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WO2000005252A1 - Vaccin comportant un derive immunogene non toxique de la neurotoxine clostridium botulinum du type d - Google Patents

Vaccin comportant un derive immunogene non toxique de la neurotoxine clostridium botulinum du type d Download PDF

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Publication number
WO2000005252A1
WO2000005252A1 PCT/IB1999/001301 IB9901301W WO0005252A1 WO 2000005252 A1 WO2000005252 A1 WO 2000005252A1 IB 9901301 W IB9901301 W IB 9901301W WO 0005252 A1 WO0005252 A1 WO 0005252A1
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WIPO (PCT)
Prior art keywords
bont
botulinum type
derivative
immunogenic
fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB1999/001301
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English (en)
Inventor
Engela Elizabeth De Bruyn
Adriaan David Botha
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Agricultural Research Council
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Agricultural Research Council
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Agricultural Research Council filed Critical Agricultural Research Council
Priority to AU46412/99A priority Critical patent/AU4641299A/en
Publication of WO2000005252A1 publication Critical patent/WO2000005252A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to immunogenic derivatives of
  • Clostridium botulinum neurotoxin DNA encoding such derivatives
  • immunogenic botulinum neurotoxin derivatives vaccines for combating C.
  • botulinum neurotoxins methods for the preparation of immunogenic derivatives of C. botulinum neurotoxin and to methods for the preparation
  • Clostridium botulinum is a species of the large bacterial genus
  • Clostridium Bacteria belonging to this genus are spore-forming anaerobic
  • Gram positive bacilli The species C. botulinum can be sub-divided into types and the different types produce several toxins, e.g. such as types A,
  • Types C and D are generally pathogenic to cattle, sheep, pigs,
  • BoNT neurotoxin
  • Pathogenesis or poisoning usually results after the ingestion of carrion or decomposed material contaminated with BoNT
  • Clostridial neurotoxins are one of the
  • the target sites of the BoNT are the cholinergic nerve endings of neurons in the animal.
  • the BoNT affects
  • BoNT is a protein with an approximate molecular
  • BoNT may be viewed as being composed of three
  • Botulism vacines currently available are generally
  • botulinium vacine production strains low levels of toxin production
  • Non-toxic is defined as the intra peritoneal injection of 0.2 ml of the
  • the derivative or immunogenic fragment thereof may carry a
  • sequence ID No. 1 or a fragment, analog or derivative thereof.
  • sequence ID No. 2 amino acids 1 to 399 or a fragment, analog or
  • the invention provides a non-toxic immunogenic
  • amino acid sequence which is at least 75%, more preferably
  • immunogenic fragment thereof is understood to be a fragment that, although not comprising the full length amino acid sequence of the
  • derivative of the type D neurotoxin still comprises regions of the derivative
  • a mutation is understood to be a change in the nucleic acid
  • the mutation may be a replacement
  • mutation can e.g. be such that one or more amino acids of the type D
  • BoNT are replaced by other amino acids, with different characteristics.
  • the invention also relates to derivatives of type D BoNT
  • Non-toxic immunogenic derivatives of type D BoNT according to the invention may be made by introducing mutations in the gene encoding the
  • the mutated DNA fragments may then be cloned in a nucleotide sequence, such as a suitable expression plasmid and
  • nucleotide sequence comprising a mutated or recombinant DNA fragment
  • the invention provides a nucleic acid characterised by
  • sequence ID No. 2 (nucleotides 58 to 1 254), or a fragment of said
  • the invention provides a nucleic acid comprising a
  • nucleotide sequence which encodes a non-toxic immunogenic derivative
  • nucleotide sequence being selected from the group consisting of sequence
  • sequence ID No. 1 sequence ID No. 2 (nucleotides 58 to 1 254), and a fragment of
  • the invention comprises a nucleotide sequence
  • polynucleotide having at least 75%, preferably 85%, identity
  • polynucleotide encoding a non-toxic immunogenic derivative of C.
  • the invention comprises a nucleotide sequence
  • polynucleotide having at least 75%, preferably 85%, identity
  • polynucleotide encoding a genetically non-toxic immunogenic
  • the polynucleotide may be DNA or RNA.
  • the polynucleotide may be DNA or RNA.
  • polynucleotide is plasmid DNA.
  • a suitable bacterial expression system for expressing the non-toxic immunogenic derivatives of type D BoNT according to the invention are
  • Gram positive bacteria such as Bacillus brevis, Bacillus subtilus or Gram negative bacteria such as Escherichia coli. It is also envisaged that other members of the plant.
  • expression systems may be used for expressing non-toxic immunogenic
  • suitable yeast expression system for example Pichia pastoris.
  • positive bacterial expression system comprising a nucleotide sequence according to the invention encoding a non-toxic immunogenic derivative
  • negative bacterial expression system comprising a nucleotide sequence according to the invention encoding a non-toxic immunogenic derivative
  • said vaccine comprising a non-toxic immunogenic derivative
  • Such vaccines may be made by admixing an immunologically sufficient amount of a non-toxic immunogenic derivative or derivatives of
  • Still another embodiment of the invention relates to vaccines for protection against botulinum caused by C. botulinum type D BoNT that
  • BoNT according to the invention or an immunogenic fragment thereof
  • one or more compounds having adjuvant activity may be any one or more compounds having adjuvant activity.
  • one or more compounds having adjuvant activity may be any one or more compounds having adjuvant activity.
  • the vaccine e.g. Alhydrogel, Alum or Saponin.
  • the vaccine may be administered to all hosts sensitive to C.
  • botulinum type D BoNT such as cattle, mules, sheep, goats, horses, birds, humans, etc.
  • dosage is administered once and may be repeated after a month or two.
  • Non-toxic immunogenic derivatives of C. botulinum type D BoNT are nucleotide sequence according to the invention.
  • Non-toxic immunogenic derivatives of C. botulinum type D BoNT are nucleotide sequence according to the invention.
  • the non-toxic immunogenic amino acids of the polypeptide or protein are acids of the polypeptide or protein.
  • the non-toxic immunogenic amino acids of the polypeptide or protein are acids of the polypeptide or protein.
  • derivatives may also be prepared by introducing mutations in the gene or
  • non-toxic immunogenic derivatives may be prepared
  • recombinant DNA or fragments thereof may then be cloned in a nucleotide
  • sequence such as a suitable expression vector
  • D BoNT poisoning which method comprises admixing a non-toxic compound
  • the vaccine may be administered by a variety of suitable routes
  • sequence of sequence ID No. 2 amino acids 1 to 399.
  • the invention provides a vector which includes a
  • nucleotide sequence according to the invention which encodes a non-toxic
  • the invention also extends to a host cell genetically engineered with
  • the host cell may be any suitable host cell as a yeast or bacterium.
  • the host cell may be B. subtilus, E. coli. or B. brevis, e.g. B.
  • the vector may be any suitable vector known in the art, such as a suitable plasmid.
  • the invention also extends to a method for preparing a non-toxic vector
  • nucleotide sequence encoding a non-toxic amino acid sequence
  • the invention also provides a method for preparing a non-toxic compound
  • the nucleotide sequence may be a polynucleotide of sequence ID
  • sequence ID No. 2 nucleotides 58 to 1 254.
  • the nucleotide sequence may be expressed under the control of its
  • the Gram positive bacterium may be selected from the group consisting of Bacillus brevis and Bacillus subtilus.
  • the Gram negative bacterium may be E. coli. According to yet another aspect of the invention, there is provided
  • substance or composition for use in a method of vaccinating an animal against C. botulinum type D BoNT, said substance or composition
  • Figure 1 shows the structure of a suitable expression-secretion
  • the closed bar indicates the 5 region of the MWP gene containing
  • MCS multiple cloning site
  • SD 1 and SD2 are the ribosome-binding sites located
  • Figure 2 is a genetic map of a gene in accordance with the invention.
  • Figure 3 is a genetic map indicating sequencing coverage
  • sequence primer location includes a list of primers used and their
  • Figure 4 is an analysis of recombinant plasmids through comparative
  • Lane 1 represents DNA molecular weight
  • Lanes 2 and 4 represent plasmid extractions from B.
  • the arrows indicating the DNA fragments of 1 207 bp, represent the genes according to the invention as digested from
  • Figure 5 is a PAGE electrophoretogram of B. brevis culture supernatant demonstrating secretion of recombinant heterologous COOH-
  • Lane 1 represents the protein profile of the culture supernatant of a B. brevis strain transformed with PNU 21 1 without the gene fragment
  • Figure 6 is a Western blot analysis of a PAGE protein profile of B.
  • Lane 1 represents the
  • Lane 4 represents the protein
  • Figure 7 is a growth curve of a fermentation culture of B. brevis
  • Sequence ID No. 1 is a nucleotide sequence of a synthetic gene
  • Sequence ID No. 2 shows a nucleic acid sequence of a gene
  • sequence ID No. 2 nucleotide and amino acid sequences
  • a novel nucleotide sequence or gene according to the invention encoding a non-toxic immunogenic derivative of C. botulinum type D toxin
  • nucleotide sequence or gene was redesigned to have the optimal codons
  • Bacterial strains and vectors Bacterial strains and vectors.
  • Bacillus brevis strain Bacillus brevis strain
  • Plasmid PNU 21 1 was obtained from S Udaka, Department of Applied Biological Sciences, Nagoya University, Japan..
  • Clones were selected by growth on LB agar plates supplemented with 50
  • Plasmid extractions were performed according to the method of Reference 1 9. Plasmids were
  • ECL Western blotting detection reagents and ECL protein molecular weight markers were obtained from Amersham International.
  • DAB substrate kit was obtained from Zymed Laboratories Inc. Difco
  • the gene according to the invention was designed
  • Hind III were included for cloning purposes.
  • the gene was synthesized and cloned into a suitable vector
  • Antiserum used for immunoblots was prepared by hyper-immunization of horses against C. botulinum serotype D neurotoxin (OBP). Antibodies
  • Bacillus brevis recombinants Bacillus brevis recombinants.
  • Clones were selected by growth on LB agar plates
  • Plasmids were digested with restriction enzymes Pst I and Hind III and analyzed on a 1 % agarose gel to confirm the presence of the gene
  • Bacillus brevis strain 47- 5Q (JMC no. 8970) was transformed with PNU 21 1 according to the instructions of the manufacturer.
  • Clones were selected by growth on T2U plates
  • the gene fragment insert were screened by immuno colony blot analysis.
  • lysing buffer 50 mM Glucose
  • botulinum antiserum type D 1000 units/ml was diluted 1 : 200 in TBS ( 1 % milk powder) and used
  • DAB tetrahydrochloride
  • Modified PY medium 100 ml was inoculated
  • PAGE electrophoresis PAGE electrophoresis (Bio-Rad Mini Protean II) on a 5% gel were
  • destain solution 300 ml ethanol, 80 ml glacial acetic acid, 620
  • antiserum type D 1000 units/ml was, after adsorption, diluted 1 :200 and
  • HRP horseradish peroxidase conjugated Protein G was diluted 1 : 3000 and used as secondary antibody.
  • LumiAnalyst Image Analysis software program (Boehringer Mannheim).
  • T2U medium 100 ml was
  • the culture supernatant was mixed in a 1 : 1 ratio with 50 % Aluminium
  • OBP Hydroxide adjuvant
  • mice weighing 1 8 g to 20 g. The mice were each injected subcutaneously with 0.2 ml. After 21 days, two groups of 5 immunized mice and 1 0 control mice each, were challenged with OBP C. botulinum type D standard
  • mice One group of immunized mice and one group control mice
  • mice were injected intraperitoneally into each of the mice with 0. 2 ml of toxin diluted in saline to give a final concentration of 0.05 U.
  • the other group
  • mice were observed for 24 h for survival, signs of botulism or death.
  • the size of the gene is 1 207 bp and the resulting protein
  • the %GC content is 34.6 %, compared to the %GC of B. brevis of 42.7 to 47 % (Reference 27) and to that of C. botulinum of
  • Bacillus brevis recombinants Bacillus brevis recombinants.
  • transformants (B. brevis 1 1 5) was cultured on modified PY-medium and
  • production medium used as a 20 % inoculum for 10 liter medium in the
  • Figure 7 represents the growth curve of a typical fermenter run.
  • the fermenter was set at maximum value for oxygen saturation.
  • polypeptide according to the invention in the formulation of a vaccine
  • mice used to vaccinate mice. Mice challenge studies with 0.03 U and 0.05 U toxin, 21 days after vaccination, resulted in death of all the control mice
  • botulinum type D BoNT botulinum type D BoNT. This method of vaccine production would involve fermenter technology, a unique method for botulism type D vaccine
  • the vaccines can be produced or manufactured relatively quickly and the
  • vaccine is non-toxic perse. Since the product of the fermentation process
  • Kiyatkin, N., Maksymowych, A. B. and Simpson, L. L. ( 1 997) .
  • serotype B expressed in the methylotrophic yeast Pichia pastoris.
  • Toxicon Vol.36, p. 1539-1548.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un dérivé immunogène non toxique de la neurotoxine C. botulinum du type D ou son fragment immunogène, ledit dérivé ou fragment comportant au moins une mutation dans sa séquence d'acides aminés que l'on ne retrouve pas dans les neurotoxines D du type sauvage. L'invention concerne aussi une séquence de nucléotides comprenant un fragment recombinant d'ADN, caractérisée en ce que ce fragment d'ADN code un dérivé immunogène non toxique de la neurotoxine C. botulinum du type D BoNT ou son fragment immunogène.
PCT/IB1999/001301 1998-07-22 1999-07-20 Vaccin comportant un derive immunogene non toxique de la neurotoxine clostridium botulinum du type d Ceased WO2000005252A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU46412/99A AU4641299A (en) 1998-07-22 1999-07-20 Vaccine comprising a non-toxic immunogenic derivative of (clostridium botulinum)type d neurotoxin

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ZA986538 1998-07-22
ZA98/6538 1998-07-22

Publications (1)

Publication Number Publication Date
WO2000005252A1 true WO2000005252A1 (fr) 2000-02-03

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008268A3 (fr) * 2000-07-21 2003-02-20 Allergan Inc Motif a base de leucine et neurotoxines clostridiales
US7491799B2 (en) 2000-07-21 2009-02-17 Allergan, Inc. Modified botulinum neurotoxins
US7691983B2 (en) 2000-07-21 2010-04-06 Allergan, Inc. Chimera botulinum toxin type E
US8445650B2 (en) 2007-09-25 2013-05-21 Thomas Jefferson University Mutant botulinum neurotoxin serotype A polypeptide and uses thereof
WO2019126542A1 (fr) * 2017-12-20 2019-06-27 Allergan, Inc. Polypeptides du domaine de liaison cellulaire de toxine botulique et procédés d'utilisation pour des traitements de troubles associés à la fibrose
WO2019152380A1 (fr) * 2018-01-30 2019-08-08 Children's Medical Center Corporation Production de neurotoxines de botulinum à l'aide de systèmes bacillus

Citations (4)

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US4292307A (en) * 1977-09-30 1981-09-29 Zemlyakova Valentina P Vaccine and method for prophylaxis and treatment of clostridioses of animals and poultry
WO1996041881A1 (fr) * 1995-06-12 1996-12-27 Microbiological Research Authority Toxine botulinique de type f et son utilisation
WO1998007864A1 (fr) * 1996-08-23 1998-02-26 Microbiological Research Authority Camr (Centre For Applied Microbiology & Research) Fragments de toxines recombines
WO1998008540A1 (fr) * 1996-08-28 1998-03-05 Ophidian Pharmaceuticals, Inc. VACCIN POLYVALENT CONTRE LA NEUROTOXINE DU $i(CLOSTRIDIUM BOTILINUM)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4292307A (en) * 1977-09-30 1981-09-29 Zemlyakova Valentina P Vaccine and method for prophylaxis and treatment of clostridioses of animals and poultry
WO1996041881A1 (fr) * 1995-06-12 1996-12-27 Microbiological Research Authority Toxine botulinique de type f et son utilisation
WO1998007864A1 (fr) * 1996-08-23 1998-02-26 Microbiological Research Authority Camr (Centre For Applied Microbiology & Research) Fragments de toxines recombines
WO1998008540A1 (fr) * 1996-08-28 1998-03-05 Ophidian Pharmaceuticals, Inc. VACCIN POLYVALENT CONTRE LA NEUROTOXINE DU $i(CLOSTRIDIUM BOTILINUM)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MORIISHI K ET AL: "Mosaic structures of neurotoxins produced from Clostridium botulinum types C and D organisms", BIOCHIMICA BIOPHYSICAL ACTA, vol. 1307, 1996, pages 123 - 6, XP000857076 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7705124B2 (en) 2000-07-21 2010-04-27 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
US7723480B2 (en) 2000-07-21 2010-05-25 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
US7393925B2 (en) 2000-07-21 2008-07-01 Allergan, Inc. Leucine-based motif and Clostridial neurotoxins
CN100457777C (zh) * 2000-07-21 2009-02-04 阿勒根公司 基于亮氨酸的基序和梭菌神经毒素
US7491799B2 (en) 2000-07-21 2009-02-17 Allergan, Inc. Modified botulinum neurotoxins
US7534863B2 (en) * 2000-07-21 2009-05-19 Allergan, Inc. Leucine-based motifs and enhanced biological persistence of clostridial neurotoxins
US7671177B2 (en) 2000-07-21 2010-03-02 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
US7691974B2 (en) 2000-07-21 2010-04-06 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
US7691983B2 (en) 2000-07-21 2010-04-06 Allergan, Inc. Chimera botulinum toxin type E
US7705125B2 (en) 2000-07-21 2010-04-27 Allergan, Inc. Leucine-based motif and Clostridial neurotoxins
EP1849801A1 (fr) * 2000-07-21 2007-10-31 Allergan Sales, Inc. Motif à base de tyrosine et neurotoxines clostridiales
US8008465B2 (en) 2000-07-21 2011-08-30 Allergan, Inc. Nucleic acids encoding chimera botulinum toxin type E
WO2002008268A3 (fr) * 2000-07-21 2003-02-20 Allergan Inc Motif a base de leucine et neurotoxines clostridiales
US8017741B2 (en) 2000-07-21 2011-09-13 Ester Fernandez-Salas Chimera botulinum toxin type E
US8206723B2 (en) 2000-07-21 2012-06-26 Allergan, Inc. Leucine-based motif and clostridial neurotoxins
US8445650B2 (en) 2007-09-25 2013-05-21 Thomas Jefferson University Mutant botulinum neurotoxin serotype A polypeptide and uses thereof
WO2019126542A1 (fr) * 2017-12-20 2019-06-27 Allergan, Inc. Polypeptides du domaine de liaison cellulaire de toxine botulique et procédés d'utilisation pour des traitements de troubles associés à la fibrose
CN112351991A (zh) * 2017-12-20 2021-02-09 阿勒根公司 肉毒杆菌毒素细胞结合结构域多肽及其用于治疗纤维化相关障碍的方法
WO2019152380A1 (fr) * 2018-01-30 2019-08-08 Children's Medical Center Corporation Production de neurotoxines de botulinum à l'aide de systèmes bacillus
CN111971294A (zh) * 2018-01-30 2020-11-20 儿童医学中心公司 使用芽孢杆菌系统产生肉毒神经毒素
US12529046B2 (en) 2018-01-30 2026-01-20 Children's Medical Center Corporation Production of botulinum neurotoxins using Bacillus systems

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