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WO2011113801A1 - Utilisation de la souche 23 de clostridium perfringens pour la protection contre une entérite nécrotique - Google Patents

Utilisation de la souche 23 de clostridium perfringens pour la protection contre une entérite nécrotique Download PDF

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Publication number
WO2011113801A1
WO2011113801A1 PCT/EP2011/053825 EP2011053825W WO2011113801A1 WO 2011113801 A1 WO2011113801 A1 WO 2011113801A1 EP 2011053825 W EP2011053825 W EP 2011053825W WO 2011113801 A1 WO2011113801 A1 WO 2011113801A1
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WIPO (PCT)
Prior art keywords
strain
composition according
necrotic enteritis
culture
clostridium perfringens
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Ceased
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PCT/EP2011/053825
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English (en)
Inventor
Filip Van Immerseel
Richard Ducatelle
Anouk Lanckriet
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Universiteit Gent
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Universiteit Gent
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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
    • A61K39/02—Bacterial antigens
    • A61K39/08—Clostridium, e.g. Clostridium tetani
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine

Definitions

  • Clostridium perfringens strain 23 to protect against necrotic enteritis
  • the present invention relates to the prevention of necrotic enteritis caused by an infection with the anaerobic bacterium Clostridium perfringens . More specifically, the invention relates to compounds produced by or derived from Clostridium perfringens strain 23 which are useful as vaccines to effectively protect against necrotic enteritis.
  • Clostridium perfringens is the causative agent of necrotic enteritis, an intestinal disease that affects industrial poultry worldwide [1 , 2]. After the ban of growth promoting antibiotics in the European Union, necrotic enteritis has become much more widespread. It is mainly contained by using curative antibiotics or ionophore anticoccidials [13, 14]. However, the use of curative antibiotics holds the risk of inducing resistance among the C. perfringens population and the rest of the intestinal microflora. Anticoccidials are meant in the first place to control coccidiosis.
  • Vaccination would be a valuable approach for the prevention of necrotic enteritis.
  • C. perfringens strains are ubiquitous and notorious for the wide range of toxins and virulence factors they excrete in their environment [3].
  • the majority of the chicken strains are toxinotype A, meaning that they carry the pic gene encoding alpha toxin [4-7].
  • this alpha toxin a metalloenzyme with lecithinase and sphingomyelinase activities, was the major virulence factor involved in necrotic enteritis [8].
  • NetB a novel pore forming toxin
  • perfringens chicken strain is still capable of inducing necrotic lesions in the gut of experimentally infected broilers, a netB deletion mutant from the same strain is not [9, 10].
  • C. perfringens secretes other proteins like metalloproteinases, growth inhibiting factors, etc... that play a role in the pathogenesis of necrotic enteritis [1 1, 12].
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • the present invention discloses an effective vaccine against necrotic enteritis.
  • a first embodiment of the present invention relates to a composition comprising at least an immunoprotective fraction of Clostridium perfringens strain 23 for use as a medicament.
  • the terms 'a composition comprising at least an immunoprotective fraction of Clostridium perfringens strain 23' relate to a composition comprising at least an antigen preparation derived of Clostridium perfringens strain 23.
  • Said antigen preparation has immunoproptective properties and includes whole-cell bacterial preparations or preparations of components produced by or derived from Clostridium perfringens strain 23 or a combination thereof.
  • Such antigen preparations may thus comprise whole -killed (inactive) bacteria, live-attenuated (weakened) bacteria or processed and/or artificial bacterial preparations, or combinations thereof.
  • Processed bacterial preparations include preparations of bacterial proteins, which are partially or completely purified. These can be used alone or in combination with artificial antigen preparations such as preparations which are either in part or entirely obtained by synthetic or recombinant methods. Also processed bacterial preparations which are in part or entirely obtained by synthetic or recombinant methods are part of the present invention.
  • the antigen preparation or immunoprotective fraction of the present invention can also be a cell lysate of Clostridium perfringens strain 23, i.e. a mixture obtained upon lysis of bacterial cells.
  • Said lysate can be detoxified or inactivated, and/or further purified, and/or further fractionated using methods known to a skilled person in order to obtain a composition of enriched or purified immunoprotective antigens.
  • said antigen preparation can be a soluble, immunoprotective fraction of a sonicated bacterial culture, e.g. obtained after filtration.
  • the composition comprising an antigen preparation or immunoprotective fraction of the present invention is an antigen preparation or immunoprotective fraction, of the supernatant of a culture of said Clostridium strain.
  • the composition of the present invention comprises a C. perfringens strain 23 culture supernatant. Said supernatant can be detoxified or inactivated, and/or further purified, and/or further fractionated using methods known to a skilled person in order to obtain a composition of enriched or purified immunoprotective antigens.
  • Said supernatant can, for example, be derived from an overnight culture of said Clostridium strain which is concentrated by dialysis (against for example a 20kDa polyethyleneglycol solution) followed by further concentration and desalting using desalting columns, and, which is subsequently diluted in PBS.
  • the antigen preparation or immunoprotective fraction of the present invention as indicated above will thus comprise immunoprotective organic compounds, preferably immunoprotective proteins or antigens, produced by Clostridium perfringens strain 23.
  • said proteins will be secreted by said bacterial strain.
  • the term 'immunoprotective' relates to a significant prevention of the development of necrotic lesions in the majority of animals vaccinated with antigen preparations derived from C. perfringens strain 23 and challenged with pathogenic C perfringens strains.
  • This can, for example, be tested as follows: 1) vaccinate a group of animals with a composition comprising at least an immunoprotective fraction of Clostridium perfringens strain 23 according to the present invention using methods known in the art.
  • said immunoprotective fraction is an antigen preparation of Clostridium perfringens strain 23, more preferably a supernatant of a culture of Clostridium perfringens strain 23.
  • necrotic lesions means that at least 50%, 60%, 70%, preferably 80%, more preferably 90% or most preferably 100% of the necrotic lesions that would develop without prior vaccination will not develop due to the vaccine of the present invention.
  • majority of animals is meant that at least 50%, 60%, 70%, preferably 80%, more preferably 90% or, most preferably 100% of all animals which are vaccinated with the vaccine of the present invention will show a significant prevention of the development of necrotic lesions.
  • the composition of the present invention is capable to reduce the number of animals having necrotic lesions with 50%, 60%, 70%, preferably 80%, more preferably 90% or most preferably 100% when compared to a group of control animals which did not receive the composition of the present invention.
  • 'Clostridium perfringens strain 23' is a type A strain (i.e. is a strain that contains alpha toxin, but not iota, beta and epsilon toxin) and corresponds to isolate number 23 as described in Table 2 on page 147 of ref n° 5.
  • the latter strain has further been deposited on January 29, 2010 with BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie - Universiteit Gent, K.L.
  • Ledeganckstraat 35, B-9000 Gent, Belgium http://bccm.belspo.be/) and has accession number LMG S-25552 and deposited on June 4, 2010 with BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie - Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium (http ://bccm.belspo .be/) and has accession number LMG P-25816 and deposited on June 4, 2010 with BCCM/LMG Bacteria Collection,
  • a 'medicament' as used above also referred to as pharmaceutical drug, medicine or medication, refers to a composition comprising at least an antigen preparation or an immunoprotective fraction of Clostridium perfringens strain 23 intended for use in the medical diagnosis, cure, treatment, or prevention of disease wherein said disease preferably is necrotic enteritis.
  • Necrotic enteritis refers to an intestinal disease that affects industrial poultry worldwide.
  • Suitable carriers, excipients or other materials are known to the skilled man.
  • the 'medicament' or composition may be administered by any suitable method within the knowledge of the skilled man.
  • a specific route of administration is oral or parenteral administration.
  • Vaccination in ovo is also possible.
  • the dosage and mode of administration will depend on the individual/animal.
  • the present invention relates in particular to a composition of the present invention for use to protect against necrotic enteritis in birds.
  • Said birds are preferably chickens, ducks or turkeys and said chickens are preferably broilers or breeders.
  • the present invention refers to any method known to the skilled person for the preparation of a vaccine against necrotic enteritis.
  • the invention provides the use of the antigen preparation or immunoprotective fraction Clostridium perfringens strain 23 in the preparation of a medicament or vaccine for protecting a subject against necrotic enteritis.
  • the latter method specifically relates to a vaccine against necrotic enteritis in birds.
  • the latter vaccine comprises a composition according to the present invention.
  • the vaccine can comprise, in addition to the antigen preparation of the present invention, a suitable adjuvant. A large number of adjuvants are known to a person skilled in the art.
  • the vaccines of the present invention can be in solid or liquid form such as tablets, capsules, powders, solutions, suspensions or emulsions.
  • the present invention further encompasses a method of administering said vaccine or composition according to the invention to a subject in need thereof.
  • the subject is thereby vaccinated by said vaccine or composition.
  • the vaccines of the present invention can be administered orally, or, in injectable dosages by solution or suspension of these materials in a physiologically acceptable diluent with or without a pharmaceutical carrier as known in the art as is for example described in PCT/EP2007/009855.
  • said vaccines can be administered as aerosols as is for example described in PCT EP2007/009855.
  • the administration of vaccines of the present invention may take place in a single dose or in a dose repeated once or several times after a certain period.
  • the appropriate dosage varies according to various parameters, for example the individual/animal treated (adult animal, young animal including one-day old animal, or, in the case of birds, animals which didn't hatch yet when in ovo administration as described by Williams C.J. and Zedek A.S. 2010 Poultry Science 89: 189-193 is envisioned), the antigen itself, the mode and frequency of administration, the presence or absence of adjuvant and the type of adjuvant.
  • birds can be vaccinated twice (on day 3 and 12 post-hatching) subcutaneously with a 200 ⁇ dose of supernatant of said bacteria containing 7 to 70 ⁇ g total protein ( possibly in addition of 50 ⁇ g/animal/vaccination adjuvant).
  • dosages can be expressed in CFUs.
  • Crude antigen preparations i.e. containing traces of culture medium
  • the vaccine of the present invention can be administered through any suitable route, such as in ovo or by mucosal (intranasal), parenteral, intramuscular, oral, intradermal, intraperitoneal, intravenous, transdermal/transcutaneous or subcutaneous administration, or any combination thereof.
  • mucosal intranasal
  • parenteral intramuscular, oral, intradermal, intraperitoneal, intravenous, transdermal/transcutaneous or subcutaneous administration, or any combination thereof.
  • the presence of the netB gene was determined by PCR using the primers AKP78 (5 '- GCTGGTGCTGGAATAAATGC-3', cf. SEQ ID NO.l) and AKP79 (5'- TCGCC ATTGAGT AGTTTCCC-3 ', cf. SEQ ID NO.2), as described by Keyburn et al. (2008) [9]. Lactate dehydrogenase cytotoxicity assays were performed as an indicator for NetB expression [9]. Overnight cultures were grown in BHI. The supematants were filter sterilized and dialyzed overnight against 10 mM Tris-HCl pH 8.5.
  • the culture supematants were added to the medium of chickens hepatoma (LMH) cells (ATCC CRL-21 17) in a 1 :4 dilution. The cells were incubated for 3 hours at 37°C and 5% C0 2 . Lactate dehydrogenase release in the supernatant was used as an indicator of cytolysis and hence NetB production and was measured using the Cytotoxicity Detection Kit (Roche Applied Sciences, Penzberg, Germany). As a positive control, 10% Triton X-100 (Sigma Aldrich, St. Louis MO, USA) was added to the cells. The negative control consisted of 1 :4 diluted BHI. Percentage cytotoxicity was determined relative to the control groups.
  • LMH chickens hepatoma
  • the in vivo necrotic enteritis challenge model was applied as described previously [15]. Groups of 27 (trial 2 and 3) or 30 (trial 1) broilers were fed a wheat/rye -based (43%/7.5%) diet, with soybean meal as protein source.
  • Nobilis Gumboro D78 Gumboro vaccine (Schering-Plough Animal
  • Table 4 shows that strain 23 is only capable of inducing lesions in half as many birds as strain 56 is.
  • ⁇ Percentage cytotoxicity of the supematants were determined relative to control wells treated with 10% Triton X-100 (positive control) or 1 :4 diluted BHI (negative control). The values are averages of 4 independent trials.
  • strain 23 produces low levels of alpha toxin in vitro whereas strain 48 produces high levels. This indicates that the immunity against necrotic enteritis cannot be attributed to alpha toxin or NetB on its own.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne la prévention d'une entérite nécrotique provoquée par une infection par la bactérie anaérobie Clostridium perfringens. De façon plus spécifique, l'invention concerne des composés produits par ou issus de la souche 23 de Clostridium perfringens qui sont utiles comme vaccins pour protéger de manière efficace contre l'entérite nécrotique.
PCT/EP2011/053825 2010-03-16 2011-03-15 Utilisation de la souche 23 de clostridium perfringens pour la protection contre une entérite nécrotique Ceased WO2011113801A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP10156632 2010-03-16
EP10156632.1 2010-03-16
EP10168278.9 2010-07-02
EP10168278 2010-07-02

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WO2011113801A1 true WO2011113801A1 (fr) 2011-09-22

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Cited By (12)

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US8906668B2 (en) 2012-11-23 2014-12-09 Seres Health, Inc. Synergistic bacterial compositions and methods of production and use thereof
US9011834B1 (en) 2013-02-04 2015-04-21 Seres Health, Inc. Compositions and methods
US9956282B2 (en) 2013-12-16 2018-05-01 Seres Therapeutics, Inc. Bacterial compositions and methods of use thereof for treatment of immune system disorders
US10076546B2 (en) 2013-03-15 2018-09-18 Seres Therapeutics, Inc. Network-based microbial compositions and methods
US10258655B2 (en) 2013-11-25 2019-04-16 Seres Therapeutics, Inc. Synergistic bacterial compositions and methods of production and use thereof
CN110741972A (zh) * 2019-09-06 2020-02-04 江苏大学 面向林木病虫害生态防治的益鸟养殖装置及方法
EP3630800A4 (fr) * 2017-05-31 2021-03-24 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Vaccin contre l'entérite nécrotique chez la volaille
US10973861B2 (en) 2013-02-04 2021-04-13 Seres Therapeutics, Inc. Compositions and methods
US11701394B2 (en) 2017-08-14 2023-07-18 Seres Therapeutics, Inc. Compositions and methods for treating cholestatic disease
US12083151B2 (en) 2012-11-23 2024-09-10 Seres Therapeutics, Inc. Synergistic bacterial compositions and methods of production and use thereof
US12214002B2 (en) 2017-10-30 2025-02-04 Seres Therapeutics, Inc. Compositions and methods for treating antibiotic resistance
CN120384029A (zh) * 2025-06-27 2025-07-29 国药集团动物保健股份有限公司 一株猪源a型产气荚膜梭菌及其在疫苗制备中的应用

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