EP1523559A1 - Mutants de paroi cellulaire pour la delivrance de composes biologiquement actifs - Google Patents

Mutants de paroi cellulaire pour la delivrance de composes biologiquement actifs

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Publication number
EP1523559A1
EP1523559A1 EP03759974A EP03759974A EP1523559A1 EP 1523559 A1 EP1523559 A1 EP 1523559A1 EP 03759974 A EP03759974 A EP 03759974A EP 03759974 A EP03759974 A EP 03759974A EP 1523559 A1 EP1523559 A1 EP 1523559A1
Authority
EP
European Patent Office
Prior art keywords
lactobacillus
mutant
recombinant
cell wall
bacterium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP03759974A
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German (de)
English (en)
Inventor
Corinne Grangette
Annick Mercenier
Jean Delcour
Pascal Hols
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Universite Catholique de Louvain UCL
Institut Pasteur de Lille
Original Assignee
Universite Catholique de Louvain UCL
Institut Pasteur de Lille
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Application filed by Universite Catholique de Louvain UCL, Institut Pasteur de Lille filed Critical Universite Catholique de Louvain UCL
Priority to EP03759974A priority Critical patent/EP1523559A1/fr
Publication of EP1523559A1 publication Critical patent/EP1523559A1/fr
Ceased legal-status Critical Current

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route

Definitions

  • the present invention relates to the field of recombinant microorganisms for delivery of compounds at mueosal surfaces.
  • the delivery at mueosal surfaces is the potentially most attractive route for administration of biologically active compounds as it involves non-invasive techniques and enjoys a high patient compliance.
  • WO 9714806 relates to the use of non-invasive gram-positive bacteria such as Lactococcus lactis for the delivery of biologically active polypeptides in the body.
  • WO 9709437 describes methods for obtaining surface expression of a desired protein or polypeptide in gram-positive bacteria such as L lactis. It also relates to the possible immobilization of the gram-positive host microorganisms on a solid phase by displaying on their cell surface molecules such as streptavidin.
  • the immune system can be divided into two functionally independent compartments: (i) the systemic, which comprises the bone marrow, spleen and lymph nodes, and (ii) the mueosal, which comprises lymphoid tissue associated with mueosal surfaces and external secretory glands. Mueosal surfaces are associated with the gastrointestinal, genitourinary and respiratory tracts. Each compartment is associated with both humoral (antibodies) and cell-mediated (for example, cytotoxic T-cells) responses, however there are differences in the nature of the immune responses induced in each compartment. Antibodies associated with the systemic compartment are predominantly of the IgG isotype, which function to neutralize pathogens in the circulatory system.
  • secretory immunoglobulins A characterizes mueosal immune responses and is recognized to be a key factor for preventing entry of pathogens at the mueosal surfaces which are the major sites at which microbial infections are initiated.
  • S-lgA secretory immunoglobulins A
  • non-pathogenic bacteria such as lactic acid bacteria (LAB)
  • LAB lactic acid bacteria
  • LAB lactic acid bacteria
  • LAB lactic acid bacteria
  • LAB lactic acid bacteria
  • LAB lactic acid bacteria
  • LAB certain strains or species of LAB belong to the natural endogenous microflora of individuals where they play a critical role in maintaining a balanced ecosystem.
  • WO 0023471 describes the construction of recombinant Lactococcus lactis strains producing IL-10 and their use in anti-inflammatory treatment.
  • EP 1084709 relates to oral vaccines comprising lactic acid bacteria as specific component for eliciting immunogenicity against a heterologous antigen.
  • Lactobacillus plantarum strains are used.
  • Said strains comprise an expression vector capable of expressing the heterologous antigen intracellularly and/or such that the heterologous antigen is exposed on the cell surface of the bacterium.
  • the invention relates to a recombinant gram-positive bacterium for use as a medicament wherein said microorganism comprises a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expresses a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component and wherein said recombinant microorganism is producing intracellularly a polypeptide for prophylactic or therapeutic applications.
  • the invention further relates to a method for the delivery of polypeptides at mueosal surfaces using said recombinant microorganism, comprising administering to a mueosal surface of an individual a composition comprising said recombinant microorganism having a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expressing a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component and wherein the polypeptide to be delivered is produced by said recombinant microorganism.
  • the invention relates to the use of said recombinant for the manufacture of a medicament for preventing or treating for example infectious, inflammatory, auto-immune or allergic diseases, metabolic deficiencies or cancers.
  • the present invention relates to use of a mutant microorganism as a vehicle for delivery of compounds or polypeptides at mueosal surfaces characterized in that said mutant microorganism comprises a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expresses a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component.
  • the present invention relates to a gram positive recombinant microorganism capable of producing a polypeptide having a prophylactic or therapeutic activity, and bearing a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expressing a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component.
  • recombinant microorganism used herein, relates to a microorganism comprising a vector or an expression vector that has been modified by the introduction of a heterologous nucleic acid or a transgenic strain carrying the heterologous nucleic acid integrated in its chromosome.
  • recombinant microorganisms express genes that are not found in identical form or mode of expression within the native (non- recombinant) microorganism.
  • heterologous nucleic acids can be obtained from any natural source and/or can be prepared synthetically using well known DNA synthesis techniques.
  • expression vector as used herein relates to a vector wherein the heterologous nucleic acid sequence comprises at least one heterologous gene operably linked to one or more control sequences allowing the expression in prokaryotic host cells.
  • Said nucleic acid sequence comprises coding sequences or open reading frames (ORF) as well as regulatory sequences or elements such as a promoter, an operator or a terminator.
  • ORF open reading frames
  • the microorganism according to the invention is a bacterial strain, preferably a non-pathogenic strain, preferably a non-invasive strain, preferably a food-grade strain, more preferably a gram-positive bacterial strain, most preferably a lactic acid bacterial strain.
  • suitable bacterial strain examples include but are not limited to Lactobacillus, Lactococcus, Bifidobacterium and non-pathogenic staphylococci species.
  • said microorganism is a Lactobacillus or a Lactococcus species.
  • suitable food grade strains can be found in Bergey's Manual of Systematic Bacteriology, Volume 2 (1986), Gram-positive Bacteria other than Actinomycetes, Williams & Wilkins, Baltimore, hereby incorporated by reference.
  • lactic acid bacterial strain preferably Lactobacillus or Lactococcus strains according to the invention
  • They can be commensal strains isolated from host-specific body cavities (humans or animals), environmental isolates or starter strains used for the production of fermented food and feed. It is becoming well established that given strains are able to persist transiently in body cavities. Moreover, some of these strains have been reported to exhibit probiotic proprieties (beneficial to the host's health) including immuno-adjuvant and immuno- modulating capacities, which vary according to the species or strains used.
  • the present invention is thus applicable to any of the Lactobacillus, Lactococcus or Streptococcus species or subspecies or strains selected from the list comprising Lactobacillus acetotolerans, Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus arizonensis, Lactobacillus aviaries, Lactobacillus aviarius subsp.
  • Lactobacillus acetotolerans Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus amylolyticus
  • Lactobacillus aviarius subsp. aviarius Lactobacillus batatas, Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus carnis, Lactobacillus casei, Lactobacillus casei subsp. alactosus, Lactobacillus casei subsp. casei, Lactobacillus casei subsp. pseudoplantarum, Lactobacillus casei subsp. rhamnosus, Lactobacillus casei subsp.
  • Lactobacillus catenaformis Lactobacillus cellobiosus, Lactobacillus coleohominis, Lactobacillus collinoides, Lactobacillus confusus, Lactobacillus coprophilus, Lactobacillus coprophilus subsp. confusus, Lactobacillus corynoides, Lactobacillus corynoides subsp. corynoides, Lactobacillus corynoides subsp. minor, Lactobacillus coryniformis, Lactobacillus coryniformis subsp. coryniformis, Lactobacillus coryniformis subsp.
  • Lactobacillus crispatus Lactobacillus curvatus, Lactobacillus curvatus subsp. curvatus, Lactobacillus curvatus subsp. melibiosus, Lactobacillus cypricasei, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp.
  • lactis Lactobacillus desidiosus, Lactobacillus diolivorans, Lactobacillus divergens, Lactobacillus equi, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus ferintoshensis, Lactobacillus fornicalis, Lactobacillus frigidus, Lactobacillus fructivorans, Lactobacillus fructosus, Lactobacillus frumenti, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus graminis, Lactobacillus halotolerans, Lactobacillus hamsteri, Lactobacillus helveticus, Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus hordniae, Lactobacillus iners, Lactobacillus intestinalis, Lacto
  • Lactobacillus paracasei Lactobacillus paracasei subsp. tolerans, Lactobacillus parakefiri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus piscicola, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus psittaci, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rimae, Lactobacillus rogosae, Lactobacillus ruminis, Lactobacillus sakei, Lactobacillus sakei subsp.
  • Lactobacillus sakei subsp. sakei Lactobacillus salivarius, Lactobacillus salivarius subsp. salicinius, Lactobacillus salivarius subsp. salivarius, Lactobacillus sanfranciscensis, Lactobacillus sharpeae, Lactobacillus suebicus, Lactobacillus trichodes, Lactobacillus uli, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus vermiforme, Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillus xylosus, Lactobacillus yamanashiensis, Lactobacillus yamanashiensis subsp.
  • the lactic acid bacterial strain is a Lactobacillus plantarum or Lactococcus lactis strain.
  • L. plantarum NCIMB8826 has a very good resistance to the gastric barrier and a very good survival in the ileum in humans, and is able to persist for about a week in the mouse intestine after oral administration.
  • the recombinant microorganism comprises a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expresses a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component.
  • a cell wall component in the present invention relates to "at least one cell wall component” whereas it may happen that a mutation in a given gene has a pleitropic effect on the biosynthesis, modification or degradation of several distinct components of the cell wall.
  • mutation refers to a sequence variation within a gene such as substitution in which one or more nucleotides are substituted with (an)other nucleotide(s), deletion in which one or more of existing nucleotides are deleted, insertion in which one or more of additional nucleotides are inserted. Also, “mutation” refers to insertion into any site of the chromosome of a nucleic acid sequence originating from a different locus or from a different genome.
  • a mutation in the present invention relates to "at least one mutation” whereas it might be necessary to introduce several mutations in a given gene to have an effect on the biosynthesis, modification or degradation of a cell wall component. Furthermore, it might be necessary to have mutations in several genes to affect a single cell wall component.
  • a mutation modulating the expression relates to a mutation which preferably has an effect on the production of the mature or functional cell wall component.
  • Said effect can be the over-production, the non-production or prevention of production or delayed or decreased production of a cell wall component or of an enzyme or structural element involved in the biosynthesis, modification or degradation of a cell wall component.
  • Said effect can also be the production of a non-functional or premature or incomplete polypeptide due to a missense or frame-shift mutation or the generation of a premature stop codon.
  • all said mutations may result in a modification of the bacterial cell wall which may lead to a variety of phenotypes, including reduction in viability, especially under stress conditions, weakening of the cell wall, leakage of cytoplasmic content, auto/co- aggregation, a change in its porosity/permeability, a change in its global physical-chemical surface properties, such as change in hydrophobycity or electric charge.
  • the expression "expressing a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component” relates to recombinant microorganisms which have been transformed with at least one foreign gene, and wherein said gene is capable of modulating the production of a cell wall component or of an enzyme or structural element involved in the biosynthesis, modification or degradation of a cell wall component.
  • said foreign gene can be responsible of the production, the over-production, the non-production or prevention of production or delayed or decreased production of an endogenous or non-endogenous polypeptide or protein interfering with the biosynthesis, modification or degradation of the microorganism cell wall.
  • foreign gene means a polynucleotide encoding a protein or fragment thereof or antisense RNA or catalytic RNA, which is foreign to the said recombinant microorganism.
  • gene relates to a nucleic acid sequence comprising coding sequences or open reading frames as well as regulatory sequences or elements such as a promoter, an operator, or a terminator.
  • regulatory sequences or elements such as a promoter, an operator, or a terminator.
  • coding sequence or "ORF” is defined as a nucleotide sequence that can be transcribed into mRNA and translated into a polypeptide when placed under the control of appropriate control sequences or regulatory sequences. Said coding sequence or ORF is bounded by a 5' translation start codon and a 3' translation stop codon.
  • a coding sequence or ORF can include, but is not limited to RNA, mRNA, cDNA, recombinant nucleotide sequences, synthetically manufactured nucleotide sequences or genomic DNA.
  • promoter refers herein to a "promoter” in its broadest context and includes the transcriptional start signal and additional regulatory or control elements (i.e. operators).
  • promoter includes the prokaryotic transcriptional start signal, in which case it may include a -35 box sequence and/or a -10 box sequence. Promoters may contain one or several copies of regulatory elements, to control expression of the associated transcription unit in response to intra or extra cellular signals or molecules.
  • terminal refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription.
  • the recombinant microorganism according to the present invention comprises a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expresses a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component, wherein the expression "cell wall component” generally refers to all structures and molecules found in a bacterial cell wall, such as peptidoglycans, teichoic acids, lipoteichoic acids, teichuronic acids, polysaccharides, and cell wall associated proteins.
  • the said microorganism In order for the recombinant microorganism to elicit a mueosal immune response, the said microorganism has to come in close contact with the cells from the body cavity mucosa which are responsible for triggering the mueosal immune response, and to deliver to them the said compounds or polypeptides.
  • the compounds or the polypeptides produced in the cytoplasmic compartment of recombinant microorganism are confined in said microorganism by the bacterial cell wall.
  • a cell wall mutant of the bacterium is used.
  • Said recombinant microorganism comprising said cell wall mutation or expressing said foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component is therefore bearing an altered cell wall which may be subject to a premature lysis and which may result in a more efficient and more controlled release of the cellular compounds or polypeptides when administered to an organism in need thereof.
  • the mutation may induce changes in the global cell wall properties such that the resulting mutant is able to interact more efficiently with the host immune system or the epithelial surface of the targeted body cavity, and/or to better persist in the targeted body cavity.
  • the recombinant microorganism according to the invention comprises a mutation in a gene involved in the biosynthesis of peptidoglycans, teichoic acids, or lipoteichoic acids, more preferably the mutated gene is involved in the biosynthesis of D-alanine, D-glutamate or D-aspartate, or in the alanylation or glucosylation of teichoic or lipoteichoic acids.
  • genes involved in the biosynthesis, modification or degradation of a cell wall component comprise the following gene products: alanine racemase, glutamate racemase, aspartate racemase, D-lactate dehydrogenase, lactate racemase, aspartate kinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, N- succinyldiaminopimelate-aminotransferase, N-succinyl-L-diaminopimelic acid desuccinylase, diaminopimelate epimerase, diaminopimelate decarboxylase, N- acetylglucosamine-1 -phosphate uridyltransferase, UDP-N-acetylglucosamine enolpyruvoyltransferase, UDP-N-
  • the recombinant microorganism used in the present invention has a mutation in the gene encoding alanine racemase (air), or glutamate racemase, or aspartate racemase, or in the dlt cluster of genes responsible for D-alanylation of teichoic acids and lipoteichoic acids, or in the tagE gene responsible for glucosylation of teichoic acids.
  • air alanine racemase
  • glutamate racemase or aspartate racemase
  • the present inventors clearly demonstrated that the use of a recombinant Air- mutant producing an antigen led to a very significant increase of specific immune responses after mueosal administration to mice when compared to the recombinant Alr+ strain.
  • said recombinant microorganism produces a least one polypeptide for prophylactic or therapeutic application.
  • Produced polypeptides can be derived from eukaryotic sources or prokaryotic sources, or from viruses.
  • polypeptide when used herein refers to amino acids in a polymeric form of any length. Said term also includes any naturally occurring post- translational modifications. Furthermore said term refers to biologically active polypeptides, wherein “biologically active” refers to ability to perform a biological function. Examples of such biologically active compounds are antigenic compounds, immunomodulators, nutritional compounds, medicaments, as well as analogues or derivatives thereof.
  • the various classes of biologically active polypeptides which can be delivered by the method of the invention include, but are not limited to any therapeutic or prophylactic compound selected from the group comprising antigens, epitopes, allergens, immune modulators including adjuvants, enzymes, receptor ligands or variants thereof.
  • anti-inflammatory compounds include analgesics, antiarthritics, antispasmodics, antidepressants, antipsychotics, tranquilizers, antianxiety compounds, narcotic antagonists, antiparkinsonism compounds, cholinergic agonists, chemotherapeutic compounds, immunosuppressive compounds, antiviral compounds, antibiotics, antimicrobial compounds, appetite suppressants, antiemetics, anticholinergics, antihistaminics, antimigraine compounds, coronary, cerebral or peripheral vasodilators, hormones, contraceptives, antithrombotic compounds, diuretics, haemostatics, hemolytics, antihypertensive compounds, cardiovascular compounds, allergoids, vitamins, antibodies, enzyme inhibitors, anti-receptors or receptor blockers, neuropeptides and the like.
  • polypeptides include but are not limited to the C subunit of tetanus toxin (TTFC) and the Urease B subunit (Urease B or UreB)
  • the produced polypeptide may be fused to another peptide, polypeptide or protein to form a chimeric protein.
  • polypeptide produced exerts a beneficial effect or activity on the health of the individual, in a prophylactic or therapeutic manner, wherein “prophylactic” means protective or preventive, and “therapeutic” means curative.
  • the invention further relates to the use of any recombinant microorganism herein described as a medicament.
  • the invention relates to a recombinant microorganism for use as a medicament wherein said recombinant microorganism comprises a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expresses a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component and wherein said recombinant microorganism is producing a polypeptide for prophylactic or therapeutic application.
  • the invention further relates to a method for the delivery of polypeptides at mueosal surfaces using said recombinant microorganism, comprising delivering to a mueosal surface of an individual an effective amount of a composition comprising said recombinant microorganism having a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expressing a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component and wherein the polypeptide to be delivered is synthesized by said recombinant microorganism.
  • the term "individual” as used herein refers to a human or an animal host. The individual will preferably be a human, but veterinary applications are also in the scope of the present invention targeting for example domestic livestock, laboratory or pet animals.
  • Mueosal surfaces include mueosal membranes such as buccal, gingival, nasal, tracheal, bronchial, gastrointestinal, rectal, urethral, ureteral, vaginal, cervical, uterine, etc.
  • Administration of said composition to a mueosal surface comprises any route of administration to the body of a human or animal for which it is not required to puncture the skin (e.g. as with intravenous, intramuscular, subcutaneously or intraperitoneal administration).
  • said composition is administered to the body via one of the body cavities, such that it comes into contact with the mucosa.
  • routes to administer said composition include nasal, oral or intragastric, genital, rectal, aural (i. e. via the ear), ocular, sublingual, buccal, bronchial, inhalatory, and urethral routes.
  • the invention also relates to the use of a recombinant microorganism as defined previously for the manufacture of a medicament for prophylactic or therapeutic application. It may for example be used for vaccine application as well as for treating illness such as infectious diseases, allergy, chronic inflammation, auto-immune diseases, metabolic deficiencies and cancers.
  • the invention relates to the use of a mutant microorganism as a vehicle for delivery of compounds or polypeptides at mueosal surfaces characterized in that said mutant microorganism comprises a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expresses a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component.
  • the compound or polypeptide to be delivered is chosen from a protein, polypeptide, or peptide.
  • the mutant microorganism is a gram-positive commensal bacterium, preferably a lactic acid bacterium, most preferably a Lactobacillus or Lactococcus strain chosen from Lactobacillus plantarum or Lactococcus lactis.
  • the mutant microorganism according to the invention comprises a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component wherein said cell wall component is preferably peptidoglycan, teichoic acid, or lipoteichoic acid. More preferably said mutation is in a gene involved in the biosynthesis of D-alanine, D-glutamate, or D-aspartate, or in the alanylation or glucosylation of teichoic or lipoteichoic acids.
  • said gene encodes alanine racemase, or aspartate racemase, or glutamate racemase, or (poly)glycerolphosphate/ (poly)ribitolphosphate D-alanyltransferase, or
  • the invention also relates to the use of a mutant microorganism as previously defined as a vaccine, wherein said mutant microorganism is expressing a nucleic acid encoding an antigen which is able to elicit an immune response when administered to a human or animal host.
  • the term "antigen" is meant to include peptides, polypeptides and proteins, encoded by a nucleic acid expressed by a recombinant microorganism of the present invention, against which an immune response can be elicited, such as an antibody response, and which can act as a trigger for immunizing the individual against the target pathogen.
  • the antigen produced is preferably a peptide, a polypeptide or a protein derived from a viral or a bacterial pathogen or from fungi or parasites or from other microorganisms capable of infecting human or animal species.
  • an antigen can also be a peptide, a polypeptide or a protein, for instance, derived from a hyperproliferative disease such as cancer, thus also including tumor antigens and auto-immune antigens. They may be post-transcriptionally derivatized antigens like glycosylated, lipidated, glycolipidated or hydroxylated antigens.
  • antigens and antigenic components including antigenic parts, fragments or epitopes thereof
  • antigenic mutants or analogs thereof - obtained synthetically or via recombinant DNA techniques - may be produced.
  • a combination of two or more such antigens may be produced, i.e. by a single type or strain of the recombinant microorganism according to the invention, or by several different types or strains said microorganism.
  • antigens produced may be chosen from the group comprising antigens derived from toxins such as toxins from diphtheria or from Clostridium (such as Clostridium septicum, Clostridium tetani, Clostridium perfringens), Pneumococcus and other Streptococcus species, Pasteurella maltocida and Corynebacterium pseudotuberculosis.
  • toxins such as toxins from diphtheria or from Clostridium (such as Clostridium septicum, Clostridium tetani, Clostridium perfringens), Pneumococcus and other Streptococcus species, Pasteurella maltocida and Corynebacterium pseudotuberculosis.
  • the uses and the methods according to one aspect of the present invention are related to the delivery of antigen at mueosal surfaces and therefore are directed to the induction of mueosal immunity
  • selected antigens produced by said recombinant microorganism can also be derived from pathogens which invade the mammal via the mucosa.
  • the pathogen may be selected from viruses: Human Papilloma Viruses (HPV), HIV, HSV2/HSV1 , influenza virus (types A, B, and C), Polio virus, RSV virus, Rhinoviruses, Rotaviruses, Hepatitis A virus, Norwalk Virus Group, Enteroviruses, Astroviruses, Measles virus, Para Influenza virus, Mumps virus, Varicella-Zoster virus, Cytomegalovirus, Epstein- Barr virus, Adenoviruses, Rubella virus, Human T-cell Lymphoma type I virus (HTLV-I), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus, Pox virus, Marbug and Ebola; bacteria: Bacillus anthracis, Bordetella pertussis, Brucella, Campylobacter; Escherichia coli, Giacardia lamblia, Klebsiellae species, Haemophil
  • PI. falciparum PI. vivax, etc.
  • Pseudomonas aeruginosa Pneumococcus, Salmonellae species, Shigellae species, Staphylococcus aureus, Treponema pallidum, Vibrio cholerae, Yersinina pestis, Donovanosis, and Actinomycosis
  • fungal pathogens including Candida and Aspergillus
  • parasitic pathogens including Taenia, Flukes, Roundworms, Antamoeba, Giardia, Leishmania, Cryptosporidium, Schistosoma, Pneumocystis carinii, Trichomonas and Trichinella.
  • the recombinant microorganism provided by the invention is readily applicable as a vaccine against any pathogen against which immunization via the mueosal route is effective. Therefore, the invention encompasses the production of antigens derived from a wide range of human or animal pathogens to which mueosal immunity is desired.
  • the term "antigen" is further intended to encompass peptide or protein analogs of known or natural antigens such as those described above, which analogs may be more soluble or more stable than wild type antigen, and which may also contain mutations or modifications rendering the antigen more immunologically active.
  • the invention is not limited by the identity of the particular antigen produced by said microorganism.
  • the immune response elicited may be prophylactic or therapeutic, wherein prophylactic immune response refers to an immune response which targets an antigen to which the individual has not yet been exposed such as a pathogen antigen in an uninfected individual, or a disease cell associated protein in an individual who does not have the disease such as a tumor associated protein in a patient who does not have a tumor.
  • prophylactic immune response refers to an immune response which targets an antigen to which the individual has not yet been exposed such as a pathogen antigen in an uninfected individual, or a disease cell associated protein in an individual who does not have the disease such as a tumor associated protein in a patient who does not have a tumor.
  • therapeutic immune response refers to an immune response which targets an antigen to which the individual has been exposed such as a pathogen antigen in an infected individual, or a disease cell associated protein in an individual who has the disease or a tumor associated protein in a patient who has a tumor.
  • an antigen to which the individual has been exposed such as a pathogen antigen in an infected individual, or a disease cell associated protein in an individual who has the disease or a tumor associated protein in a patient who has a tumor.
  • the recombinant microorganism of the present invention can be used to modulate an immune response not only to stimulate (i.e., elicit, produce, and/or enhance) a protective immune response but also to suppress (i.e., reduce, inhibit, block) an overactive, or harmful immune response (induction of tolerance against an allergen, reduction of inflammation, restoration of the immune homeostasis).
  • a response (e.g. an antibody or cellular immune response) is considered significant when it leads to a detectable change or response in a human or animal, and in particular to a detectable immunological change or response, such as the production of antibodies, cytokines, chemokines, cytotoxic or helper T cell responses, etc.
  • a significant response as used herein may be, but is not necessarily, also a protective response.
  • a response (e.g. an immunological response against a pathogen or an antigen) is considered protective when it is capable of protecting the individual which shows said response against said pathogen and/or against a pathogen associated with said antigen.
  • the vaccines according to the invention can be formulated such that a single dose is sufficient. However embodiments where multiple applications over a period of time, e.g. with a view to the persistence of the bacterial host in the gastro-intestinal tract, are also envisaged as falling within the scope of the invention.
  • the provision of booster vaccinations is also envisaged as a potential embodiment with the vaccine formulations according to the invention.
  • the invention further relates to a method for the preparation of a vaccine comprising admixing a recombinant microorganism as defined previously with a pharmaceutically acceptable carrier.
  • Said vaccine or said medicament may also contain one or more adjuvants, including immune adjuvants such as, but not limited to cytokines, co- stimulatory molecules, as long as these are compatible with the recombinant microorganism and do not interfere with its desired immunogenic properties.
  • the adjuvants may be a lactic acid bacterium, such as the bacterial host strain itself, or one of the other lactic strains mentioned herein.
  • said vaccine may be prepared or formulated/processed as in use in the food fermentation technology. It can be formulated such as to obtain a fermented product.
  • the invention further relates to a method for the preparation of a medicament comprising admixing a recombinant microorganism as defined previously with a pharmaceutically acceptable carrier or formulated/processed as in use in the food fermentation technology.
  • Pharmaceutically acceptable carriers may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions suitable for ingestion, inhalation, or administration as a suppository to the rectum or vagina.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and certain organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • antimicrobials for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
  • said vaccine or medicament comprising recombinant microorganisms may be lyophilized, freeze dried, dry sprayed or submitted to any formulation or process in use in the starter technology or for probiotic preparations, according to the route of administration, for example inhalation, absorption, genital administration etc....
  • the vaccine composition or the medicament may suitably be provided in the form of a spray, an aerosol, a mixture, tablets (entero-or not-enterocoated), capsule (hard or soft, entero-or not-enterocoated), a suspension, a dispersion, granules, a powder, a solution, an emulsion, chewable tablets, tablets for dissolution, drops, a gel, a paste, a syrup, a cream, a lozenge (powder, granulate, tablets), an instillation fluid, a gas, a vapor, an ointment, a stick, implants (ear, eye, skin, nose, rectal, or vaginal), vagitories, suppositories, or uteritories suitable for administration via the oral, nasal, vaginal, sublingual, ocular, rectal, urinal, intramammal, pulmonary, otolar, or buccal route.
  • the vaccine composition or the medicament may also be in the form of
  • the invention further relates to the use of a recombinant microorganism comprising a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expressing a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component, as a vehicle for delivery of therapeutic or prophylactic compounds or polypeptides at mueosal surfaces, wherein said therapeutic or prophylactic compound or polypeptide is produced by said microorganism and is chosen from a protein, polypeptide, or peptide.
  • said therapeutic or prophylactic compound or polypeptide is able to elicit or modulate an immune response when administered to a human or animal host.
  • the invention further relates to the use of a mutant microorganism comprising a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expressing a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component, as a vehicle for delivery of DNA molecules such as for example a vector carrying immunostimulatory DNA sequences ISS-DNA, also known as CpG-DNA.
  • a mutant microorganism comprising a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component or expressing a foreign gene interfering with the biosynthesis, modification or degradation of a cell wall component, as a vehicle for delivery of DNA molecules such as for example a vector carrying immunostimulatory DNA sequences ISS-DNA, also known as CpG-DNA.
  • the present invention further encompasses the mutant microorganism according to the invention for use as a medicament, wherein said mutant microorganism comprises a mutation modulating the expression of a gene involved in the biosynthesis, modification or degradation of a cell wall component.
  • said cell wall component is preferably peptidoglycan, teichoic acid, or lipoteichoic acid.
  • said mutation is in a gene involved in the biosynthesis of D-alanine, D-glutamate, or D-aspartate, or in the alanylation or glucosylation of teichoic or lipoteichoic acids.
  • said gene encodes alanine racemase, or aspartate racemase, or glutamate racemase, or (poly)glycerolphosphate/ (poly)ribitolphosphate D-alanyltransferase, or
  • suitable bacterial strain examples are the same as that described above.
  • said microorganism is a gram-positive bacterium, such as a lactic acid bacterial strain.
  • said lactic acid bacterial strain is a Lactobacillus plantarum or Lactococcus lactis strain.
  • the invention also relates to the use of a mutant microorganism as previously defined as a medicament.
  • a cell wall mutant microorganism for therapeutic application may result in the prevention, reduction or curing of a disease.
  • said mutant microorganism can be used to modulate an immune response not only to stimulate (i.e., elicit, produce, and/or enhance) a protective immune response but also to suppress (i.e., reduce, inhibit, block) an overactive, or harmful immune response (induction of tolerance against an allergen, reduction of inflammation, restoration of the immune homeostasis and the like).
  • the mutant microorganism used in the present invention has a mutation in the gene encoding alanine racemase (air), or glutamate racemase (glr), or aspartate racemase, or in the dlt cluster of genes responsible for D-alanylation of teichoic acids and lipoteichoic acids, or in the tagE gene responsible for glucosylation of teichoic acids.
  • the present inventors clearly demonstrated that a Dlt- mutant of L. plantarum NCIMB8826 was able to strongly elicit regulatory cytokines such as IL-10, without having pro-inflammatory properties (i.e. reduced induction of pro- inflammatory cytokines), compared to the wild type strain. Furthermore, the use of said Dlt- mutant led to a more effective prevention of chemically induced colitis when administered to mice, as compared to the wild type strain.
  • the invention also relates to the use of a mutant microorganism according to the invention for the manufacture of a medicament for prophylactic or therapeutic application. It may for example be used for preventing or treating illness such as allergy, chronic inflammation, chronic infection (HCV), auto-immune diseases, metabolic deficiencies and cancers. Such mutants might also be used as mueosal adjuvants.
  • a mutant microorganism according to the invention for the manufacture of a medicament for prophylactic or therapeutic application. It may for example be used for preventing or treating illness such as allergy, chronic inflammation, chronic infection (HCV), auto-immune diseases, metabolic deficiencies and cancers. Such mutants might also be used as mueosal adjuvants.
  • said mutant microorganism can be used for the manufacture of a medicament for the prevention or treatment of various intestinal disorders.
  • said mutant microorganism can be used for the manufacture of a medicament for the prevention or treatment of inflammatory bowel disease.
  • the invention further relates to a method for the preparation of a medicament comprising admixing a cell wall mutant microorganism as defined previously with a pharmaceutically acceptable carrier or formulated/processed as in use in the food fermentation technology.
  • a pharmaceutically acceptable carrier or formulated/processed as in use in the food fermentation technology.
  • Suitable examples of pharmaceutically acceptable carriers are the same as that described above.
  • Preservatives and other additives may also be present as described above.
  • Suitable forms for said medicament are the same as that described above.
  • Figure 1 corresponds to a Western Blot illustrating the intracellular production level of the C subunit of tetanus toxin (TTFC) in different host strains.
  • the blot was obtained by immunoblotting cell extracts (equivalent to 10 8 CFU) obtained from control strain NCIMB8826/ pTG2247 (1) or MG1363/ pTX (8) or TTFC-producing strains NCIMB8826/ pMEC4 (2), NCIMB8826 Air-/ pMEC4 (3), NCIMB8826/ pMEC46 (4), NCIMB8826 Air-/ pMEC46 (5), NCIMB8826/ pMEC127 (6), NCIMB8826 Air-/ pMEC127 (7), MG1363/ pMEC46 (9), MG1363 Air-/ pMEC46 (10) or from 50 ng purified TTFC (11).
  • Figure 2 is a bar graph depicting the effect of immunization with different strains on serum anti-TTFC IgG titers.
  • Anti-TTFC serum IgG responses were measured following intragastric immunization with wild type NCIMB8826/ pMEC4, NCIMB8826/ pMEC46, NCIMB8826/ pMEC127 or mutant NCIMB8826 Air-/ pMEC4, NCIMB8826 Air-/ pMEC46 or NCIMB8826 Air-/ pMEC127 recombinant Lactobacillus plantarum strains, in comparison to control non-expressor strain [Lactobacillus plantarum (pTG2247)] or buffer alone. Individual sera were collected 10 days after the first administration (I), the first ( ⁇ ), the second (D) or the third (ED) boost.
  • Figure 3 is a bar graph depicting the effect of immunization with different strains on specific anti-TTFC IgA titers.
  • Anti-TTFC IgA levels were measured in intestinal lavages from groups of eight mice immunized intragastrically with recombinant mutant Lactobacillus plantarum NCIMB8826 Air-/ pMEC127 in comparison to control non- expressor strain NCIMB8826/ pTG2247 or buffer alone. Intestinal lavages were performed 10 days after the last administration (third boost).
  • Figure 4 is a bar graph depicting the effect of immunization with different strains on serum anti-TTFC IgG titers.
  • Anti-TTFC IgG response were measured, following intragastric immunization with wild type NCIMB8826/ pMEC127 or mutant NCIMB8826 Air-/ pMEC127 recombinant Lactobacillus plantarum and wild type MG1363/ pMEC46 or mutant MG1363 Air-/ pMEC46 recombinant Lactococcus lactis strains in comparison to control non- expressor strains [NCIMB8826/ pTG2247 or MG1363/ pTX] or buffer alone. Individual sera were collected 10 days after the first administration (M), the first ( ⁇ ), or the second (D) boost.
  • Figure 5 is a bar graph depicting the effect of immunization with different strains on serum anti-TTFC IgG titers.
  • Anti-TTFC IgG response were measured following intravaginal immunization with wild type NCIMB8826/ pMEC127 or mutant NCIMB8826 Air-/ pMEC127 recombinant Lactobacillus plantarum and wild type MG1363/ pMEC46 or mutant MG1363 Air-/ pMEC46 recombinant Lactococcus lactis strains in comparison to control non- expressor strains [NCIMB8826/ pTG2247 or MG1363/ pTX] or buffer alone. Individual sera were collected 10 days after the first administration (H), the first ( ⁇ ) or the second (EH) boost.
  • Figure 6 is a bar graph depicting the effect of immunization with different strains on serum anti-TTFC IgG titers.
  • Anti-TTFc IgG response were measured following intrarectal immunization with wild type NC1MB8826/ pMEC127 or mutant NCIMB8826 Air-/ pMEC127 recombinant Lactobacillus plantarum and wild type MG1363/ pMEC46 or mutant MG1363 Air-/ pMEC46 recombinant L. lactis strains in comparison to control non-expressor strains [NCIMB8826/ pTG2247 or MG1363/ pTX] or buffer alone. Individual sera were collected 10 days after the first administration (H), the first ⁇ ffl), or the second (D) boost.
  • Figure 7 is a bar graph depicting the effect of immunization with different strains on specific anti-TTFC fecal IgA titers.
  • Anti-TTFC IgA levels were measured in feces from groups of mice immunized intrarectally with wild type NCIMB8826/ pMEC127 or mutant NCIMB8826 Air-/ pMEC127 recombinant Lactobacillus plantarum strain or wild type MG1363/ pMEC46 or mutant MG1363 Air-/ pMEC46 recombinant L lactis strains in comparison to control non-expressor strain [NCIMB8826/ pTG2247 or MG1363/ pTX] or buffer alone. Feces were collected from pooled groups of 3 mice, 10 days after the first (_ ⁇ ) or second (H) boost.
  • Figure 8A corresponds to a Western Blot illustrating the intracellular production level of UreB in different Lactobacillus plantarum strains.
  • the blot was obtained by immunoblotting 5 ⁇ g (1) or 10 ⁇ g (2) of recombinant UreB (rUreB) and cell extracts (equivalent to 10 6 CFU) obtained from UreB-producing strains NCIMB8826/ pMEC142 and NCIMB8826 Air-/ pMEC142 prepared at day 1 , and stored at 4°C until day 2 or 3.
  • Figure 8B and 8C are bar graphs depicting the effect of immunization with different strains on specific anti-UreB serum IgA (B) and IgG (C).
  • Anti-UreB antibody responses were measured following intragastric immunization with wild type NCIMB8826/ pMEC142, or mutant NCIMB8826 Air-/ pMEC142 recombinant Lactobacillus plantarum strains, in comparison to control non-expressor strain Lactobacillus plantarum I pTG2247 or medium alone.
  • mice were immunized with 50 ⁇ g recombinant UreB + 10 ⁇ g cholera toxin (CT).
  • Results are expressed as OD units using 1 :200 (IgA) or 1:1 '000 (IgG) dilutions of mouse sera, respectively.
  • Figure 9 represents genomic DNA amplification by PCR resolved on 2 % agarose gels containing ethidium bromide. Representative examples for mice vaccinated with Lactobacillus plantarum NCIMB8826 Air-/ pMEC142 (A) and UreB/CT (B) are shown. Single band (marked by a star) detected by UN. fluorescence was considered as PCR- positive. End-points of analysis were set at 32 and 40 cycles with constant amount of template genomic D ⁇ A.
  • Figure 10 corresponds to a Western Blot illustrating the intracellular production level of TTFC in different host strains.
  • the blot was obtained by immunoblotting cell extracts (equivalent to 10 8 CFU) obtained from the control strain Lactococcus lactis MG1363 / pTX (1) or TTFC-producing Lactococcus lactis strains ⁇ Z3900/ pMEC46 (2), MG1363 DltD-/ pMEC46 (3) or 200 ng purified TTFC (4).
  • Figure 11 is a bar graph depicting the effect of immunization with different strains on serum anti-TTFC IgG responses.
  • Anti-TTFC IgG titers were measured, following intragastric immunization with the wild type (NZ3900/ pMEC46) or mutant (MG1363 DltD-/ pMEC46) recombinant Lactococcus lactis strains in comparison to control non-expressor strain (MG1363/ pTX) or buffer alone.
  • Individual sera were collected 10 days after the first administration (I), the first ( ⁇ ), or the second (11) boost.
  • Figure 12 is a bar graph depicting the effect of immunization with recombinant Lactococcus lactis strains on specific anti-TTFC intestinal IgA titers.
  • Anti-TTFc IgA levels were measured in intestinal lavages from groups of mice immunized intragastrically with wild type NZ3900/ pMEC46 or mutant MG1363 DltD-/ pMEC46 recombinant Lc. lactis strains in comparison to control non-expressor strain (MG1363/ pTX) or buffer alone. Intestinal lavages were collected individually 10 days after the last boost.
  • Figure 13 is a bar graph depicting the cytokine response of human PBMC (2 x 10 ⁇ cells / ml) to Lactobacillus plantarum NCIMB8826 wild type and Dlt- mutant (NCIMB8826 DltB-) strains.
  • PBMC from healthy human donors were stimulated with or without (medium only)
  • Figure 14 is a bar graph depicting the cytokine response of human monocytes (0.5 x 10 6 cells / ml) to Lactobacillus plantarum NCIMB8826 wild type and DltB-mutant strains. Monocytes were stimulated with or without (medium only) 10 7 CFU / ml of thawed bacteria. IL-10, IL-12 (p70), TNF ⁇ and IFN ⁇ were measured by ELISA in monocytes supernatants collected after 24h stimulation. The results represent the mean + SEM for the response of 4 donors to each bacteria strain.
  • Figure 15 is a bar graph depicting the effect of preventive administration of Lactobacillus plantarum NCIMB8826 wild type and DltB- mutant strains on acute TNBS-induced colitis in Balb/c mice.
  • A Weight variation between day 5 (TNBS administration) and day 7 (sacrifice).
  • B Wallace and
  • C Ameho inflammation scores. Results are means ⁇ SEM of two separated experiments (9 mice/ group). Significant p values ⁇ 0.05 ** or ⁇ 0.1*, as compared to the TNBS control group that received no bacteria.
  • Lactobacillus plantarum (Lb. plantarum) NCIMB8826 and Lactococcus lactis (Lc. lactis) MG1363 were used.
  • the Lb. plantarum NCIMB8826 Air- (MD007) (with defective gene coding for alanine racemase) and the Lc. lactis MG1363 Air- (PH3960) were obtained as described in Bron P. et al, Appl. Environ. Microbiol. 2002 Nov; 68 (11): 5663-70, both strains were deposited on June 16, 2003 at the Belgian Coordinated Collections of Microorganisms (BCCM).
  • pMEC127 was obtained by deleting the sequence coding for 25 amino acids of the N-terminus of the LDH gene (Reveneau et al., Vaccine, 2002, 20(13): 1769-1777).
  • the replicative plasmid (pMEC46) carrying the TTFC-encoding gene under the control of nisin inducible promoter (pNis A, Pavan S. et al., Appl. Environ.
  • lactis MG1363/ pMEC46 lactis MG1363/ pMEC46.
  • the levels of TTFC produced by Lb. plantarum NCIMB8826/ pMEC4, NCIMB8826/ pMEC46 and NCIMB8826/ pMEC127 were respectively low, moderate or high.
  • the level of TTFC production observed for the Lc. lactis MG1363/ pMEC46 strain was similar to that obtained with the NCIMB8826/ pMEC127 strain. No antigen production was detected in the control strains NCIMB8826/ pTG2247 and MG 1363/ pTX.
  • Example 2 In vivo delivery of TTFC by lactic acid bacteria
  • Wild type strain Lb. plantarum harboring the control plasmid (NCIMB8826/ pTG2247) and TTFC-producing wild type strains (NCIMB8826/ pMEC4, NCIMB8826/ pMEC46 and NCIMB8826/ pMEC 27) were grown in MRS broth (Difco, Detroit, Michigan), while the recombinant Air- mutant strains (NCIMB8826 Air-/ pMEC4, NCIMB8826 Air-/ pMEC46 and NCIMB8826 Air-/ pMEC127) required the addition of D-alanine (200 ⁇ g/ml) for their growth.
  • lactis wild type strains (MG1363/ pTX and MG1363/ pMEC46) were grown in M17 (Difco) supplemented with 0.5% glucose; the Air- strain (MG1363 Air-/ pMEC46) required the addition of D-alanine (400 ⁇ g/ml) in the growth medium.
  • the pMEC46 plasmid expression of the TTFC encoding gene was induced by adding nisin at 20 ng/ml for 4h or nisin at 5 ng/ml for 3h in the case of Lb. plantarum or Lc. lactis strains, respectively.
  • the bacteria were resuspended in a gavage buffer (for intra-gastric administration) allowing buffering of the gastric pH (PBS containing 0.2 M NaHCO 3 , 0.5% casein hydrolysate and 0.5% glucose) or in PBS (for intra-vaginal and intra-rectal administration) and the concentrations were adjusted to 10 10 CFU (colony forming unit)/ml, 10 11 CFU/ml or 2 10 10 CFU/ml for intra-gastric, intra-vaginal or intra-rectal administration respectively.
  • PBS gastric pH
  • PBS for intra-vaginal and intra-rectal administration
  • mice C57/B16 were immunized either by intra-gastric gavage with 100 ⁇ l of bacterial suspension (10 9 CFU) or 100 ⁇ l buffer, or by intra-vaginal or intra-rectal administration with, respectively, 10 ⁇ l or 50 ⁇ l of bacterial suspension (10 9 CFU) or with PBS.
  • An aliquot of each suspension was stored at -20°C and analyzed by Western blot to check the antigen dose actually administered to mice.
  • the immunization protocol consisted of 3 consecutive doses of 10 9 CFU every 24 h, followed by 2 boosts at 3-week-intervals. Serum samples were collected by retro-orbital bleeding of the mice 10 days after each administration. The intestinal washes were performed 10 days after the last administration.
  • the intestine of individual mice was cut in its length and washed with 1 ml of PBS containing protease inhibitors (Complete Protease Inhibitor Cocktail, Boehringer). Each homogenate was further cleared by 2 consecutive centrifugations first at 3000 rpm and further at 12000 rpm.
  • the feces were collected from 3 mice per group, pooled, weighed and homogenized in PBS containing protease inhibitors at a final suspension (Weight/Volume) of 100 mg feces per ml. Each homogenate was further cleared by 2 consecutive centrifugations first at 3000 rpm and further at 12000 rpm (labtop centrifuge eppendorf).
  • Example 3 Evaluation of TTFC specific IgG and IgA response by ELISA .
  • TTFC antigen was coated onto a plastic support (Microtiter plates 96 wells Immulon I Dynatech) by incubation overnight at 4°C, of 100 ⁇ l per well of a 2 ⁇ g /ml solution of TTFC in 0.1 M NaHCO 3 /Na 2 CO 3 buffer pH 9.5.
  • the serum samples or intestinal wash samples were added in a 2 fold serial dilution in 1% PBS/BSA buffer from 100 ⁇ l of a 1/50 dilution (sera) or a 1/4 dilution (intestinal wash, fecal suspensions). After 2 hours incubation at room temperature, the plates were washed three times in 0.1% PBS/Tween.
  • BSA bovine serum albumin
  • the bound antibody was detected by addition of 100 ⁇ l of mice biotintylated anti-lgG (1/10000) or anti-lgA (1/2000) (Southern Biotechnology) in 1% PBS/BSA-0.1% Tween buffer, and incubation for 1 hour at room temperature. After 4 washes in 1% PBS/Tween buffer, 100 ⁇ l of streptavidin peroxidase conjugate (Amersham) were added at a 1/2000 dilution in 1% PBS/Tween buffer.
  • the concentration of specific IgA in the intestinal washes was calculated by taking into account the total IgA concentration in each sample.
  • Total IgA levels were determined by ELISA, as described above, with microplates coated with non-labeled anti-lgA from mice (Sigma).
  • the concentration of IgA was calculated using a standard curve of mouse lgA k (Sigma) with consecutive serial dilutions from 100 to 0.78 ng/ml.
  • the results were expressed as specific activity, calculated by dividing the endpoint titer by the level of total IgA concentration expressed in ⁇ g/ml. The statistical significance was evaluated by Mann-Whitney U test.
  • Example 4 Analysis of the in vivo immune response induced by intragastric administration of recombinant TTFC-producing wild-type and mutant Lb. plantarum strains
  • the heterologous gene was placed under the control of a constitutive promoter (L-LDH promoter).
  • L-LDH promoter a constitutive promoter
  • Deletion of the sequence coding for 25 amino acids of the N-terminus of the LDH gene resulted in the second-generation plasmid pMEC127 which, after transformation in Lb. plantarum NCIMB8826, resulted in a recombinant strain (NCIMB8826/ pMEC127) capable of producing much higher TTFC amounts than those produced by the NCIMB8826/pMEC4 strain (Reveneau et; al., Vaccine, 2001, 20(13): 1769-1777).
  • the heterologous gene was under the control of an inducible promoter (pnisA). Transformation of pMEC46 in Lb. plantarum NCIMB8826 resulted in a recombinant strain NCIMB8826/ pMEC46 which after induction by nisin produced more TTFC than strain NCIMB8826/ pMEC4 but less than strain NCIMB8826/ pMEC127. Nasal administration of these strains led to very strong systemic immune responses (Grangette C. et al. Infect. Immun. 2001, 69(3): 1547-53).
  • strain NCIMB8826/ pMEC127 was capable to induce excellent systemic and local responses after intra-gastric administration of 10 9 CFU ( Figure 2), leading to anti-TTFC serum IgG end point titers of 2.10 4 .
  • Figure 2 no significant response was induced by either the first generation strain NCIMB8826/ pMEC4 or the NCIMB8826/ pMEC46 strain.
  • the use of Air- mutant led to a very clear increase of specific immune responses after intra-gastric administration to mice for the two strains synthesizing medium or high amounts of antigen (NCIMB8826 Air-/ pMEC46 or NCIMB8826 Air-/ pMEC127, respectively) when compared to the wild type homologue.
  • the TTFC coding gene is under the control of the LDH constitutive promoter in the second generation vector (pMEC127)
  • the recombinant Alr- mutant strain elicited the serum IgG end point titers twenty times higher (p ⁇ 0.05) than those obtained with the recombinant wild type strain.
  • the serum IgG titers were equal to 4 x 10 5 and 2 x 10 4 for strains NCIMB8826 Air-/ pMEC127 and NCIMB8826/ pMEC127, respectively ( Figure 2).
  • the NCIMB8826 Air-/ pMEC127 strain was moreover able to induce a significant response (titer 2 x 10 3 ) already after priming. Moreover, when the TTFC-coding gene was under the control of the nisin-inducible promoter (pMEC46), only the recombinant Air- mutant strain (NCIMB8826 Air-/ pMEC46) was able to induce a significant immune response. Furthermore, intra-gastric administration of the recombinant mutant strain (NCIMB8826 Air-/ pMEC127) induced significant local IgA response (p ⁇ 0.005) in intestinal lavages of immunized mouse (Figure 3). In contrast, no local response was observed in the intestinal lavages of mice who received the buffer or the control strain (NCIMB8826/ pTG2247).
  • Example 5 In vivo comparative analysis of the immune response elicited by intra-gastric administration of wild type and mutant recombinant Lb. plantarum and Lc. lactis strains
  • MG 1363 Air-/ pMEC46 0,04-0,08 a Sera from intragastrically immunized mice were collected and pooled 10 days after the 2 nd boost and used in the tetanus toxin (TT) neutralization assay (McComb J.A., N. Engl. J. Med., 1964, 270: 175-178). Protection against tetanus toxin requires a minimum neutralizing antibody titer of 0.01 lU/ml (International Units).
  • Example 6 Construction of recombinant Lb. plantarum strains producing Urease B from Helicobacter pylori
  • the replicative plasmid pMEC142 carrying the ureB gene (encoding Urease B) from Helicobacter pylori, encoding gene under the control of a constitutive promoter, the LDH promoter, was constructed and introduced by electroporation in wild type and Air- mutant strains of Lb. plantarum NCIMB8826.
  • the intracellular production of the heterologous antigen (UreB) was verified by Western blot ( Figure 8A). It was observed that the recombinant wild type (NCIMB8826/ pMEC142) and mutant (NCIMB8826 Air-/ pMEC142) strains produced similar quantities of antigen. No antigen production was detected in the control strain NCIMB8826/ pTG2247 (data not shown).
  • Example 7 In vivo delivery of UreB by recombinant Lb. plantarum strains (Wild type and Air- mutant strains)
  • the wild type strain Lb. plantarum harboring the control plasmid (NCIMB8826/ pTG2247, non-expressor strain) and the UreB producing wild type strain (NCIMB8826/ pMEC142) were grown in MRS broth (Difco) supplemented with 5 ⁇ g/ml erythromycin or 10 ⁇ g/ml chloramphenicol, respectively.
  • the recombinant Air- mutant strain (NCIMB8826 Air-/ pMEC142) required the addition of D-alanine (200 ⁇ g/ml) for its growth. However, the expression of the altered phenotype linked to the air mutation requires growing the mutant strain without D-alanine.
  • Wild type NCIMB8826/ pMEC142 was harvested at exponential growth phase (OD 600 of 1-2), while the mutant NCIMB8826 Air-/ pMEC142 was harvested after starvation. After 2 washes in PBS, the latter was subsequently starved by incubation for 3 hours in medium without D-alanine. After two washes in the corresponding medium, the bacteria were resuspended in a gavage buffer allowing buffering of the gastric pH (PBS containing 0.2 M NaHCO 3 , 0.5% casein and 0.5% glucose) and the concentrations were adjusted to 10 10 CFU (colony forming unit)/ml.
  • PBS gastric pH
  • CFU colony forming unit
  • mice Balb/c were immunized by intra-gastric gavage with 200 ⁇ l of bacterial suspension containing 10 9 CFU or 200 ⁇ l of medium. An aliquot of each suspension was stored at -20°C and analyzed by Western blot to check the antigen dose actually administered to mice (Fig. 8A).
  • the immunization protocol consisted of a priming of 3 consecutive doses of 10 9 CFU every 24 h, followed by 2 boosts (triple doses at day 0, 1 and 2) at 3-week-intervals.
  • mice were immunized intragastrically 3 times at weekly intervals with 50 ⁇ g recombinant UreB (rUreB) and 10 ⁇ g cholera toxin (CT) in a total volume of 200 ⁇ l PBS. Serum samples were collected by tail bleeding of the mice 3 days after the last administration. Twelve days after the last immunization, mice were infected with freshly cultured H. felis (5 x 10 7 CFU) by orogastric intubation with polyethylene tubing introduced at a fixed distance of 4.5 cm from the incisors, under light anesthesia with isofluran (Baxter, Switzerland).
  • rUreB recombinant UreB
  • CT cholera toxin
  • mice were sacrificed 2 weeks later and stomachs were collected to examine the degree of protection.
  • the presence of Helicobacter in gastric tissue was assessed by urease test (UT) (Jatrox-test, Procter & Gamble, Rothstadt, Germany).
  • UT urease test
  • One half of the stomach was immersed in 500 ⁇ l of reconstituted mix according to the supplier's recommendation and incubated at 37°C for 3 h.
  • Specimen were centrifuged and the supernatant was used for spectrophotometric quantification at an optical density of 550 nm, as described previously (Corthesy-Theulaz et al., 1995).
  • the cut-off value of urease test used to discriminate between infection and cure corresponded to the mean ⁇ 2 Standard Deviation of the absorbance values obtained with gastric tissues of naive mice.
  • the other half of the stomach was fixed in neutral buffered 10% formalin prior to processing for genomic DNA extraction.
  • About 20 mg stomach was cut into small pieces, placed into a 1.5-ml plastic tube with 180 ⁇ l of ATL buffer (Qiagen, Basel, Switzerland), and homogenized. Following digestion with proteinase K (1.5 mg/ml final concentration) for 30 min at 55°C, 200 ⁇ l of AL buffer (Qiagen, Basel, Switzerland) was added.
  • Example 8 Evaluation of UreB specific serum IgG and IgA responses by ELISA
  • Example 9 Analysis of the in vivo immune response induced by intragastric administration of recombinant UreB-producing wild type and Air- mutant Lb. plantarum strains
  • the immunogenicity of the UreB producing Air- mutant strain was compared to the immune responses elicited by the recombinant wild type strain (NCIMB8826/ pMEC142).
  • Negative controls included mice receiving the non- expressor strain (NCIMB8826/ pTG2247) or medium only. Mice of the positive control group received rUreB adjuvanted with CT. All the expressor strains were able to induce a significant serum IgA ( Figure 8B) and IgG ( Figure 8C) response as compared to those induced in the control groups (non-expressor strain or medium) after intra-gastric administration.
  • the serum IgA response elicited by the recombinant mutant strain was higher than that induced by the recombinant wild type strain (NCIMB8826/ pMEC142) or the adjuvanted pure antigen (rUreB + CT).
  • Significant IgG responses were elicited in all mice immunized with either one of the recombinant strains (NCIMB8826/ pMEC142 or NCIMB882 Air-/ pMEC142) or rUreB + CT. Infection with H. felis did not induce the production of more antibodies, at least during the 2 weeks preceding mouse sacrifice (Figs. 8 B and C).
  • Example 10 Analysis of the protection against H. felis infection after intragastric administration of recombinant UreB-producing wild type and mutant Lb. plantarum strains
  • mice described in Example 9 were subsequently infected with H. felis.
  • a markedly slower color development was observed for samples of the group of mice immunized with the recombinant Air- mutant strain (NCIMB8826 Air-/ pMEC142) as compared to the negative control groups or mice immunized with the NCIMB8826/ pMEC142 strain. Consistent with this observation, end-point values measured at 3h were significantly lower for the group of mice fed with the Lb.
  • Example 11 DltD- mutant of Lc. lactis as antigen delivery vehicle: Construction of recombinant Lc. lactis strains producing TTFC
  • NisRK genes were further introduced in the chromosomal pepN gene by homologous recombination as described in Ruyter et al. J. Bacteriol. 1996; 178: 3434-39.
  • the replicative plasmid (pMEC46) carrying the TTFC-encoding gene under the control of nisin inducible promoter (pNisA, Pavan S et al, Appl. Env. Microbiol., 2000; 66 (10): 4417- 32) was introduced by electroporation in the NZ3900 wild type and in the MG1363 DltD- mutant strains of Lc. lactis.
  • the production of heterologous antigen (TTFC) was verified by Western blot ( Figure 10).
  • the blot was obtained by immunoblotting cell extracts (equivalent to 10 8 CFU) obtained from control strain MG1363 / pTX (1) or TTFC-producing strains NZ3900/ pMEC46 (2), MG1363 DltD-/ pMEC46 (3) or 200 ng purified TTFC (4).
  • the TTFC level observed for the mutant (MG1363 DltD-/ pMEC46) (3) seemed to be higher than that obtained for the wild type strain (NZ3900/ pMEC46) (2). No antigen production was detected in the control strain (MG1363/ pTX) (1).
  • Example 12 In vivo delivery of TTFC by recombinant L. lactis strains
  • All Lc. lactis strains were grown in M17 (Difco) supplemented with 0.5% glucose. Erythromycin 10 ⁇ g/ml was added for the growth of the wild type Lc. lactis strain harboring the control plasmid (MG1363/ pTX) and chloramphenicol at 20 ⁇ g/ ml was added for the growth of the TTFC-producing wild type (Lc. lactis NZ3900/ pMEC46) and mutant (Lc. lactis MG1363 DltD-/ pMEC46) strains.
  • TTFC encoding gene For strains harboring the pMEC46 plasmid, expression of the TTFC encoding gene was induced by adding nisin at 5 ng / ml when the culture reached an OD 600 of 0.5, followed by 3h incubation at 30°C.
  • the preparation of bacterial inocula before intra-gastric administration was done by growth and harvest of the bacteria after nisin induction. After two washes in PBS, the bacteria were resuspended in the gavage buffer (as previously described) at the concentration of 10 10 CFU / ml. Groups of 8 mice C57/B16 were immunized by intra-gastric gavage with 100 ⁇ l of bacterial suspension (10 9 CFU) or 100 ⁇ l buffer.
  • the immunization protocol consisted of a priming of 3 consecutive doses of 10 9 CFU every 24h, followed by 2 boosts (triple doses) at 3-week intervals. Serum samples were collected by retro-orbital bleeding of the mice 10 days after each administration. The intestinal washes were performed 10 days after the last administration as previously described.
  • Example 13 Comparative analysis of the immune response induced by intra-gastric administration of the wild type and DltD- recombinant Lc. lactis strains producing TTFC.
  • Example 14 Lb. plantarum NCIMB8826 DltB- mutant and wild type strains
  • the Lb. plantarum NCIMB8826 DltB- mutant (EP007) deposited on June 16, 2003 at the Belgian Coordinated Collections of Microorganisms (BCCM), was obtained by using a suicide knockout vector containing an internal fragment of the dltB gene by single-step homologous recombination. Disruption of the locus was confirmed by PCR and Northern blotting and by an in vitro radioactive D-Alanine incorporation test (data not shown). Wild type and mutant strains were grown at 37°C in MRS broth (Difco). Erythromycin (5 ⁇ g/ml) was added for the mutant NCIMB8826 DltB- strain.
  • Example 15 In vitro evaluation of the immunomodulation capacity of the wild type and the DltB- mutant strains of Lb. plantarum NCIMB8826
  • L. plantarum NCIMB8826 wild type and DltB- mutant (NCIMB8826 DltB-) strains were grown in MRS medium for 48h and the cells were harvested by centrifugation, washed twice in sterile PBS pH 7.2, resuspended at 10 9 CFU / ml in PBS containing 20% glycerol and stored at -80°C until used for stimulation assays.
  • PBMC Peripheral Blood Mononuclear Cell
  • Monocytes (CD14 + cells) were purified by using CD14 antibodies coupled to magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's recommendations. Briefly, PBMC (300 x 10 6 cells) were incubated on ice for 30 min with 300 ⁇ l of CD14 antibodies coupled to magnetic microbeads. After washings they were applied onto a column placed in magnetic field of a MidiMACS separation unit (Miltenyi Biotec). After elution of the column, the cells obtained by positive selection were adjusted at 0.5 X 10 6 cells per ml in complete RPMI medium.
  • cytokine release was performed by seeding PBMC (2 X 10 6 cells/ ml) or monocytes (0.5 X 10 6 cells/ml) in 24-well tissue culture plates. Ten ⁇ l of thawed bacterial suspension at 10 9 bacteria/ml were then added. PBS containing 20% glycerol was used as negative (non stimulated) control. After 24h stimulation at 37°C in 5% CO2, culture supernatants were collected, clarified by centrifugation and stored at -20°C until cytokine analysis.
  • Cytokines were measured by ELISA using BD Pharmingen (San Diego, Ca, USA) antibody pairs for TNF ⁇ , IL10 and IFN ⁇ and the Diaclone Eli-pair (Besancon, France) for IL12, according to the respective manufacturer's recommendations.
  • the Lb. plantarum NCIMB8826 wild type and DltB- mutant strains differentially induced IL10, IL12, TNF ⁇ , and INF ⁇ secretion from stimulated PBMC and monocytes.
  • the effect of the DltB- mutant strain on cytokine release after PBMC stimulation was compared to that of the wild type strain (Fig 13).
  • the Dlt- mutant strain induced largely increased levels of IL10 secretion by stimulated PBMC, altogether with a significantly decreased release of the Th1 /pro-inflammatory cytokines (IFN ⁇ , IL12 and TNF ⁇ ).
  • the effect of the wild type and Dlt- mutant Lb. plantarum strains was evaluated on monocyte stimulation, since among PBMC, monocytes are the most likely candidates for the production of IL12 (after bacterial stimulation). Similar results as those obtained with PBMC were obtained for the monocytes (Fig 14).
  • Human PBMC and monocytes were purified and stimulated with the wild type or the mutant Dlt- Lb. plantarum strains at a concentration of 1 x 10 8 CFU/ml.
  • IL12 and IL10 concentrations were measured by ELISA in the PBMC culture supernatant after 24h. The results are represented as the ratio of IL10/ IL12 taking the mean of each set of concentrations into account.
  • the ratio of IL10 to IL12 production (Table 3) for the wild type strain was equal to 1.14 for PBMCs and 3.45 for monocytes.
  • the ratio of IL10 to IL 12 production was dramatically higher for the DltB- mutant (160.8 for PBMC, 122 for monocytes) than for the wild type strain.
  • Table 3 IL10/IL12 ratio in the cytokine response of human PBMC and monocytes to thewild type and Dlt- mutant strains of Lb. plantarum.
  • Example 16 Analysis of the beneficial effect of the DltB- mutant of Lb. plantarum NCIMB8826 in a murine colitis model.
  • mice from Iffa Credo St Germain sur I'Arbresle, France
  • mice from Iffa Credo received an intrarectal administration of 40 ⁇ l of a solution of trinitrobenzene sulphonic acid (TNBS) (100 mg/kg,
  • mice Fluka, Saint Quentin Fallavier, France) dissolved in 0.9% NaCI/ethanol (60/40 v/v). Control mice received 40% ethanol. Animals were killed by cervical dislocation 2 days after TNBS administration.
  • L. plantarum NCIMB8826 wild type and DltB- mutant strains were grown for 48h, washed and collected by centrifugation before resuspension at 10 8 CFU / ml in a 0.2 M NaHCO 3 buffer containing 2% glucose.
  • 10 7 CFU of bacteria were administered once daily by oral gavage, starting 5 days before and until 1 day after TNBS administration. Mice were weighed before colitis induction and immediately after sacrifice by cervical dislocation. Comparisons between the different animal groups were analyzed by the nonparametric Wilcoxon analysis of variance (ANOVA) test.
  • Macroscopic lesions of the colon were evaluated according to the Wallace criteria (Wallace et al. 1989, Gastroenterology 96: 29-36), reflecting both the intensity of inflammation and the extend of the lesions.
  • a colon sample located in the most injured area was fixed in 4% paraformaldehyde acid and embedded in paraffin.
  • Four micrometers-sections were stained with May Gr ⁇ nwald-Giemsa and blindly scored according to the Ameho criteria (Ameho, OK., et al. 1997. Gut 41:487-493). This grading on a scale from 0 to 6 takes into account the degree of inflammatory infiltrate, the presence of erosion, ulceration or necrosis, and the depth and surface extension of the lesion.
  • mice displaying diarrhea after TNBS administration were much lower in the group of mice gavaged with the DltB- mutant (11,1%) than in the group of mice receiving the Lb. plantarum wild type strain or no bacteria (55,5% in both cases) (data not shown).
  • the weight loss was significantly reduced in mice receiving the Dlt- mutant as compared with mice receiving no bacteria or the wild type strain (Fig 15A).
  • a milder but not significantly different level of inflammation was observed in mice gavaged with the wild type Lb. plantarum bacteria (Figs. 15B and 15C).
  • mice gavaged with the DltB- mutant of Lb. plantarum displayed less severe lesions.
  • the others displayed only hyperaemia and thickening of the intestinal wall (Wallace score of 1.6 ⁇ 0.6) (Fig 15B).
  • mice with the Lb. plantarum NCIMB8826 DltB- mutant before induction of the colitis reduced the severe loss of body weight associated with TNBS administration.
  • This correlated to the significantly lower inflammation scores of mice which received the DltB- mutant strain before TNBS administration as compared to mice gavaged with the wild type strain or receiving no bacteria.
  • cell wall mutant strains can be used as effective agents for the treatment of various inflammatory diseases such as for example inflammatory bowel disease and the like.

Abstract

La présente invention a trait à un micro-organisme recombinant comportant une mutation modulant l'expression d'un gène impliqué dans la biosynthèse, la modification ou la dégradation d'un constituant de paroi cellulaire ou exprimant un gène étranger perturbant la biosynthèse, la modification ou la dégradation d'un constituant de paroi cellulaire et comportant un vecteur d'expression conduisant à la production d'un polypeptide présentant une activité prophylactique ou thérapeutique. L'invention a également trait à un procédé permettant la délivrance de polypeptides à des muqueuses, comprenant l'administration à une muqueuse d'un sujet (humain ou animal) d'une composition comportant un micro-organisme recombinant comportant une mutation modulant l'expression d'un gène impliqué dans la biosynthèse, la modification ou la dégradation d'un constituant de paroi cellulaire ou exprimant un gène étranger perturbant la biosynthèse, la modification ou la dégradation d'un constituant de paroi cellulaire et dans lequel le polypeptide à délivrer est produit par ledit micro-organisme recombinant.
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GRANGETTE CORINNE ET AL: "Enhanced anti inflammatory capacity of a Lactobacillus plantarum mutant synthesizing modified teichoic acids", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 102, no. 29, July 2005 (2005-07-01), pages 10321 - 10326, XP002563271, ISSN: 0027-8424 *
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ZEGERS N.D. ET AL: "Expression of the protective antigen of Bacillus anthracis by Lactobacillus casei: Towards the development of an oral vaccine against anthrax", JOURNAL OF APPLIED MICROBIOLOGY, OXFORD, GB LNKD- DOI:10.1046/J.1365-2672.1999.00900.X, vol. 87, no. 2, 1 August 1999 (1999-08-01), pages 309 - 314, XP002324747, ISSN: 1364-5072 *

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