US20060062807A1 - Gram positive bacteria preparations for the treatment of diseases comprising an immune dysregulation - Google Patents

Gram positive bacteria preparations for the treatment of diseases comprising an immune dysregulation Download PDF

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US20060062807A1
US20060062807A1 US10/497,703 US49770305A US2006062807A1 US 20060062807 A1 US20060062807 A1 US 20060062807A1 US 49770305 A US49770305 A US 49770305A US 2006062807 A1 US2006062807 A1 US 2006062807A1
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bcg
killed
bacterial preparation
extended freeze
dried
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Inventor
Marie-Anne Nahori
Micheline Lagranderie
Gilles Marchal
Bernado Vargaftig
Jean Lefort
Felix Romain
Georges Hekimian
Philippe Peltre
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Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
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Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
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Assigned to INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE, INSTITUT PASTEUR reassignment INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HEKIMIAN, GEORGES, PELTRE, PHILIPPE, LEFORT, JEAN, VARGAFTIG, BERNARDO BORIS, MARCHAL, GILLES, ROMAIN, FELIX, LAGRANDERIE, MICHELINE, NAHORI, MARIE-ANNE
Publication of US20060062807A1 publication Critical patent/US20060062807A1/en
Priority to US11/600,907 priority Critical patent/US7871627B2/en
Priority to US12/582,834 priority patent/US8404250B2/en
Abandoned legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to compositions comprising components prepared from Gram positive bacteria such as Gram positive facultative intra-cellular bacteria, for example mycobacteria, for the treatment of disorders comprising an immune dysregulation, in humans and animals.
  • the invention also relates to the preparation of said components and compositions.
  • Diseases comprising an immune dysregulation include different cancers, autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, Crohn disease and diabetes mellitus, and allergic disorders such as asthma, allergic rhinitis, conjonctivis and atopic dermatitis.
  • autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, Crohn disease and diabetes mellitus
  • allergic disorders such as asthma, allergic rhinitis, conjonctivis and atopic dermatitis.
  • Allergic asthma is a common disease, which involves airway allergen-induced inflammation; the inflammatory leukocytes, eosinophils and sometimes neutrophils, are recruited to airways as a consequence of the presence and activation of T lymphocytes that react with the inhaled allergens in the respiratory tract.
  • CD4+ T lymphocytes play a major role in initiating allergic airways inflammation via the production of Th2-type cytokines, which trigger the recruitment of eosinophils to the airways and possibly their subsequent activation. It has been proposed that an imbalance between Th2 and Th1 effectors drives the pathogenesis of asthma.
  • extrinsic asthma or atopic asthma is associated with the enhancement of a Th2 type immune response with the production of specific immunoglobulin E (IgE), positive skin tests to common aeroallergens and/or atopic symptoms.
  • IgE immunoglobulin E
  • the airflow obstruction in extrinsic asthma involves a non-specific bronchial hyperresponsiveness (BHR) caused by inflammation of the airways. This inflammation is mediated by chemicals released by a variety of inflammatory cells including mast cells, eosinophils and lymphocytes. The actions of these mediators result in vascular permeability, mucus secretion and bronchial smooth muscle constriction.
  • atopic asthma the immune response producing airway inflammation is brought about by Th2 class of T cells which secrete IL-4 and IL-5.
  • Intrinsic or cryptogenic asthma is reported to develop after upper respiratory tract infections, but can arise de novo in middle-aged or older people, in whom it is more difficult to treat than extrinsic asthma.
  • Th1 cells may also participate in these pathologies; for instance, Th1 cells have been identified in chronic atopic skin lesions. Such a Th1-Th2 imbalance has also been found in other disorders such as some cancers, especially bladder cancer or in autoimmune diseases.
  • One of the treatment strategies is to down regulate the Th2 component by inducing a T helper 1 (Th1) response to the relevant allergen or antigen, because Th1 and Th2 cytokines are thought to be mutually antagonistic.
  • Th1 T helper 1
  • compositions including mycobacteria have been tested:
  • live BCG vaccines exhibit several drawbacks; first, they cannot be given to immuno-compromised subjects due to their residual virulence; second, there is a local reaction to intradermal BCG vaccination which is proportional to the total bacterial mass and can lead to local ulceration, or in the case of accidental subcutaneous injection, to more severe reactions (abscess). Therefore, such a vaccine, especially if it was given by the intranasal or aerosol route, would not be adapted for repeated administration over a significant period for the immunotherapy of allergic disorders of the respiratory tract, or other disorders involving a Th1-Th2 imbalance. Administration intranasally or by aerosol could lead to inadmissible adverse effects of the live BCG vaccine in the lungs;
  • vaccae may potentially be helpful in down-regulating an IgE response (F. Tukenmez and coll., Pediatr. Allergy Immunol., 1999, 10, 2, 107-111; F. Tukernez and coll., J. Asthma, 2000, 37, 4, 329-334; Nahori and coll., precited);
  • compositions derived from Gram positive bacteria such as Gram positive facultative intracellular bacteria, for example mycobacteria
  • diseases comprising an immune dysregulation such as a Th1-Th2 imbalance, for example allergic disorders
  • said compositions being well-tolerated and not having the drawbacks exposed here above.
  • composition must allow the identification of the active components present in the preparation.
  • the heat-killed preparations which include heating at 120° C. during 10 to 30 minutes would never allow identification of heat labile components.
  • a novel killed Gram positive bacterial preparation such as killed Gram positive facultative intracellular bacterial preparation, for example a killed mycobacterial preparation and compositions containing it, useful in the treatment of diseases comprising an immune dysregulation such as a Th1-Th2 imbalance, for example cancer, autoimmune diseases and allergic disorders without important adverse reactions.
  • Gram positive bacteria such as Gram positive facultative intracellular bacteria, for example mycobacteria which are killed by “soft methods” which do not denature the molecules from the bacteria cells are able to stimulate leukocytic regulatory cells (CD4+ CD25+ T cells and/or B cells and/or dendritic cells) in vivo when they are administered to subjects suffering from asthma, or other immune dysregulation.
  • the stimulation of these regulatory cells produces a reshaping of the immune reactivity of the asthmatic subjects which has a local and a systemic effect for a prolonged period (several weeks); as a result, the allergic subjects treated with these killed Gram positive intracellular bacterial preparations are protected from asthma for a prolonged period.
  • the present invention relates to a bacterial preparation, characterized in that:
  • Immunomodulatory preparation means a preparation which is able to modify the ratio between immune regulatory and immune helper cells before or after the induction of an immune response against any antigen (self antigen or foreign antigen).
  • Immune regulatory cells include leukocytic regulatory cells such as CD4+ CD25+ T cells and/or B cells and/or dendritic cells.
  • CD4+ CD25+ T cells are described in Schevach et al., Nat. Rev. Immunol., 2002, 2, 389-400.
  • said bacterial preparation contains killed Gram positive facultative intracellular bacteria.
  • Gram positive facultative intracellular bacteria means Gram positive bacteria with the capacity of growing in synthetic medium in vitro as well as of infecting eukaryotic cells from a mammalian or non-mammalian host, in vivo and multiplying in those cells, for example macrophages.
  • said bacterial preparation contains killed Gram positive facultative intracellular bacteria chosen from Listeria sp., Corynobacterium sp., and Actinomycetes comprising Mycobacteria sp., Nocardia sp. and Rhodococcus sp.
  • said bacterial preparation contains Mycobacteria bovis, more preferably Mycobacteria bovis BCG.
  • a process which does not denature the structure of the molecules from the bacteria cells means a process which results in no extensive denaturation of the spatial configuration of the molecules; preferably, said process preserves the three-dimensional structure of the macromolecules from the bacteria cells such as proteins, polysaccharides and lipids.
  • processes which are denominated “soft processes” include with no limitation the use of physical means which disrupt the bacteria cell membranes while preserving the structure of its macromolecular components. These processes include with no limitation: extended freeze-drying, grinding in the presence of silica or zirconium beads, use of the so-called “French press”, sonication and gamma-rays irradiation. Other processes which may be used for obtaining the killed bacterial preparation as defined above are known to those of ordinary skill in the art.
  • extended freeze-dried killed bacterial preparation means that essentially all the water has been removed from said preparation; thus, the extended freeze-dried killed bacterial preparation contains less than 1.5% of residual water, preferably less than 1% and more preferably less than 0.5%.
  • the preparations of freeze-dried bacteria contain more residual water (about 10%), i.e. all the bacteria are not killed, killing of the residual living bacteria is alternatively obtained by contacting said preparations with air (atmospheric pressure); such preparations have the same properties and activity as the above described extended freeze-dried killed bacterial preparations.
  • the residual water in the extended freeze-dried killed bacterial preparation is for instance determined by the coulometric method of Karl Fisher.
  • the absence of denaturation of the molecules from the killed Gram positive bacterial preparation according to the invention are verified by any method well-known in the art.
  • the structure of the proteins can be verified by gel electrophoresis of the killed Gram positive bacterial preparation extracts, in denaturing and non-denaturing conditions according to Laemmli, Nature, 1970, 277, 680-, by comparison with protein extracts obtained from living bacteria; the proteins are visualised either directly, by staining of the gel with an appropriate dye or after transfer of the proteins onto membranes and staining with appropriate antibodies directed to the Gram positive bacteria proteins.
  • Other methods such as gel filtration or mass spectrometry can also be used to verify the structure of purified molecules from the killed Gram positive bacterial preparation according to the invention.
  • said bacterial preparation contains extended-freeze-dried killed bacteria obtainable by an extended freeze-drying process.
  • said extended-freeze-dried killed bacterial preparation is prepared by:
  • said extended-freeze-dried killed bacterial preparation is prepared by a process as defined above with the exception that the washing of the bacteria cells in (ii) is omitted.
  • step (iv) is performed at a drying chamber pressure of about 0.02 mBar to 0.2 mBar; more preferably 0.06 mBar to 0.1 mBar, during at least 10 to 12 hours, more preferably over several days with a low heat input and a low cold vapor trap temperature to ensure low temperature of dried killed bacterial preparation throughout the drying (no denaturation of the bacterial cells molecules).
  • the pressure in the drying chamber may be in the range of 0.06-0.120 mBar, usually of about 0.09 mBar, for obtaining effectively extended freeze-dried killed bacteria.
  • the invention relates also to different fractions of the killed bacterial preparation according to the invention, selected in the group consisting of:
  • fraction A consisting of an organic solvent extract of said killed-bacterial preparation, to eliminate phospholipids
  • a fraction B consisting of a glycosidase-treated extract of said killed bacterial preparation, to eliminate glyco-derived components, such as peptidoglycans,
  • fraction D consisting of a protease-treated extract of said killed bacterial preparation, to eliminate proteins
  • a fraction E consisting of a extract of said killed bacterial preparation successively treated by an organic solvent, a glycosidase, a DNase and/or a RNase, and finally a protease.
  • fraction A is obtained by extraction with a methanol/chloroform mixture.
  • said fraction B is obtained by digestion with lysozyme.
  • said fraction C is obtained by digestion with DNase I and/or RNase A.
  • said fraction D is obtained by digestion with subtilisin.
  • fractions they are isolated from an extended freeze-dried bacterial preparation, as defined above.
  • fractions consist of mycobacteria fractions, preferably Mycobacteria bovis fractions, more preferably Mycobacteria bovis BCG fractions.
  • the invention also relates to a pharmaceutical composition for the prevention or the treatment of diseases comprising an immune dysregulation such as a Th1-Th2 imbalance, comprising an effective amount of killed bacterial preparation and/or at least one fraction thereof, as defined above, a pharmaceutically acceptable carrier and/or an additive, and/or an adjuvant, and/or an immunostimulant and/or an immunomodulator distinct from the bacterial preparation according to the invention.
  • an immune dysregulation such as a Th1-Th2 imbalance
  • Adjuvant means a natural or synthetic product which potentiates the specific immune response to an antigen (antibody production, B and T cell activation) when administered in association with said antigen.
  • Immunostimulant means a natural or synthetic product which induces a non-specific immune response when administered in association with an antigen (for example an increase of the phagocytosis of the antigen).
  • said composition may advantageously be used for treating cancers, autoimmune diseases such as multiple sclerosis, rhumato ⁇ d arthritis, Crohn disease and diabetes mellitus, and allergic disorders such as asthma, allergic rhinitis and atopic dermatitis.
  • autoimmune diseases such as multiple sclerosis, rhumato ⁇ d arthritis, Crohn disease and diabetes mellitus
  • allergic disorders such as asthma, allergic rhinitis and atopic dermatitis.
  • composition it consists of mycobacteria, preferably Mycobacteria bovis, more preferably Mycobacteria bovis BCG.
  • composition it consists of extended-freeze-dried killed bacteria obtainable by an extended freeze-drying process as defined above; preferably said extended-freeze-dried killed bacteria are obtained by a freeze-drying process as defined above.
  • composition it is in a form suitable to be administered by the intranasal route.
  • composition it is in a form suitable to be administered by the oral or sublingual routes.
  • composition may be administered by parenteral injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g. by aspiration or nebulization), orally, sublingually, or topically, through the skin or through the rectum.
  • parenteral injection e.g., intradermal, intramuscular, intravenous or subcutaneous
  • intranasally e.g. by aspiration or nebulization
  • parenteral injection e.g., intradermal, intramuscular, intravenous or subcutaneous
  • intranasally e.g. by aspiration or nebulization
  • sublingually e.g. by aspiration or nebulization
  • the composition of the present invention is in a form suitable for delivery to the bronchopulmonary mucosal surfaces.
  • the composition may be suspended in a liquid formulation for delivery to a patient in an aerosol form or by means of a nebuliser device similar to those currently employed in the treatment of asthma.
  • the composition of the present invention is in a form suitable for oral administration.
  • the composition may be in the form of tablets, ordinary capsules, gelatin capsules or syrup for oral administration.
  • These gelatin capsule, ordinary capsule and tablet forms can contain excipients conventionally used in pharmaceutical formulation, such as adjuvants or binders like starches, gums and gelatin, adjuvants like calcium phosphate, disintegrating agents like cornstarch or alginic acids, a lubricant like magnesium stearate, sweeteners or flavourings.
  • Solutions or suspensions can be prepared in aqueous or non-aqueous media by the addition of pharmacologically compatible solvents. These include glycols, polyglycols, propylene glycols, polyglycol ether, DMSO and ethanol.
  • the composition may additionally contain a pharmaceutically acceptable carrier and/or an additive and/or an immunostimulant and/or an adjuvant such as a liposome containing the bacteria cells or fraction(s) thereof according to the present invention; said one or more additives used for preparing pharmaceutical compositions may be chosen among antiaggregating agents, antioxidants, dyes, flavor enhancers, or smoothing, assembling or isolating agents, and in general among any excipient conventionally used in the pharmaceutical industry.
  • the carrier preferably comprises water, saline buffer, lactose, glutamate, a fat or a wax.
  • the carrier preferably comprises water, saline buffer, lactose, glutamate, a fat or a wax.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres e.g. polylactic galactide
  • suitable biodegradable microspheres are disclosed, for example in U.S. Pat. Nos. 4,897,268 and 5,075,109.
  • adjuvants may be employed in the compositions of the present invention to enhance the immune response.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism or to create controlled inflammatory reactions, such as aluminium hydroxide or mineral oil, and a non-specific stimulator of immune response, such as lipid A, Bordetella pertussis toxin.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's complete adjuvant which can not be used for injection in human.
  • Other suitable adjuvants which can be used in human include aluminium hydroxide, biodegradable microspheres, monophosphoryl A and Quil A.
  • the preferred frequency of administration and effective dosage will vary from one species and one subject to another.
  • the amount of extended freeze-dried killed mycobacterial preparation or fraction(s) thereof is in a dose ranges which is equivalent in mice to about 1 ⁇ g to 10000 ⁇ g material (approximately 10 6 to 10 10 CFU); preferably from 10 ⁇ g to about 1000 ⁇ g; more preferably from about 10 ⁇ g to 100 ⁇ g.
  • the doses may be higher or lower, depending on the body surface of the species or the subjects and on the frequency of administration. For example, the administration of one or two doses once every two months may be more efficient in the treatment of asthma.
  • the invention also relates to products containing a bacterial preparation according to the invention or fractions thereof and a product selected from the group consisting of anticancer, anti-diabetes and immunomodulatory drugs, as a combined preparation for simultaneous, separate or sequential use in the prevention and/or the treatment of diseases comprising an immune dysregulation.
  • said products are chosen from anti-histaminic and anti-inflammatory drugs.
  • the invention also relates to the use of a bacterial preparation according to the invention or fractions thereof for the preparation of a medicament for the treatment of asthma, to be administered by the oral, sublingual, parenteral or intranasal route, in a dose range which is equivalent in mice to 1 ⁇ g to 10 000 ⁇ g, preferably 10 ⁇ g to 1 000 ⁇ g, most preferably 10 ⁇ g to 100 ⁇ g, at two months intervals, starting from at least two weeks before the usual period of allergen exposure.
  • the invention also relates to a use of a bacterial preparation according to the invention or fractions thereof for the preparation of a medicament for the prevention of asthma in human baby, to be administered by the oral, sublingual, parenteral or intranasal route, in a dose range which is equivalent in mice to 1 ⁇ g to 10 000 ⁇ g, preferably 10 ⁇ g to 1 000 ⁇ g, most preferably 10 ⁇ g to 100 ⁇ g.
  • the instant invention also relates to a method for the preparation of said extended freeze-dried killed bacterial preparation, characterized in that it comprises at least the steps of:
  • An alternative method for the preparation of said extended freeze-dried killed bacterial preparation comprises steps (i), (iii), (iv) and (v); the washing of the bacteria cells in step (ii) is omitted.
  • step (iv) is performed at a drying chamber pressure of about 0.02 mBar to 0.2 mBar; more preferably 0.06 mBar to 0.1 mBar, during at least 10 to 12 hours, in order to remove at least 98.5% of the water while preventing melting of the frozen bacteria and maintaining the dried killed bacteria below the bacterial cells molecules denaturation temperature.
  • said method consists of the preparation of extended freeze-dried killed mycobacteria.
  • the instant invention also relates to the use of said killed bacteria cells preparation or fraction(s) thereof for the preparation of a medicament for the prevention and/or the treatment of diseases comprising an immune dysregulation such as a Th1-Th2 imbalance, e.g. cancers, autoimmune diseases such as multiple sclerosis, diabetes mellitus and Crohn disease, allergic disorders, such as asthma, allergic rhinitis or atopic dermatitis.
  • an immune dysregulation such as a Th1-Th2 imbalance
  • autoimmune diseases such as multiple sclerosis, diabetes mellitus and Crohn disease
  • allergic disorders such as asthma, allergic rhinitis or atopic dermatitis.
  • said killed bacterial preparation or fraction(s) thereof are associated with a pharmaceutically acceptable carrier, and/or an immunostimulant, and/or an adjuvant and/or any conventional additives as defined hereabove.
  • mycobacteria preferably Mycobacteria bovis, more preferably Mycobacteria bovis BCG are used.
  • extended-freeze-dried killed bacteria obtainable by an extended freeze-drying process are used.
  • Th1-Th2 imbalance e.g. asthma for example
  • the present invention also relates to a kit for the treatment of a disease comprising an immune dysregulation, characterised in that it comprises at least a pharmaceutical composition according to the invention, means of administration of said pharmaceutical composition and/or means for insuring compliance with the prescribed treatment.
  • FIG. 1 illustrates the standard method of preparation of freeze-dried living BCG vaccine (A) by comparison with the method of preparation of extended freeze-dried killed BCG according to the present invention (B): the pressure in mBar (- ⁇ -) and the temperature in degree Celsius of the samples (- ⁇ -) or of the trays (- ⁇ -) are indicated over the time in hours.
  • FIG. 2 illustrates the protective effect of extended freeze-dried killed BCG (Lyoph killed BCG) on broncho-pulmonary hyper-reactivity in a particularly stringent (highly Th2) BP2 mice model of asthma as assayed by the reduction in ventilation.
  • the BP2 strain of mice is highly responsive along the Th2/asthma axis.
  • the mice are also sensitized with OVA in combination with alum, known to be a Th2 stimulant.
  • OVA in combination with alum, known to be a Th2 stimulant.
  • the area under the curve (cm 2 ) is presented: individual (- ⁇ -), mean ⁇ SEM per group of mice (- ⁇ -).
  • FIG. 3 illustrates the protective effect of extended freeze-dried killed BCG (Lyoph killed BCG) on broncho-pulmonary hyper-reactivity in the BP2 mice model of asthma using OVA as allergen, as assayed by broncho-pulmonary resistance.
  • extended freeze-dried killed BCG Locus BCG
  • FIG. 3 illustrates the protective effect of extended freeze-dried killed BCG (Lyoph killed BCG) on broncho-pulmonary hyper-reactivity in the BP2 mice model of asthma using OVA as allergen, as assayed by broncho-pulmonary resistance.
  • Enhanced pause (Penh) measured at 1 minute interval are presented: individual data (- ⁇ -), mean ⁇ SEM per group of mice (- ⁇ -).
  • FIG. 4 illustrates the protective effect of extended freeze-dried killed BCG (Lyoph killed BCG) against the increase of eosinophils in the lungs, in the BP2 mice model of asthma, as assessed by cell numeration of the bronchoalveolar lavages (BAL).
  • BAL cell numeration of the bronchoalveolar lavages
  • FIG. 5 illustrates the protective effect of extended freeze-dried killed BCG (Lyoph killed BCG) against the increase of neutrophils in the lungs, in the BP2 mice model of asthma, as assessed by cell numeration of the bronchoalveolar lavages.
  • BCG extended freeze-dried killed BCG
  • FIG. 6 illustrates the advantageous absence of lung inflammation or tissue damage, with extended freeze-dried killed BCG (Lyoph killed BCG) in the BP2 mice model of asthma, as assayed by the very low level of fibronectin in bronchoalveolar lavages.
  • FIG. 7 illustrates the level of IL-5 in bronchoalveolar ravages from groups of mice that have been treated with living BCG, heat-killed BCG, extended freeze-dried killed BCG (Lyoph killed BCG), or non-treated. Data are expressed in pg/ml; individual data (- ⁇ -), mean ⁇ SEM per group of mice (- ⁇ -).
  • FIG. 8 illustrates the level of IL-5 in the serum from groups of mice that have been treated with living BCG, heat-killed BCG, extended freeze-dried killed BCG (Lyoph killed BCG), or non-treated. Data are expressed in pg/ml; individual data (- ⁇ -), mean ⁇ SEM per group of mice (- ⁇ -).
  • FIG. 9 illustrates the level of IFN- ⁇ (pg/ml) in lung cultures from groups of mice that have been treated with living BCG, heat-killed BCG, extended freeze-dried killed BCG (Lyoph killed BCG), or non-treated; the cultures have been stimulated in vitro with purified BCG secreted protein (A) or with anti-CD3 anti-bodies (B). Individual data (- ⁇ -) and mean ⁇ SEM per group of mice (- ⁇ -) are presented.
  • FIG. 10 illustrates the level of OVA-specific IgE in the serum of mice that have been treated in a curative protocol with living BCG, heat-killed BCG, extended freeze-dried killed BCG (Lyoph killed BCG) or non-treated. Individual data (- ⁇ -) and mean ⁇ SEM per group of mice (- ⁇ -) are presented.
  • FIGS. 11A and 12A illustrate the production of IL-12 (p70 and p40) by alveolar macrophages from respectively BP2 and BALB/c mice immunised with OVA, that have been stimulated in vitro by either heat-killed BCG (Heated) (- ⁇ -), or extended freeze-dried killed BCG (Lyophilized) (.... ⁇ .). Data are expressed in pg/ml per 100 000 cells.
  • FIGS. 11B (BP2) and 12 B (BALB/c) illustrate the absence of production of TNF- ⁇ in the macrophages from mice stimulated by extended freeze-dried killed (Lyophilized) compared to significant level of TNF- ⁇ produced in the macrophages from mice stimulated by heat-killed BCG (Heated). Data are expressed in or ng/ml per 100 000 cells.
  • FIG. 13 illustrates the absence of delayed type hypersensitivity (DTH) to BCG purified proteins derivatives (PPD) in mice that have been treated with extended freeze-dried killed BCG (Lyoph killed BCG).
  • DTH delayed type hypersensitivity
  • PPD BCG purified proteins derivatives
  • FIGS. 14 illustrate the protective effect of extended freeze-dried killed BCG (EFD-BCG) on broncho-pulmonary hyper-reactivity to histamine in a guinea-pig model of asthma.
  • Groups of OVA immunised animals were treated with 10 ⁇ g ( FIG. 14B ) or 100 ⁇ g ( FIG. 14C ) of extended freeze-dried killed BCG, or non-treated ( FIG. 14A ).
  • Concentration of histamine able to create broncho-constriction before (black symbols) and after (white symbols) aerosol administration of OVA are presented for each animal.
  • *** Statistically significant protective effect in the group treated with 10 ⁇ g (p ⁇ 0.01) or 100 ⁇ g (p ⁇ 0.001) extended freeze-dried killed BCG (Kruskal-Wallis non parametric test).
  • FIG. 15A illustrates the protective effect of extended freeze-dried killed BCG (EFD1) against the increase of eosinophils in the lungs, in the BP2 mice model of asthma, as assessed by cell numeration of the bronchoalveolar lavages.
  • EFD1 extended freeze-dried killed BCG
  • FIG. 15B illustrates the level of IL-5 in bronchoalveolar ravages from groups of mice that have been treated with living BCG, heat-killed BCG, extended freeze-dried killed BCG (EFD1), or non-treated.
  • FIG. 16 illustrates the preservation of the structure of the proteins in the extended freeze-dried killed BCG preparations as assessed by SDS-PAGE and (A) staining with anti- mycobacteria antibodies after transfer onto PVDF membranes or (B) staining with Aurodye.
  • the proteins appear as discrete bands without smear.
  • FIG. 17 illustrates the protective effect of extended freeze-dried killed BCG (EFD1) on broncho-pulmonary hyper-reactivity in the BP2 mice model of asthma using a water soluble ray-grass pollen extract as allergen, as assayed by broncho-pulmonary hyper-reactivity.
  • EFD1 extended freeze-dried killed BCG
  • FIGS. 18A and B illustrates the protective effect of extended freeze-dried killed BCG (EFD1) against the increase of polymorphonuclears and eosinophils in the lungs, in the BP2 mice model of asthma, as assayed by cell numeration of the bronchoalveolar lavages.
  • the data are expressed as mean ⁇ SEM per group of mice.
  • FIG. 18C illustrates the correlation between the decrease of the number of polymorphonuclears and eosinophils in the lungs of the extended freeze-dried BCG (EFD1) treated groups (10 ⁇ g, 100 ⁇ g, 1 mg and 10 mg) , and the decrease of IL-4 production as assessed by cells and cytokines analysis in the bronchoalveolar lavages.
  • the data are expressed as mean ⁇ SEM per group of mice.
  • Statistically significant reduction of the level of IL-4 ** p ⁇ 0.01) in the groups treated with 10 ⁇ g, 100 ⁇ g, 1 mg and 10 mg of extended freeze-dried killed BCG.
  • FIG. 19 illustrates the prevention of leukocyte infiltration of the lungs in the extended freeze-dried killed BCG (EFD1) treated groups, in the BP2 mice model of asthma, as assayed by lungs cellular infiltrate numeration.
  • FIG. 20 illustrates the prevention of leukocyte infiltration of the lungs in the extended freeze-dried killed BCG (EFD) treated groups, in the BP2 mice model of asthma, as assayed by flow cytometry analysis of the macrophages (CD11b+), the plymorphonuclear cells (Gr1+) and the dendritic cells (CD11c+).
  • FIG. 22 illustrates the absence of anemia (A) and thrombopenia (B) after intravenous administration of extended freeze-dried killed BCG (EFD-BCG), contrary to administration of living BCG or heat-killed BCG by the same route.
  • A anemia
  • B thrombopenia
  • FIG. 23 illustrates the minimal inflammatory reaction at the site of injection after subcutaneous injection of extended freeze-dried killed BCG (EFD1), by comparison with living BCG and heat-killed BCG, as assessed by footpad increase measurement every week for 10 weeks after the injection. Arrow indicate the day of injection; D0 and D36 for EFD and heat-killed BCG, D0 only for living BCG.
  • EFD1 extended freeze-dried killed BCG
  • FIG. 24 illustrates the reduced TNF- ⁇ production after LPS stimulation of lung explants 18 days after intravenous injection of extended freeze-dried killed BCG (END1), by comparison with injection of living BCG and heat-killed BCG by the same route.
  • END1 extended freeze-dried killed BCG
  • significant production of TNF- ⁇ is observed in the living BCG and heat-killed BCG treated groups.
  • FIG. 25 illustrates the increase of the CD11c + Gr1 + B220 + plasmacytoid dendritic cells number in the spleen from BALB/c mice immunized 90 days previously with ovalbumine, treated or not with extended freeze-dried killed BCG (EFD1) at days 45 and 65 and challenged or not with ovalbumine.
  • EFD1 extended freeze-dried killed BCG
  • FIG. 26 illustrates the increase of the CD4 + CD 25 + IL-10 + cells number in the spleen from BALB/c mice immunized 90 days previously with ovalbumine, treated or not with extended freeze-dried killed BCG (EFD1) at days 45 and 65 and challenged or not with ovalbumine; spleen cells were stimulated in vitro with OVA or BCG culture supernatant or non-stimulated before analysis of the leukocyte populations. *** Statistically significant increase of the CD4 + CD25 + IL-10 + cells number (p ⁇ 0.001) in the treated groups.
  • FIG. 27 illustrates the protective effect of extended freeze-dried killed BCG (EFD1) given by the oral route, in the mouse model of asthma, as assessed by lungs cell infiltrate numeration. *** Statistically significant decrease of the lungs cell number in the treated group (p ⁇ 0.001).
  • FIG. 28 illustrates the protective effect of one dose of extended freeze-dried killed BCG (EFD) injected by the subcutaneous route, as assessed by the prevention of bronchopulmonary hyper-reactivity in the BALB/c model of asthma.
  • EFD extended freeze-dried killed BCG
  • FIG. 29 illustrates the delay of action of extended freeze-dried killed BCG (EFD) injected by the subcutaneous route, as assessed by the prevention of bronchopulmonary hyper-reactivity in the BP2 model of asthma.
  • EFD extended freeze-dried killed BCG
  • FIG. 30 illustrates the duration of action of extended freeze-dried killed BCG (EFD1), injected by the subcutaneous route, as assessed by the prevention of bronchopulmonary hyper-reactivity in the BP2 model of asthma; the extended freeze-dried killed BCG protective effect persists at least for two months.
  • EFD1 extended freeze-dried killed BCG
  • the Mycobacterium bovis BCG cells (BCG Pasteur vaccine strain 1173P2, deposited at the Collection Nationale de Cultures de Microorganismes, 25 rue du Dondel Roux, 75724 PARIS Cedex 15 (France), on Apr. 24, 1978, under n o I-059 (M. Gheorghiu and coll., 1983, Bull. Inst.
  • Pasteur 81, 281-288 are grown in sterile Sauton medium (HOOCCH(NH 2 O)CH 2 CONH 2 H 2 O (asparagine), 4 g/l; C 6 H 8 O 7 ; H 2 O (citric acid), 2 g/l; K 2 HPO 4 (di-potassium hydrogenophosphate), 0.5 g/l; MgSO 4 —H 2 O (magnesium sulphate), 0.50 g/l; FeIII citrate, 0.05 g/l; glycerol, 60 ml; Zinc sulphate solution (0.155 g Zinc sulphate in 10 ml pyrogen free water), 240 ⁇ l/l, pH 7). More precisely the 1173 P2 strain is grown in 250 ml spherical culture flask containing 130 ml sterile Sauton medium, at 37° C., for 14 days, corresponding to the time the culture ends its exponential phase.
  • sterile Sauton medium HO
  • the culture is then centrifuged at 2000 g for 10 minutes, at 4° C. to pellet the BCG cells.
  • the cell culture supernatant is discarded and the pellet is washed extensively (three times) in distilled water; each wash consists in resuspending said pellet in distilled water (20 volumes of water per volume of cell-pellet) and centrifuging the cells at 2000 g for 10 minutes, at 4° C.
  • the pellet is resuspended in a volume of distilled water equivalent or twice that of the volume of the pellet.
  • 20 g of bacteria in 50 ml of water is layered and frozen on the wall of bottles, in order to form a layer of approximately 1 cm in thickness.
  • the mixture is freezed at ⁇ 60° C.
  • the frozen suspension of cells in water is then extended freeze-dried under the conditions of the extended freeze-drying protocol described in FIG. 1B , to remove essentially all the water (residual water ⁇ 1.5%).
  • the pressure is rapidly decreased to 0.150 mBar, the temperature of the ice condenser being approximately— ⁇ 50° C. to ⁇ 54° C., and the freeze-drying process begins.
  • the samples remain frozen, the temperature being not exactly monitored.
  • the pressure is then maintained at 0.1 mBar for 34 h, in order to remove essentially all the water (residual water ⁇ 1.5%). There is a progressive increase up to room temperature (approximately 20° C.).
  • the pressure is then slowly returned to atmospheric pressure with air, at room temperature (approximately 20° C.) and the extended freeze-dried killed BCG cells (approximately 2 g) are harvested and stored at room temperature, under air atmospheric pressure.
  • the residual water present in the extended freeze-dried killed BCG preparation is determined using the coulometric method from Karl Fisher and a 756 KF Coulometer (METHROM), according to the manufacturer's instructions.
  • CFU colony forming units
  • CFDA-SE Carboxyfluorescein diacetate-Succimidyl Ester; MOLECULAR PROBES reference number C-1157
  • CFDA-SE Carboxyfluorescein diacetate-Succimidyl Ester
  • MOLECULAR PROBES reference number C-1157 which stains viable cells only
  • 1 ml of a suspension of the extended freeze-dried killed preparation (approximately 10 8 extended freeze-dried killed BCG cells in water) are mixed with 100 ⁇ l of a 1/100 dilution in PBS of CFDA reagent (stock solution: 1 mg/ml in DMSO).
  • PBS CFDA reagent
  • the mixture is incubated in the dark at room temperature for 60 min. Labeled bacteria are centrifuged at 3000 rpm for 15 minutes, washed twice in PBS and resuspended in the same buffer. Then, the presence of viable cells was examined by fluorescence microscopy.
  • BCG cells were extended freeze-dried killed following the extended freeze-drying protocol described in material and methods and summarized in figure 1B .
  • BCG cells were freeze-dried following the standard method for preparation of live BCG vaccine: BCG cells (strain 1173P2) were cultured in Sauton medium at 37° C., harvested at the end of the exponential phase by centrifugation and the BCG cell pellet was resuspended in sodium glutamate (1.5 g/100 ml H 2 O), at the concentration of 4 mg/ml of BCG).
  • the BCG suspension was distributed in vials (0.25 ml/vial), freeze-dried according to the protocol illustrated in FIG. 1A and each vial containing 1.5 ⁇ 0.5% H 2 O was stored at room temperature under vacuum (0.06 mBar).
  • 200 mg of extended freeze-dried killed BCG preparation contained 1.073 mg if water in a representative determination, which is equivalent to less than 0.5% residual water.
  • a preparation of extended freeze-dried killed BCG preparation (10 mg) was suspended in 1 ml of 4% butanol in water and distributed in two eppendorf tubes (500 ⁇ l per tube) containing 1 g of zirconia/silica beads (0.1 mm) (BIOSPEC PRODUCTS, INC.).
  • the tubes were then placed in a Mixer Mills (MM301, RESCH) at a frequency of 25 during 5, 15, 30, 60, 120, 180, 240, 300 or 470 minutes.
  • the tubes were then centrifuged at 10000 rpm for 30 min; the supernatant was recovered, filtered (0.22 ⁇ m filter, MILLIPORE) and the protein, the amino acids and the polysaccharide concentrations were determined as follows:
  • total polysaccharide concentration was determined using anthrone method, according to S. Melvin, Anal. Biochem. 1953, 25, 1656-.
  • the bacteria extracts collected after 5, 60 or 300 min of extraction contains respectively 280, 500 and 1258 ⁇ g of proteins; 190, 420 and 880 ⁇ g of amino acids and 697, 1267 and 1765 ⁇ g of polysaccharides.
  • results presented in FIG. 16 show that the extended freeze-drying process preserves the structure of the proteins which are present in the killed BCG preparations as indicated by the presence of discrete protein bands without smear; the protein profile observed with the extended freeze-dried killed BCG preparation extracts is comparable to that obtained with living BCG preparations.
  • mice Male adult (6 to 7 weeks old) BP2 (H-2 q ) mice were obtained from the Centre d'élevage R. Janvier (Le Genest, Saint Isle, France) and were maintained in animal facilities in specific pathogen free-conditions.
  • mice Male adult BP2 mice were immunized by the subcutaneous route (in the dorsal part of the neck) with 1 ⁇ g ovalbumin (OVA; ICN Laboratories) and 1.6 mg aluminium hydroxide adjuvant in saline (0.4 ml final volume), on days 0 (D 0 ) and 7 (D 7 ).
  • OVA ovalbumin
  • saline 0.4 ml final volume
  • the allergic response is provoked by intranasal challenge with 10 ⁇ g OVA in 50 ⁇ l saline on day 96-98.
  • a water soluble ray-grass pollen extract is used as an allergen, in the same conditions as for OVA except that 10 ⁇ g is used for immunization.
  • Extended freeze-dried killed BCG prepared as described in example 1 were tested as an asthma vaccine in the BP2 mouse, by comparison with the following BCG preparations:
  • the Mycobacterium bovis BCG Pasteur vaccine strain 1173P2 was grown as dispersed bacilli in Beck-Proskauer medium (Gheorghiu and coll., 1988, J. Biol. Standard., 16, 15-26)) supplemented with 0.05% Triton WR 1339 (SIGMA) and 6% glucose.
  • the bacteria were harvested at the exponential phase (5 to 7 days) and stored at ⁇ 70° C. in Beck-Proskauer medium supplemented with 0.05% Triton and 6% glycerol.
  • the number of colony forming unit (CFU) per ml was determined by plating suitable dilutions in phosphate-buffered saline (PBS) on Middlebrook 7H10 agar medium (DIFCO). The suspension was diluted at the concentration of 10 8, 10 9 or 10 10 CFU/ml in PBS just before its injection (100 ⁇ l).
  • Living BCG prepared as above described was centrifuged at 3000 rpm for 15 minutes and the supernatant was discarded.
  • the pellet was resuspended in saline, for example in a buffer containing borate (Na 2 B 4 O 7 10H 2 O 0.363%; H 3 BO 3 0.525%; NaCl 0.619% and Tween 20 0.0005% in distilled water, pH 8), and the suspension was autoclaved for 15 min at 115° C.
  • mice Male adult BP2 mice are immunised with OVA as described above. Groups of 25 mice were injected with 100 ⁇ l of the BCG compositions prepared as described above.
  • the allergic response is provoked by intranasal challenge with 100 ⁇ g OVA in 50 ⁇ l saline on day 96-98; PBS alone is administered intranasally for comparison.
  • a barometric plethysmographic equipment (BLUXCO) that allows BHR study on unanesthetised animals was used. Animals immunized with OVA, treated with the different BCG preparations or non-treated were tested 24 h after the challenge with OVA or water soluble ray-grass pollen extract. The animals were placed in a plethysmographic chamber. Their basal ventilation parameters were recorded and during 20 s or 1 minute they received inhalation of methacholin (ALDRICH), 100 mM in H 2 O by using standard nebuliser, and their ventilation parameters were recorded during 10 minutes after methacholin aerosol administration.
  • ADRICH methacholin
  • mice were anesthetised by the intraperitoneal route with a lethal dose of urethane (1.5 g/kg; SIGMA). Circulating blood was removed from the abdominal aorte and a canula connected to a syringe was placed in the trachea, and the lungs were washed three times with 0.7 ml PBS and the lavage was collected in a tube and kept on ice.
  • SIGMA Steml bovine serum
  • the number of cells present in the BAL was determined in an automatic counting equipment (ZBI), provided by COULTER. To determine the percentages of the different cell types (eosinophils, neutrophils, macrophages), the BAL cells which have been cytospinnned on glass slides, were stained with DIFF QUICK (BAXTER).
  • the lungs were cut in small pieces of approximately 1 mm per 3 mm. Four or five pieces from each lung were placed in a well of a tissue culture plate containing 1 ml of AIM V culture medium (Life Technologies). The pulmonary explants were cultured in medium alone for basal level determination; alternatively BCG culture filtrate (10 ⁇ g/ml) was added to the culture to reveal mycobacterial specific cells, or anti-CD3 monoclonal antibody was added to the culture to reveal reactivity of T lymphocytes. 24 h later, the presence of different lymphokines (IFN- ⁇ , TNF- ⁇ , IL-10 . . . ) was assayed by ELISA using commercial kits, according to the manufacturer's instructions.
  • IFN- ⁇ , TNF- ⁇ , IL-10 . . . lymphokines
  • the entire lung (without trachea) was dissociated in collagenase and DNase medium to enumerate the infiltrating cells on a Flow cytometer using various monoclonal antibodies labelled with different fluorochromes.
  • the entire lungs were fixed in buffered formaldehyde and processed for histopathological analysis.
  • the Schiff staining was used to stain the mucus and the mucus containing cells.
  • the hematoxy-eosin stain was used as a counter staining.
  • the slides were examined and an evaluation of cell infiltration and mucus content was performed by one observer who scored the slide without knowledge of the origin of the sample (blind assay).
  • cytokines which are either present in the serum and the BAL samples from the groups of immunised mice, or secreted by the lung explants cultures from these mice, were assayed as follows: IFN- ⁇ , IL-10 and IL-12 were tested by ELISA; TNF- ⁇ and IL-5 were tested by EIA (Enzyme Immunometric Assay).
  • OVA-specific IgE present in the serum was assayed by ELISA, following standard protocols (Hansen and coll., J. Immunol., 2000, 223-230).
  • the number (n) of animals per group was 4 ⁇ n ⁇ 8.
  • the student's two-tailed (t) test was performed for independent events.
  • the histopathological analysis of the lungs shows that the treatment with extended freeze-dried killed BCG (EFD) prevents leukocyte infiltration of the lungs and mucosal metaplasia of bronchial epithelia.
  • EFD extended freeze-dried killed BCG
  • Non-specific stimulation of T lymphocytes with anti-CD3 antibodies shows similar levels of IFN- ⁇ in the different groups (treated or non-treated with BCG) suggesting that immunisation with BCG does not modify the number of T lymphocytes present in the lungs ( FIG. 9B ).
  • results presented in FIG. 21 show also a statistically significant (p ⁇ 0.001) higher level of IL-10 in the extended freeze-dried killed BCG treated groups compared to living BCG and heat-inactivated BCG treated groups.
  • the differences of the cytokines expression profile in the lungs between the extended freeze-dried killed BCG treated groups and the groups treated with the other BCG preparations indicate that extended freeze-dried killed BCG activity is correlated with modifications in the leukocyte populations present in the lung tissues after allergen delivery.
  • the high amount of IL-10 and low amount of IFN- ⁇ released by lung explants could assert the presence of regulatory cells in lung tissues of the extended freeze-dried killed BCG groups, after allergen delivery.
  • mice obtained and maintained as mentioned in example 3 were immunized with OVA as described in example 3b.
  • OVA a lethal dose of urethane (1.5 g/kg; SIGMA).
  • Circulating blood was removed from the abdominal aorte and a canula connected to a syringe was placed in the trachea, and the lungs were washed twice with 0.5 ml and 5 times with 1 ml of HBSS medium (Life Technologies) containing 2% of foetal calf serum.
  • the number of macrophages present in the BAL was determined in an automatic counting equipment (ZBI), provided by COULTER.
  • ZBI automatic counting equipment
  • the macrophages were centrifuged at 1000 rpm for 20 min at room temperature and their concentration was adjusted to 340. 10 3 cells/ml in RPMI 1640 medium containing 3% foetal. calf serum, 2 mM glutamine, 100 UI/ml penicilline and 10 ⁇ l/ml streptomycine.
  • the macrophages were then distributed in a 96 well plate for tissue-culture (100 000 cells /well) and incubated for at least 2 hours at 37° C. in the presence of 5% CO 2 .
  • macrophages were cultured in the presence of the heat-killed BCG or extended freeze-dried killed BCG (5 equivalent CFU per macrophage e.g. 1.6 10 6 CFU/ml for both BCG preparations); E. coli lipopolysaccharide (LPS; 1 ⁇ l/ml) and OVA (10 ⁇ g/ml) were used as controls.
  • the cell culture supernatant were harvested 2, 4, 6, 8, 12, 24, 48, 72 and 96 hours after addition of the different preparations and frozen immediately at ⁇ 20° C.
  • IL-12 which is involved in the maturation of the Th1 cells and the production of anti-allergic cytokines (IFN- ⁇ ) is produced by BALB/c and BP2 mice alveolar macrophages stimulated by extended freeze-dried killed and heat-killed BCG preparations,
  • TNF- ⁇ a cytokine that can be associated with important inflammatory side-effects (necrosis, ulceration . . . ) is produced only by BALB/c and BP2 mice alveolar macrophages stimulated by heat-killed BCG preparations; no TNF- ⁇ is produced by alveolar macrophages stimulated by extended freeze-dried killed BCG preparations at the concentrations used in the present experiments.
  • mice were immunised with OVA and treated with different preparations of BCG, or non-treated, as described in example 3d.
  • DTH delayed type hypersensitivity
  • PPD BCG purified proteins derivatives
  • mice immunised with extended freeze-dried killed BCG do not develop a delayed type hypersensitivity to BCG purified proteins derivatives as shown by a negative skin-test.
  • mice immunised with living BCG or heat-killed BCG even at low doses (10 7 CFU of living BCG or equivalent to 10 8 bacterial corpses for heat-inactivated BCG) develop a delayed type hypersensitivity to BCG purified proteins derivatives as shown by a positive skin-test.
  • guinea-pigs (3 groups of 10 animals) were injected intradermally with either extended freeze-dried killed BCG (10 7 or 10 8 CFU equivalent i.e. 10 ⁇ g or 100 ⁇ g), or PBS (control).
  • broncho-pulmonary reactivity is assayed on guinea-pigs with a method different to that used for mice; the sensitivity of each animal was evaluated with increasing doses of aerosolized histamine (20, 50, 100 or 400 ⁇ g), 24 h before allergen challenge, and the highest concentration of histamine giving elevated Penh values was considered to be the basal level of sensitivity to histamine.
  • the dosage of histamine triggering a clear bronchoconstriction is measured, for each guinea-pig, with a barometric plethysmograph.
  • Each guinea-pig is its own control, knowing that the basal reactivity is determined the 1 st day of the test.
  • ovalbumin increases the broncho-pulmonary hyper-reactivity to histamine.
  • FIG. 14 show that 5/6 and 6/6 guinea-pigs that have been treated respectively with 10 ⁇ g (10 7 CFU equivalent) ( FIG. 14B ) or 100 ⁇ g (10 8 CFU equivalent) ( FIG. 14C ) extended freeze-dried killed BCG according to the invention, are protected against broncho-pulmonary hyper-reactivity (no increase of the histamine sensitization) whereas in the control group ( FIG. 14A ) (no treatment with BCG) there is a significant increase of broncho-pulmonary hyper-reactivity to histamine ( ⁇ 4).
  • mice were injected intravenously with extended freeze-dried killed BCG (10 ⁇ g) or the different BCG preparations as described in example 3d, or non-treated. 18 days after the injection, red blood cells and platelets numbers were determined from a blood sample.
  • results presented in FIG. 22 show that, contrary to the living BCG and the heat-killed BCG treated groups, no anemia and no thrombopenia were observed in the extended freeze-dried BCG treated group.
  • the different BCG preparations were injected subcutaneously (in the footpad area) to groups of mice.
  • Living BCG was injected once only and heat-killed and extended freeze-dried killed BCG were injected twice (day 0 and day 36).
  • the foot pad increase was then measured every week for ten weeks.
  • mice treated 18 days previously with the different BCG preparations as described above were maintained in vitro in presence or not of LPS, and TNF- ⁇ production was measured by ELISA using commercial kits, according to the manufacturer's instructions.
  • the results presented in FIG. 24 show that, contrary to living BCG and heat-killed BCG treatment, extended freeze-dried killed BCG treatment does not induce the production and/or the activation of macrophages.
  • mice treated 48 hours previously by the subcutaneous route, with the different BCG preparations as described in example 3d, were analysed by flow cytometry using monoclonal antibodies directed to cell surface markers of different leukocyte populations, labelled with various fluorochromes.
  • mice The cells present in the spleen of mice treated by the subcutaneous route with extended freeze-dried killed BCG, were analysed as above described, after in vitro stimulation in the presence of OVA or BCG culture supernatant or no stimulation.
  • the analysis of the cells present in the draining lymph nodes show that by comparison with the living BCG and the heat-killed BCG treated groups, the cells present in the lymph nodes were three to five times more numerous 48 h after subcutaneous injection of extended freeze-dried killed. BCG.
  • a kinetic analysis of the different leukocyte populations for four days after the injection of extended freeze-dried killed BCG show a transient increase of the dendritic cells (CD11c + ) number at 48 hours, as well as an increasing number of B220+ and CD4+ lymphocytes from 6 hours to 96 hours.
  • mice model of asthma extended freeze-dried killed BCG is able to induce a significant increase in the number of cells which induce immunomodulatory cells (CD11c + Gr1 + B220 + plasmacytoid dendritic cells) or are themselves immunomodulatory cells (CD4 + CD25 + IL-10 + cells) in the mice which have been previously sensitized to an allergen (OVA or water soluble ray grass pollen), which results in a protective effect against the symptoms of asthma.
  • immunomodulatory cells CD11c + Gr1 + B220 + plasmacytoid dendritic cells
  • CD4 + CD25 + IL-10 + cells immunomodulatory cells
  • Extended Freeze-Dried Killed BCG is Active by the Oral Route
  • mice which have been immunized previously with OVA, as described in example 3d were treated orally with 1 mg extended freeze-dried at days 42, 44, 46 and 62, 64 and 66 or non-treated and then challenged or not with OVA at day 96 .
  • the protective effect of extended freeze-dried killed BCG was assessed by numeration of lungs cells infiltrate as described in example 3.
  • the results show a significant decrease p ⁇ 0.001 of the total cell number in the lungs of the extended freeze-dried killed BCG treated group, by comparison with the untreated group ( FIG. 27 ).
  • Analysis of the leukocyte populations show a significant decrease of the macrophages, polynuclears and dendritic cells numbers in the lungs of the treated group.
  • Extended freeze-dried killed BCG was injected by the subcutaneous route to mice previously immunized and then challenged with OVA, and its activity was then tested as described in example 3.
  • mice were sensitized with OVA at days 0 and 7, injected by the subcutaneous route with extended freeze-dried killed BCG once at day 45 or twice at days 45 and 65, and then challenged with ovalbumine at day 96.
  • the activity of extended freeze-dried killed BCG was tested by the prevention of bronchopulmonary hyper-reactivity in the BP2 model of asthma as described in example 3.
  • mice (6 per group) were sensitized with OVA at days 0 and 7, injected subcutaneously with 100 ⁇ g extended freeze-dried killed BCG at day 14 and then challenged with OVA at day 21, 28 or 35.
  • the activity of extended freeze-dried killed BCG was tested by the prevention of bronchopulmonary hyper-reactivity in the BP2 model of asthma as described in example 3.
  • mice were treated as follows:
  • mice (6 to 7 weeks old; 6 mice per group) were injected. subcutaneously with extended freeze-dried killed BCG (100 ⁇ g) at days 14, 21 or 28 before OVA immunization (days ⁇ 14, ⁇ 21 and ⁇ 28), immunized with OVA at days 0 and 7 and then challengeed with OVA at day 14.
  • BCG freeze-dried killed BCG
  • mice 7 to 8 days old, 6 to 8 mice per group were injected subcutaneously or intranasally with extended freeze-dried killed BCG (100 ⁇ g) at day 56 before OVA immunization (day ⁇ 56), immunized with OVA at days 0 and 7 and then challenged with OVA at day 14.
  • the activity of extended freeze-dried killed BCG in the preventive protocol was assessed by the preventive effect on broncho-pulmonary hyper-reactivity in the BP2 model of asthma, as described in example 3.
  • mice show a protective effect of extended freeze-dried BCG in a preventive protocol, only when the preparation is injected some days before sensitization (2 weeks and 3 weeks).
  • mice show a protective effect of extended freeze-dried BCG in a preventive protocol, only with the subcutaneous administration; the intranasal route is poorly active in these conditions.

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ES2471241T3 (es) * 2002-09-06 2014-06-25 Ucl Business Plc Células bacterianas completas como inmunomodulador
ES2347566T3 (es) 2003-11-14 2010-11-02 Ucl Business Plc Inmunomodulador que comprende celulas enteras de bacterias tsukamurella.
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US9107864B2 (en) 2004-06-07 2015-08-18 Qu Biologics Inc. Tissue targeted antigenic activation of the immune response to treat cancers
US8034359B2 (en) 2004-06-07 2011-10-11 Qu Biologics Inc. Tissue targeted antigenic activation of the immune response to cancers
US8501198B2 (en) 2004-06-07 2013-08-06 Qu Biologics Inc. Tissue targeted antigenic activation of the immune response to treat cancers
ES2404695T3 (es) * 2004-06-07 2013-05-28 Qu Biologics Inc Composiciones bacterianas para el tratamiento del cáncer
EP1761631B1 (fr) 2004-06-24 2009-10-28 Yissum Research Development Company Utilisation de mimotopes peptidiques de lipoglycanes mannosylates mycobacteriens pour traiter une inflammation
CA2531261A1 (fr) * 2005-12-21 2007-06-21 Institut Pasteur Traitement de syndromes d'inflammation intestinale a l'aide d'une preparation a base de bacteries tuees ou non infectieuses
NZ576590A (en) * 2006-10-27 2012-08-31 Qu Biolog Inc Tissue targeted antigenic activation of the immune response to treat cancers
GB0716778D0 (en) * 2007-08-29 2007-10-10 Bioeos Ltd Use
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CA2629057A1 (fr) * 2008-04-14 2009-10-14 Institut Pasteur Controle de maladies associees a une diminution des cellules t suppressives au moyen d'une preparation de bacteries mortes lyophilisees etendue
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EP2292260A1 (fr) 2009-08-13 2011-03-09 Institut Pasteur Utilisation de mycobactérie bois tuée par lyophilisation étendue pour la prévention ou le traitement de l'athérosclérose
EP2620159A1 (fr) 2012-01-24 2013-07-31 Institut Pasteur Traitement du cancer amélioré par immunothérapie par BCG ou des mycobactéries non pathogènes apparentées du point de vue des antigènes
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CA3015688A1 (fr) * 2016-02-25 2017-08-31 Thomas Julius Borody Compositions et procedes de traitement de maladies infectieuses chroniques
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US20070190076A1 (en) 2007-08-16
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