WO2004002521A1 - Preparation of vaccines - Google Patents

Preparation of vaccines Download PDF

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Publication number
WO2004002521A1
WO2004002521A1 PCT/DK2003/000425 DK0300425W WO2004002521A1 WO 2004002521 A1 WO2004002521 A1 WO 2004002521A1 DK 0300425 W DK0300425 W DK 0300425W WO 2004002521 A1 WO2004002521 A1 WO 2004002521A1
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WO
WIPO (PCT)
Prior art keywords
oxidoreductase
biological material
vaccine composition
haloperoxidase
source
Prior art date
Application number
PCT/DK2003/000425
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French (fr)
Inventor
Steffen Danielsen
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Novozymes A/S
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Publication date
Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to AU2003239773A priority Critical patent/AU2003239773A1/en
Publication of WO2004002521A1 publication Critical patent/WO2004002521A1/en

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    • 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
    • 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
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)

Definitions

  • the p resent i nvention relates to m ethods for p reparation of vaccines from b iological material (such as microorganisms or spores) by contacting it with an oxidoreductase enzyme capable of inactivating living biological material.
  • the present invention provides a method for producing a vaccine composition from biological material, comprising inactivating the biological material by contacting it with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions.
  • the inactivated biological material is formulated into a vaccine composition.
  • a container comprising the vaccine composition, and use of the vaccine composition as a medicament.
  • the oxidoreductase is an enzyme from the enzyme class EC 1.-.-.- as set out by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) or a compound exhibiting a corresponding enzymatic activity, which is capable of inactivating the biological material of the method and composition of the invention.
  • the oxidoreductase of the invention is a phenol oxidizing enzyme.
  • the phenol oxidizing enzyme is an enzyme or a compound capable of oxidizing phenolic compounds.
  • the oxidoreductase may be a peroxidase or a compound exhibiting peroxidase activity
  • laccase such as a haloperoxidase or a compound exhibiting haloperoxidase activity
  • a laccase or a compound exhibiting laccase activity or an activity corresponding to laccase activity, such as a catechol oxidase ( EC 1 .10.3.1), a n o-aminophenol oxidase ( EC 1 .10.3.4), a nd/or a b ilirubin oxidase (EC 1.3.3.5).
  • catechol oxidase EC 1 .10.3.1
  • a n o-aminophenol oxidase EC 1 .10.3.4
  • a nd/or a b ilirubin oxidase EC 1.3.3.5
  • laccase related enzymes Compounds exhibiting an activity corresponding to laccase activity are referred to below as laccase related enzymes.
  • the oxidoreductase is capable of generating free radicals.
  • the oxidoreductase of the invention may typically be present in concentrations of from
  • microgram enzyme protein per liter aqueous solution preferably of from 5 to 2000 microgram enzyme protein per liter aqueous solution, more preferably of from 5 to 1000 microgram enzyme protein per liter aqueous solution, and most preferably of from 1 to 500 microgram enzyme protein per liter aqueous solution.
  • Assays for determining the activity of these enzymes are well known to a person skilled in the art.
  • oxidoreductase variants e.g. produced by recombinant techniques are included within the meaning of the term oxidoreductase.
  • Compounds exhibiting laccase activity may be any laccase enzyme comprised by the enzyme classification EC 1.10.3.2, or any fragment derived therefrom, exhibiting laccase activity.
  • Preferred laccase enzymes and/or laccase related enzymes are enzymes of microbial origin.
  • the enzymes may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts).
  • Suitable examples from fungi include a laccase derivable from a strain of Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinus, e.g., C. cinereus, C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g., P.
  • papilionaceus Myceliophthora, e.g., M. thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radita (WO 92/01046), or Coriolus, e.g., C. hirsutus (JP 2-238885).
  • Suitable examples from bacteria include a laccase derivable from a strain of Bacillus.
  • Rhizoctonia is preferred; in particular a laccase derived from Coprinus cinereus, Myceliophthora thermophila, Polyporus pinsitus, Scytalidium thermophilum or Rhizoctonia solani.
  • the laccase or the compound exhibiting laccase activity may furthermore be one which is producible by a method comprising cultivating a host cell transformed with a recombinant
  • DNA vector which carries a DNA sequence encoding said laccase or the compound exhibiting laccase activity as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the laccase or the compound exhibiting laccase activity, in a culture medium under conditions permitting the expression of the laccase enzyme or the compound exhibiting laccase activity, and recovering the laccase or the compound exhibiting laccase activity from the culture.
  • LACU Laccase Activity
  • Laccase activity (particularly suitable for Polyporus laccases) may be determined from the oxidation of syringaldazin under aerobic conditions.
  • the violet colour produced is photometered at 530 nm.
  • the analytical conditions are 19 mM syringaldazin, 23 mM acetate buffer, pH 5.5, 30°C, 1 min. reaction time.
  • LACU laccase unit
  • Laccase activity may be determined from the oxidation of syringaldazin under aerobic conditions. The violet colour produced is measured at 530 nm. The analytical conditions are 19 mM syringaldazin, 23 mM Tris/maleate buffer, pH 7.5, 30°C, 1 min. reaction time.
  • LAMU laccase unit
  • Compounds exhibiting peroxidase activity may be any peroxidase enzyme comprised by the enzyme classification (EC 1.11.1.7), or any fragment derived therefrom, exhibiting peroxidase activity.
  • compounds exhibiting peroxidase activity comprise peroxidase active fragments derived from cytochromes, haemoglobin or peroxidase enzymes (such as microperoxidases).
  • the peroxidase employed in the composition of the invention is producible by plants (e.g. horseradish or soybean peroxidase) or microorganisms such as fungi or bacteria.
  • Some preferred fungi include strains belonging to the subdivision Deuteromycotina, class Hyphomycetes, e.g., Fusarium, Humicola, Trichoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embeliisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, Myrothecium verrucaria (IFO 6113), Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embeliisia alii or Dreschlera
  • fungi include strains belonging to the subdivision Basidiomycotina, class Basidiomycetes, e.g., Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371 ), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (previously called Polyporus), e.g., T. versicolor (e.g. PR4 28-A).
  • Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Basidiomycetes, e.g., Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371 ), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or
  • Mycoraceae e.g., Rhizopus or Mucor, in particular Mucor hiemalis.
  • Some preferred bacteria include strains of the order Actinomycetales, e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium.
  • Other preferred bacteria include Bacillus pumilus (ATCC 12905), Bacillus stearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11).
  • Further preferred bacteria include strains belonging to Myxococcus, e.g., M. virescens.
  • the peroxidase may furthermore be one which is producible by a method comprising cultivating a host cell transformed with a recombinant DNA vector which carries a DNA sequence encoding said peroxidase as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the peroxidase, in a culture medium under conditions permitting the expression of the peroxidase and recovering the peroxidase from the culture.
  • a recombinantly produced peroxidase is a peroxidase derived from a
  • Coprinus sp. in particular C. macrorhizus or C. cinereus according to WO 92/16634.
  • PXU peroxidase unit
  • ABTS 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonate)
  • the reaction is followed for 60 seconds (15 seconds after mixing) by the change in absorbance at 418 nm, which should be in the range 0.15 to 0.30.
  • For calculation of activity is used an absorption coefficient of oxidized ABTS of 36 mM “1 cm “1 and a stoichiometry of one micromole H 2 O 2 converted per two micromole ABTS oxidized.
  • haloperoxidases suitable for being incorporated in the method of the invention include chloroperoxidases, bromoperoxidases and compounds exhibiting chloroperoxidase or bromoperoxidase activity.
  • Haloperoxidases form a class of enzymes, which are capable of oxidizing halides (CI-, Br-, I-) in the presence of hydrogen peroxide or a hydrogen peroxide generating system to the corresponding hypohalous acids according to:
  • Haloperoxidases are classified according to their specificity for halide ions: - Chloroperoxidases (E.C. 1.11.1.10) which are capable of catalyzing formation of hypochlorite from chloride ions, hypobromite from bromide ions and hypoiodite from iodide ions; and
  • Bromoperoxidases which are capable of catalyzing formation of hypobromite from bromide ions and hypoiodite from iodide ions. Hypoiodite, however, undergoes spontaneous disproportionation to iodine and thus iodine is the observed product. These hypohalite compounds may subsequently react with other compounds forming halogenated compounds.
  • Haloperoxidases have been isolated from various organisms: mammals, marine animals, plants, algae, lichen, fungi and bacteria. It is generally accepted that haloperoxidases are the enzymes responsible for the formation of halogenated compounds in nature, although other enzymes may be involved.
  • Haloperoxidases have been isolated from many different fungi, in particular from the fungus group dematiaceous hyphomycetes, such as Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.
  • Caldariomyces e.g., C. fumago
  • Alternaria Curvularia
  • Curvularia e.g., C. verruculosa and C. inaequalis
  • Drechslera Ulocladium and Botrytis.
  • Haloperoxidases have also been isolated from bacteria such as Pseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S. aureofaciens.
  • the haloperoxidase is a vanadium haloperoxidase (i.e. a vanadium or vanadate containing haloperoxidase) derivable from Curvularia sp., in particular Curvularia verruculosa and Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046, e.g. a vanadium haloperoxidase encoded by the DNA sequence of WO 95/27046, figure 2 all incorporated by reference; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97/04102.
  • vanadium haloperoxidase i.e. a vanadium or vanadate containing haloperoxidase
  • the haloperoxidase is a vanadium containing haloperoxidase, such as a vanadium chloroperoxidase.
  • the vanadium chloroperoxidase may be derivable from Drechslera hartlebii as described in WO 01/79459, Dendryphiella salina as described in WO 01/79458, Phaeotrichoconis crotalarie as described in WO 01/79461 , or Geniculosporium sp. as described in WO 01/79460.
  • the vanadium haloperoxidase is more preferably derivable from Drechslera hartlebii (DSM 13444), Dendryphiella salina (DSM 13443), Phaeotrichoconis crotalarie (DSM 13441 ) or Geniculosporium sp. (DSM 13442).
  • the haloperoxidase is horseradish peroxidase, myeloperoxidase or eosinophil peroxidase.
  • concentration of the haloperoxidase is typically in the range of 0.01-100 ppm enzyme protein, preferably 0.05-50 ppm enzyme protein, more preferably 0.1-20 ppm enzyme protein, and most preferably 0.5-10 ppm enzyme protein.
  • Microtiter assays are performed by mixing 100 ⁇ l of haloperoxidase sample (about 0.2 ⁇ g/ml) and 100 ⁇ l of 0.3 M sodium phosphate pH 7 buffer - 0.5 M potassium bromide - 0.008% phenol red, adding the solution to 10 ⁇ l of 0.3% H 2 O 2 , and measuring the absorption at 595 nm as a function of time.
  • Assays are performed in 0.1 M sodium phosphate or 0.1 M sodium acetate, 50 ⁇ M monochlorodimedone, 10 mM KBr/KCI, and 1 mM H 2 O 2 using a haloperoxidase concentration of about 1 ⁇ g/ml.
  • One HU is defined as 1 micromol of monochlorodimedone chlorinated or brominated per minute at pH 5 and 30°C.
  • the oxidoreductase of the methods and compositions of the invention requires a source of hydrogen peroxide as an oxidizing agent to inactivate the biological material in the methods and compositions (e.g. peroxidases and haloperoxidases), the oxidizing agent may be a source of hydrogen peroxide.
  • the source of hydrogen peroxide may be hydrogen peroxide or a hydrogen peroxide precursor for i n s itu p roduction of h ydrogen p eroxide.
  • a ny s olid e ntity which I iberates u pon dissolution a peroxide, which is useable by haloperoxidase, can serve as the source of hydrogen peroxide.
  • Compounds which yield hydrogen peroxide upon dissolution in water or an appropriate aqueous based medium include but are not limited to metal peroxides, percarbonates, persulphates, perphosphates, peroxyacids, alkyperoxides, acylperoxides, peroxyesters, urea peroxide, perborates and peroxycarboxylic acids or salts thereof.
  • Any compound, which generates a peroxide that peroxidase can use as an electron acceptor, is an acceptable source of hydrogen peroxide for this invention; this includes a large number of compounds as one skilled in the art will recognize. Mixtures of two or more of these substances can also be used.
  • Another source of hydrogen peroxide is a hydrogen peroxide generating enzyme system, such as an oxidase together with a substrate for the oxidase.
  • oxidase and substrate comprise, but are not limited to, amino acid oxidase (see e.g. US 6,248,575) and a suitable amino acid, glucose oxidase (see e.g. WO 95/29996) and glucose, lactate oxidase and lactate, galactose oxidase (see e.g. WO 00/50606) and galactose, and aldose oxidase (see e.g. WO 99/31990) and a suitable aldose.
  • the source of hydrogen peroxide may be added at the beginning of or during the process, e.g., typically in an amount corresponding to levels of from 0.001 mM to 25 mM, preferably to levels of from 0.005 mM to 5 mM, and particularly to levels of from 0.01 to 1 mM.
  • the oxidoreductase of the methods and compositions of the invention requires a source of molecular oxygen as an oxidizing agent to inactivate the biological material in the methods and compositions (e.g. laccases), the oxidizing agent may be a source of molecular oxygen.
  • the source of molecular oxygen may be molecular oxygen from the atmosphere or a precursor thereof for in situ production of molecular oxygen. Molecular oxygen from the atmosphere will usually be present in sufficient quantity. If more O 2 is needed, additional oxygen may be added.
  • the source of iodide ions needed for the reaction with the haloperoxidase may be achieved in many different ways, such as by adding a salt of iodide.
  • the salt of iodide is sodium iodide or potassium iodide, or a mixture thereof.
  • the concentration of the source of iodide ions will typically correspond to a concentration of iodide ions of from 0.01 mM to 1000 mM, preferably in the range of from 0.05 mM to 500 mM.
  • an improved inactivation of the biological material may be obtained by including an enhancing agent in the method of the invention.
  • the enhancing agents may be Preferably the enhancing agent is an amine compound, which may be a compound of the following formula:
  • substituent groups R1 and R2. which may be identical or different, represent any of the following radicals: hydrogen, phenyl, and C 1-6 -alkyl; which phenyl and C 1-6 -alkyl groups may be unsubstituted or substituted with one or more independent substituent groups R3; which substituent group R3 represents any of the following radicals: hydroxy, halogen, formyl, carboxy and esters and salts thereof, carbamoyl, sulfo and esters and salts thereof, sulfamoyl, nitro, amino, phenyl, acyl, C 1-6 -alkyl, and C 1-6 -alkoxy; which carbamoyl, sulfamoyl, amino, phenyl, C 1-6 -alkyl, C 1-6 -alkoxy and acyl groups may furthermore b e u nsubstituted o r s u
  • C 1-n -alkyl wherein n can be from 2 through 6, as used herein, represents a saturated or unsaturated, and branched or straight alkyl group having from one to the specified number of carbon atoms (n).
  • Typical C 1-6 -alkyl groups include, but are not limited to, methyl, ethyl, ethenyl (vinyl), n-propyl, isopropyl, propenyl, isopropenyl, butyl, isobutyl, sec-butyl, tert- butyl, crotyl, methallyl, pentyl, isopentyl, propenyl, prenyl, hexyl, isohexyl, and the like.
  • C 1-n -alkoxy wherein n can be from 2 through 6, as used herein, represents a C 1-n -alkyl group linked through an ether group; such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, and the like.
  • acyl refers to a monovalent substituent comprising a C 1-6 - alkyl group linked through a carbonyl group; such as acetyl, propionyl, butyryl, isobutyryl, pivaloyl, valeryl, and the like.
  • the amine compounds may be in their cationic form.
  • the enhancing agent is not an alpha amino acid.
  • R1 is hydrogen
  • R1 is hydrogen and R2 is an alcohol (amino alcohol), e.g., ethanolamine.
  • the amine compound is an ammonium salt, i.e. any ammonium salt known in the art: e.g., diammonium sulphate, ammonium chloride, ammonium bromide, or ammonium iodide.
  • the enhancing agent may be present in the composition in a concentration in the range of from 0.01 m M to 1000 mM, preferably in the range of from 0.05 m M to 500 mM, more preferably in the range of from 0.1 mM to 100 mM, and most preferably in the range of from 0.1 mM to 50 mM.
  • the biological material of the method and composition of the invention comprises all kinds of bacteria, fungi and virus.
  • the bacteria and fungi may be in a vegetative and/or a sporulated f orm.
  • the bacteria, f ungi a nd virus are capable of being reproduced.
  • the bacteria, fungi and virus are capable of inducing infection or disease.
  • the spores are endospores, such as all Clostridium sp. spores,
  • Brevibacillus sp. spores and Bacillus sp. spores e.g. spores from Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus putida, and Bacillus pumila.
  • the spores are exospores, such as Actinomycetales spores, e.g. spores from Actinomyves sp., Streptomyces sp., Thermoactinomyces sp., Saccharomonospora sp., and Saccharopylospora sp.
  • Actinomycetales spores e.g. spores from Actinomyves sp., Streptomyces sp., Thermoactinomyces sp., Saccharomonospora sp., and Saccharopylospora sp.
  • the spores are bacterial spores.
  • bacterial spores include, but are not limited to, all Clostridium sp. spores and Bacillus sp. spores as mentioned above.
  • the spores are fungal spores.
  • fungal spores include (in addition to those mentioned above), but are not limited to, conidiospores, such as spores from Aspergillus sp., and Penicillium sp.
  • the present invention provides a vaccine composition
  • a vaccine composition comprising biological material, which has been inactivated by contacting it with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions.
  • the vaccine composition may be stored in a small container, such as a vial, e.g. a glass vial.
  • the vaccine composition is capable of immunizing an animal or a human being against infections or diseases induced by the active biological material.
  • the vaccine composition of the invention may further comprise an inactivated microbial toxin or fragment thereof or a microbial toxin analogue.
  • the microbial toxin is a protein toxin.
  • the microbial toxin is a fungal toxin or a bacterial toxin, such as the bacterial protein toxins described in "The Comprehensive Sourcebook of Bacterial Protein Toxins", 2 nd edition (1999), Academic Press, ISBN 0120530759, edited by Alouf and Freer.
  • the microbial toxins may be inactivated by being contacted with chemical agents such as formaldehyde (formalin), or by using molecular biological methods to change the amino acid sequence of a protein toxin into a non-toxic sequence as described in WO 89/09617.
  • chemical agents such as formaldehyde (formalin)
  • molecular biological methods to change the amino acid sequence of a protein toxin into a non-toxic sequence as described in WO 89/09617.
  • toxin is intended to mean any poisonous agent, especially a poisonous substance produced by one living organism that is poisonous to other organisms; e.g., cholera toxin, shigella toxin, anthrax toxin, diphteria toxin, tetanus toxin or botulinum toxins.
  • the present invention provides a method for producing a vaccine from biological material, comprising inactivating the biological material by contacting it with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions. Subsequently the inactivated biological material may be formulated into a vaccine composition, which may be stored in vials in liquid form or as a lyophilized powder.
  • inactivating or “inactivated” in relation to the biological material or toxin is intended to mean that the biological material or toxin is not capable of causing infection or disease.
  • the oxidoreductase and/or the suitable oxidizing agent and/or the source of halide ions may be formulated as a liquid (e.g. aqueous) or a dry product formulation.
  • the dry product formulation may subsequently be re-hydrated to form an active liquid or semi-liquid formulation usable in the method of the invention.
  • the components may be mixed, arranged in discrete layers or packaged separately.
  • the invention also relates to the use of a vaccine composition of the invention as a medicament.
  • a vaccine composition of the invention may also be used for the manufacture of a medicament for controlling or combating microorganisms, such as fungi or bacteria.
  • the vaccine composition may be used as a veterinarian or human therapeutic or prophylactic agent.
  • the vaccine composition may be used in the preparation of veterinarian or human therapeutic agents or prophylactic agents for the treatment of a microbial, such as a fungal or bacterial infection.

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Abstract

The invention provides a method for producing a vaccine composition from biological material by inactivating the biological material with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions. Further, a vaccine composition is provided and use of the vaccine composition as a medicament.

Description

PREPARATION OF VACCINES
FIELD OF THE INVENTION
The p resent i nvention relates to m ethods for p reparation of vaccines from b iological material (such as microorganisms or spores) by contacting it with an oxidoreductase enzyme capable of inactivating living biological material.
BACKGROUND
It is an object of the present invention to provide methods for preparing vaccines which do not involve using chemical agents, such as formaldehyde. It is further an object of the invention to provide vaccines which do not contain remnants of harsh chemicals such as formaldehyde.
SUMMARY The present invention provides a method for producing a vaccine composition from biological material, comprising inactivating the biological material by contacting it with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions.
In an embodiment the inactivated biological material is formulated into a vaccine composition.
In a s econd aspect, the i nvention p rovides a vaccine composition o btainable by the method of the invention.
In further aspects are provided a container comprising the vaccine composition, and use of the vaccine composition as a medicament.
DETAILED DESCRIPTION Qxidoreductases
In the context of the present invention the oxidoreductase is an enzyme from the enzyme class EC 1.-.-.- as set out by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) or a compound exhibiting a corresponding enzymatic activity, which is capable of inactivating the biological material of the method and composition of the invention. In an embodiment, the oxidoreductase of the invention is a phenol oxidizing enzyme. In the context of the present invention the phenol oxidizing enzyme is an enzyme or a compound capable of oxidizing phenolic compounds. The oxidoreductase may be a peroxidase or a compound exhibiting peroxidase activity
(such as a haloperoxidase or a compound exhibiting haloperoxidase activity), a laccase, or a compound exhibiting laccase activity or an activity corresponding to laccase activity, such as a catechol oxidase ( EC 1 .10.3.1), a n o-aminophenol oxidase ( EC 1 .10.3.4), a nd/or a b ilirubin oxidase (EC 1.3.3.5). Compounds exhibiting an activity corresponding to laccase activity are referred to below as laccase related enzymes.
In an embodiment the oxidoreductase is capable of generating free radicals. The oxidoreductase of the invention may typically be present in concentrations of from
1 to 10000 microgram enzyme protein per liter aqueous solution, preferably of from 5 to 2000 microgram enzyme protein per liter aqueous solution, more preferably of from 5 to 1000 microgram enzyme protein per liter aqueous solution, and most preferably of from 1 to 500 microgram enzyme protein per liter aqueous solution. Assays for determining the activity of these enzymes are well known to a person skilled in the art.
It is to be understood that oxidoreductase variants (e.g. produced by recombinant techniques) are included within the meaning of the term oxidoreductase.
Laccases and Compounds Exhibiting Laccase Activity
Compounds exhibiting laccase activity may be any laccase enzyme comprised by the enzyme classification EC 1.10.3.2, or any fragment derived therefrom, exhibiting laccase activity.
Preferred laccase enzymes and/or laccase related enzymes are enzymes of microbial origin. The enzymes may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts).
Suitable examples from fungi include a laccase derivable from a strain of Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinus, e.g., C. cinereus, C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M. thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radita (WO 92/01046), or Coriolus, e.g., C. hirsutus (JP 2-238885).
Suitable examples from bacteria include a laccase derivable from a strain of Bacillus. A laccase derived from Coprinus, Myceliophthora, Polyporus, Scytalidium or
Rhizoctonia is preferred; in particular a laccase derived from Coprinus cinereus, Myceliophthora thermophila, Polyporus pinsitus, Scytalidium thermophilum or Rhizoctonia solani.
The laccase or the compound exhibiting laccase activity may furthermore be one which is producible by a method comprising cultivating a host cell transformed with a recombinant
DNA vector which carries a DNA sequence encoding said laccase or the compound exhibiting laccase activity as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the laccase or the compound exhibiting laccase activity, in a culture medium under conditions permitting the expression of the laccase enzyme or the compound exhibiting laccase activity, and recovering the laccase or the compound exhibiting laccase activity from the culture.
Determination of Laccase Activity (LACU)
Laccase activity (particularly suitable for Polyporus laccases) may be determined from the oxidation of syringaldazin under aerobic conditions. The violet colour produced is photometered at 530 nm. The analytical conditions are 19 mM syringaldazin, 23 mM acetate buffer, pH 5.5, 30°C, 1 min. reaction time.
1 laccase unit (LACU) is the amount of enzyme that catalyses the conversion of 1.0 mmole syringaldazin per minute at these conditions.
Determination of Laccase Activity (LAMU) Laccase activity may be determined from the oxidation of syringaldazin under aerobic conditions. The violet colour produced is measured at 530 nm. The analytical conditions are 19 mM syringaldazin, 23 mM Tris/maleate buffer, pH 7.5, 30°C, 1 min. reaction time.
1 laccase unit (LAMU) is the amount of enzyme that catalyses the conversion of 1.0 mmole syringaldazin per minute at these conditions.
Peroxidases and Compounds Exhibiting Peroxidase Activity
Compounds exhibiting peroxidase activity may be any peroxidase enzyme comprised by the enzyme classification (EC 1.11.1.7), or any fragment derived therefrom, exhibiting peroxidase activity. In the context of this invention, compounds exhibiting peroxidase activity comprise peroxidase active fragments derived from cytochromes, haemoglobin or peroxidase enzymes (such as microperoxidases).
Preferably, the peroxidase employed in the composition of the invention is producible by plants (e.g. horseradish or soybean peroxidase) or microorganisms such as fungi or bacteria. Some preferred fungi include strains belonging to the subdivision Deuteromycotina, class Hyphomycetes, e.g., Fusarium, Humicola, Trichoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embeliisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, Myrothecium verrucaria (IFO 6113), Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embeliisia alii or Dreschlera halodes.
Other preferred fungi include strains belonging to the subdivision Basidiomycotina, class Basidiomycetes, e.g., Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371 ), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (previously called Polyporus), e.g., T. versicolor (e.g. PR4 28-A). Further preferred fungi include strains belonging to the subdivision Zygomycotina, class
Mycoraceae, e.g., Rhizopus or Mucor, in particular Mucor hiemalis.
Some preferred bacteria include strains of the order Actinomycetales, e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382) or Streptoverticillum verticillium ssp. verticillium. Other preferred bacteria include Bacillus pumilus (ATCC 12905), Bacillus stearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11).
Further preferred bacteria include strains belonging to Myxococcus, e.g., M. virescens. The peroxidase may furthermore be one which is producible by a method comprising cultivating a host cell transformed with a recombinant DNA vector which carries a DNA sequence encoding said peroxidase as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the peroxidase, in a culture medium under conditions permitting the expression of the peroxidase and recovering the peroxidase from the culture. Particularly, a recombinantly produced peroxidase is a peroxidase derived from a
Coprinus sp., in particular C. macrorhizus or C. cinereus according to WO 92/16634.
Determination of Peroxidase Activity (POXU)
One peroxidase unit (POXU) is the amount of enzyme which under the following conditions catalyze the conversion of 1 micromole hydrogen peroxide per minute:
0.1 M phosphate buffer pH 7.0, 0.88 mM hydrogen peroxide, 1.67 mM 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonate) (ABTS) and 30°C.
The reaction is followed for 60 seconds (15 seconds after mixing) by the change in absorbance at 418 nm, which should be in the range 0.15 to 0.30. For calculation of activity is used an absorption coefficient of oxidized ABTS of 36 mM"1 cm"1 and a stoichiometry of one micromole H2O2 converted per two micromole ABTS oxidized.
Haloperoxidases and Compounds Exhibiting Haloperoxidase Activity
The haloperoxidases suitable for being incorporated in the method of the invention include chloroperoxidases, bromoperoxidases and compounds exhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidases form a class of enzymes, which are capable of oxidizing halides (CI-, Br-, I-) in the presence of hydrogen peroxide or a hydrogen peroxide generating system to the corresponding hypohalous acids according to:
H2O2 + X- + H+ -> H2O + HOX wherein X- is a halide and HOX is a hypohalous acid.
Haloperoxidases are classified according to their specificity for halide ions: - Chloroperoxidases (E.C. 1.11.1.10) which are capable of catalyzing formation of hypochlorite from chloride ions, hypobromite from bromide ions and hypoiodite from iodide ions; and
Bromoperoxidases which are capable of catalyzing formation of hypobromite from bromide ions and hypoiodite from iodide ions. Hypoiodite, however, undergoes spontaneous disproportionation to iodine and thus iodine is the observed product. These hypohalite compounds may subsequently react with other compounds forming halogenated compounds.
Haloperoxidases have been isolated from various organisms: mammals, marine animals, plants, algae, lichen, fungi and bacteria. It is generally accepted that haloperoxidases are the enzymes responsible for the formation of halogenated compounds in nature, although other enzymes may be involved.
Haloperoxidases have been isolated from many different fungi, in particular from the fungus group dematiaceous hyphomycetes, such as Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.
Haloperoxidases have also been isolated from bacteria such as Pseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S. aureofaciens.
In a preferred embodiment the haloperoxidase is a vanadium haloperoxidase (i.e. a vanadium or vanadate containing haloperoxidase) derivable from Curvularia sp., in particular Curvularia verruculosa and Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046, e.g. a vanadium haloperoxidase encoded by the DNA sequence of WO 95/27046, figure 2 all incorporated by reference; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97/04102.
In another preferred embodiment the haloperoxidase is a vanadium containing haloperoxidase, such as a vanadium chloroperoxidase. The vanadium chloroperoxidase may be derivable from Drechslera hartlebii as described in WO 01/79459, Dendryphiella salina as described in WO 01/79458, Phaeotrichoconis crotalarie as described in WO 01/79461 , or Geniculosporium sp. as described in WO 01/79460. The vanadium haloperoxidase is more preferably derivable from Drechslera hartlebii (DSM 13444), Dendryphiella salina (DSM 13443), Phaeotrichoconis crotalarie (DSM 13441 ) or Geniculosporium sp. (DSM 13442).
In yet another embodiment the haloperoxidase is horseradish peroxidase, myeloperoxidase or eosinophil peroxidase. The concentration of the haloperoxidase is typically in the range of 0.01-100 ppm enzyme protein, preferably 0.05-50 ppm enzyme protein, more preferably 0.1-20 ppm enzyme protein, and most preferably 0.5-10 ppm enzyme protein.
Determination of Haloperoxidase Activity
Microtiter assays are performed by mixing 100 μl of haloperoxidase sample (about 0.2 μg/ml) and 100 μl of 0.3 M sodium phosphate pH 7 buffer - 0.5 M potassium bromide - 0.008% phenol red, adding the solution to 10 μl of 0.3% H2O2, and measuring the absorption at 595 nm as a function of time. Assays using monochlorodimedone (Sigma M4632, ε = 20000 M"1cm"1 at 290 nm) as a substrate are performed as described below. The decrease in absorption at 290 nm is measured as a function of time. Assays are performed in 0.1 M sodium phosphate or 0.1 M sodium acetate, 50 μM monochlorodimedone, 10 mM KBr/KCI, and 1 mM H2O2 using a haloperoxidase concentration of about 1 μg/ml. One HU is defined as 1 micromol of monochlorodimedone chlorinated or brominated per minute at pH 5 and 30°C.
Oxidizing agent
If the oxidoreductase of the methods and compositions of the invention requires a source of hydrogen peroxide as an oxidizing agent to inactivate the biological material in the methods and compositions (e.g. peroxidases and haloperoxidases), the oxidizing agent may be a source of hydrogen peroxide.
The source of hydrogen peroxide may be hydrogen peroxide or a hydrogen peroxide precursor for i n s itu p roduction of h ydrogen p eroxide. A ny s olid e ntity which I iberates u pon dissolution a peroxide, which is useable by haloperoxidase, can serve as the source of hydrogen peroxide. Compounds which yield hydrogen peroxide upon dissolution in water or an appropriate aqueous based medium include but are not limited to metal peroxides, percarbonates, persulphates, perphosphates, peroxyacids, alkyperoxides, acylperoxides, peroxyesters, urea peroxide, perborates and peroxycarboxylic acids or salts thereof. Any compound, which generates a peroxide that peroxidase can use as an electron acceptor, is an acceptable source of hydrogen peroxide for this invention; this includes a large number of compounds as one skilled in the art will recognize. Mixtures of two or more of these substances can also be used.
Another source of hydrogen peroxide is a hydrogen peroxide generating enzyme system, such as an oxidase together with a substrate for the oxidase. Examples of combinations of oxidase and substrate comprise, but are not limited to, amino acid oxidase (see e.g. US 6,248,575) and a suitable amino acid, glucose oxidase (see e.g. WO 95/29996) and glucose, lactate oxidase and lactate, galactose oxidase (see e.g. WO 00/50606) and galactose, and aldose oxidase (see e.g. WO 99/31990) and a suitable aldose.
By studying EC 1.1.3.-, EC 1.2.3.-, EC 1.4.3.-, and EC 1.5.3.- or similar classes (under the International Union of Biochemistry and Molecular Biology), other examples of such combinations of oxidases and substrates are easily recognized by one skilled in the art. The source of hydrogen peroxide may be added at the beginning of or during the process, e.g., typically in an amount corresponding to levels of from 0.001 mM to 25 mM, preferably to levels of from 0.005 mM to 5 mM, and particularly to levels of from 0.01 to 1 mM.
If the oxidoreductase of the methods and compositions of the invention requires a source of molecular oxygen as an oxidizing agent to inactivate the biological material in the methods and compositions (e.g. laccases), the oxidizing agent may be a source of molecular oxygen.
The source of molecular oxygen may be molecular oxygen from the atmosphere or a precursor thereof for in situ production of molecular oxygen. Molecular oxygen from the atmosphere will usually be present in sufficient quantity. If more O2 is needed, additional oxygen may be added.
Source of Halide ions
According to the invention the source of iodide ions needed for the reaction with the haloperoxidase may be achieved in many different ways, such as by adding a salt of iodide. In a preferred embodiment the salt of iodide is sodium iodide or potassium iodide, or a mixture thereof.
The concentration of the source of iodide ions will typically correspond to a concentration of iodide ions of from 0.01 mM to 1000 mM, preferably in the range of from 0.05 mM to 500 mM.
Enhancing agent
We have observed that an improved inactivation of the biological material may be obtained by including an enhancing agent in the method of the invention. The enhancing agents may be Preferably the enhancing agent is an amine compound, which may be a compound of the following formula:
,R1 HN XR2 wherein the substituent groups R1 and R2. which may be identical or different, represent any of the following radicals: hydrogen, phenyl, and C1-6-alkyl; which phenyl and C1-6-alkyl groups may be unsubstituted or substituted with one or more independent substituent groups R3; which substituent group R3 represents any of the following radicals: hydroxy, halogen, formyl, carboxy and esters and salts thereof, carbamoyl, sulfo and esters and salts thereof, sulfamoyl, nitro, amino, phenyl, acyl, C1-6-alkyl, and C1-6-alkoxy; which carbamoyl, sulfamoyl, amino, phenyl, C1-6-alkyl, C1-6-alkoxy and acyl groups may furthermore b e u nsubstituted o r s ubstituted with o ne o r m ore i ndependent s ubstituent groups R4; which substituent group R4 represents any of the following radicals: halogen, hydroxy, formyl, carboxy and esters and salts thereof, carbamoyl, sulfo and esters and salts thereof, sulfamoyl, nitro, amino, phenyl, acyl, C1-6-alkyl, and C1-6-alkoxy; which carbamoyl, sulfamoyl, and amino groups may furthermore be unsubstituted or substituted independently once or twice with hydroxy, C1-4-alkyl and C1-4-alkoxy; and which phenyl, C1-6-alkyl, C1-6-alkoxy and acyl groups may furthermore be unsubstituted or substituted independently with one or more of the following groups: halogen, hydroxy, amino, formyl, carboxy and esters a nd salts thereof, carbamoyl, s ulfo and esters a nd salts thereof, and sulfamoyl. In an embodiment, when the substituent group R3 is a carboxy group or an ester thereof, R3 cannot be directly connected to a carbon atom which is directly connected to the nitrogen atom in the above formula.
The term "C1-n-alkyl" wherein n can be from 2 through 6, as used herein, represents a saturated or unsaturated, and branched or straight alkyl group having from one to the specified number of carbon atoms (n). Typical C1-6-alkyl groups include, but are not limited to, methyl, ethyl, ethenyl (vinyl), n-propyl, isopropyl, propenyl, isopropenyl, butyl, isobutyl, sec-butyl, tert- butyl, crotyl, methallyl, pentyl, isopentyl, propenyl, prenyl, hexyl, isohexyl, and the like.
The term "C1-n-alkoxy" wherein n can be from 2 through 6, as used herein, represents a C1-n-alkyl group linked through an ether group; such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, and the like.
The term "acyl" as used herein refers to a monovalent substituent comprising a C1-6- alkyl group linked through a carbonyl group; such as acetyl, propionyl, butyryl, isobutyryl, pivaloyl, valeryl, and the like.
As used herein, the amine compounds may be in their cationic form. In an embodiment the enhancing agent is not an alpha amino acid.
In a preferred embodiment R1 is hydrogen.
In another preferred embodiment R1 is hydrogen and R2 is an alcohol (amino alcohol), e.g., ethanolamine.
In a further preferred embodiment the amine compound is an ammonium salt, i.e. any ammonium salt known in the art: e.g., diammonium sulphate, ammonium chloride, ammonium bromide, or ammonium iodide.
The enhancing agent may be present in the composition in a concentration in the range of from 0.01 m M to 1000 mM, preferably in the range of from 0.05 m M to 500 mM, more preferably in the range of from 0.1 mM to 100 mM, and most preferably in the range of from 0.1 mM to 50 mM.
Biological material
The biological material of the method and composition of the invention comprises all kinds of bacteria, fungi and virus. The bacteria and fungi may be in a vegetative and/or a sporulated f orm. Preferably, the bacteria, f ungi a nd virus are capable of being reproduced. More preferably, the bacteria, fungi and virus are capable of inducing infection or disease. In an embodiment the spores are endospores, such as all Clostridium sp. spores,
Brevibacillus sp. spores and Bacillus sp. spores, e.g. spores from Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus putida, and Bacillus pumila.
In another embodiment the spores are exospores, such as Actinomycetales spores, e.g. spores from Actinomyves sp., Streptomyces sp., Thermoactinomyces sp., Saccharomonospora sp., and Saccharopylospora sp.
In another embodiment the spores are bacterial spores. Examples of bacterial spores include, but are not limited to, all Clostridium sp. spores and Bacillus sp. spores as mentioned above.
In yet another embodiment the spores are fungal spores. Examples of fungal spores include (in addition to those mentioned above), but are not limited to, conidiospores, such as spores from Aspergillus sp., and Penicillium sp.
Compositions
The present invention provides a vaccine composition comprising biological material, which has been inactivated by contacting it with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions.
Until use the vaccine composition may be stored in a small container, such as a vial, e.g. a glass vial.
The vaccine composition is capable of immunizing an animal or a human being against infections or diseases induced by the active biological material.
The vaccine composition of the invention may further comprise an inactivated microbial toxin or fragment thereof or a microbial toxin analogue. In an embodiment the microbial toxin is a protein toxin. In another embodiment the microbial toxin is a fungal toxin or a bacterial toxin, such as the bacterial protein toxins described in "The Comprehensive Sourcebook of Bacterial Protein Toxins", 2nd edition (1999), Academic Press, ISBN 0120530759, edited by Alouf and Freer. The microbial toxins may be inactivated by being contacted with chemical agents such as formaldehyde (formalin), or by using molecular biological methods to change the amino acid sequence of a protein toxin into a non-toxic sequence as described in WO 89/09617.
In the context of the present invention the term "toxin" is intended to mean any poisonous agent, especially a poisonous substance produced by one living organism that is poisonous to other organisms; e.g., cholera toxin, shigella toxin, anthrax toxin, diphteria toxin, tetanus toxin or botulinum toxins.
Methods and Uses
The present invention provides a method for producing a vaccine from biological material, comprising inactivating the biological material by contacting it with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions. Subsequently the inactivated biological material may be formulated into a vaccine composition, which may be stored in vials in liquid form or as a lyophilized powder.
In the context of the present invention the term "inactivating" or "inactivated" in relation to the biological material or toxin is intended to mean that the biological material or toxin is not capable of causing infection or disease.
The oxidoreductase and/or the suitable oxidizing agent and/or the source of halide ions may be formulated as a liquid (e.g. aqueous) or a dry product formulation. The dry product formulation may subsequently be re-hydrated to form an active liquid or semi-liquid formulation usable in the method of the invention. In a dry product formulation, the components may be mixed, arranged in discrete layers or packaged separately.
The invention also relates to the use of a vaccine composition of the invention as a medicament. Further, a vaccine composition of the invention may also be used for the manufacture of a medicament for controlling or combating microorganisms, such as fungi or bacteria. The vaccine composition may be used as a veterinarian or human therapeutic or prophylactic agent. Thus, the vaccine composition may be used in the preparation of veterinarian or human therapeutic agents or prophylactic agents for the treatment of a microbial, such as a fungal or bacterial infection.

Claims

1. A method for producing a vaccine composition from biological material, comprising inactivating the biological material by contacting it with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions.
2. The method of claim 1 , which further comprises formulating the inactivated biological material into a vaccine.
3. The method of claims 1 or 2, wherein the biological material is protein or DNA derived from microorganisms or viruses.
4. A vaccine composition obtainable by the method of claim 2.
5. A vaccine composition, comprising biological material which has been inactivated by being contacted with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions.
6. A container comprising the vaccine composition of claim 5.
7. The vaccine composition of claim 5 for use as a medicament.
8. The vaccine composition of claim 5 for use as a veterinarian or human therapeutic agent for the treatment of a microbial infection or for prophylactic use.
9. Use of the vaccine composition of claim 5 as a veterinarian or human therapeutic agent for the treatment of a microbial infection or for prophylactic use.
10. Use of biological material for vaccination, wherein the biological material has been inactivated by being contacted with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions.
1 . Use of biological material for producing a vaccine, wherein the biological material has been inactivated by being contacted with an oxidoreductase, a suitable oxidizing agent and, if the oxidoreductase is a haloperoxidase, a source of halide ions.
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