimens and homogenize in 1 - 3 volumes of 0.05% peptone using a sterile stainless-steel blender with 1 - 3 volumes of peptone. Serially dilute (10-fold) approximately 1 gram of the specimen in a pre-reduced anaerobic sterilized (PRAS) dilution blank (Anaerobe Systems). Weigh a separate approximately 1 gram aliquot, dry overnight in vacuo, and reweigh to calculate the amount on a dry weight basis. To select Clostridiales bacteria including Blautia species, plate 100 μL of the homogenized stool sample dilution series onto brain heart infusion blood agar medium (SBA, Becton Dickinson) supplemented with 4 μg / mL trimethoprim (Sigma Chemical) and 1 μg / mL sulfamethoxazole (Sigma), brucella blood agar medium (BAP, Anaeobe Systems), CDC ANA blood agar medium, (BBL Microbiology Systems), and egg yolk agar medium (EYA, Anaeobe Systems) (Finegold SM, Molitoris D, Song Y, Liu C, Vaisanen ML, Bolte E, McTeague M, Sandler R, Wexler H, Marlowe EM, Collins MD, Lawson PA, Summanen P, Baysallar M, Tomzynski TJ, Read E, Johnson E, Rolfe R, Nasir P, Shah H, Haake DA, Manning P, Kaul A, 2002. Gastrointestinal microflora studies in Late-onset autism. Clin Infect Dis 1:35). To select spores, the dilution may be heated at 70-80 °C for 10-20 minutes and plated in the same manner as the unheated homogenized stool sample. After 5 days of growth at 37 °C in an anaerobic chamber, single colonies are selected. The selected single colonies are streaked, grown as described above, and the colony purification process is repeated by reselecting single colonies. The single colonies are frozen in 15%-25% glycerol in cryotubes and stored at -80 °C.

[0085] Administration of Brachyspira / consortium to mitigate experimental GVHD C57BL / 6 mice purchased from The Jackson Laboratory (Bar Harbor, Maine) were treated with oral vancomycin and ampicillin. Following decontamination, the mice were housed under autoclave conditions (caging, bedding, water, and food) to eliminate nearly all endogenous Clostridium present in the mouse flora. The mice were then treated with enteral nutrition with either a liquid suspension of cultured Enterococcus faecalis or a Brachyspira isolate as a control. The mice were then exposed to a single dose of myeloablative total body irradiation (TBI, 11 Gy) and then transplanted with bone marrow and purified T cells from fully MHC-incompatible B10.BR mice (H2k to H2b) by intravenous injection. The effects on intestinal pathology and overall survival were evaluated as described. Mice colonized with Brachyspira were protected from GVHD and had improved survival compared to those with Enterococcus (Figure 9).

[0086] GVHD clinical and histological scoring Mice were monitored daily for survival and weekly for GVHD clinical score (see Cooke, K.R., L. Kobzik, T.R. Martin, J. Brewer, J. Delmonte Jr., J.M. Crawford, and J.L. Ferrara. 1996. An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin. Blood. 88:3230 - 3239). Small intestine, large intestine, and liver samples were histologically evaluated for evidence of GVHD and scored as previously described (see Hill, G.R., J.M.Crawford, K.R. Cooke, Y.S. Brinson, L. Pan, and J.L. Ferrara. 1997. Total body irradiation and acute graft - versus - host disease: the role of gastrointestinal damage and inflammatory cytokines. Blood. 90:3204 - 3213).

[0087] Measurement of Paneth cell number and function The lumens of the small intestines of adult mice are rinsed with ice - cold water and dissected. Crypts are eluted first by turning the sections inside - out and then by agitating them in PBS containing 30 mM EDTA and lacking Ca2+ and Mg2+. The eluted villi and crypts are pelleted at 700 x g, resuspended in PBS, and transferred to silicon - treated microtubes using a capillary pipette. In preparation for exposure to secretory stimuli, the crypts are resuspended in iPIPES buffer (10 mM PIPES (pH 7.4) and 137 mM NaCl).

[0088] Crypts are incubated in 30 μl of iPIPES containing 1000 bacterial (Clostridiales) CFU per crypt for 30 min at 37°C. Cellular components are pelleted by brief centrifugation and the supernatant transferred to a sterile microtube and stored at -20°C. This method may be scaled up to approximately 3000 crypts in 2 ml iPIPES (more or less Clostridiales bacteria). Crypts are pelleted and 10 μL of the supernatant is analyzed for bactericidal activity against Clostridiales and Enterococcus bacteria in liquid medium or on agar plates. Proteins are extracted from the remaining supernatant as well as from the crypts using 30% acetic acid. Total proteins extracted from each fraction were resolved by AU-PAGE and subjected to Western blot analysis using anti-cryptdin-1 as follows: Proteins from AU-PAGE are transferred to a nitrocellulose membrane. The membrane was then blocked with 5% nonfat milk and incubated sequentially with anti-rabbit mouse scriptin-1 (1:500), horseradish peroxidase-conjugated anti-rabbit IgG (1:20,000), and chemiluminescent substrate (SuperSignal, Pierce, Rockland, IL) for visualization (Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME, Ouellette AJ, 2000. Secretion of microbicidal α-defensins by intestinal Paneth cells. in response to bacteria.Nature Immunology 1:113-118).

[0089] Methods for measuring levels of microorganisms in distal organs (liver, thymus, lungs, kidneys) Quantitative PCR (qPCR) of bacterial 16Sr RNA genes was performed in tissue samples using the DyNAmo SYBR Green qPCR kit (Finnzymes) and 0.2 μM of the universal bacterial primer 8F (5'-AGAGTTTGATCCTGGCTCAG-3' SEQ ID NO:1) and broad-range bacterial primer 338R (5'-TGCTGCCTCCCGTAGGAGT-3' SEQ ID NO:2). A standard curve was generated by serial dilutions of a PCR blunt vector (Invitrogen) containing one copy of the 16Sr RNA gene.

[0090] Methods for measuring the effects of microorganisms on bacterial metabolites such as SCFA levels Short chain fatty acids (SCFAs) are produced by many bacteria as by-products of carbohydrate fermentation. SCFAs have been found to be important regulators of the immune system. They are abundantly produced by Blautia and related bacteria from the class Clostridium. To evaluate how Blautia and related bacteria alleviate the suppression of GVHD, fecal pellets were collected to quantify SCFA levels, specifically acetate, propionate, or butyrate. SCFAs, creatine, and hydroxy-SCFAs were quantified by alkalizing the stool samples and using 1D 1H NMR on a Bruker Avance-600 MHz spectrometer to obtain a fingerprint of the metabolic composition of the samples and analyzed with supervised multivariate statistical methods using the Chenomx NMR Suite software.

[0091] Administration of SCFAs to mitigate GVHD. Preliminary experiments were conducted to test the effect of SCFA administration on murine GVHD. No significant efficacy was observed for propionate administered via drinking water (data not shown), or butyrate administered via drinking water or enema (data not shown), but marked efficacy was observed for acetate administration via drinking water. Sodium acetate (150 mM) is delivered via the drinking water of mice starting 2 weeks before BMT. The mice are then treated with radiation and transplanted with continued supplementation of sodium acetate. Prognoses evaluated in the mice include GVHD clinical score, survival rate, and histopathology on days 14 and 28. Kaplan-Meier curves show the overall survival of the two groups. In addition, the area under the curve (AUC) is the sum of the weekly total GVHD scores for each mouse from the time of injection to week 13. The mice are euthanized to evaluate pathological evidence of GVHD and to quantify and characterize colonic Tregs and alloreactive effector T cells by flow cytometry on days 14 and 28.

[0092] Method for measuring the effect of microbial metabolites (SCFA) on intestinal crypt regeneration An intestinal epithelial crypt culture system as previously described (Toshiro Sato, Robert We used (G.Vries, Hugo J. Snippert, Marc van de Wetering, Nick Barker, Daniel E. Stange, Johan H. van Es, Arie Abo, Pekka Kujala, Peter J. Peters & Hans Clevers, 2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche, Nature, 459:262-265). Four hundred crypts per well were suspended at 4°C in liquefied growth factor-reduced Matrigel (Corning) (25% Advanced DMEM / F12 medium (Gibco), 75% growth factor-reduced Matrigel). They were then plated into pre-warmed Delta Surface Nunc 24-well plates at 50 μL drops for the small intestine and 30 μL drops for the large intestine (each containing approximately 100-500 crypts). After the Matrigel drops polymerized, 500 μL of complete crypt medium, small intestine crypt medium (ENR-medium: advanced DMEM / F12 (Sigma), 2 mM L-glutamine (Sigma), 10 mM HEPES (Sigma), 100 U / ml penicillin / 100 μg / ml streptomycin (Sigma), 1 mM N-acetylcysteine (Sigma), 1× B27 supplement (Invitrogen,), 1× N2 supplement (Invitrogen), 50 ng / ml mEGF (Peprotech), 100 ng / ml mNoggin (Peprotech) and 10% human R-spondin-1 conditioned medium from HEK 293T cells transfected with hR-spondin-1 were added. In some experiments to evaluate budding, hR-spondin-1 was reduced to 1.25 - 5%. Colonic crypts were cultured in WENR medium containing 50% Wnt3a conditioned medium in addition to the aforementioned proteins and 1% bovine serum albumin (Sigma). In the case of colonic medium, 10 μM SB202190 (Sigma, Cat.nr.S7067) and the ALK5 inhibitor (A83-01, Tocris) were added to WENR. All plates were incubated at 37 °C / 5% CO2 and the medium was replaced every 2 - 3 days. Control wells were left untreated and, where appropriate, different concentrations of bacterial metabolites were added to the treated wells along with medium changes. Crypts were passaged on day 7 by mechanically disrupting them with a serum pipette, washing away Matrigel by centrifuging the crypts in excess medium, and replating them after reconstituting the pellet in liquefied Matrigel.

[0093] Selection of oligosaccharides for increasing Blautia Using a Blautia isolate from C57BL / 6 and Lactobacillus johnsonii from C57BL / 6, their ability to ferment various sugars was evaluated using pH and optical density to assess cell growth in medium lacking glucose. Lactobacillus johnsonii was evaluated because this bacterium expands at the expense of Clostridium in calorie-restricted situations and is thus likely a direct competitor for nutrients in the mouse intestine. Two sugars, rhamnose and xylose, fermented by Blautia rather than Lactobacillus were identified from this analysis.

[0094] Administration of oligosaccharides to increase Blautia and alleviate experimental GVHD C57BL / 6 mice were orally inoculated with a murine Blautia isolate known to ferment xylose. Subsequently, some mice were administered xylose in drinking water (10 g / L) starting 7 days before BMT using B10.BR BM and T cells. These mice receiving xylose exhibited an increase in Blautia in the gut flora 14 days after BMT despite the presence of GVHD (measured by 16S deep sequencing) (Figure 9). Interestingly, long-term administration of xylose also resulted in an improvement in the survival rate of mice with GVHD.

[0095] Further studies show the growth of strains from a Clostridium mixture in BHI medium without glucose to which various sugars are added. Glucose yielded the best results and was able to support the growth of 10 out of a total of 17 strains. However, glucose has limited effectiveness as it probably does not confer a selective advantage to beneficial bacteria. Raffinose was able to support 5 strains, while cellobiose seemed to support 4 strains not supported by raffinose. Thus, in one embodiment, a mixture of raffinose and cellobiose can be administered to provide support for at least a portion of beneficial bacteria.

[0096] Detection of metabolites produced by cultured Blautia. Fingerprints of the metabolic composition of the samples were obtained using 1D 1H NMR on a Bruker Avance-600 MHz spectrometer, and bacterial metabolites including SCFA, creatine, and hydroxy-SCFA were quantified by analyzing with multivariate statistical methods under monitoring using Chenomx NMR Suite software.

[0097] In vitro assay to show suppression of Blautia by microorganisms with a dominant gut microbiota dysbiosis due to free radical production By studying the effect of calorie restriction on the gut microbiota composition, it was observed that the abundance of obligate anaerobes (phylum Bacteroidetes, Clostridium spp.) decreased, while the facultative anaerobes (phylum Proteobacteria, order Lactobacillales) expanded. The production of free radicals by Escherichia coli in the starvation situation has been previously described (Saby S, et al. Appl Environ Microbiol. 1999;5600-5603.). The experiments were designed to test in vitro whether Lactobacillus johnsonii can suppress the growth of our mouse Blautia isolates under starvation conditions and to further investigate whether the production of free radicals can be a contributing factor. Specifically, when Blautia was cultured either alone or together with L. johnsonii, it was observed that L. johnsonii actually suppressed the growth of Blautia, but could not do so when additional medium was added to prevent starvation (Figure 11). The effect of the medium that supported the growth of L. johnsonii until the stationary phase was further investigated after sterilization. The lacto-conditioned medium did not affect the growth of Lactobacillus when mixed with fresh medium at a 1:1 ratio, but could suppress the growth of Blautia. Under starvation conditions, Lactobacillus was shown to release a substance that suppresses the growth of Blautia, rather than Lactobacillus itself. Next, it was examined whether a reducing agent could rescue Blautia from lacto-conditioned-media-mediated suppression. It was found that L-cysteine, ascorbic acid, and sodium thioglycolate could all support Blautia growth in the presence of lacto-conditioned medium. In summary, these results suggest that Lactobacillus can gain a competitive advantage over Blautia in a limited nutrient situation due to the production of free radicals that selectively harm obligate anaerobic bacteria due to the lack of defensive enzymes including glutathione transferase, catalase, and superoxide dismutase.

[0098] In vitro combination of Blautia + xylose and its effect on bacterial metabolites Brachyarcha was cultured for 48 hours in liquid PY medium alone or supplemented with glucose or xylose (10 g / L). The medium was centrifuged, and the supernatant was evaluated for SCFA levels.

[0099] Antibiotic classification The antibiotics used during transplantation hospitalization were divided into those with significant activity against anaerobic bacteria (piperacillin-tazobactam, ticarcillin-clavulanic acid, imipenem-cilastatin, meropenem, metronidazole, oral vancomycin, and clindamycin) and those with reduced activity (intravenous vancomycin, ceftriaxone, ceftazidime, cefepime, aztreonam, trimethoprim-sulfamethoxazole). 19 into two groups.

[0100] Transplantation practice As per institutional practice, patients receiving ciprofloxacin prophylaxis and patients receiving more intensive preconditioning followed by a non-myeloablative regimen also received intravenous vancomycin prophylaxis starting on day 2 and lasting until day 7. 20 Antibiotic prophylaxis against Pneumocystis jirovecii (trimethoprim-sulfamethoxazole, inhaled pentamidine, or atovaquone) was given at the discretion of the transplant physician.

[0101] Statistical analysis The incidences of acute GVHD and GVHD-related death were estimated using the cumulative incidence function, treating relapse and GVHD-unrelated death as competing events, and compared between factors using Gray’s test. Overall survival probability was estimated using the Kaplan–Meier methodology and compared using the log-rank test. Comparison of bacterial abundance was performed using the Mann–Whitney U for unpaired tests. In the mouse experiments, data were presented as mean ± SEM. Survival curves were analyzed by the Mantel–Cox log-rank test. For other comparisons, the nonparametric Mann–Whitney U test was used. Statistical significance for all analyses was defined as P < 0.05 based on two-sided tests. Statistical analyses were performed using R version 3.1.0 (The R Foundation for Statistical Computing, Vienna, Austria) and GraphPad Prism version 6.00 for Macintosh (GraphPad Software, San Diego, California, USA).

[0102] Composition of the gut flora and GVHD-related death—the influence of diversity and identification of predictive subsets of bacteria Our group has recently reported that increased bacterial diversity at engraftment is associated with improved overall survival and transplant-related death after allo BMT, but in that heterogeneous patient population, we were unable to determine whether diversity was associated with a decrease in GVHD 13In the present study, the inventors began by asking whether bacterial flora diversity can predict fatal GVHD in a more homogeneous population of patients at high risk of developing GVHD. The inventors utilized stored stool samples collected from patients who received allo BMT at their facility. The inventors identified a cohort of 64 patients who provided stool samples after conventional allo BMT without T cell depletion, both after BMT infusion and prior to discharge (days 8 - 16 after collection, median day 12, clinical characteristics summarized in Table 2). The inventors analyzed the flora composition of these stool samples by sequencing the V1 - V3 regions of the 16S rRNA gene using the 454 platform and clinically followed the patients for the occurrence of GVHD - related death.

Table 2

[0103] The inventors ranked the patients into two equal - sized groups by the median - method Shannon diversity index and found that an increase in bacterial diversity was actually associated with a decrease in GVHD lethality (Figure 1A, p = 0.005). To identify bacterial subsets associated with either an increase or decrease in GVHD - related death, the inventors used linear discriminant analysis (LDA) effect size (LEfSe) 27 as a hypothesis - generating approach to compare the abundances of bacterial genera from patients who died or did not die from GVHD. The inventors found that bacteria belonging to the genus Blautia were most significantly associated with a decrease in GVHD - related death (Figure 1B, p = 0.01). The genus Blautia includes, among others, anaerobic gut symbiotic organisms within the class Clostridia 28、29 .

[0104] We assessed Blautia abundance as a predictor of GVHD-related mortality by stratifying patients by median Blautia abundance of 0.05% and found that higher Blautia abundance was associated with reduced GVHD-related mortality (Figure 1C, p=0.04). We repeated this analysis in a subsequent cohort of 51 patients (clinical characteristics summarized in Table 3). [Table 3]

[0105] Includes analysis of V4-V5 of the 16S rRNA gene and is compatible with the MiSeq platform. Despite the differences in sequencing methodology used, this independent cohort showed similar results, with median Blautia abundance again at 0.05% and confirmation of the association of Blautia abundance with lower GVHD lethality (Figure 1C, p=0.01). By evaluating the combined cohort, we found that Blautia abundance robustly predicted improved overall survival after allo BMT. This was largely determined by reduced GVHD-related mortality and, to a lesser extent, reduced relapse-related mortality (p=0.03), with no difference in non-GVHD treatment-related mortality (Figure 2). Similar results were obtained when the cohorts were analyzed separately (data not shown). By adjusting for the two most easily modifiable risk factors in acute GVHD, graft source and conditioning intensity, we found that Blautia abundance maintained its association with reduced GVHD-related mortality (HR 0.13 [0.04-0.46], p=0.001). For recurrence-related mortality, an adjusted model taking into account disease risk and graft source showed a reduced association with Blautia abundance (p=0.055).

[0106] We evaluated the association of other bacteria with GVHD-related mortality. 30Since it can be associated, the inventors evaluated whether Enterococcus, or potentially beneficial bacteria (Lactobacillus and Bacteroides), were associated with GVHD-related death in their patient population. The inventors also evaluated Bacteroides, which was predicted to be associated with an increase in GVHD-related death by LEfSe analysis of the first patient flora cohort (p = 0.047). The inventors' results indicate that none of these bacterial taxa predicted GVHD-related death in the combined cohort (Figure 5).

[0107] The inventors also asked whether bacterial subtypes associated with Blautia could predict a decrease in lethal GVHD. Bacteria of the genus Blautia are classified as follows: family - Ruminococcaceae, order - Clostridiales, class - Clostridia, and phylum - Firmicutes 28 Analyzing patients by the abundance of bacteria from Ruminococcaceae, Clostridiales, and Clostridia all showed an association with a decrease in the incidence of lethal GVHD, suggesting that members of Blautia and potentially its close relatives contribute to a protective effect against lethal GVHD (data not shown). At the species level, similar to the results at the genus level, three Blautia taxa were associated with a decrease in GVHD-related death.

[0108] By identifying Blautia as a promising biomarker for GVHD-related death, the inventors asked whether it also correlates with a decrease in clinically acute GVHD. The inventors' results show that the Blautia abundance can be associated with a decrease in the incidence of acute GVHD grades 2-4, but this did not reach statistical significance (p = 0.1), and there was no association with acute GVHD grades 3-4 (Figure 3A). However, the Blautia abundance predicted a decrease in the onset of acute GVHD requiring treatment with systemic corticosteroids (p = 0.01), suggesting that the loss of Blautia is associated with acute GVHD that does not respond to topical corticosteroids alone. Regarding standard acute GVHD target organs, an increase in Blautia abundance was not associated with skin GVHD or upper gastrointestinal GVHD (Figure 3B). It tended to be associated with a decrease in lower gastrointestinal GVHD (p = 0.1), and although the number of events was small, it was associated with a decrease in liver GVHD (p = 0.02).

[0109] The inventors further examined the association between the abundance of Blautia and the prognosis of GVHD while adjusting for clinical risk factors. After adjusting for two of the most readily modifiable risk factors in acute GVHD, the source of the graft and the intensity of the pretreatment, the inventors found that the abundance of Blautia maintained an association with a decrease in GVHD that caused treatment with systemic steroids (HR 0.39 [0.19 - 0.78], p = 0.009) and death (HR 0.13 [0.04 - 0.46], p = 0.001). The limited number of events in the inventors' patient population made it impossible to adjust for additional factors. To support this analysis, the inventors found that in patients grouped by pretreatment intensity, Blautia remained predictive of protection against lethal GVHD in patients who received non-myeloablative pretreatment (Figure 7A, p = 0.02), and there was a tendency for it to be associated with protection in patients who received myeloablative and reduced-intensity pretreatment (p = 0.1 and p = 0.1). Among patients grouped by the source of the graft, patients who received peripheral blood stem cell grafts showed a strong association between the abundance of Blautia and lethal GVHD (Figure 7B, p = 0.002), while patients who received cord blood stem cell grafts had a tendency to show this association (p = 0.2). Collectively, these results suggest that studies of larger cohorts of patients may demonstrate an association between the abundance of Blautia and a decrease in GVHD lethality across different pretreatment intensities and sources of grafts.

[0110] The abundance of Blautia is independent of known clinical acute GVHD risk factors To determine whether the abundance of Blautia provides additional prognostic information, the inventors investigated the potential of the abundance of Blautia and known risk factors for acute GVHD 31~33The inventors found that pretreatment intensity, patient age, performance status, donor / patient gender, CMV status, and disease risk were not associated with the presence of Blautia (Table 4). Although limited in number, Asian or Latin American patients appeared to have lower levels of Blautia. Finally, evaluating Blautia presence and graft source was not relevant. In summary, the inventors' analysis indicates that Blautia presence does not appear to be associated with known risk factors for acute GVHD.

Table 4-1

Table 4-2

Table 4-3

[0111] Identification of potential clinical determinants of Blautia presence during allo BMT hospitalization To better understand the heterogeneity of Blautia presence in the inventors' patient population, the inventors attempted to identify determinants of Blautia presence. Analysis of all stool samples from both flow cohorts showed that most patients had relatively large amounts of Blautia at the time of transplant hospitalization application (>0.1 (10%) median abundance) (Figure 4A). However, in many patients, Blautia levels rapidly decreased during the hospitalization period. As expected, the inventors found that patients not exposed to antibiotics with anaerobic coverage tended to have increased levels of Blautia (Figure 4B).

[0112] Due to post-treatment nausea and mucositis, allo BMT patients commonly experience a significant reduction in oral intake over a long period and are treated with adjunctive TPN. The inventors used the duration of TPN supplementation as an indicator of oral nutrition and examined its association with the abundance of Blautia. Interestingly, patients who had received TPN for less than 10 days (showing TPN therapy that had been delayed, interrupted, or discontinued since consideration of initiation on day 2, and stool samples were collected on average on day 12) had increased levels of Blautia (Figure 4B). The duration of TPN continuation was also associated with loss of Blautia even in patients who had avoided treatment with anaerobic-acting antibiotics (Figure 4C). Collectively, these results indicate that both anaerobic antibiotic therapy and inadequate oral nutritional intake appear to alleviate the suppression of Blautia in the intestinal tract.

[0113] In this study, the inventors began by discovering in two independent cohorts of allo BMT recipients that the bacterial genus from stool samples most strongly associated with a reduction in GVHD-related death was Blautia. Patients with more Blautia also showed a decreased incidence of acute GVHD requiring treatment with systemic corticosteroids and an improvement in overall survival. To demonstrate these relationships, the inventors ranked patients by their Blautia abundance and stratified them by the median (which was fortuitously 0.05% in both cohorts).

[0114] Surprisingly, despite its association with GVHD-related death, Blautia abundance did not characterize the incidence of acute GVHD grades 3 - 4, which is known to identify patients who are less responsive to steroids and result in lower survival rates. 34 . However, a subset of patients is known to initially present with grade 2 acute GVHD, and these patients remain poor, which is a new GVHD grading system 34 or a new biomarker 35can be identified by. Further investigation of additional patient cohorts may determine whether the presence of Blautia can similarly be added to the predictive utility of clinical acute GVHD grading.

[0115] The abundance of bacteria of the class Clostridia, including Blautia, has also been shown to predict a decrease in GVHD-related deaths in the inventors' patients. Interestingly, some of the 17 Clostridia isolates included a strain with a 16S sequence that most closely matched that of the 16S sequence of Blautia producta species (ATCC R 27340-DSM2950, American Type Culture Collection, Manassas, VA), a very close relative of members of the genus Blautia. The beneficial anti-inflammatory relevance of Blautia has been observed in other clinical situations including colorectal cancer 36 , inflammatory ileitis after ileoanal anastomosis 36 , and cirrhosis 37 .

[0116] Treatment with antibiotics with increased activity against anaerobic bacteria has been associated with an increase in GVHD-related deaths and a decrease in the order Clostridiales in allo-HSCT patients who develop neutropenic fever. In a second study, the inventors began by asking whether treatment with antibiotics targeting anaerobic bacteria is associated with a clinical difference in GVHD-related mortality. At the inventors' facility, Allo-HSCT patients receive a prophylactic regimen of antibiotics, including short-term trimethoprim-sulfamethoxazole to prevent Pneumocystis jirovecii pneumonia, as well as intravenous vancomycin and ciprofloxacin over the period of neutropenia. In particular, the inventors discovered that this regimen usually results in only mild perturbation of the composition of the gut microbiota (14). In the second half of the Allo-HSCT period, patients who develop neutropenic fever are treated with experimental antibiotics, the selection of which can vary due to drug allergy history or patient-specific considerations. Some patients who develop persistent fever, develop abdominal symptoms, or have microbiological evidence of infection with resistant bacteria may often receive a second antibiotic that is more active against anaerobic bacteria. Finally, Allo-HSCT patients also commonly are diagnosed with and treated for Clostridium difficile colitis, which rapidly causes a loss of anaerobic intestinal commensalism, during Allo-HSCT hospitalization (17, 18).

[0117] The inventors retrospectively identified a cohort of 538 adult patients who underwent allo-HSCT at the inventors' facility continuously from 1994 to 2013 and who met the inventors' inclusion criteria of being at standard risk of GVHD (i.e., without ex vivo T-cell depletion) and receiving treatment for neutropenic fever. Patients who received a second antibiotic or an antibiotic for the treatment of Clostridium difficile colitis (either metronidazole intravenously or orally, or vancomycin orally) were excluded. The remaining 283 patients were classified into patients who received an antibiotic more active against anaerobic bacteria (mainly piperacillin-tazobactam (pip / tazo) and imipenem-cilastatin (imipenem)), or patients who were treated with an antibiotic with low activity against anaerobic bacteria (mainly cefepime and aztreonam) (19), and the clinical characteristics are provided in Table 2. The inventors found that 225 patients who received an antibiotic with anaerobic activity had a significant increase in the incidence of GVHD-related death in the first year after allo-HSCT (Figure 1A, p = 0.04). Univariate analysis of previously identified GVHD risk factors did not show a significant association with GVHD-related death in this dataset (Table 3), suggesting that the type of antibiotic exposed could be a novel predictor of GVHD-related death. The inventors also performed a multivariate analysis to evaluate the association between the type of antibiotic exposed and GVHD-related death while adjusting for GVHD risk factors associated with GVHD-related death in the inventors' patient population using a significance criterion of p < 0.1. The inventors found that the type of antibiotic exposed remained significant after adjustment for donor / HLA compatibility (p = 0.047) (Table 3). These results support the possibility that selecting an antibiotic that preserves anaerobic symbiosis could reduce the risk of GVHD. The alternative hypothesis, that patients with a history of allergy to penicillin (and thus likely to receive cephalosporin or aztreonam instead of penicillin and carbapenem) could be protected against GVHD, appears to have low biological plausibility.

[0118] HSCT patients were associated with the intestinal bacterial composition. In 2009, the inventors' facility began collecting stool samples from patients receiving allo-HSCT on a weekly basis in anticipation of the future. From this specimen bank, the inventors identified pairs of stool samples collected from patients before and after the initiation of specific antibiotics during the allo-HSCT period. Representative cases of patients treated for neutropenic fever and patients who did not require therapeutic antibiotics (but received prophylactic antibiotics) are shown in FIGS. 15B-G. Using 16S rRNA gene deep sequencing, the inventors evaluated the impact of these antibiotics on the microbial composition. The inventors focused on changes in the abundance of the Clostridiales, a major order of anaerobic Gram-positive commensal bacteria that includes many species associated with intestinal health (8, 13, 20).

[0119] The inventors found that patients often showed a loss of the Clostridiales, which transiently coincided with the initiation of treatment with imipenem, pip / tazo, or metronidazole, while treatment with aztreonam often resulted in relative preservation of the Clostridiales abundance (FIGS. 15B-G). By quantifying the change in Clostridiales abundance before and after the initiation of specific antibiotics, the inventors found that all patients treated with imipenem, pip / tazo, and metronidazole had significantly lower Clostridiales abundance compared to patients treated with aztreonam (FIG. 15H).

[0120] Treatment with imipenem (compared to aztreonam) has been associated with increased disruption of the intestinal microbiota and exacerbation of GVHD in mice. To further explore the causal relationship and mechanism of the effect of antibiotics with anaerobic activity on GVHD, the inventors switched to experiments in mice. First, the inventors treated healthy C57BL / 6 mice with either an antibiotic with increased activity against anaerobic bacteria (pip / tazo, imipenem, and metronidazole) or an antibiotic with decreased activity (aztreonam and cefepime). The mice were treated twice a day for 2 days by subcutaneous (SC) injection of each antibiotic (500 mg / kg for pip / tazo and 100 mg / kg for the others), fecal samples were collected, and subsequently 16S rRNA gene amplification and sequence analysis were performed. The inventors found that systemic treatment with imipenem or metronidazole significantly decreased the abundance of Clostridiales and increased the abundance of Enterococcus, while treatment with aztreonam or cefepime did not harm Clostridiales (Figure 16A). Interestingly, treatment with pip / tazo did not yield culturable bacterial DNA 2 days after treatment and showed almost complete decontamination in the mice (data not shown).

[0121] The inventors next investigated the impact of antibiotic treatment in a clinically relevant MHC-mismatched minor antigen-incompatible allo-HSCT model (from C57BL / 6 to 129S1). The inventors chose not to administer prophylactic antibiotics such as IV vancomycin or ciprofloxacin that minimally disrupt the gut microbiota composition, and focused on comparing the impact of aztreonam, which did not pose a risk to Clostridiales in both patients and mice, with imipenem, which eliminated Clostridiales in both patients and mice, when given in the first few weeks after allo-HSCT, similar to a common clinical scenario of post-transplant fever / neutropenia. Lethally irradiated 129S1 recipients were transplanted with C57BL / 6 T cell-depleted bone marrow (TCD-BM) cells and 1×106 C57BL / 6 splenic T cells. Control recipients received TCD-BM only. Recipients were treated three times a week starting 10 days after allo-HSCT with either aztreonam or imipenem SC. Notably, the inventors observed a marked increase in mortality in recipients treated with imipenem within 2 weeks of starting treatment (Figure 16B). Control recipients without T cell transplantation (no GVHD control) showed 100% survival, indicating that antibiotic treatment itself had no adverse effect on BM engraftment or survival after myeloablative irradiation. These results were reproducible in three consecutive and independent experiments. Histological examination of GVHD target organs 21 days after allo-HSCT (11 days after starting antibiotic therapy) revealed an increase in GVHD pathology in mice treated with imipenem. Interestingly, this was localized to the colon (Figures 16C and 18), and other common GVHD target organs such as the skin, liver, and small intestine showed no significant differences in the degree of inflammation and injury. Principal component analysis following 16S rRNA gene sequencing of fecal samples from mice with GVHD showed that aztreonam and imipenem therapies resulted in distinct patterns of microbiota composition (Figure 16D). Taxonomic groups that best explained the differences between these groups are depicted in Figures 16E and F, as analyzed by linear discriminant analysis effect size (LEfSe) (21).Transplanted mice treated with imipenem showed a loss of Clostridiales, corroborating the inventors' results in patients and non-transplanted mice.

[0122] Increase in Akkermansia muciniphila Interestingly, the increase in Akkermansia muciniphila was consistently observed in six experiments in these animals (Figures 16G and H). The inventors evaluated the effect of imipenem treatment on T cell invasion and STAT3 phosphorylation in the colon of mice with GVHD, and found an increase in the number of T cells invading the colon by both flow cytometry and histopathology, along with a significantly high level of phosphorylated STAT3 found in T cells in situ by fluorescence microscopy, supporting the idea that the elevated IL-23 level was involved in the recruitment and activation of donor T cells, which was highly likely to contribute to exacerbated GVHD, particularly in the colon. In mice with GVHD, imipenem treatment resulted in the expansion of Akkermansia muciniphila, a common commensal bacterium found in the gut of humans, mice, and other animals. Notably, this bacterium is exceptional in terms of its ability to utilize mucin as a source of carbon and nitrogen (33). The disruption of the colonic mucus layer has been observed after single colonization of germ-free mice with Akkermansia muciniphila, suggesting that Akkermansia muciniphila can reduce mucin in vivo as well as in vitro (51). However, what the effect of Akkermansia muciniphila on intestinal homeostasis is remains unclear and is likely to be environment-dependent. In a mouse obesity model, treatment with Akkermansia muciniphila resulted in the improvement of metabolic disorders and a decrease in the level of systemic endotoxin, suggesting that Akkermansia muciniphila improved the intestinal barrier function in this situation (52). However, the Salmonella typhimurium-infected notobiotic mouse model showed that the presence of Akkermansia muciniphila led to the exacerbation of intestinal inflammation, which was the cause of colonic mucus disruption (53). Examination of the colon by the inventors in imipenem-treated animals showed almost complete disappearance of the mucus layer. The inventors also detected the presence of bacteria within the colonic lamina propria beyond the disrupted mucus layer, which is consistent with the mucus layer that provides a critical first line of defense against intestinal mucosal invasion (54).Why Akkermansia expands in the colon of transplanted mice treated with imipenem is unclear, and to the inventors' knowledge, it has not been previously reported that Akkermansia isolates are resistant to imipenem or other related antibiotics. The competitive interaction between Akkermansia and the Clostridiales order has also not been previously explained to the inventors' knowledge, although multiple studies have observed a similar increase in Akkermansia after treatment of mice with other antibiotics that suppress the Clostridiales order, such as clindamycin (55). These results suggest that selecting antibiotics with a more limited spectrum of activity (especially against anaerobes) may prevent microbiota damage and reduce GVHD.

[0123] Clostridium is identified as a major producer of short-chain fatty acids (SCFAs), which play an important role in maintaining colonic homeostasis and health (23, 24). Surprisingly, despite a large difference in the abundance of the Clostridiales order, the inventors did not observe a significant change in the levels of SCFAs in the colon compared to samples from recipients treated with aztreonam or imipenem (data not shown).

[0124] To obtain further resolution of the bacterial composition between aztreonam-treated and imipenem-treated subject samples, the inventors performed metagenomic shotgun sequencing using stool samples collected 21 days after allo-HSCT. The inventors' findings, consistent with the 16S sequencing results, revealed that imipenem treatment, but not aztreonam treatment, led to an increase in the abundance of Akkermansia muciniphila (Figure 16I). However, since the most abundant reads from the analysis were determined to be unclassified, there may be further significant differences in bacterial species composition between the two antibiotic treatment types. Metagenomic shotgun sequencing analysis also revealed differences in gene content between microbiome samples from mice treated with aztreonam and imipenem, as depicted by principal component analysis of gene orthologs (Figure 16J). LEfSe analysis of gene pathways showed that microbiome genes in mice treated with imipenem were enriched in processes including lipopolysaccharide synthesis and relatively depleted in several processes including D-alanine metabolism (data not shown). Interestingly, lipopolysaccharides are well known to induce pro-inflammatory cascades in many disease processes including GVHD (25), while a decrease in the D-alanine content of lipoteichoic acid can enhance the anti-inflammatory properties of lactic acid bacteria (26, 27).

[0125] As described above, the inventors used 16S rRNA deep sequencing to detect an increase in Akkermansia muciniphila in the flora of imipenem-treated mice (Figure 16H). This bacterium has the ability to degrade mucus as a carbohydrate source (33, 34). Using the inventors' metagenomic shotgun sequencing results, the inventors asked whether genes predicted to encode secreted mucus-degrading enzymes were differentially present in mice treated with each antibiotic. The identification and characterization of bacterial mucus-degrading enzymes is still a relatively new field, but recent studies have examined the entire genomic sequence of Akkermansia muciniphila ATCC BAA-835 isolated from human feces (34). The authors identified two strong candidates for mucus degradation, Amuc_0953, a sulfatase, and Amuc_2164, a glycosyl hydrolase, both containing a predicted signal peptide cleavage site and a predicted mucin-binding domain. The inventors quantified the presence of sequences homologous to these two genes and found that both were significantly more abundant in samples from imipenem-treated mice (Figure 17G). The inventors then aimed to characterize the mucus layer of the colon in antibiotic-treated transplanted mice. Using periodic acid-Schiff staining, the inventors observed a significant decrease in the thickness of the mucus layer in recipients treated with imipenem on day 21 compared to recipients treated with aztreonam (Figures 17H and I). No difference in the number of mucus-producing goblet cells was seen between aztreonam-treated and imipenem-treated recipients, suggesting that mucus production was not impaired (Figure 17J). Furthermore, by using a general bacterial 16S rRNA probe (EUB338) (35) in addition to Muc2 staining, the inventors directly visualized the inner mucus layer in the colon of antibiotic-treated recipients and confirmed a dramatic thinning of the mucus layer in mice treated with imipenem. Notably, the inventors also histologically observed the dissemination of bacteria through the colonic epithelial barrier in imipenem-treated mice (Figure 17K), which was not seen in aztreonam-treated mice.In summary, these results indicate that imipenem treatment can exacerbate GVHD through a combination of factors such as thinning of the protective mucus layer and barrier dysfunction due to a decrease in the number of colonic B cells, increased infiltration by granulocytes, elevated levels of IL-23, and increased numbers and activity of donor effector CD4+ T cells.

[0126] One question arises as to whether the abundance of Blautia and other related bacteria as early as 12 days after allo BMT can biologically affect acute GVHD and GVHD-related death (which can occur months or, in the case of death, years after allo BMT). However, there is a precedent where, despite most of the symptoms of acute GVHD occurring after day 30, the serum cyclosporine concentration during the first week after allo BMT predicted the symptoms of acute GVHD 38 Similarly, the serum level of the biomarker ST2 predicted steroid-refractory GVHD, and levels as early as day 14 were associated with 6-month mortality without recurrence 35 In summary, these studies suggest that the early post-BMT pathology can influence the onset of GVHD and determine the ultimate severity of the GVHD process, although this may take months to fully clarify, likely due to partial suppression of inflammation by continuing immunosuppressant administration in the form of GVHD prevention and therapy.

[0127] Interestingly, while our results suggest that Blautia is associated with a decrease in GVHD, an increase in Blautia abundance was not associated with an increase in relapse-related mortality. This suggests that Blautia may be primarily associated with a local anti-inflammatory effect, a possibility supported by our finding of the lack of association with cutaneous GVHD. Collectively, these data suggest that targeting the microbiota may enable a reduction in GVHD without concurrently compromising the graft-versus-tumor effect. Indeed, we found an association between Blautia abundance and a decrease in relapse-related mortality, but this association was lost after adjusting for disease risk and graft source. Further studies are needed to more thoroughly examine the impact of the microbiota on relapse.

[0128] By characterizing the abundance of Blautia over the course of our patients' transplant hospitalizations, we found that most patients initially had relatively large amounts of Blautia, but that in many patients, Blautia species were subsequently lost dramatically. We identified two potential risk factors associated with Blautia loss: receipt of antibiotics with anaerobic coverage and a longer duration of TPN therapy. The finding of a decrease in Blautia with antibiotic administration is not surprising, but the association with long-term TPN was not predicted. Although pretreatment intensity is known to be associated with the duration of TPN dependence 39 we found no significant association between pretreatment intensity and Blautia abundance (data not shown). This suggests that inadequate enteral nutrition may contribute to Blautia loss more than more intensive pretreatment. This explanation is supported by findings in a mouse model in which myeloablative pretreatment was associated only with mild perturbation in the flora composition, compared with greater perturbation characterized by loss of Clostridiales, a potent inducer of anorexia seen in both mice and humans with signs of GVHD. 40。The pattern of loss of members of the order Clostridiales, including Roseburia, Faecalibacterium, Luminococcus, and Blautia species, can also be observed in volunteers on a high-protein and low-carbohydrate diet 41 or a diet derived entirely from animal-based foods. 42 。

[0129] From the foregoing detailed description of specific embodiments of the present invention, it should be readily apparent that a unique methodology for the use of Clostridiales bacteria to reduce risk and / or treat GVHD after bone marrow or hematopoietic stem cell transplantation has been described. While specific embodiments have been disclosed in detail herein, these have been made by way of example for purposes of illustration and are not intended to be limiting with respect to the appended claims. References References 1. Pasquini MC WZ. Current use and outcome of hematopoietic stem cell transplantation. CIBMTR Summary Slides, 2013. Available at: http: / / www.cibmtr.org. 2. Jones JM, Wilson R, Bealmear PM. Mortality and gross pathology of secondary disease in germfree mouse radiation chimeras. Radiat Res. 1971;45(3):577-588. 3. van Bekkum DW, Roodenburg J, Heidt PJ, van der Waaij D. 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Claims

1. A therapeutic composition for the prevention and / or treatment of graft-versus-host disease (GVHD) after bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), comprising two or more purified populations of bacteria, wherein the first purified population of bacteria in the composition comprises Dorea longicatena, and the second purified population of bacteria in the composition comprises bacteria selected from the group consisting of Lachnoclostridium ovalum, Lachnoclostridium lactaris (Blautia producta), Clostridium haswaei, Eubacterium contortum, Holdemanella filiformis, Eubacterium desmolans, Lachnoclostridium faecis, Clostridium sordellii, and combinations or mixtures thereof.

2. Does the Lachnoclostridium ovalum contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 1? Does the Clostridium haswaei contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 3? Does the Eubacterium desmolans contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 4? Does the Dorea longicatena contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 5? Does the Lachnoclostridium lactaris (Blautia producta) contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 7? Does the Eubacterium contortum contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 8? Does the Lachnoclostridium faecis contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 9? Does the Holdemanella filiformis contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 12 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 12? Or, The Clostridium sordellii includes 16S rDNA having a nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence that is about 98% to 100% identical to SEQ ID NO:

15. A therapeutic composition for the use according to claim 1.

3. The therapeutic composition for the use according to claim 1 or 2, wherein the bacteria in the composition are live bacteria, frozen bacteria, germinable spores, or a combination thereof.

4. The bacterium in the composition is present in a single dose of 10 ４ to 10 １０ CFU, and the therapeutic composition is for the use according to any one of claims 1 to 3.

5. The bacterium in the composition is present in a single dose of 10 ５ to 10 ９ CFU, and the therapeutic composition is for the use according to any one of claims 1 to 3.

6. The bacterium in the composition is present in a single dose of 10 ６ to 10 ８ CFU, and it is a therapeutic composition for the use according to any one of claims 1 to 3.

7. The therapeutic composition for the use according to any one of claims 1 to 6, wherein at least one of the bacteria in the composition ferments oligosaccharides selected from xylose, raffinose, cellobiose, melizitose, and combinations thereof.

8. The therapeutic composition for the use according to any one of claims 1 to 7, formulated for oral administration.

9. The therapeutic composition for the use according to any one of claims 1 to 7, formulated for colonic / rectal administration.

10. A therapeutic composition comprising a population of two or more purified bacteria for reducing the risk of developing graft-versus-host disease (GVHD) and / or treating GVHD in a subject undergoing bone marrow or hematopoietic stem cell transplantation, wherein a first purified population of bacteria in the composition comprises Dorea longicatena, and a second purified population of bacteria in the composition comprises bacteria selected from the group consisting of Lachnoclostridium ovalum, Lachnoclostridium lactaris (Blautia producta), Clostridium haswaei, Eubacterium contortum, Holdemania filiformis, Eubacterium desmolans, Lachnoclostridium faecis, Clostridium sordellii, and combinations or mixtures thereof.

11. The composition is (i) promoting the growth or activity of one or more bacterial taxa that are overwhelmingly low in the microbiota of the subject either before or after transplantation, or (ii) suppressing the growth or activity of one or more bacterial taxa that are overwhelmingly abundant in the microbiota of the subject. The therapeutic composition for the use according to claim 10.

12. The therapeutic composition for the use according to claim 10 or 11, characterized in that it is administered to the subject.

13. The therapeutic composition for use according to claim 12, wherein the composition is administered to the subject about 1 day to about 2 weeks after discontinuation of the treatment of the subject with an antibiotic having high activity against anaerobic bacteria.

14. The therapeutic composition for use according to claim 12 or 13, wherein the composition is administered to the subject about 7 to 10 days before allogeneic BMT or allogeneic HSCT.

15. The therapeutic composition for use according to claim 12 or 13, wherein the composition is administered to the subject about 1 day to about 1 week before allogeneic BMT or allogeneic HSCT.

16. A therapeutic composition comprising two or more purified bacterial populations for use in the prevention, risk reduction, and / or treatment of GVHD in an individual undergoing allogeneic BMT or allogeneic HSCT, wherein the first purified population of bacteria in the composition comprises Dorea longicatena, and the second purified population of bacteria in the composition comprises bacteria selected from the group consisting of Lachnoclostridium ovalum, Lachnoclostridium lactaris (Blautia producta), Clostridium haswaei, Eubacterium contortum, Holdemania filiformis, Eubacterium desmolans, Lachnoclostridium faecis, Clostridium sordellii, and combinations or mixtures thereof.

17. Does the Lachnoclostridium ovalum contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 1? Does the Clostridium haswaei contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 3? Does the Eubacterium desmolans contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 4? Does the Dorea longicatena contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence about 98% to 100% identical to SEQ ID NO: 5? Does the Luminococcus lactaris (Brouchothrix producta) contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence that is about 98% to 100% identical to SEQ ID NO: 7, Does the Eubacterium contortum contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence that is about 98% to 100% identical to SEQ ID NO: 8, Does the Luminococcus faecis contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence that is about 98% to 100% identical to SEQ ID NO: 9, Does the Holdemania filiformis contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 12 or a nucleotide sequence that is about 98% to 100% identical to SEQ ID NO: 12, or Does the Clostridium sordellii contain 16S rDNA having the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence that is about 98% to 100% identical to SEQ ID NO: 15? A therapeutic composition for the use according to claim 16.

18. The therapeutic composition for the use according to claim 16 or 17, wherein the bacteria in the composition are live bacteria, frozen bacteria, germinable spores, or a combination thereof.

19. A method for determining the abundance of Dorea longicatena in a sample of fecal material from a subject as an indicator of the risk of developing GVHD after bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT), the method comprising determining the abundance of Dorea longicatena in a sample of fecal material from the subject, wherein when the abundance of Dorea longicatena in the sample of fecal material from the subject is less than the abundance of Dorea longicatena in a sample of fecal material from a subject at no risk of developing GVHD, the subject has an increased risk of developing GVHD.

20. The method according to claim 19, wherein the abundance of Dorea longicatena is from 0.5% to less than 0.01%. The method according to claim 19 or 20, wherein the Dorea longicatena in the sample of fecal material from the subject contains 16S rDNA having the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence that is about 98% to 100% identical to the nucleotide sequence of SEQ ID NO:

5.

22. A composition comprising a therapeutically effective amount of two or more purified populations of bacteria, wherein the first purified population of bacteria in the composition comprises Dorea longicatena, and the second purified population of bacteria in the composition comprises bacteria selected from the group consisting of Lachnoclostridium ovalum, Lachnoclostridium lactaris (Blautia producta), Clostridium haswaei, Eubacterium contortum, Holdemanella filiformis, Eubacterium desmolans, Lachnoclostridium faecis, Clostridium sordellii, and combinations or mixtures thereof, A composition that is suitable for reducing the likelihood, incidence, or severity of, or preventing or otherwise treating, graft-versus-host disease (GVHD) in a subject after bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT).

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