US20120195859A1 - Recombinant probiotic bacteria for the prevention and treatment of inflammatory bowel disease (ibd) and irritable bowel syndrome (ibs) - Google Patents

Recombinant probiotic bacteria for the prevention and treatment of inflammatory bowel disease (ibd) and irritable bowel syndrome (ibs) Download PDF

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US20120195859A1
US20120195859A1 US13/357,063 US201213357063A US2012195859A1 US 20120195859 A1 US20120195859 A1 US 20120195859A1 US 201213357063 A US201213357063 A US 201213357063A US 2012195859 A1 US2012195859 A1 US 2012195859A1
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inflammatory
bacterium
gene
protein
active fraction
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Nathalie Vergnolle
Jean-Michel Sallenave
Philippe Langella
Luis Bermudez-Humaran
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Institut National de la Recherche Agronomique INRA
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Diderot Paris 7
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to the general field of therapy of gut inflammatory diseases such as Inflammatory Bowel Diseases (IBD), pulmonary diseases such as cystic fibrosis and broncho-pulmonary chronic obstructive diseases (BPCO), inflammatory articular disease (such as osteoarthritis), inflammatory urogenital disease, and diseases associated with chronic visceral pain symptoms, such as Irritable Bowel Syndrome (IBS).
  • IBD Inflammatory Bowel Diseases
  • pulmonary diseases such as cystic fibrosis and broncho-pulmonary chronic obstructive diseases (BPCO)
  • BPCO broncho-pulmonary chronic obstructive diseases
  • IBS Irritable Bowel Syndrome
  • IBD chronic inflammatory disorders
  • GIT gastrointestinal tract
  • the inventors propose that delivery of those protease inhibitors into the GIT, could be used for the treatment of an IBD and/or irritable Bowel syndrome (IBS).
  • IBS irritable Bowel syndrome
  • probiotics for the treatment of IBD has now been proposed for several years and different studies have reported some beneficial effects of these probiotic bacteria tested alone or in combination [Hedin, C. et al., 2007; Sartor, R. B. 2004].
  • the strategy of using recombinant non-pathogenic food-grade bacteria as delivery vehicles of anti-inflammatory molecules at the mucosal level has already been used to deliver the anti-inflammatory cytokine IL-10 [Steidler, L., et al., 2000].
  • Phase I clinical trials have demonstrated that orally given Lactococcus lactis strain expressing IL-10 cytokine, was safe as no serious side-effects occurred in those patients [Braat, H., et al., 2006].
  • the invention is based on the discovery that the use of a food-grade bacterium to deliver an anti-inflammatory molecule such as trappin-2 provides a safety and better efficiency than existing treatments. A better choice in the nature of the anti-inflammatory molecule to be delivered by L. lactis , could thus considerably improve the efficacy of treatment.
  • the inventors propose to use a food-grade bacterium to express and deliver anti-protease trappin-2 into the gut.
  • the invention relates to a molecule selected from the trappin-2 protein or an active fraction of the trappin-2 protein, a member of the WAP family proteins or an active fraction of a member of the WAP family proteins or a member of the Serpin family proteins or an active fraction of a member of the Serpin family proteins for the treatment of Irritable Bowel Syndrome (IBS).
  • IBS Irritable Bowel Syndrome
  • a further object of the invention relates to a recombinant food-grade bacterium comprising a gene selected from a gene coding for the trappin-2 protein or an active fraction of the trappin-2 protein, a gene coding for a member of the WAP family proteins or an active fraction of a member of the WAP family proteins, or a gene coding for a member of the Serpin family proteins or an active fraction of a member of the Serpin family proteins.
  • Another aspect of the invention relates to a therapeutic composition
  • a therapeutic composition comprising a recombinant food-grade bacterium as defined above.
  • FIG. 1 Weight differences (A), macroscopic score (B), Wall thickness (C), and myeloperoxydase (MPO) activity (D) in colonic tissues from mice that had water or water+DSS (3%) in their drinking bottles, and that received daily oral treatments for 7-days with wild-type L. lactis , or recombinant L. lactis strains expressing either Elafin or IL-10.
  • Significant differences compared to PBS-treated mice that have received DSS were noted * for p ⁇ 0.05, ** for p ⁇ 0.01, and * * * for p ⁇ 0.005.
  • Significant differences compared to wild-type L. lactis -treated mice were noted ⁇ for p ⁇ 0.05, ⁇ for p ⁇ 0.01, and ⁇ for p ⁇ 0.05.
  • FIG. 2 Trypsin-like activity (A) and elastase activity (B) in washes of the colonic lumen of mice that had water or water+DSS (3%) in their drinking bottles, and that received daily oral treatments for 7-days with wild-type L. lactis , or recombinant L. lactis strains expressing either Elafin or IL-10.
  • ⁇ means undetectable levels.
  • Significant differences compared to PBS-treated mice that have received DSS were noted * for p ⁇ 0.05, ** for p ⁇ 0.01.
  • Significant differences compared to wild-type L. lactis -treated mice were noted ⁇ for p ⁇ 0.05.
  • Significant differences between PBS+DSS group and the PBS-water group were noted # for p ⁇ 0.05, ## for p ⁇ 0.01, and ### for p ⁇ 0.005.
  • FIG. 3 Macroscopic score (A), Wall thickness (B), and myeloperoxydase (MPO) activity (C) in colonic tissues from mice that had water or water+DSS (3%) in their drinking bottles, and that received daily oral treatments for 7-days with wild-type Lb. casei , or recombinant Lb. casei strains expressing Elafin.
  • Significant differences compared to PBS-treated mice that have received DSS were noted * for p ⁇ 0.05, ** for p ⁇ 0.01, and *** for p ⁇ 0.005.
  • Significant differences compared to wild-type Lb. casei -treated mice were noted ⁇ for p ⁇ 0.05, ⁇ p ⁇ 0.01, and ⁇ for p ⁇ 0.005.
  • FIG. 4 Trypsin-like activity (A) and elastase activity (B) in washes of the colonic lumen of mice that had water or water+DSS (3%) in their drinking bottles, and that received daily oral treatments for 7-days with wild-type Lb. casei , or recombinant Lb. casei expressing elafin.
  • Significant differences compared to PBS-treated mice that have received DSS were noted * for p ⁇ 0.05, and significant differences compared to wild-type Lb. casei -treated mice were noted ⁇ for p ⁇ 0.05, and TT for p ⁇ 0.01.
  • FIG. 5 Protein concentration of RANTES (A), TNF ⁇ (B), IL-6 (C), MCP-1 (D), KC (E), INF ⁇ (F) and IL-17 (G) detected in colonic tissues from mice that had water or water+DSS (3%) in their clinking bottles, and that received daily oral treatments for 7-days with wild-type Lb. casei , or recombinant Lb. casei expressing Elafin. ⁇ means undetectable levels. Significant differences compared to PBS-treated mice that have received DSS were noted * for p ⁇ 0.05, ** for p ⁇ 0.01, and *** for p ⁇ 0.005. ⁇ showed significant differences for p ⁇ 0.05, compared to mice treated with wild-type L. lactis . Significant differences between PBS+DSS group and the PBS-water group were noted # for p ⁇ 0.05, ## for p ⁇ 0.01, and ### for p ⁇ 0.005.
  • FIG. 6 Protein concentration of IL-2 (A), IL-4 (B), IL-5 (C), IL-10 (D) and IL-13 (E) detected in colonic tissues from mice that had water or water+DSS (3%) in their clinking bottles, and that received daily oral treatments for 7-days with wild-type Lb. casei , or recombinant Lb. casei expressing Elafin. Significant differences compared to PBS-treated mice that have received DSS were noted * for p ⁇ 0.05, and ** for p ⁇ 0.01. Significant differences compared to mice treated with wild-type Lb. casei were noted ⁇ for p ⁇ 0.05, and ⁇ for p ⁇ 0.01.
  • Significant differences compared to PBS-treated mice that have received mustard oil were noted * for p ⁇ 0.05, ** for p ⁇ 0.01, and *** for p ⁇ 0.005.
  • ⁇ showed significant differences for p ⁇ 0.05, compared to mice treated with wild-type L. lactis.
  • FIG. 8 Elafin secreted by L. lactis wt and htrA strains. Western blot experiments performed with antibodies anti-elafin on cellular (C) and supernatant (S) extracts of wild type (wt) or htrA (htrA) strains. Elafin production was induced by nisin from exponential-phase cultures of wt or htrA strains (both containing the expression vector where elafin gene expression may be induced by nisin addition).
  • FIG. 9 Protective effects of L. lactis wt and htrA mutant strains in DSS 5%-induced colitis model. Macroscopic (A), histological damages (B) and MPO activities (C) were evaluated in different groups of 10 mice treated either with water (negative control) or with DSS 5%. Two first control groups were treated i) with water and orally fed with PBS (negative control group) and ii) with DSS 5% and orally fed with PBS (positive control group). The other groups were all treated with DSS 5% and with either wt strain (WT), wt strain expressing elafin (Elafin) and htrA mutant strain expressing elafin (Elafin+).
  • WT wt strain
  • Elafin wt strain expressing elafin
  • Elafin+ wtrA mutant strain expressing elafin
  • FIG. 10 Protective effects of L. casei wt strain and SOD-expressing, elafin-expressing and IL-10-expressing L. casei strains in DSS 5%-induced colitis model. Macroscopic (A), histological damages (B) and MPO activities (C) were evaluated in different groups of 10 mice treated either with water (negative control) or with DSS 5%. Two first control groups were treated i) with water and orally fed with PBS (negative control group) and ii) with DSS 5% and orally fed with PBS (positive control group). The other groups were all treated with DSS 5% and with either L. casei wt strain (WT) or L. casei strains expressing superoxide dismutase (SOD), elafin (Elafin) or IL-10. * and ** indicate that the data are significantly different (P ⁇ 0.05) from the data obtained with L. casei wt.
  • WT L. casei strain
  • SOD superoxide dismutase
  • FIG. 11 Myeloperoxydase (MPO) activity (D) in colonic tissues from mice.
  • Myeloperoxydase (MPO) activity (D) was measured in colonic tissues from mice that had water or water+DSS (3%) in their dinking bottles, and that received daily oral treatments for 7-days with i) WT L. lactis , recombinant L. lactis and L. lactis htrA strains expressing either Elafin or IL-10 and with ii) L. casei wt strain and SOD-expressing, elafin-expressing and IL-10-expressing L. casei strains. * and ** indicate that the data are significantly different (P ⁇ 0.05) from the data obtained with L. casei wt.
  • FIG. 12 Elafin secretion in a L. lactis WT strain where the elafin gene is expressed under the control of an EDTA-inducible promoter [Llull D and Poquet I. 2004; EP 1 537 215 and FR 98 16462].
  • lactis WT strain expressing elafin was grown overnight in the presence (+) or not ( ⁇ ) of EDTA, a chelator agent (in this construct, elafin gene expression is controlled by a lactococcal promoter that may be induced by EDTA addition: on the chromosome, this promoter controls the expression of genes encoding an ABC uptake system specific for zinc and is derepressed under zinc starvation conditions that may be mimicked by EDTA addition). Proteins were then extracted and fractionated between cell (C) and supernatant fractions (S) and Western blot experiments were performed using antibodies anti-elafin.
  • C cell
  • S supernatant fractions
  • trappin-2 also known as elafin, elafin-specific inhibitor (ESI) or SKALP for skin anti-leucoprotease
  • WAP WAP family
  • HNE human neutrophil elastase
  • proteinase 3 proteinase 3 which is secreted in the respiratory tract [Sallenave et al., 1991 and 1993].
  • HNE human neutrophil elastase
  • trappin-2 comprises an integral part of the ‘anti-elastase shield’ in the lung.
  • An exemplary sequence for human trappin-2 gene is deposited in the database Genbank under accession number S58717.
  • WAP family for “Whey Acidic Protein” denotes a family of protein containing the trappin-2, and the ps20.
  • Serpin family for SERine Protease INhibitors denotes a family of serine proteinase inhibitors which are similar in amino acid sequence and mechanism of inhibition, but differ in their specificity toward proteolytic enzymes.
  • This family includes alpha 1-antitrypsin (A1-Pi), angiotensinogen, ovalbumin, antiplasmin, alpha 1-antichymotrypsin, thyroxine-binding protein, complement 1 inactivators, antithrombin III, heparin cofactor II, plasminogen inactivators, gene Y protein, placental plasminogen activator inhibitor, and barley Z protein.
  • This family does not include the secretory leukocyte proteinase inhibitor (SLPI) [Gold Moreau et al., 2008]. Some members of the Serpin family may be substrates rather than inhibitors of serine endopeptidases, and some serpins occur in plants where their function is not known.
  • SLPI secretory leukocyte proteinase inhibitor
  • alpha 1-antitrypsin protein denotes a glycoprotein.
  • Alpha 1-antitrypsin is also referred to as alpha-1 proteinase inhibitor (A1PI) because it is a serine protease inhibitor (serpin), inhibiting a wide variety of proteases. It protects tissues from enzymes of inflammatory cells, especially elastase.
  • A1PI alpha-1 proteinase inhibitor
  • serpin serine protease inhibitor
  • An exemplary sequence for human Alpha 1-antitrypsin gene is deposited in the database Genbank under accession number NC008290.
  • an active fraction of denotes a fraction of a protein with the activity of the complete protein”.
  • an active fraction of the trappin-2 protein denotes a fraction of the protein which conserves the capacity to inhibit the HNE or an active fraction of the Serpin family proteins denotes a fraction of the protein which conserves the capacity of inhibition.
  • the term “food-grade bacterium” denotes a bacterium that is widely used in fermented foods and possesses a perfect safety profile recognized by the GRAS (Generally Recognized As Safe) and QPS (Qualified Presumption of Safety) status in USA and European Community, respectively. Such bacterium may be safely in functional foods or food additives with allegations concerning maintain in good health and well-being or prevention of disease.
  • probiotic bacterium denotes a bacterium which ingested live in adequate quantities may exert beneficial effects on the human health. They are now widely used as a food additive for their health-promoting effects. Most of the probiotic bacteria are Lactic Acid Bacterium (LAB) and among them strains of the genera Lactobacillus and Bifidobacterium are the most widely used probiotic bacteria.
  • LAB Lactic Acid Bacterium
  • thyA gene denotes, the gene coding for thymidylate synthase which is an enzyme generating thymidine monophosphate (dTMP), which is subsequently phosphorylated to thymidine triphosphate used in DNA synthesis and repair.
  • dTMP thymidine monophosphate
  • IBS Irritable Bowel Syndrome
  • IBD inflammatory bowel diseases
  • a first object of the invention relates to a molecule selected from the trappin-2 protein or an active fraction of the trappin-2 protein, a member of the WAP family proteins or an active fraction of a member of the WAP family proteins, or a molecule selected from the Serpin family or an active fraction of the Serpin family for the treatment of Irritable Bowel Syndrome (IBS).
  • IBS Irritable Bowel Syndrome
  • the member of the Serpin family may be the alpha 1-antitrypsin protein.
  • said fraction of the protein comprises at least about 75% identity over said protein, even more preferably at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%.
  • said protein or fraction of the protein thereof may be used in combination with an anti-inflammatory agent.
  • Proteins of the invention or fractions of the proteins thereof may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination(s).
  • amino acid sequence of the desired sequence may readily produce a relevant part of the said proteins or fraction of the protein, by standard techniques for production of proteins. For instance, they may be synthesized using well-known solid phase method, preferably using a commercially available protein synthesis apparatus (such as that made by Applied Biosystems, Foster City, Calif.) and following the manufacturer's instructions.
  • proteins or fraction of the proteins of the invention thereof may be synthesized by recombinant DNA techniques as is now well-known in the art.
  • these fragments may be obtained as DNA expression products after incorporation of DNA sequences encoding the desired polypeptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired protein or fraction of the protein, from which they may be later using well-known techniques.
  • Proteins or fraction of the proteins of the invention thereof may be used in an (e.g., purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).
  • a vector such as a membrane or lipid vesicle (e.g. a liposome).
  • a further object of the invention relates to a recombinant food-grade bacterium comprising a gene selected from a gene coding for the trappin-2 protein or an active fraction of the trappin-2 protein, a gene coding for a member of the WAP family proteins or an active fraction of a member of the WAP family proteins, or a gene coding for a member of the Serpin family proteins or an active fraction of a member of the Serpin family proteins.
  • the food-grade bacterium according to the invention may be a probiotic bacterium.
  • the probiotic bacterium according to the invention comprises a defective auxotrophic gene, whereby survival of said bacterium may be strictly dependent upon the presence of specific compounds.
  • the auxotrophic gene according to the invention may be the thyA gene encoding the thymidylate synthase.
  • the auxotrophic gene according to the invention may be the alanine racemase (alr) gene [Bron et al, 2002].
  • thyA gene of the probiotic bacterium renders it auxotrophic to thymidine which is absent from the gastrointestinal tract (GIT).
  • GIT gastrointestinal tract
  • the selected gene may be inserted in the thyA gene.
  • the recombinant gene may be located in the chromosome into the thyA gene locus which may be thus inactivated by gene disruption.
  • gene disruption denotes disruption by insertion of a DNA fragment, disruption by deletion of the gene, or a part thereof, as well as exchange of the gene or a part thereof by another DNA fragment, and the disruption may be induced by recombinant DNA techniques, and not by spontaneous mutation.
  • disruption is the exchange of the gene, or a part thereof, by another functional gene.
  • the defective recombinant thyA gene may be a non-reverting mutant gene.
  • the term “non-reverting mutant” denotes that the reversion frequency may be lower than about 10 ⁇ 8 , preferably the reversion frequency may be lower than about 10 ⁇ 10 , even more preferably, the reversion frequency may be lower than about 10 ⁇ 12 , even more preferably, the reversion frequency may be lower than about 10 ⁇ 14 , most preferably, the reversion frequency may not be detectable using the routine methods known to the person skilled in the art.
  • the gene according to the invention codes for the alpha 1-antitrypsin protein, or another members of the Serpin family such as, but not limited to, antiplasmin, alpha 1-antichymotrypsin.
  • the food-grade bacterium strain according to the invention may be a L. lactis strain or a Lactobacillus casei strain or a L. lactis htrA strain [Poquet et al., 2000] or a Lactobacillus plantarum strain of a Bifidobacterium longum strain.
  • the food-grade bacterium strain according to the invention may be a Lactobacillus casei strain.
  • the gene according to the invention codes for trappin-2.
  • trappin-2 may be naturally expressed in the human colonic mucosa, with a prominent expression in intestinal epithelial cells [Motta et al.] and that, patients with IBD show a down-regulation of trappin-2 in tissues compared to healthy subjects [Motta et al.].
  • trappin-2 overexpression may be protective against the development of colitis (in constitutive and transient expression). Moreover, trappin-2 overexpression in models of colitis may be able to completely inhibit the increase of elastase and trypsin-like activities associated with colitis.
  • trappin-2 overexpression in mice may be also able to significantly inhibit colitis-induced increases of pro-inflammatory cytokines and chemokines (such as, but not limited to, IL-6, Il-17A, TNF-alpha, Interferon-gamma, MCP-1 and KC).
  • pro-inflammatory cytokines and chemokines such as, but not limited to, IL-6, Il-17A, TNF-alpha, Interferon-gamma, MCP-1 and KC.
  • the gene according to the invention codes for the alpha 1-antitrypsin protein.
  • alpha 1-antitrypsin protein inhibits trypsin-like activities associated with IBD (like colitis) and thus has similar effects as trappin-2.
  • the food-grade bacterium strain according to the invention may be a Lactobacillus casei strain which comprises a gene coding for trappin-2.
  • the food-grade bacterium strain according to the invention may be a Lactobacillus casei strain which comprises a gene coding for trappin-2 inserted in the thyA gene.
  • the food-grade bacterium according to the invention is useful for the treatment of intestinal inflammatory conditions.
  • the food-grade bacterium according to the invention is useful for the treatment of an IBD and/or IBS.
  • the inflammatory conditions may be selected from IBD, IBS, inflammatory pulmonary disease, inflammatory articular disease or inflammatory urogenital disease.
  • Another object of the invention relates to a therapeutic composition comprising a food-grade bacterium according to the invention.
  • therapeutic composition according to the invention is intended for mucosal administration to a subject.
  • compositions are intended for oral administration to a subject.
  • compositions may be in the form of a suspension, tablet, pill, capsule, granulate or powder.
  • the food-grade bacterium according to the invention is present, free and not immobilized, in suspension.
  • the suspension has a composition which ensures physiological conditions for a probiotic bacterium, so that in particular the osmotic pressure within the cell does not lead to lysis.
  • the food-grade bacterium according to the invention may be present in free, preferably lyophilized form, or in immobilized form.
  • the food-grade bacterium according to the invention may be enclosed in a gel matrix which provides protection for the cells.
  • a solid therapeutic composition intended for oral administration and containing the food-grade bacterium according to the invention in immobilized or non-immobilized form is preferably provided with a coating resistant to gastric juice. It is thereby ensured that the food-grade bacterium contained in the therapeutic composition may pass through the stomach unhindered and undamaged and the release of the food-grade bacterium first takes place in the upper intestinal regions.
  • the therapeutic composition contains sufficient colony-forming units (CFU) of the food-grade bacterium capable of forming the protein according to the invention so that with multiple administration of the therapeutic composition according to a patient, the state of the IBD or IBS is healed, the progression of the IBD or the IBS is stopped, and/or the symptoms of the IBD or IBS may be alleviated.
  • CFU colony-forming units
  • a therapeutic composition contains about 1 ⁇ 10 8 -1 ⁇ 10 11 , preferably about 1 ⁇ 10 9 to about 1 ⁇ 10 10 CFU of the food-grade bacterium according to the invention.
  • the therapeutic composition containing the food-grade bacterium may be administered intrarectally.
  • a rectal administration preferably takes place in the form of a suppository, enema or foam.
  • Intrarectal administration may be particularly suitable for chronic inflammatory intestinal diseases which affect the lower intestinal sections, for example the colon.
  • Intranasal administrations are also suitable to treat chronic pulmonary diseases such as, but not limited to, cystic fibrosis and BPCO.
  • the invention relates to a food composition
  • a food composition comprising a food-grade bacterium according to the invention.
  • compositions according to the invention are intended for oral administration to a subject.
  • compositions may be in the form of a suspension, tablet, pill, capsule, granulate, powder or yogurt.
  • the food composition may contain 1 ⁇ 10 8 -1 ⁇ 10 11 , preferably 1 ⁇ 10 9 -1 ⁇ 10 10 CFU of the food-grade bacterium according to the invention.
  • the food composition may be administered to the patient at a daily dose of 10 10 bacteria.
  • Gene coding for elafin was PCR amplified from plasmid DK6-elafin. Sequences of primers used were: 5′ forward Elafin (CCAATGCATCAGCAGCTGTCACGGG AGTTCC) (SEQ ID No 1) and 3′ reverse-Elafin (GGACTAGTCCTCACTGGGGAACGAAACA GGCC) (SEQ ID No 2). Primers were designed to eliminate first codons of elafin region encoding for signal peptide (SP) and was replaced by the SP of Usp45 protein (PS Usp45 ), the main secreted protein from L. lactis .
  • SP signal peptide
  • PS Usp45 the SP of Usp45 protein
  • PCR product was digested, purified, and cloned in pSEC, a L. lactis secretion vector.
  • pSEC a L. lactis secretion vector.
  • elafin is fused in frame with a DNA fragment encoding for RBS and PS Usp45 .
  • Expression of the cassette is controlled by the inducible promoter P nisA , the activity of which depends upon the concentration of nisin used.
  • This plasmid was then introduced in a L. lactis strain bearing the nisin regulatory genes nisR et nisK ( L. lactis NZ9000) to give rise the recombinant strain: NZ(pSEC:elafin).
  • lactobacilli strains such as, but not limited to, Lactobacillus casei and Lb. plantarum .
  • These two strains (each bearing the genes nisRK on their chromosome) have been chosen because of their persistence ability in the digestive tract (up to 4 days, as opposed to 24 to 48 hr in L. lactis .
  • Lb. casei BL23 strain possesses anti-inflammatory properties in a DSS-induced colitis model [Rochat et al, 2007].
  • mice (6-8 weeks old) were obtained from Janvier (St Quentin Fallavier) and were kept at room temperature, under 12 h light/dark cycles and having free access to food and water, except the day before the induction of colitis, where they were fasted for 12 h. All procedures were approved by Institutional animal care committee and veterinary services.
  • DSS Dextran Sodium Sulfate
  • the first treatment started at the same time DSS was added to drinking water and the last treatment was on the day of the sacrifice (day 7). Body weight and survival rate were measured daily after the induction of colitis. On day 7 after adding DSS to their drinking water, mice were sacrificed and colons were harvested for measure of several parameters of inflammation: macroscopic score, bowel thickness, myeloperoxydase (MPO) activity, proteolytic activity, cytokine expression.
  • MPO myeloperoxydase
  • Macroscopic damage was evaluated as follows. Briefly, when observed, the following parameters were given a score of 1: haemorrhage, edema, stricture, ulceration, fecal blood, mucus, and diarrhoea. Erythema was scored a maximum of 2 depending on the length of the area being affected (0: absent, 1: less than 1 cm, 2: more than 1 cm). Adhesion was scored based on its severity (0: absent, 1: moderate, 2: severe).
  • MPO was measured as an index of granulocyte infiltration in colonic tissues harvested at the time of the sacrifice.
  • Tissue samples were homogenized in a solution of 0.5% hexadecyltrimethylamonium bromide in phosphate buffer (pH 6), and centrifuged at 13000 ⁇ G for 2 min. Supernatants were added to a buffer containing 1% hydrogen peroxide and O-dianisidine dihydrochloride. Optical density readings for the enzymatic solution were read for 2 min at 450 nm.
  • cytokine and chemokine protein measures frozen colonic samples harvested at sacrifice were homogenized using a polytron for 30 s at 4° C. in 500 ⁇ l of cell lysis buffer (20 mM Tris-Hcl, pH 7.5, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophospate, 1 mM beta-glycerophosphate, 1 mM Na 3 VO 4 , 1 ⁇ g/ml leupeptin; Cell Signalling, Sigma) supplemented with anti-proteases (Roche Diagnostics, Meylan, France) cocktail.
  • cell lysis buffer (20 mM Tris-Hcl, pH 7.5, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophospate, 1 mM beta-gly
  • Protein concentration in the lumenal washes was determined using colorimetric dosage of bicinchoninic acid on microplate (BCA Kit®, Pierce, Thermo Scientific, Courtaboeuf, France) and was used to standardize the proteolytic activity in each samples.
  • mice were resuspended in sterile PBS at a final concentration of 5 ⁇ 10 10 cfu/mL.
  • Groups of 4 to 8 mice were treated daily with 100 ⁇ L (5 ⁇ 10 9 cfu) of bacterial suspension by intragastric administration for seven days.
  • mice were administered with 50 pt of PBS or mustard oil (0.01% (v/v) in ethanol 70%) by intracolonic instillation, performed under slight isoflurane anesthesia.
  • the number of pain-related behavioral responses were then counted for 20 min.
  • DSS-induced colitis caused severe weight loss in all groups of mice compared to control mice that drank water. None of the lactic acid bacteria treatments significantly modified this weight loss ( FIG. 1A ). DSS in drinking water also caused macroscopic damage, increased wall thickness and increased MPO activity in colonic tissues ( FIGS. 1B , C, D). Mice that were treated with wild-type L. lactis did not show significant decrease in colonic wall thickness and MPO activity, only a slight decrease in macroscopic damage score was observed in that group, compared to DSS alone-treated mice. In contrast, mice treated with recombinant L.
  • lactis expressing elafin showed after the induction of DSS colitis a significantly reduced macroscopic damage score and significant less increase in colonic wall thickness, but MPO activity was not different from DSS alone group ( FIGS. 1B , C, D).
  • mice treated with recombinant L. lactis expressing IL-10 cytokine showed reduced macroscopic damage score and MPO activity after the induction of DSS colitis, but wall thickness was not modified by this treatment compared to DSS alone ( FIGS. 1B , C, D).
  • none of the treatments modified the inflammatory parameters compared to na ⁇ ve control mice.
  • DSS-induced increase in trypsin-like activity was significantly reduced in mice treated with recombinant L. lactis expressing Elafin, but was not changed in mice treated with wild-type L. lactis or recombinant L. lactis expressing IL-10 ( FIG. 2A ). Only treatment with recombinant L. lactis expressing elafin was able to significantly reduce DSS-induced increase in elastase activity ( FIG. 2B ).
  • DSS-induced increase in trypsin-like activity was significantly reduced in mice treated with recombinant Lb. casei expressing Elafin, compared to mice treated with wild-type Lb. casei ( FIG. 4A ).
  • the level of elastase activity was also significantly reduced in mice with colitis (DSS) treated with recombinant Lb. casei expressing elafin, compared to inflamed (colitis) mice treated with wild-type Lb. casei , or even compared to inflamed mice treated with PBS ( FIG. 4B ).
  • Intracolonic administration of mustard oil caused a significant increase in the number of pain behaviors: both the number of abdominal retractions and the number of integrated pain behaviors such as licking, stretching, and squashing against the floor ( FIGS. 7 A and B).
  • treatment with wild-type, IL-10 recombinant or elafin-secreting L. lactis had no effect ( FIG. 7A ).
  • elafin-, but not IL-10-recombinant L. lactis or wild-type significantly reduced the number of pain behaviors ( FIG. 7B ).
  • only recombinant L. lactis expressing elafin induced a notable decrease in the number of pain behaviors compared both to PBS treatment or wild-type L. lactis treatment ( FIG. 7B ).
  • L. lactis expresses only one housekeeping extracellular protease called htrA which degrades all the unfolded exported proteins] Poquet et al, 2000 and U.S. Pat. Nos. 6,994,997 and FR2787810].
  • a L. lactis mutant strain inactivated in htrA gene was constructed and allowed increasing the production rate of several heterologous secreted proteins in L. lactis [Poquet et al, 2000 and Miyoshi et al, 2002]. According to the invention, the elafin expression cassette was cloned in the htrA mutant.
  • the pleiotropic anti-microbial/anti-inflammatory activity of elafin makes it a very good therapeutic molecule candidate as compared to IL-10.
  • IBS Irritable Bowel Syndrome
  • a recombinant food-grade bacterium comprising a gene selected from a gene coding for the trappin-2 protein or an active fraction of the trappin-2 protein, a gene coding for a member of the WAP family proteins or an active fraction of a member of the WAP family proteins or a gene coding for a member of the Serpin family proteins or an active fraction of a member of the Serpin family proteins.
  • a probiotic bacterium according to any one of paragraphs 3 to 5 wherein the gene coding for the WAP or Serpin family progene is trappin-2 or the alpha I-antitrypsin protein.
  • a food-grade bacterium according to any one of paragraph 3 to 6 selected from Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum.
  • a food-grade bacterium according to paragraph 10 wherein the inflammatory condition is selected from Inflammatory Bowel Disease, Irritable Bowel Syndrome, inflammatory pulmonary disease, inflammatory articular disease or inflammatory urogenital disease.
  • a therapeutic composition comprising a food-grade bacterium according to any one of paragraphs 3 to 9.
  • a food composition comprising a food-grade bacterium according to any one of paragraphs 3 to 9.
  • IBS Irritable Bowel Syndrome
  • a molecule according to the paragraph 21 expressed by a genetically engineered host cell.
  • a recombinant food-grade bacterium comprising a recombinant gene selected from a gene coding for the trappin-2 protein or an active fraction of the trappin-2 protein, a gene coding for a member of the WAP family proteins or an active fraction of a member of the WAP family proteins or a gene coding for a member of the Serpin family proteins or an active fraction of a member of the Serpin family proteins.
  • a probiotic bacterium according to any one of paragraphs 23 to 25 wherein the gene coding for the WAP or Serpin family protein encodes the trappin-2 protein or the alpha 1-antitrypsin protein.
  • a food-grade bacterium according to any one of paragraphs 23 to 26 selected from Lactic Acid Bacterium, Bifidobacterium, Lactococcus or Lactobacillus.
  • a food-grade bacterium according to any one of paragraphs 23 to 26 selected from Lactococcus lactis, Lactococcus lactis htrA, Lactobacillus casei, Lactobacillus plantarum , and Bifidobacterium longum.
  • a food-grade bacterium according to any one of paragraphs 23 to 30 for the treatment of an inflammatory condition is provided.
  • a therapeutic composition comprising a food-grade bacterium according to any one of paragraphs 23 to 30.
  • composition intended for mucosal, oral, intranasal or rectal administration to a subject.

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US20130344033A1 (en) 2013-12-26
BR112012016982B1 (pt) 2021-02-09

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