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    RECOMBINANT PROBIOTIC BACTERIA FOR THE PREVENTION AND TREATMENT OF INFLAMMATORY INTESTINE DISEASE (IBD) AND IRRITABLE INTESTINE SYNDROME (IBS) The present invention relates to the general field of inflammatory bowel disease (IBD) and / or irritable bowel syndrome (IBD) IBS). Thus, the invention relates to a molecule selected from the trappin-2 protein or an active fraction of the trappin-2 protein, an element of the proteins of the WAP family, or an active fraction of the element of the proteins of the WAP family, or an element of the proteins of the Serpina family, or an active fraction of an element of the proteins of the Serpina family for the treatment of irritable bowel syndrome (IBS). The invention also relates to a food-grade recombinant bacterium that includes a gene selected from a gene encoding the trappin-2 protein, or an active fraction of the trappin-2 protein, a gene encoding an element of the proteins of the WAP family, or an active fraction of an element of the WAP family proteins, or a gene that codes for an element of the proteins of the serpine family, or an active fraction of an element of the proteins of the serpine family.
    公开号:BR112012016982B1
    申请号:R112012016982-3
    申请日:2011-01-14
    公开日:2021-02-09
    发明作者:Nathalie Vergnolle；Jean-Michel Sallenave；Philippe Langella；Luis Bermudez-Humaran
    申请人:Institut National De La Recherche Agronomique (Inra)；Institut Pasteur；Université Paris Diderot - Paris 7；Institut National De La Sante Et De La Recherche Medicale (I.N.S.E.R.M.)；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    [001] The present invention relates to the general field of therapy for inflammatory bowel diseases, such as inflammatory bowel diseases (IBD), lung diseases such as cystic fibrosis and chronic bronchopulmonary obstructive diseases (BPCO), inflammatory joint disease (for example , osteoarthritis), inflammatory urogenital diseases and diseases associated with symptoms of chronic visceral pain, such as irritable bowel syndrome (IBS). BACKGROUND OF THE INVENTION
    [002] The treatment of chronic inflammatory disorders such as IBD represents a great medical challenge since they afflict several million people. Its highest incidence is among developed countries and has steadily increased over the past 3 decades. Current therapies for IBD need to be improved a lot, a high percentage of patients (between 20 and 40%) resistant to any forms of treatments, serious side effects and high costs, and also associated with currently available drugs (glucocorticoids and monoclonal antibody therapies) . In addition, the mechanisms involved in the pathogenesis of IBD are not fully understood, and the development of more effective treatments or even cures for IBD depends on a better understanding of the regulation of the inflammatory response. Several studies have demonstrated a crucial role for proteases in maintaining the chronic inflammatory response of the gastrointestinal tract [Vergnolle, N. 2005 .; Cenac, N. et al., 2007; Hyun, E., et al, 2008; Vergnolle, N., et al, 2004]. Therefore, endogenous protease inhibitors appear to be crucial for controlling intestinal inflammatory responses.
    [003] Based on this knowledge, inventors propose that the delivery of protease inhibitors to GIT, could be used for the treatment of an IBD and / or irritable bowel syndrome (IBS).
    [004] The use of probiotics for the treatment of IBD has now been proposed for several years and several studies have reported some beneficial effects that these probiotic bacteria tested alone or in combination [Hedin, C. et al, 2007; Sartor, R. B. 2004]. The strategy of using non-pathogenic food-grade recombinant bacteria as delivery vehicles for mucosal anti-inflammatory molecules has already been used to deliver the anti-inflammatory cytokine IL-10 [Steidler, L., et al., 2000] . Phase 1 clinical trials have shown that the Lactococcus lactis strain administered orally expressing cytokine IL-10, was safe, with no serious side effects occurring in these patients [Braat, H., et al., 2006]. However, the decreased disease activity in Crohn's disease patients treated with recombinant IL-10 L. lactis was somehow limited. This limited efficacy could be explained by the fact that delivery of IL-10 has always been reported to have only mild beneficial effects against the development of colitis [Braat, H. et al., 2003]. A better choice in the nature of the anti-inflammatory molecule delivered by L. lactis, could thus considerably improve the effectiveness of the treatment. Here, the inventors propose to use a food-grade bacterium to express and deliver trappin-2 anti-protease in the intestine. SUMMARY OF THE INVENTION
    [005] 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 better safety and efficiency than existing treatments. Thus, 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 the member of the WAP family proteins or a member of the WAP family proteins. Serpina family or an active fraction of a family member other proteins for the treatment of irritable bowel syndrome (IBS).
    [006] An object of invention concerns a food-grade recombinant bacterium that includes a gene selected from a gene encoding the trappin-2 protein or an active fraction of the trappin-2 protein, a gene encoding a member of the WAP family proteins or an active fraction of a member of the WAP family proteins or a gene encoding a member of the Serpina family proteins or an active fraction of a member of the Serpina family proteins.
    [007] Another aspect of the invention relates to a therapeutic composition composed of recombinant food-grade bacteria, as defined above. DETAILED DESCRIPTION OF THE INVENTION: DEFINITIONS
    [008] As used here, the term "trappin-2" (also known as elafina, specific inhibitor of elafine (ESI) or SKALP for skin anti-leukoprotease) of the WAP family, denotes a low molecular weight inhibitor (9, 9 kDa) HNE inhibitor (human neutrophil elastase) and proteinase 3, which is secreted in the respiratory tract [Sallenave et al., 1991 and 1993]. Together with (Al-Pi) and SLPI, trappin-2 comprises an integral part of the "anti-elastase shield" in the lung. An exemplary human trappin-2 gene sequence is deposited in the Genbank database under the membership number S58717.
    [009] As used here, the term "WAP Family" for "Whey acidic protein" means a family of proteins containing trappin-2 and ps20.
    [010] As used here, the term "Serpine family" for serine protease inhibitors denotes a family of serine protease inhibitors that resemble the amino acid sequence and inhibition mechanism, but differ in their specificity for proteolytic enzymes. This family includes alpha 1-antitrypsin (Al-Pi), angiotensinogen, ovalbumin, antiplasmin, alpha 1-antiquimotrypsin protein, thyroxine-binding protein, complement 1 inactivators, antithrombin III, heparin II cofactor, plasminogen inactivators, plasma protein gene Y, placental plasminogen activator inhibitor and barley Z protein. This family does not include the secretory leukocyte protease inhibitor (SLPI) [Thierry Moreau et al, 2008]. Some members of the serpine family may be substrates rather than inhibitors of serine endopeptidases, and some serpins occur in plants where their function is not known.
    [011] In this document, the term "alpha 1 protein - antitrypsin" denotes a glycoprotein. Alpha 1-antitrypsin is also referred to as an alpha-1 protease inhibitor (A1PI) because it is a serine protease inhibitor (serpine), inhibiting a wide variety of proteases. It protects tissues from inflammatory cell enzymes, especially elastase. An exemplary human alpha 1-antitrypsin gene sequence is deposited in the Genbank database under the membership number NC008290.
    [012] In this document, the term "an active fraction denotes a fraction of a protein with the activity of the complete protein". For example, an active fraction of the trappin-2 protein denotes a fraction of the protein that retains the ability to inhibit FINE or an active fraction of the proteins in the Serpina families denotes a fraction of the protein that retains the inhibition capacity.
    [013] In this document, the term "food-grade bacteria" denotes a bacterium that is widely used in fermented foods and has a perfect safety profile recognized by GRAS (generally recognized as safe) and QPS status (qualified safety presumption) in USA and European Community, respectively. This bacterium can be safe in functional foods or food additives, with claims regarding maintaining good health and well-being or preventing disease.
    [014] As used in this document, the term "probiotic bacteria" denotes a bacterium that eaten alive in adequate quantities can have beneficial effects on human health. They are now widely used as food additives for their health-promoting effects. Most probiotic bacteria are lactic acid bacteria (LAB), and among them strains of the genera Lactobacillus and Bifidobacterium are the most widely used probiotic bacteria.
    [015] In this document, the term "thyA gene" denotes the gene that codes for thymidylate synthase, which is an enzyme generating thymidine monophosphate (dTMP), which is later phosphorylated to thymidine triphosphate, used in DNA synthesis and repair .
    [016] As used in this document, the term "Irritable bowel syndrome (IBS)" is a term for a variety of pathological conditions, causing discomfort in the gastrointestinal tract. It is a functional intestinal disorder characterized by chronic abdominal pain, discomfort, swelling and altered bowel habits, in the absence of any organic cause.
    [017] As used here, the term "inflammatory bowel disease (IBD)" is a group of inflammatory diseases of the colon and small intestine. The main types of IBD are Crohn's disease, ulcerative colitis and pouchitis. Proteins and their uses:
    [018] A first object of the invention relates to a molecule selected from the protein of trappin-2 or an active fraction of the protein trappin-2, a member of the proteins WAP families or an active fraction of the member the proteins of families of WAP, or a molecule selected from the serpin family or an active fraction of the Serpina family for the treatment of irritable bowel syndrome (IBS).
    [019] In a preferred embodiment, the member of the Serpina family is alpha1-antitrypsin protein.
    [020] In a preferred embodiment, said fraction of the protein consists of at least 75% of the identity of such a protein, preferably even more, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%.
    [021] Usually, such a protein or protein fraction thereof can be used in combination with an anti-inflammatory agent.
    [022] Proteins of the invention or fractions of the proteins thereof can be produced by any technique known in the art such as, without limitation, any chemical, biological, genetic or enzymatic technique, alone or in combination (s). The. Knowing the amino acid sequence of the desired sequence, a person skilled in the art can readily produce a relevant part of said proteins or fraction of the protein, by standard techniques for the production of proteins. For example, they can be synthesized using a known solid phase method, preferably using a commercially available protein synthesis apparatus (such as Applied Biosystems, Foster City, California) and following the manufacturer's instructions.
    [023] Alternatively, the proteins or protein fraction of the invention thereof can be synthesized by recombinant DNA techniques as are well known in the art. For example, these fragments can be obtained as DNA expression products after incorporating the DNA sequences encoding the desired polypeptide into expression vectors and introducing such vectors into suitable prokaryotic or eukaryotic hosts that will express the desired protein or fraction of the protein, from which well-known techniques can later be used.
    [024] Proteins or protein fraction of the invention can be used in a form (eg, purified) or contained in a vector, such as a lipid membrane or vesicle (eg, liposomes). Food-grade bacteria
    [025] An object of invention concerns a food-grade recombinant bacterium that includes a gene selected from a gene encoding the trappin-2 protein or an active fraction of the trappin-2 protein, a gene encoding a member of the WAP family proteins or an active fraction of a member of the WAP family proteins or a gene encoding a member of the Serpina family proteins or an active fraction of a member of the Serpina family proteins.
    [026] In a preferred embodiment, the food-grade bacterium according to the invention is a probiotic bacterium.
    [027] In a preferred embodiment, the probiotic bacterium according to the invention comprises a defective auxotrophic gene, whereby the survival of such a bacterium is strictly dependent on the presence of specific compounds.
    [028] In another preferred embodiment, the auxotrophic gene according to the invention is the thyA gene encoding thymidylate synthase.
    [029] In another preferred embodiment, the auxotrophic gene according to the invention is the alanine racemase (ar) gene (Bron et al, 2002).
    [030] Inactivation of the thyA gene of the probiotic bacterium according to the invention yields this auxotrophic for thymidine which is absent from the gastrointestinal tract (GIT). This recombinant thyA mutant will be able to deliver its protein of interest, but it will not survive and thus, it persists in the GIT limiting its diffusion and providing the requested biological containment for recombinant bacteria. Similar results can be obtained with the aerial gene.
    [031] In another preferred embodiment, the selected gene is inserted into the thyA gene.
    [032] Preferably, the recombinant gene is located on the chromosome at the locus of the thyA gene, which is thus inactivated by the disruption of the gene. As used in this document, the term "gene disorder" denotes interruption by insertion of a DNA fragment, disruption by deletion of the gene, or a part of it, as well as the exchange of the gene or part by another DNA fragment and the interruption is induced by recombinant DNA techniques and not by spontaneous mutation. Preferably, the interruption is the exchange of the gene, or a part of it, for another functional gene. Preferably, the defective recombinant thyA gene is a non-reversible mutant gene.
    [033] As used in this document, the term "non-reversible mutant" denotes that the reversal frequency is less than 10 "8, preferably the reversal frequency is less than 10" 10, even more preferably, the reversal frequency is less than 10 "12, even more preferably, the reversal frequency is less than 10 ~ 14, more preferably, the reversal frequency is not detectable using routine methods known to the person skilled in the art.
    [034] In a preferred embodiment, the gene according to the invention codes for the alpha 1-antitrypsin protein, or other members of the Serpine family such as antiplasmin, alpha 1-antiquymotrypsin.
    [035] In a preferred embodiment, the food-grade bacterial strain according to the invention is an L. lactis strain or Lactobacillus casei strain or an L. lactis htrA strain [Poquet et al., 2000] or a Lactobacillus plantarum strain of a Bifidobacterium longum strain.
    [036] In a preferred embodiment, the food-grade bacterial strain according to the invention is a strain of Lactobacillus casei.
    [037] In a more preferred embodiment, the gene according to the invention codes for trappin-2.
    [038] In fact, the inventors have shown that trappin-2 manifests itself naturally in human colonic mucosa, with a prominent expression in intestinal epithelial cells (Motta et al.) And that, patients with IBD show low regulation of trappin-2 in the tissues compared to healthy individuals (Motta et al).
    [039] In addition, the inventors demonstrated in different models of colitis, that overexpression of trappin-2 is protective against the development of colitis (in the constitutive and transient expression). In addition, overexpression of trappin-2 in colitis models is able to completely inhibit the increase in elastase and trypsin, as activities associated with colitis.
    [040] Finally, overexpression of trappin-2 in mice is also able to significantly inhibit colitis-induced increase in proinflammatory cytokines and chemokines (IL-6, I1-17A, TNF-alpha, Interferon-gamma, MCP- 1 and KC).
    [041] All of these results are in favor of the delivery of trappin-2 and other proteases of the WAP or Serpina family that have similar properties to treat IBD. According to the results below, food-grade bacteria are the safest and most efficient means of delivering this type of proteases to the intestine.
    [042] In another preferred embodiment, the gene according to the invention codes for the alpha 1-antitrypsin protein.
    [043] In fact, alpha 1-antitrypsin protein inhibits activities such as trypsin associated with IBD (such as colitis) and therefore has similar effects as trappin-2.
    [044] In another preferred embodiment, the food-grade bacterial strain according to the invention is a Lactobacillus casei strain, which comprises a gene encoding trappin-2.
    [045] In another preferred embodiment, the food-grade bacterial strain according to the invention is a strain of Lactobacillus casei, which comprises a gene encoding trappin-2 inserted into the thyA gene.
    [046] In another embodiment, the food-grade bacteria according to the invention is useful for the treatment of inflammatory bowel conditions.
    [047] In another preferred embodiment, the food-grade bacterium according to the invention is useful for the treatment of IBD and / or IBS.
    [048] Inflammatory conditions can be selected from IBD, IBS, inflammatory lung disease, inflammatory joint disease, or urogenital inflammatory disease. Compositions
    [049] Another object of the invention relates to a therapeutic composition composed of food-grade bacteria according to the invention.
    [050] In a preferred embodiment, the therapeutic composition according to the invention is intended for mucous administration to an object.
    [051] In the other preferred administration, the therapeutic composition according to the invention is intended for oral administration for an object. For example, the compositions can be in the form of a suspension, tablet, capsule, pill, granulate or powder.
    [052] In a therapeutic liquid composition, the food-grade bacteria according to the invention is present, free and not immobilized, in suspension. The suspension has a composition that guarantees physiological conditions for a probiotic bacterium, so that in particular the osmotic pressure within the cell does not cause lysis.
    [053] In a solid therapeutic composition, the food-grade bacteria according to the invention can be present in free form, preferably lyophilized, or in immobilized form. For example, the food-grade bacteria according to the invention can be placed in a gel matrix that protects the cells.
    [054] A solid therapeutic composition intended for oral administration and containing the food grade bacteria according to the invention in immobilized or non-immobilized form is preferably provided with a gastric juice resistant coating. Thus, it is ensured that the food-grade bacteria contained in the therapeutic composition can pass through the stomach without hindrance and without damage and the release of the food-grade bacteria occurs first in the upper intestinal regions.
    [055] In another aspect of the invention, the therapeutic composition contains sufficient colony-forming units (CFU) of the food-grade bacteria capable of forming the protein according to the invention, so that with multiple administration of the therapeutic composition according to the patient , the status of IBD or IBS is improved, the progression of IBD or IBS is stopped, and / or the symptoms of IBD or IBS can be relieved. According to the invention, in particular a therapeutic composition is provided which contains 1x10 8-1x1011, preferably 1x10 9-1x1010 CFU of the food-grade bacteria according to the invention.
    [056] In a more preferred embodiment of the invention, the therapeutic composition containing the food grade bacteria is administered intraretally. Rectal administration is preferably carried out in the form of a suppository, enema or foam. Intra-rectal administration is particularly suitable for chronic inflammatory bowel diseases that affect the lower intestinal sections, for example, the colon. Intranasal administrations are also suitable for treating chronic lung diseases, such as cystic fibrosis and BPCO.
    [057] In another aspect, the invention relates to a food composition composed of food-grade bacteria according to the invention.
    [058] In a preferred embodiment, the food composition according to the invention is intended for oral administration to an object. For example, compositions can be in the form of a suspension, tablet, capsule, pill, granulate, powder or yogurt.
    [059] In a preferred embodiment, the food composition may contain 1x108-1x1011, preferably 1x109-1x1010 CFU of the food-grade bacteria according to the invention.
    [060] In a preferred embodiment, the composition of the food can be administered to the patient in a daily dose of 1010 bacteria.
    [061] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way that limits the scope of the present invention. FIGURES
    [062] Figure 1: differences in weight (A), macroscopic score (B), wall thickness (C) and myeloperoxidase activity (MPO) (D) in the colon tissues of rats that had water or water + DSS (3 %) in their drinking fountains, and who received daily oral treatments for 7 days with wild type L. lactis, or recombinant L. lactis strains expressing elfin or IL-10. Significant differences were observed in relation to mice treated with PBS that received DSS * for p <0.05, ** for p <0.01, and *** for p <0.005. Significant differences in relation to the mouse treated with wild type L. / actw were noted Φ for P <0.05, Φ Φ for P <0.01 and Φ Φ Φ for P <0.05. Significant differences between the PBS + DSS group and the PBS-water group were noted # for p <0.05, # # for p <0.01, and # # # for p <0.005. Significant differences in relation to the recombinant IL-10 L. lactis group were noted w for p <0.05, w w for p <0.01 and w w w for p <0.005.
    [063] Figure 2: trypsin-like activity (A) and elastase activity (B) in colon lumen washes of mice that had water or water + DSS (3%) in their drinkers, and that received daily oral treatments for 7 days with wild type L. lactis, recombinant strains L. lactis expressing Elafina or IL-10. ε means undetectable levels. Significant differences were observed in relation to mice treated with PBS that received DSS * for p <0.05, ** for p <0.01. Significant differences in relation to mice treated with wild-type L. lactis were noted Φ for p <0.05. Significant differences between the PBS + DSS group and the PBS-water group were noted # for p <0.05, # # for p <0.01, and # # # for p <0.005.
    [064] Figure 3: Macroscopic score (A), wall thickness (B), and myeloperoxidase activity (MPO) (C) in the colon tissues of mice that had water or water + DSS (3%) in their drinking troughs, and who received daily oral treatments for 7 days with Lb. wild type, or Lb strains. I married recombinants expressing Elafina. Significant differences were observed in relation to mice treated with PBS that received DSS * for p <0.05, ** for p <0.01, and *** for p <0.005. Significant differences were observed in relation to the mice treated with Lb. I married wild type Φ for p <0.05, Φ Φ for p <0.01, and Φ Φ Φ for p & lt p <0.005.
    [065] Figure 4: trypsin-like activity (A) and elastase activity (B) in colon lumen washes of mice that had water or water + DSS (3%) in their drinkers, and that received daily oral treatments for 7 days with wild type Lb. casei, recombinant strains Lb. I married expressing Elafina. Significant differences were observed in relation to mice treated with PBS that received DSS * for p <0.05 and significant differences in relation to mice treated with Lb. I married wild type Φ for p <0.05 and Φ Φ for p <0.01.
    [066] Figure 5: RANTES (A), TNFα (B), IL-6 (C), MCP-1 D, KC (E), INF / (F) and IL-17 (G) protein concentration detected in colon tissues of mice that had water or water + DSS (3%) in their drinking fountains, and that received daily oral treatments for 7 days with wild type Lb. casein or recombinant Lb. I married expressing Elafina. ε means undetectable levels. Significant differences were observed in relation to mice treated with PBS that received DSS * for p <0.05, ** for p <0.01, and *** for p <0.005. Φ showed significant differences for p <0.05, in comparison with mice treated with wild type L. lactis. Significant differences between the PBS + DSS group and the PBS-water group were noted # for p <0.05, # # for p <0.01, and # # # for p <0.005.
    [067] Figure 5: IL-2 (A), IL-4 (B), IL-5 (C), IL-10 (D) and IL-13 (E) protein concentration detected in the colon tissues of mice that had water or water + DSS (3%) in their drinkers, and that received daily oral treatments for 7 days with Lb. married wild type or Lb. recombinant case expressing Elafina. Significant differences were observed in relation to mice treated with PBS that received DSS * for p <0.05, ** for p <0.01. Significant differences were observed in relation to the mice treated with Lb. I married wild type Φ for p <0.05, Φ Φ for P <0.01.
    [068] Figure 7: Total number of pain behaviors (A) or the number of abdominal contractions and licking, stretching and crushing behavior (B) in mice that received intracolonial PBS (n = 5), or mustard oil (0 , 01% (v / v) in 70% ethanol) and who received for 7 days prior, pretreatments by oral intubation of PBS (n = 8), L. lactis wild type (n = 8), or L. recombinant lactis expressing Elafin (n = 8) or IL-10 (n = 5). Significant differences were observed in relation to mice treated with PBS that received mustard oil * for p <0.05, ** for p <0.01, and *** for p <0.005. Φ showed significant differences for p <0.05, in comparison with mice treated with wild type L. lactis.
    [069] Figure 8: Elafine secreted by L. lactis wild type and htrA strains. Western blot experiments carried out with anti-elafine antibodies in cell extracts (C) and supernatants (S) of wild type (ts) or htrA (htrA) strains. Elafin production was induced by nisin from cultures of the exponential phase of ts or htrA strains (both containing the expression vector where the expression of the elastin gene can be induced by the addition of nisin).
    [070] Figure 9: Protective effects of L. lactis ts and mutant htrA strains in 5% DSS - induced by the colitis model. Macroscopic activities (A), histological damage (B) and MPO (C) were evaluated in different groups of 10 mice treated with water (negative control) or with 5% DSS. The first two control groups were treated i) with water and fed orally with PBS (negative control group) and ii) with 5% DSS and orally fed with PBS (positive control group). Other groups were treated with DSS 5%> and with any ts strain (TS), ts strain expressing elfin (Elafina) and the mutant htrA strain expressing elfin (Elafina +).
    [071] Figure 10: Protective effects of L. casei ts strain and L. casei strains expressing SOD, expressing elfin and expressing IL-10 model of colitis in induced by the 5% DSS>. Macroscopic activities (A), histological damage (B) and MPO (C) were evaluated in different groups of 10 mice treated with water (negative control) or with 5% DSS. The first two control groups were treated i) with water and fed orally with PBS (negative control group) and ii) with 5% DSS and orally fed with PBS (positive control group). All other groups were treated with 5% DSS> and with L. casei ts (TS) or L. casei strains expressing superoxide dismutase (SOD), elafine (Elafina) or IL-10. * and * indicate that the data are significantly different (P <0.05) from the data obtained with L. casei ts.
    [072] Figure 11: Myeloperoxidase (MPO) activity (D) in the colon tissues of mice. Myeloperoxidase (MPO) activity (D) was measured in the colon tissues of mice that had water or water + DSS (3%) in their drinking fountains, and that received daily oral treatment for 7 days i) L. lactis TS, L. recombination lactis and htrA L. lactis strains expressing Elafina or IL-10 and with ii) L. casei TS strain and expressing SOD, expressing elfin and L. casei strains expressing IL-10. * and * indicate that the data are significantly different (p <0.05) from the data obtained with L. casei ts.
    [073] Figure 12: Elafin secretion in an L. lactis TS strain where the elafine gene manifests itself under the control of an inducible EDTA promoter - [Llull D and Poquet I.2004; EP 1 537 215 and FR 98 16462]. The strain L. lactis TS expressing elafina was grown overnight in the presence (+) or not (-) of EDTA, a chelating agent (in this construction, the expression of the elafina gene is controlled by a lactococcal promoter that can be induced by addition of EDTA: on the chromosome, this promoter controls the expression of genes that encode a specific ABC absorption system for zinc and is depressed under conditions of absence of zinc that can be mimicked by the addition of EDTA). Proteins were then extracted and fractionated between cells (C) and supernatant fractions (S) and Western blot experiments were performed using anti-elafine antibodies. Example Material and methods
    [074] Elafin cloning in recombinant acid lactic acid bacteria (Lactococcus lactis and Lactobacillus casei)
    [075] Cloning and expression of elafine in lactic acid bacteria
    [076] The gene encoding elafine was PCR amplified from plasmid DK6-elafine (14). Sequences of the primers used were: a. 5 'ahead of Elafina (CCAATGCATCAGCAGCTGTCACGGG AGTTCC) (SEQ ID NO: 1); and 3 'reverse Elafina (GGACTAGTCCTCACTGGGGAACGAAACA CG) (SEQ ID NO: 2). Primers were designed to eliminate the first codons from the signal peptide (PS) coding elafine region and were replaced by PS from the Usp45 protein (PSusp4s), the main secreted protein of L. lactis. For this purpose, PCR product was digested, purified and cloned in pSEC, a L. lactis secretion vector. In the resulting pSEC of the plasmid: elafina, elafina is fused into the structure with a DNA fragment encoding RBS and PSusP45- Expression from the cassette is controlled by the inducible promoter VnisA, whose activity depends on the concentration of nisin used. This plasmid was then introduced into a strain of L. lactis comprising the regulatory genes of nisin nisR et nisK (L. lactis NZ9000) to give rise to the recombinant strain: NZ (pSEC: elafm). The tools used (replicons, promoter, RBS and SP) are functional in strains of lactobacilli, such as Lactobacillus casei and Lb. plantarum. These two strains (each comprising nisRK genes on their chromosomes) were chosen because of their ability to persist in the digestive tract (up to 4 days, as opposed to 24 to 48 fir in L. lactis. In addition, we recently demonstrated that the strain Lb. casei BL23 has anti-inflammatory properties in a model of DSS-induced colitis [Rochat et al., 2007]. Therefore, we have weakly (L. lactis) and strongly (Lb. plantarum) persistent as highly persistent immuno-modulating strains (Lb. casei), allowing us to assess the feasibility of combining the intrinsic anti-inflammatory effects of the strains used with the overexpressed molecules.
    [077] For the induction of the PnisA promoter, recombinant L. lactis were cultured to OD600 = ~ 04-0.6 and then induced with 10 ng / ml nisin (Sigma) for 1h. The functionality of this induction was then tested as follows: NZ cultures (pSEC: elafine) (final OD600 = ~ 1) were separated into granules and supernatant fractions and the elastin content was measured by ELISA and / or Western Blot. Animals
    [078] C57B16 mice (6-8 weeks old) were obtained from Janvier (St Quentin Fallavier) and kept at room temperature, under 12 h of light / dark cycles and having free access to food and water, except in day before the induction of colitis, where they fasted for 12h. All procedures were approved by the institutional Committee for animal care and veterinary services. Colitis induction and study design
    [079] Colon inflammation was induced by treatments with Dextran Sodium Sulfate (DSS). In detail, DSS was dissolved in drinking water (3 or 5% weight / vol.) And the animals were free to drink this solution for 7 days. Water consumption was measured in groups treated with DSS and compared to groups of naive mice drinking drinking water: no difference was observed for the volume of liquid consumed between mice that drank water and drank DSS. The mice were treated daily orally, with 100 μl of 5,109 colony-forming units (cfu) of wild type, L. lactis and Lb. I married recombinant with elafine or bacterial medium alone. The first treatment started at the same time that DSS was added to drinking water and the last treatment was on the day of sacrifice (day 7). Survival rate and body weight were measured daily after induction of colitis. On day 7 after the addition of DSS in drinking water, the mice were sacrificed and two colons were harvested to measure various parameters of inflammation: macroscopic classification, intestinal thickness, myeloperoxidase activity (MPO), proteolytic activity, cytokine expression. Measurement of inflammatory parameters
    [080] Macroscopic damage was assessed as previously described (5'15'16). Briefly, when observed, the following parameters received a score of 1: hemorrhage, edema, stenosis, ulceration, fecal blood, mucus and diarrhea. Erythema 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). Adherence was marked based on its severity (0: absent, 1: moderate, 2: severe).
    [081] MPO was measured as an index of granulocyte infiltration, as previously described (5'15'16), in the colon tissues collected at the time of sacrifice. Tissue samples were homogenized in a 0.5% solution of hexadecyltrimethylammonium bromide in phosphate buffer (pH 6) and centrifuged at 13,000 X G for 2 min. Supernatants were added to a buffer containing 1% hydrogen peroxide and O-dianisidine dihydro chloride. Optical density values of the enzyme solution were read for 2 min. at 450 nm.
    [082] For cytokine and chemokine protein measurements, frozen colonic samples collected at sacrifice were homogenized using a 30 s polyester at 4 ° C in 500μ1 cell lysis buffer (20mM Tris-Hcl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, ImM beta-glycerophosphate, 1 mM Na3V04, 1 μg / ml leupeptin; Cell Signalling, Sigma) supplemented with anti-protease cocktail (Roche Diagnostics, Meylan, France). After centrifugation (10,000 Xg, 10 min, 4 ° C), supernatants were filtered on QIAshredder columns (Qiagen, France) and fifty microliters of this homogenate were used for simultaneous dosing of cytokines and chemokines using cytometric sphere matrix in the cell classifier fluorescent lamp FACSCalibur. Crude values were normalized for tissue weight (average 30 to 50 mg) and cytokine concentrations were inferred from standard curves with the help of the FCAP Array® software. In accordance with the manufacturer's information, only values above the limit of cytokine detection were considered. Serine protease activity in colon tissues and luminal lavage
    [083] As previously described (17), after sacrifice, the entire colon and 1 ml were excised.
    [084] PBS was instilled and washed twice through the lumen. Proteolytic activities (trypsin-like and elastase activity) were measured in those luminous washes. Trypsin and elastase-like activities were measured using Tosil-Gly-Pro-Arg-nitroanilide (150 μM, Sigma) and MeO-sucini 1- Ala-Ala-Pro-Val-nitroanilide (100 μM, Sigma, Saint Quentin Fallavier, France ) respectively as substrates. Samples (20 μl for trypsin activity or 10 μl for elastase activity) were resuspended in their respective buffer: 100 nmM Tris / HCl, ImM CaCl2, pH = 8 for trypsin activity and 50 mM Tris-HCl, 500 mM NaCl, 0.1% Triton X100 for elastase activity. The change in absorbance at 405 nm was determined for 30 minutes at 37 ° C with a NOVOstar ™ microplate reader (BMG Labtech, France). The activity was compared with the standard known dilution of porcine trypsin from the porcine pancreas (Sigma) or human neutrophil elastase (Sigma). The protein concentration in luminous washes was determined using the colorimetric dosage of bicinchoninic acid in a microplate (BCA kit®, Pierce, Thermo Scientific, Courtaboeuf, France) and was used to standardize the proteolytic activity in each sample.
    [085] Induction and measurement of visceral pain behaviors in response to mustard oil
    [086] Cultures of the three L. lactis strains (L. lactis wt, L. lactis-Elaim, L. lactis-lL-10) were carried out in M17 medium (Oxoid) supplemented with glucose (0.5%>) complemented with chloramphenicol (10 μg / mL) at 30 ° C without stirring. Bacteria from overnight cultures were grown in fresh medium at 1/50 (w / w) until OD6oo = 0.4 to 0.6. Bacteria were then grown for one more spruce with nisin (1 ng / mL), added to allow expression of the recombinant protein. Bacteria were collected by centrifugation at 450 g and washed with sterile PBS. The pellets were resuspended in sterile PBS with a final concentration of 5x1010 cfu / ml. Groups of 4 to 8 mice were treated daily with 100μl (5x10 cfu) of bacterial suspension by intragastric administration for seven days. On day 8, the mice were administered 50 μl of PBS or mustard oil (0.01% (w / w) in 70% ethanol) by intracolonic instillation, performed under light anesthesia with isoflurane. The number of behavioral responses related to pain (abdominal retractions, abdominal discomfort, stretching, and crushing the lower abdomen against the floor) were then counted for 20 min. Statistics
    [087] Group comparisons were made using a 2-tailed Student t test with Bonferroni correction. Data are expressed as mean ± SEM, and a P value less than 0.05 was considered significant. Results of
    [088] Recombinant Lactococcus lactis expressing elafine protects against the development of DSS colitis in mice.
    [089] As expected, DSS-induced colitis (5% DSS) caused severe weight loss in all groups of mice compared to control mice that drank water. None of the treatments for lactic acid bacteria significantly changed this weight loss (Fig. 1A). DSS in drinking water also caused macroscopic damage, increased wall thickness and increased MPO activity in the colon tissues (Fig. 1 B, C, D). Mice that were treated with L. lactis wild type showed no significant decrease in colon wall thickness and MPO activity, only a slight decrease in macroscopic damage score was observed in this group, compared to mice treated with DSS alone. In contrast, mice treated with recombinant L. lactis expressing elafine showed, after induction of DSS colitis, a significantly reduced macroscopic damage score and less significant increase in colon wall thickness, but MPO activity was no different from the DSS group alone. (Fig. 1, B, C, D). In addition, mice treated with recombinant L. lactis expressing IL-10 cytokines showed reduced macroscopic damage score and MPO activity after induction of DSS colitis, but the wall thickness was not changed by this treatment, compared to DSS alone (Fig. 1, B, C, D). In non-inflamed mice, none of the treatments changed the inflammatory parameters compared to naive control mice.
    [090] The DSS-induced increase in trypsin-like activity was significantly reduced in mice treated with recombinant L. lactis expressing elfin, but was not altered in mice treated with wild-type L. lactis or recombinant L. lactis expressing IL-10 ( Fig. 2A). Treatment with recombinant L. lactis expressing elafine alone was able to significantly reduce the DSS-induced increase in elastase activity (Fig. 2B).
    [091] Lactobacillus casei recombinant expressing any elafine protects against the development of DSS colitis in mice.
    [092] Although the treatment of mice with Lb. If wild-type casei has significantly reduced the macroscopic scores observed after induction of DSS colitis, this treatment has failed to reduce the greater wall thickness and MPO activity compared to DSS alone (Fig. 3 A, B, C). On the other hand, treatments with Lb. recombinant casei expressing elafine significantly reduced all parameters of inflammation: macroscopic damage score, colon wall thickness and MPO activity (Fig. 3A, B, C).
    [093] The DSS-induced increase in trypsin-like activity was significantly reduced in mice treated with Lb. recombinant casei expressing elafine, compared to mice treated with Lb. I married wild type (Fig. 4A). The level of elastase activity was also significantly reduced in mice with colitis (DSS) treated with Lb. recombinant casei expressing elafine, compared to inflamed mice (colitis) treated with Lb. I married wild type, or even compared to inflamed mice treated with PBS (Fig. 4B).
    [094] Protein expression of RANTES chemokine was not significantly increased by DSS colitis at the observed point (7 days after starting DSS treatment). Lb. wild type or elafine secretory was not able to modify the level of RANTES expression in mice treated with DSS (Fig. 5A). TNFa, IL-6, MCPl, KC, INFy and IL-17A were significantly increased by DSS colitis, 7 days after induction (Fig. 5B to G). The treatment of mice with Lb. recombinant casei expressing elafine significantly reduced the expression of IL-6, MCPl, KC and IL-17 proteins (Fig. 5C, D, E, G), but failed to reduce the level of expression of other proinflammatory cytokines, such as such as TNFa and INFy (Fig. 5B and F). Interestingly, while DSS colitis did not cause any increase in cytokines IL-2, IL-4, IL-5, IL-10 and IL-13 (Fig. 6 A to E), the treatment of mice with Lb. recombinant casein for elafine significantly increased the expression of these cytokines, but only in a context of colitis (after DSS treatment). This increase in Th2 cytokines in response to Lb. recombinant casein for elafine could explain, at least in part, the anti-inflammatory effects of this recombinant bacterium. The levels of IL2, IL-4, IL-5, IL-10 and IL-13 were not modified by any of the other treatments in non-inflamed or inflamed mice (DSS) (Fig. 6 A to E).
    [095] Recombinant lactococcus lactis for expression of elafine decreases visceral pain behavior
    [096] The 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 discomfort, stretching and crushing against the floor (Fig. 7 and B ). Considering all behaviors together, treatment with recombinant wild-type IL-10 or L. lactis-secreting elafine had no effect (Fig. 7A). When considering only integrated pain behaviors, L. lactis elafina-, but not recombinant or wild-type IL-10- significantly reduced the number of pain behaviors (Fig. 7B). In addition, only recombinant L. lactis expressing elafine induced a notable decrease in the number of pain behaviors compared to the treatment of PBS or the treatment of wild-type L. lactis (Fig. 7B). Results with htrA strain
    [097] L. lactis expresses only a maintenance extracellular protease called htrA, which degrades all unfolded export proteins (Poquet et al, 2000 and US patent applications 6,994,997 and FR2787810). A mutant L. lactis strain inactivated in the htrA gene was constructed and allowed to increase the rate of production of various heterologous proteins secreted in L. lactis (Poquet et al, 2000 and Miyoshi et al, 2002). In accordance with the invention, the elafine expression cassette has been cloned into an htrA mutant. Levels of production of elafine in mutant htrA and the wild type strain (wt) were compared by Western blot experiments (Fig. 8). A significant increase in elafine secreted in the supernatant of the htrA mutant was observed compared to the wt strain. Thus, the htrA strain will allow higher levels of production and secretion of elafine.
    [098] These two strains were then tested in the DSS-induced colitis model and we confirmed in vivo that the htrA mutant protects mice against colitis damage better than the wt strain (Fig. 9 A, B and C). Comparative results
    [099] The protective effects of IL-10, superoxide dismutase (SOD) and L. casei strains that produce elafine were evaluated in parallel in a 5% -induced DSS colitis model. It should be remembered that L. casei has two major differences in relation to L. lactis: i) greater persistence in the GIT and ii) intrinsic anti-inflammatory properties (Rochat et al, 2007; Watterlot et al, 2010). As shown in Fig. 10A / B / C, the best protective effects on the three criteria (macroscopic, histological and MPO activity) are obtained with L. casei strain producing elf, followed by L. casei strain producing SOD. The IL-10 producing L. casei strain provided only weak effects.
    [0100] Furthermore, L. casei producer of elafina provided better protection than the two L. lactis strains (L. Lactis wt and htrA strain) (Fig. 11).
    [0101] These results are more surprising, considering the fact that elafine has antibacterial activities in vitro and in vivo [Simpson AJ et al, 1999]. Thus, one skilled in the art would expect poor or no production by the host of the bacterium. On the contrary, the results obtained by the inventors show a very good production of elafine by the probiotic and, therefore, a therapeutic effect.
    [0102] In addition, in vitro and in vivo studies (including clinical studies) have shown that the lack of the host antimicrobial shield is potentially deleterious in colon diseases [Salzman NH et al, 2003 and Bevins CL et al, 2009].
    [0103] Thus, the pleiotropic anti-microbialanti-inflammatory activity of elafine makes it a very good candidate therapeutic molecule compared to IL-10.
    [0104] EDTA induction of zinc Promoter (PZn) zitR- controlled expression in L. lactis.
    [0105] The production of elafine in L. lactis driven by PZn zitR [Llull D and Poquet I. 2004] was tested by Western blot analysis after 1 h of induction with 1 mM EDTA. Samples from non-induced cultures, cell pellet (C) and supernatant (S), produce very low levels of production and secretion of elafine, while induced cultures result in higher levels of expression and secretion (Fig. 12). REFERENCES:
    [0106] Throughout this application, several references describe the state of the art to which this invention belongs. Disclosures of these references are incorporated by reference into this disclosure.
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    权利要求:
    Claims (7)
    [0001]
    1. Food-grade recombinant lactic acid bacteria, characterized by the fact that it comprises a recombinant gene that codes for the elastin protein or an active fraction of the elastin protein.
    [0002]
    2. Food-grade recombinant lactic acid bacteria according to claim 1, characterized by the fact that the lactic acid bacteria comprises a defective auxotrophic gene, whereby the survival of said lactic acid bacteria is strictly dependent on the presence of specific compounds.
    [0003]
    3. Food-grade recombinant lactic acid bacteria according to claim 2, characterized by the fact that the defective auxotrophic gene is thyA.
    [0004]
    4. Food-grade recombinant lactic acid bacteria according to claim 3, characterized by the fact that the selected gene is inserted in place of the defective auxotrophic gene.
    [0005]
    Food-grade recombinant lactic acid bacteria according to any one of claims 1 to 4, characterized in that it is selected from Bifidobacterium, Lactococcus lactis or Lactobacillus.
    [0006]
    Food-grade recombinant lactic acid bacteria according to any one of claims 1 to 5, characterized by the fact that it is selected from Lactococcus lactis htrA, Lactobacillus casei, Lactobacillus plantarum and Bifidobacterium longum.
    [0007]
    7. Therapeutic composition, characterized by the fact that it comprises a food-grade recombinant lactic acid bacterium, as defined in any one of claims 1 to 6.
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    法律状态:
    2017-10-24| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
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    申请号 | 申请日 | 专利标题
    FR10305045.6|2010-01-14|
    EP10305045|2010-01-14|
    PCT/EP2011/050489|WO2011086172A1|2010-01-14|2011-01-14|Recombinant probiotic bacteria for the prevention and treatment of inflammatory bowel diseaseand irritable bowel syndrome |
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