as) United States
`a2) Patent Application Publication (10) Pub. No.: US 2012/0276143 Al
` O’MAHONYetal. (43) Pub. Date: Nov.1, 2012
`
`
`
`US 20120276143A1
`
`(54) PROBIOTIC BIFIDOBACTERIUM STRAIN
`
`(76)
`
`Inventors:
`
`Liam O’MAHONY,Cork (IE);
`Barry Kiely, Cork (IE); John
`Francis Cryan, Cork (IE);
`Timothy Dinan, Cork (IE); Eileen
`Frances Murphy, Cork (IE)
`
`(21) Appl. No.:
`
`13/468,374
`
`(22)
`
`Filed:
`
`May10, 2012
`
`Related U.S. Application Data
`(63) Continuation ofapplication No. PCT/IE2010/000066,
`filed on Nov. 11, 2010.
`;
`(60) Provisional application No. 61/344,030,filed on May
`11, 2010.
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`A6IK 39/02
`AGIP 1/00
`aon Oe
`AGIP 37/00
`AG6IP 1/12
`AG6IP 25/00
`AGIP 25/28
`AG6IP 3/10
`AGIP 37/06
`
`(2006.01)
`(2006.01)
`(3006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`(2006.01)
`AG6IP 31/04
`(2006.01)
`AG6IP 31/12
`(2006.01)
`AG6IP 31/10
`(2006.01)
`A6IP 1/02
`(2006.01)
`A6IP 15/00
`(2006.01)
`A6IP 31/18
`(2006.01)
`A6IP 35/04
`(2006.01)
`AG6IP 3/04
`(2006.01)
`AGIP 17/02
`(2006.01)
`AG6IP 37/08
`(2006.01)
`A61P 11/06
`(2006.01)
`AG6IP 11/00
`(2006.01)
`AGIP 9/00
`(2006.01)
`A6IP 7/06
`(2006.01)
`A6IP 7/02
`(2006.01)
`AG6IP 13/12
`(2006.01)
`A6IP 1/16
`(2006.01)
`A6IP 9/10
`(2006.01)
`AGIP 19/10
`(2006.01)
`A6IP 5/00
`(2006.01)
`AG6IP 17/00
`(2006.01)
`AGIP 17/06
`(2006.01)
`A6IP 17/10
`(2006.01)
`CI2N 1/20
`(2006.01)
`AG6IP 29/00
`(52) US. Cd. wees ceteeeeeees 424/234.1; 435/252.1
`(57)
`ABSTRACT
`
`Probiotic Bifidobacterium strain AH1714 is significantly
`immunomodulatory following oral consumption. The strain
`is useful as an immunomodulatory biotherapeutic agent.
`
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`Patent Application Publication
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`Nov. 1, 2012 Sheet 1 of 9
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`US 2012/0276143 Al
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`Patent Application Publication
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`Nov. 1, 2012 Sheet 2 of 9
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`US 2012/0276143 Al
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`US 2012/0276143 Al
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`Nov. 1, 2012
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`PROBIOTIC BIFIDOBACTERIUM STRAIN
`
`CROSS REFERENCE TO RELATED
`APPLICATION
`
`[0001] This application is a continuation application of
`prior International Application No. PCT/IE2010/000066
`filed on Nov. 11, 2010 (aka 12424& WO) which claims the
`benefit ofU.S. application Ser. No. 12/616,752 (aka 11484M)
`filed Nov. 11, 2009 and U.S. Provisional Application No.
`61/344,030 (aka 12424P&)filed May 11, 2010.
`
`INTRODUCTION
`
`[0002] Theinvention relates to a Bifidobacterium strain and
`its use as a probiotic bacteria in particular as an immuno-
`modulatory biotherapeutic agent.
`[0003] The defense mechanismsto protect the human gas-
`trointestinal tract from colonization by intestinal bacteria are
`highly complex and involve both immunological and non-
`immunological aspects (1).
`Innate defense mechanisms
`include the low pH of the stomach, bile salts, peristalsis,
`mucin layers and anti-microbial compounds
`such as
`lysozyme (2). Immunological mechanisms include special-
`ized lymphoid aggregates, underlying M cells, called peyers
`patches which are distributed throughout the small intestine
`and colon (3). Luminal antigens presentedat these sites result
`in stimulation of appropriate T and B cell subsets with estab-
`lishment of cytokine networks and secretion of antibodies
`into the gastrointestinaltract (4). In addition, antigen presen-
`tation may occur viaepithelial cells to intraepithelial lympho-
`cytes and to the underlying lamina propria immunecells (5).
`Therefore, the host invests substantially in immunological
`defense of the gastrointestinal tract. However, as the gas-
`trointestinal mucosa is the largest surface at which the host
`interacts with the external environment, specific control
`mechanisms mustbe in place to regulate immuneresponsive-
`ness to the 100 tons of food which is handled by the gas-
`trointestinal tract over an average lifetime. Furthermore, the
`gut is colonized by over 500 species of bacteria numbering
`10''-10'/g in the colon. Thus, these control mechanisms
`must be capable of distinguishing non-pathogenic adherent
`bacteria from invasive pathogens, which would causesignifi-
`cant damageto the host. In fact, the intestinal flora contributes
`to defense of the host by competing with newly ingested
`potentially pathogenic micro-organisms.
`[0004] Bacteria present in the humangastrointestinal tract
`can promote inflammation. Aberrant immune responses to
`the indigenous microflora have been implicated in certain
`disease states, such as inflammatory boweldisease. Antigens
`associated with the normalflora usually lead to immunologi-
`cal tolerance and failure to achieve this tolerance is a major
`mechanism of mucosal inflammation (6). Evidence for this
`breakdown in tolerance includes an increase in antibody lev-
`els directed against the gut flora in patients with inflammatory
`bowel disease IBD).
`[0005] The present invention is directed towards a Bifido-
`bacterium strain which has been shown to have immuno-
`modulatory effects, by modulating cytokine levels or by
`antagonizing and excluding pro-inflammatory micro-organ-
`isms from the gastrointestinal tract.
`
`STATEMENTSOF INVENTION
`
`[0006] The invention provides an isolated strain of Bifido-
`bacterium NCIMB41676.
`
`[0007] The Bifidobacterium strain may be in the form of
`viable cells. The Bifidobacterium strain may be in the form of
`non-viable cells. The Bifidobacterium may beisolated from
`colonic biopsy tissue from a healthy human subject. The
`Bifidobacterium strain maybe significantly immunomodula-
`tory following oral consumption in humans.
`[0008] The invention also provides a formulation which
`comprises a Bifidobacterium strain as described herein. The
`formulation may further comprise a probiotic material. The
`formulation may further comprise a prebiotic material. The
`formulation may further comprise an ingestable carrier. The
`ingestable carrier may a pharmaceutically acceptable carrier
`suchas acapsule, tablet or powder. The ingestable carrier may
`be a food product such as acidified milk, yoghurt, frozen
`yoghurt, milk powder, milk concentrate, cheese spreads,
`dressings or beverages. The formulation may further com-
`prise a protein and/or peptide, in particular proteins and/or
`peptides that are rich in glutamine/glutamate, a lipid, a car-
`bohydrate, a vitamin, mineral and/or trace element. The Bifi-
`dobacterium strain maybe present in an amount ofmore than
`10° cfu per gram of the formulation. The formulation may
`further comprise an adjuvant. The formulation may further
`comprise a bacterial component. The formulation may further
`comprise a drug entity. The formulation may further comprise
`a biological compound. The formulation may be used for
`immunisation and vaccination protocols.
`[0009] Theinventionalso provides a Bifidobacterium strain
`or a formulation as described herein for use in foodstuffs.
`
`[0010] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein for use as a medicament.
`
`[0011] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein for usein the prophylaxis
`and/or treatment of undesirable inflammatory activity.
`[0012] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein for usein the prophylaxis
`and/or treatment of undesirable gastrointestinal inflamma-
`tory activity such as inflammatory bowel disease eg. Crohns
`disease or ulcerative colitis, irritable bowel syndrome; pou-
`chitis; or post infection colitis.
`[0013] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein for usein the prophylaxis
`and/or treatment of gastrointestinal cancer(s).
`[0014] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein for usein the prophylaxis
`and/or treatment of systemic disease such as rheumatoid
`arthritis.
`
`[0015] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein for usein the prophylaxis
`and/or treatment of autoimmunedisorders due to undesirable
`inflammatory activity.
`[0016] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein for usein the prophylaxis
`and/or treatment of cancer due to undesirable inflammatory
`activity.
`[0017] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein for usein the prophylaxis
`of cancer.
`
`[0018] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein for usein the prophylaxis
`and/or treatment of diarrhoeal disease due to undesirable
`
`inflammatory activity, such as Clostridium difficile associated
`diarrhoea, Rotavirus associated diarrhoea or post infective
`diarrhoea or diarrhoeal disease due to an infectious agent,
`such as E.coli.
`
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`[0019] Theinventionalso provides a Bifidobacterium strain
`or a formulation as described herein for use in the preparation
`of anti-inflammatory biotherapeutic agents for the prophy-
`laxis and/or treatment of undesirable inflammatory activity.
`[0020] Bifidobacterium strains as described herein may be
`used in the preparation of a panel of biotherapeutic agents for
`modifying the levels of IL-10.
`[0021] Theinventionalso provides a Bifidobacterium strain
`or a formulation as described herein for use in the prevention
`and/or treatment of inflammatory disorders,
`immunodefi-
`ciency,
`inflammatory bowel disease,
`irritable bowel syn-
`drome, cancer
`(particularly of the gastrointestinal and
`immune systems), diarrhoeal disease, antibiotic associated
`diarrhoea, paediatric diarrhoea, appendicitis, autoimmune
`disorders, multiple sclerosis, Alzheimer’s disease, rheuma-
`toid arthritis, coeliac disease, diabetes mellitus, organ trans-
`plantation, bacterial infections, viral infections, fungal infec-
`tions, periodontal disease, urogenital disease,
`sexually
`transmitted disease, HIV infection, HIV replication, HIV
`associated diarrhoea, surgical associated trauma, surgical-
`induced metastatic disease, sepsis, weight loss, anorexia,
`fever control, cachexia, wound healing, ulcers, gut barrier
`function, allergy, asthma, respiratory disorders, circulatory
`disorders, coronary heart disease, anaemia, disorders of the
`blood coagulation system,renal disease, disorders ofthe cen-
`tral nervous system, hepatic disease, ischaemia, nutritional
`disorders, osteoporosis, endocrine disorders, epidermal dis-
`orders, psoriasis, acne vulgaris, panic disorder, behavioral
`disorder and/or post traumatic stress disorders.
`[0022] The Bifidobacterium strain as described herein may
`act by antagonising and excluding proinflammatory micro-
`organisms from the gastrointestinaltract.
`[0023] Theinventionalso provides a Bifidobacterium strain
`or a formulation as described herein for use in the preparation
`of anti-inflammatory biotherapeutic agents for reducing the
`levels of pro inflammatory cytokines.
`[0024] The Bifidobacterium strain as described herein may
`be used as an anti-infective probiotic strain.
`[0025] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein foruse in the prophylaxis
`and/or treatment of bipolar illness, depression, mood disor-
`ders, and/or anxiety disorders.
`[0026] Theinventionalso provides a Bifidobacterium strain
`or a formulation as described herein may be used as a cogna-
`tive enhancer for the prophylaxis and/or treatment of disor-
`ders of the central nervous system such as Alzheimer’s dis-
`ease, schizophrenia and/or mild cognative disorder.
`[0027] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein foruse in the prophylaxis
`and/or treatment of obesity related inflammation.
`[0028] Theinventionalso provides a Bifidobacterium strain
`ora formulation as described herein foruse in the prophylaxis
`and/or treatment of obesity related metabolic dysregulation.
`[0029] We describe Bifidobacterium strain AH1714
`(NCIMB 41676) or mutants or variants thereof. The mutant
`maybe a genetically modified mutant. The variant may be a
`naturally occurring variant of Bifidobacterium. Also
`described is a rifampicin resistant variant of strain AH1714.
`The strain may be a probiotic. It may be in the form of a
`biologically pure culture.
`[0030] Wealso describe an isolated strain of Bifidobacte-
`rium NCIMB 41676. The Bifidobacterium strains may be in
`the form ofviable cells. Alternatively Bifidobacterium strains
`are in the form of non-viable cells. The general use of probi-
`
`otic bacteria is in the form ofviable cells. However, it can also
`be extended to non-viable cells such as killed cultures or
`
`compositions containing beneficial factors expressed by the
`probiotic bacteria. This could include thermally killed micro-
`organisms or micro-organisms killed by exposure to altered
`pH or subjection to pressure or gammairradiation. With non-
`viable cells product preparation is simpler, cells may be incor-
`porated easily into pharmaceuticals and storage requirements
`are muchless limited than viable cells. Lactobacillus casei
`
`YIT 9018 offers an example ofthe effective use ofheat killed
`cells as a method for the treatment and/or prevention of
`tumour growth as described in U.S. Pat. No. 4,347,240.
`[0031] The Bifidobacterium strains may be isolated from
`colonic biopsy tissue from healthy human subjects, the Bifi-
`dobacterium strains being significantly immunomodulatory
`following oral consumption in humans.
`[0032] Wealso describe a formulation which comprises the
`Bifidobacterium strain as described herein. The formulation
`mayinclude another probiotic material. The formulation may
`include a prebiotic material. Preferably the formulation
`includesan ingestable carrier. The ingestable carrier may bea
`pharmaceutically acceptable carrier such as a capsule, tablet
`or powder. Preferably the ingestable carrier is a food product
`such as acidified milk, yoghurt, frozen yoghurt, milk powder,
`milk concentrate, cheese spreads, dressings or beverages. The
`formulation may further comprise a protein and/or peptide, in
`particular proteins and/or peptides that are rich in glutamine/
`glutamate, a lipid, a carbohydrate, a vitamin, mineral and/or
`trace element. The Bifidobacterium strain may be present in
`the formulation at more than 10° cfu per gram ofdelivery
`system. Preferably the formulation includes any one or more
`of an adjuvant, a bacterial component, a drug entity or a
`biological compound.
`[0033] We also describe a Bifidobacterium strain or a for-
`mulation for use as foodstuffs, as a medicament, for use in the
`prophylaxis and/or treatment of undesirable inflammatory
`activity, for use in the prophylaxis and/or treatment of unde-
`sirable respiratory inflammatory activity such as asthma, for
`use in the prophylaxis and/or treatment of undesirable gas-
`trointestinal
`inflammatory activity such as inflammatory
`bowel disease eg. Crohns disease or ulcerative colitis, irri-
`table bowel syndrome,pouchitis, or post infectioncolitis, for
`use in the prophylaxis and/or treatment of gastrointestinal
`cancer(s), for use in the prophylaxis and/or treatmentof sys-
`temic disease such as rheumatoid arthritis, for use in the
`prophylaxis and/or treatment of autoimmunedisorders due to
`undesirable inflammatory activity, for use in the prophylaxis
`and/or treatment of cancer due to undesirable inflammatory
`activity, for use in the prophylaxis of cancer, for use in the
`prophylaxis and/or treatment of diarrhoeal disease due to
`undesirable inflammatory activity, such as Clostridium diffi-
`cile associated diarrhoea, Rotavirus associated diarrhoea or
`post infective diarrhoea, for use in the prophylaxis and/or
`treatment of diarrhoeal disease due to an infectious agent,
`such as E.coli.
`
`[0034] We also describe a Bifidobacterium strain or a for-
`mulation of the invention for use in the preparation of an
`anti-inflammatory biotherapeutic agent for the prophylaxis
`and/or treatment of undesirable inflammatory activity or for
`use in the preparation of anti-inflammatory biotherapeutic
`agents for the prophylaxis and/or treatment of undesirable
`inflammatory activity. The strain mayact by antagonising and
`excluding proinflammatory micro-organisms from the gas-
`trointestinaltract.
`
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`[0035] Wealso describe a Bifidobacterium strain or a for-
`mulation for use in the preparation of anti-inflammatory bio-
`therapeutic agents for reducing the levels of pro-inflamma-
`tory cytokines.
`[0036] The Bifidobacterium strain may be used in the
`preparation of anti-inflammatory biotherapeutic agents for
`modifying the levels of IL-10.
`[0037] The Bifidobacterium strain may be usedas a anti-
`infective probiotic due to their ability to antagonise the
`growth of pathogenic species.
`[0038] We have foundthat particular strains of Bifidobac-
`terium elicit immunomodulatory effects in vitro.
`[0039] The invention is therefore of major potential thera-
`peutic value in the prophylaxis or treatment of dysregulated
`immune responses, such as undesirable inflammatory reac-
`tions for example asthma.
`[0040] Bifidobacterium are commensal microorganisms.
`They have been isolated from the microbial flora within the
`human gastrointestinal tract. The immune system within the
`gastrointestinal tract cannot have a pronouncedreaction to
`membersofthis flora, as the resulting inflammatory activity
`would also destroy host cells and tissue function. Therefore,
`some mechanism(s) exist whereby the immune system can
`recognize commensal non-pathogenic members of the gas-
`trointestinal flora as being different to pathogenic organisms.
`This ensures that damage to hosttissues is restricted and a
`defensivebarrieris still maintained.
`
`[0041] A deposit of Bifidobacterium longum strain
`AH1714 was madeat the National Collections of Industrial
`
`and Marine Bacteria Limited (NCIMB) Ferguson Building,
`Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scot-
`land, UK on Nov.5, 2009 and accorded the accession number
`NCIMB41676.
`
`[0042] The Bifidobacterium longum may be a genetically
`modified mutant or it may be a naturally occurring variant
`thereof.
`
`Preferably the Bifidobacterium longum may be in
`[0043]
`the form ofviable cells.
`
`[0044] Alternatively the Bifidobacterium longum may be in
`the form of non-viablecells.
`
`It will be appreciated that the specific Bifidobacte-
`[0045]
`rium strain of the invention may be administered to animals
`(including humans)in anorally ingestible form in a conven-
`tional preparation such as capsules, microcapsules, tablets,
`granules, powder, troches, pills, suppositories, suspensions
`and syrups. Suitable formulations may be prepared by meth-
`ods commonly employed using conventional organic and
`inorganic additives. The amount of active ingredient in the
`medical composition may beat a level that will exercise the
`desired therapeutic effect.
`[0046] The formulation mayalso include a bacterial com-
`ponent, a drug entity or a biological compound.
`[0047]
`In addition a vaccine comprising the strains of the
`invention maybe prepared using any suitable known method
`and may include a pharmaceutically acceptable carrier or
`adjuvant.
`[0048] Throughoutthe specification the terms mutant, vari-
`ant and genetically modified mutant includea strain of Bifi-
`dobacteria whose genetic and/or phenotypic properties are
`altered compared to the parent strain. Naturally occurring
`variant of Bifidobacterium longum includes the spontaneous
`alterations of targeted properties selectively isolated. Delib-
`erate alteration of parent strain properties is accomplished by
`conventional (in vitro) genetic manipulation technologies,
`
`such as gene disruption, conjugative transfer, etc. Genetic
`modification includes introduction of exogenous and/or
`endogenous DNA sequencesinto the genomeofa Bifidobac-
`teria strain, for example by insertion into the genomeof the
`bacterial strain by vectors, including plasmid DNA,or bac-
`teriophages.
`[0049] Natural or induced mutationsincludeat least single
`base alterations such as deletion, insertion, transversion or
`other DNA modifications which mayresult in alteration ofthe
`amino acid sequence encoded by the DNA sequence.
`[0050] The terms mutant, variant and genetically modified
`mutant also include a strain of Bifidobacteria that has under-
`gone genetic alterations that accumulate in a genomeata rate
`whichis consistent in nature for all micro-organisms and/or
`genetic alterations which occur through spontaneous muta-
`tion and/or acquisition of genes and/or loss of genes which is
`not achieved by deliberate (in vitro) manipulation of the
`genomebutis achieved throughthe natural selection of vari-
`ants and/or mutants that provide a selective advantage to
`support the survival of the bactertum when exposed to envi-
`ronmental pressures such as antibiotics. A mutant can be
`created by the deliberate (in vitro) insertion of specific genes
`into the genome which do not fundamentally alter the bio-
`chemical functionality of the organism but whose products
`can be used foridentification or selection ofthe bacterium,for
`example antibiotic resistance.
`[0051] A person skilled in the art would appreciate that
`mutantor variantstrains ofBifidobacteria can beidentified by
`DNA sequence homology analysis with the parent strain.
`Strains of Bifidobacteria having a close sequence identity
`with the parent strain are considered to be mutant or variant
`strains. A Bifidobacteria strain with a sequence identity (ho-
`mology) of 96% or more, such as 97% or more, or 98% or
`more, or 99% or more with the parent DNA sequence may be
`considered to be a mutant or variant. Sequence homology
`may be determined using on-line homology algorithm
`“BLAST”program, publicly available at http://(www.ncbi.
`nlm.nih,gov/BLAST/.
`[0052] Mutants of the parent strain also include derived
`Bifidobacteria strains having at least 85% sequence homol-
`ogy, such as at least 90% sequence homology,or at least 95%
`sequence homology to the 16s-23s intergenic spacer poly-
`nucleotide sequenceof the parent strain. These mutants may
`further comprise DNA mutations in other DNA sequences in
`the bacterial genome.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0053] The invention will be more clearly understood from
`the following description thereof given by way of example
`only with reference to the accompanying drawings in
`which;—
`[0054]
`FIG. 1 is a graphillustrating transit of B. Jongum
`AH1714 through the gastrointestinal tract;
`[0055]
`FIG. 2isa photograph ofB. longum AH1714 grown
`on a Congo Red Agarplate;
`[0056]
`FIG. 3 is a bar chart illustrating the IL-10:IL-12p70
`ratio for PBMCsstimulated with Bifidobacterium longum
`strain 1714 (Bifidobacterium 1714);
`[0057]
`FIG. 4 isa bar chart showing the induction profile of
`IL-10 in splenocytes isolated from both 1714 and PBS fed
`mice with and without in vivo LPS challenge 1 mg/kg.In vitro
`cells are either unstimulated (A), stimulated with LPS (B) or
`stimulated with antiCD3/CD28 (C). Data is shown as Average
`& SEM;
`
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`FIG. 5is a bar chart showing the induction profile of
`[0058]
`TNF-a in splenocytes isolated from both 1714 and PBS fed
`mice with and without in vivo LPS challenge 1 mg/kg. In vitro
`cells are either unstimulated (A) or stimulated with antiCD3/
`CD28 (B). Data is shown as Average & SEM;
`[0059]
`FIG. 6 is a bar chart showing the induction profile of
`IFN-y in splenocytes isolated from both 1714 and PBS fed
`mice with and without in vivo LPS challenge 1 mg/kg. In vitro
`cells are either unstimulated (A) or stimulated with antiCD3/
`CD28 (B). Data is shown as Average & SEM.
`[0060]
`FIG. 7 is abar chart showing the induction profile of
`IL-12p70 in splenocytes isolated from both 1714 and PBS fed
`mice with and without in vivo LPS challenge 1 mg/kg. In vitro
`cells are either unstimulated (A) or stimulated with antiCD3/
`CD28 (B). Data is shown as Average & SEM;
`[0061]
`FIG. 8is a bar chart showing the induction profile of
`TNF-a (A) and IL-10 (B) in serum sampled from both 1714
`and PBS fed mice post 2H in vivo challenge with LPS 1
`mg/kg. Data is shown as Average & SEM;
`[0062]
`FIG. 9 is a bar chart showing NFkB activity (Pho-
`tons/second) from isolated spleen 3 hours post challenge with
`a single 0.5 mg/kg dose of LPS, from Placebo and 1714-fed
`animals (** designates p<0.01);
`[0063]
`FIG. 10 is a bar chart (A) showing NFkBactivity
`(Photons/second) from whole body imaging 1.5 hours post
`challenge with a single 0.5 mg/kg dose of LPS, from Placebo
`and 1714-fed animals ((B) and (C) are whole body represen-
`tative images in black and white and colour;
`[0064] FIG.11 is abar chart representing the time ofimmo-
`bility displayed by the mice over a 6-mintest;
`[0065]
`FIG. 12 is a line graph representing the freezing
`percentage in response to the fearful stimulus (context) for
`day 1 (acquisition), day 2 (memory/extinction) and day 3
`(extinction);
`[0066]
`FIG. 13 is a line graph representing the freezing
`percentage in responseto the fearful stimulus (cue) for day 1
`(acquisition), day 2 (memory/extinction) and day 3 (extinc-
`tion);
`FIG. 14 is a bar chart representing the number of
`[0067]
`marbles buried by the mice over a 30-min session;
`[0068]
`FIG. 15 is a bar chart representing the body tem-
`perature variation (AT) mice displayed following handling;
`and
`FIG. 16 is a bar chart showing the changesin cytok-
`[0069]
`ine levels in stimulated splenocytes from the Diet Induced
`Mouse model.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0070] A deposit of Bifidobacterium longum strain
`AH1714 was madeat the National Collections of Industrial
`and Marine Bacteria Limited (NCIMB) Ferguson Building,
`Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scot-
`land, UK on Nov.5, 2009 and accorded the accession number
`NCIMB41676.
`
`[0071] A deposit of Bifidobacterium longum strain
`UCC35624 was made at the National Collections of Indus-
`trial and Marine Bacteria Limited (NCIMB)Ferguson Build-
`ing, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA,
`Scotland, UK on Jan. 13, 1999 and accorded the accession
`number NCIMB41003.
`
`EXAMPLES
`
`[0072] The following examples further describe and dem-
`onstrate embodiments within the scope of the invention. The
`examples are given solely for the purposeof illustration and
`are not to be construed as limitationsof the present invention,
`
`as many variations thereof are possible without departing
`from the spirit and scope of the invention.
`
`Example 1
`
`Isolation of Bifidobacterium longum AH1714
`
`[0073] Bifidobacterium longum strain AH1714 was iso-
`lated from colonic biopsy tissue from healthy human sub-
`jects.
`Sections of the large of the human G.LT, obtained
`[0074]
`during colorectal scoping, were screened for probiotic bacte-
`rial strains. Musocal tissue from the humangastrointestinal
`tract was transferred to a collection tube containing Phos-
`phate Buffered Saline (PBS), supplemented with 0.05% cys-
`teine-HCl). Triton X-100 (0.05%) was added to release the
`adherent microorganisms from the tissue sample. Tissue
`samples were then incubated for 10 min. The samples were
`vortexed vigorously and adherent Lactobacilli and Bifidobac-
`teria isolated from the gastrointestinal tissue by plating on
`selective agar (De Man, Rogosa and Sharpe (MRS) agar+
`Vancomycin and Wilkins-Chalgren Agar+Mupirocin, respec-
`tively). Isolated colonies were picked from the plates and
`re-streaked three times to ensure purity. Microscope exami-
`nation, Gram staining, Catalase testing, Fructose-6-Phos-
`phate Phosphoketolase assessment were used to determine
`presumptive Bifidobacteria species and isolates were stocked
`in 40% glycerol and stored at -20° and -80° C. 16S intergenic
`spacer region sequencing were usedto confirm the identity of
`the newly isolated strains.
`[0075]
`Following isolation of a pure bifidobacteria strain,
`assignedthe designation AH1714, microbiological character-
`istics were assessed and are summarized in Table 1 below.
`AH1714 is a gram positive, catalase negative pleomorphic
`shaped bacterium which is Fructose-6-Phoshate Phosphoke-
`tolase positive, confirming its identity as a bifidobacterium.
`
`TABLE1
`
`Physiochemical characteristics ofB. longum AH1714
`
`Strain Characteristics
`Gram Stain
`Catalase
`Motility
`F6PPK*
`
`B. longum AH1714
`+
`-
`-
`+
`
`16s-23s intergenic spacer (IGS) sequencing was performed to
`identify the species of Bifidobacteria isolated. Briefly, DNA
`wasisolated from AH1714 using 100 wl of Extraction Solu-
`tion and 25 ul of Tissue Preparation solution (Sigma, XNAT2
`Kit). The samples were incubated for 5 minutes at room
`temperature followed by 2 hrs at 95° C. and then 100 ul of
`Neutralization Solution (Sigma, XNAT2 kit) was added.
`Genomic DNA solution was quantified using a Nanodrop
`spectrophotometer and stored at 4° C. PCR was performed
`using the IGS primers. The primer pairs used were IGS R
`5'-CTGGTGCCAAGGCATCCA-3' (SEQ ID No. 4) and IGS
`L 5'-GCTGGATCACCTCCTTTCT-3' (SEQ ID No. 3). The
`cycling conditions were 94° C. for 4 min (1 cycle), 94° C. for
`45 sec, 53° C. for 45 sec, 72° C. for 45 sec (28 cycles). The
`PCR reaction contained 2 wl (100 ng) of DNA, PCR mix
`(Sigma, Red Taq), 0.025 nM IGS L and R primer (MWG
`Biotech, Germany). The PCR reactions were performed on a
`Biotherma thermocycler. The PCR products (10 wl) were ran
`alongside a molecular weight marker (100 bp Ladder, Roche)
`on a 2% agarose EtBr stained gel in TAF, to determine the
`IGSprofile. PCR products of Bifidobacterium (single band)
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`were purified using the Promega Wizard PCRpurification kit.
`The purified PCR products were sequenced using the primer
`sequences (above) for the intergenic spacer region. Sequence
`data was then searched against the NCBInucleotide database
`to determine the identity of the strain by nucleotide homol-
`ogy. The resultant DNA sequence data was subjected to the
`NCBIstandard nucleotide-to-nucleotide homology BLAST
`search engine (http://www.ncbi.nlm.nih.gov/BLAST/). The
`nearest match to the sequence was identified and then the
`sequences were aligned for comparison using DNASTAR
`MegAlign software. The sequences (SEQ ID NO.
`1
`[IGS
`forward sequence] and SEQ ID NO. 2 [IGS reverse
`sequence]) obtained can be viewed in the sequence listing.
`Searching the NCIMBdatabase revealed that AH1714 has a
`unique IGS (SEQ ID NO. 1 [forward sequence] and SEQ ID
`NO. 2 [reverse sequence]) sequence with its closest sequence
`homologyto a Bifidobacterium longum.
`[0076]
`In order to develop a barcode PCR profile for
`AH1714, PCR was performed using BOX primers (8). The
`cycling conditions were 94° C. for 7 min (1 cycle); 94° C. for
`1 minute, 53°C. for 45 secs, 65° C. for 8 minutes, (30 cycles)
`and 65° C. for