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WO1992003553A1 - Vaccin destine a lutter contre la maladie de pietin - Google Patents

Vaccin destine a lutter contre la maladie de pietin Download PDF

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Publication number
WO1992003553A1
WO1992003553A1 PCT/AU1991/000366 AU9100366W WO9203553A1 WO 1992003553 A1 WO1992003553 A1 WO 1992003553A1 AU 9100366 W AU9100366 W AU 9100366W WO 9203553 A1 WO9203553 A1 WO 9203553A1
Authority
WO
WIPO (PCT)
Prior art keywords
nodosus
vaccine
antigen
proteins
omp
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/AU1991/000366
Other languages
English (en)
Inventor
Eric Keith Moses
Robert Trygve Good
Christopher John Langford
Julian Ian Rood
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.)
Agriculture Victoria Services Pty Ltd
Original Assignee
Daratech Pty Ltd
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 Daratech Pty Ltd filed Critical Daratech Pty Ltd
Publication of WO1992003553A1 publication Critical patent/WO1992003553A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1257—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Bacteridaceae (F)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

  • This invention relates to veterinary vaccines and more particularly to recombinant vaccines for the treatment or prevention of footrot in ruminants caused by Dichelobacter nodosus previously referred to as Bacteroides nodosus.
  • the invention also relates to a method of treating ruminants to prevent footrot caused by D.nodosus.
  • the invention provides aids for diagnosing virulent, benign and intermediate strains of footrot.
  • Ruminant footrot is an economically important disease particularly in sheep.
  • the disease is characterised by separation of the hoof from the underlying epidermal tissues.
  • Various treatments have been proposed including foot bathing with formalin and zinc sulphate solutions as well as the use of antibiotics. More recently preventative treatments using vaccines have been proposed.
  • the present invention provides in one form a veterinary vaccine wherein the antigen is genetically engineered and wherein the antigen is substantially homologous to secreted outer membrane proteins of D.nodosus.
  • a vaccine containing one or more of these genetically engineered antigens or closely related molecules is able to stimulate cross-protective immunity in sheep to infection with D.nodosus strains from various serogroups.
  • a veterinary vaccine comprising protein antigens of D.nodosus which are characterised by being members of a family of closely related molecules which are synthesised as 70-75 kDa proteins which are major components of the outer membrane of D.nodosus.
  • a diagnostic test based on the nucleotide sequences of the genes described herein which encode the family of outer membrane proteins.
  • the DNA from any strain of D.nodosus can be rapidly amplified using the polymerase chain reaction (PCR) technique (see R.K. Saiki et al., Science 239, 487-491 (1987).) from samples taken directly from the hoof of ruminants.
  • PCR polymerase chain reaction
  • This amplified DNA can then be specifically fragmented by digestion with any one of a number of restriction endonucleases to generate a discrete pattern of DNA molecules of varying sizes which can be visualised by a variety of standard procedures as shown Figure 14.
  • Each strain of D.nodosus produces a unique pattern (or fingerprint) which is recorded and stored in a computer data base.
  • the fingerprints of strains or isolates which share the same phenotypic characteristics can be grouped together. New isolates with the same fingerprints can be immediately assigned to one of the defined phenotypic groups. This technique provides a method for typing virulent, intermediate and benign strains of D.nodosus.
  • Figure 1 describes the linear restriction maps of cloned D.nodosus genomic DNA fragments. Arrows indicate the location of the coding region of the OMP genes on these DNA fragments. The size of the cloned DNA fragments is indicated by the scale at the bottom.
  • Figure 2 describes one arrangement of the E, B, H and C OMP genes in the D.nodosus genome. Also shown is the location of the promoter sequences (P), sequences encoding the hydrophobic leader peptide (hatched) and direction of transcription of each gene (arrows). The circles indicate some of the regions of genome which invert resulting in alternative arrangements of the OMP genes and to the altered expression of the OMP antigens on the surface of the D.nodosus organism.
  • P promoter sequences
  • H hydrophobic leader peptide
  • Figures 3-7 illustrate the entire nucleotide sequence of the 8.5kB DNA fragment described in Figure 2.
  • the coding regions for the OMP antigens are indicated and are located at the following positions. E (26 to 2146), B
  • hydrophobic leader sequence of the H and E genes is shown underlined.
  • the break points which are generated by the process of DNA inversion are indicated with an asterisk.
  • Figure 8 illustrates the multiple alignment of the deduced amino acid sequences encoded by the E, H, B and C OMP genes.
  • the alignment includes the full sequence of the B and C genes which are generated by the addition of the sequences at the NH 2 -terminal end of the sequences described in Figures 3-7 as a result of the DNA inversion process described in Figure 2.
  • Asterisks indicate positions at which the amino acid sequences are conserved in all four OMP proteins. It is noted that regions of homology flank regions of amino acid sequence which are not homologous.
  • Arrowed lines labelled oligo A and oligo C indicate the location of 18 and 16 nucleotide long sequences respectively which are identical in all four OMP genes from D.nodosus strain A198.
  • Figure 9 shows SDS-PAGE analysis of the recombinant B, E, H and C OMP proteins expressed in E. coli .
  • ROMP C/B is a hybrid antigen comprised of the first 448 amino acids of OMP C fused to the last 221 amino acids of OMP B.
  • ROMP 3'C is the last 355 amino acids of OMP C expressed as a fusion protein.
  • the DNA sequences encoding the E, B, C/B and H OMP antigens were cloned into the IPTG inducible expression vector PMMB66EH.
  • Figure 11 shows the reactivity of antibodies raised against rOMP C/B antigen.
  • Fimbriae and proteases purified from D.nodosus strain A198 and D.nodosus A198 whole cells were solubilized by boiling in SDS and subjected to SDS- PAGE. The gel was transferred to nitrocellulose membrane and blotted with the rOMP C/B antibodies.
  • the antibodies react strongly with a 75-80 kDa protein which co-purifies with the fimbriae (arrowed in lane 1) and 75-80 kDa proteins from whole cells (arrowed in lane 2).
  • FIG. 12 shows the Coomassie blue protein staining profiles and Western blotting profiles of cell envelope proteins from virulent and benign D.nodosus strains within eight serogroups.
  • Cell envelope proteins were subjected to SDS-PAGE on 10-17.5% linear gradient gels and stained with Coomassie blue R250.
  • a duplicate gel was subjected to Western transfer and blotting with rOMP C/B antibodies.
  • the antibodies cross-react with major cell envelope proteins of D.nodosus irrespective of serogroup.
  • Figure 13 shows PCR analysis of OMP genes from various D.nodosus strains.
  • oligo A and oligo C Two oligonucleotide primers oligo A and oligo C that are based on conserved nucleotide sequences found in all four OMP genes from D.nodosus strain A198 (1168-1186, 3193-3210 reverse and complement, 5511-5528, 7525-7542 reverse and complement, 1676-1691 reverse and complement, 2688-2703, 6012-6027 reverse and complement and 7044-7059 in Figures 3-7), were used to amplify, by PCR, intervening DNA sequences from the genomic DNA of 16 different D.nodosus strains representative of 9 serogroups. The observation that DNA fragments could be synthesized from all strains tested indicates that the conserved sequences defined by oligo A and oligo C in strain A198 are also conserved in other strains of D.nodosus.
  • Figure 14 shows DNA fingerprints from various D.nodosus strains. These fingerprints were generated by PCR with oligonucleotide primers oligo A and oligo C followed by digestion with the restriction endonuclease Sau3A and agarose gel electrophoresis. A unique DNA fingerprint was generated from each of the D.nodosus strains tested.
  • Figure 15 descibes the homology between the predicted amino acid sequence of gene E ( Figure 2) and two ttryptic peptides derived from the 35-40kDa fraction of proteins isolated from the supernatant of a D.nodosus strain 198 culture.
  • the antigen is preferably a polypeptide which includes the whole part or analogues of the amino acid sequences set out in Figures 3-8 or an antigenic fragment thereof prepared by recombinant techniques.
  • part or analogues of the amino acid sequences we mean molecules which have a high homology with those regions of the sequences which are conserved between the OMP proteins. These are indicated by asterisks in Figures 8.
  • high homology we mean generally at least 80%.
  • closely related molecules means molecules which have a high level of homology in the regions where the sequences are conserved between the outer membrane proteins of D.nodosus.
  • the vaccine preferably also comprises a pharmaceutically acceptable carrier or diluent, and more preferably an adjuvant. Suitable carriers, diluents and adjuvants are known to those skilled in the art.
  • the invention also provides in an alternative form a method for immunising ruminants against D.nodosus infection.
  • This method comprises administering to the ruminant an effective amount of antigen as defined above or antigenic fragment(s) thereof either alone or in combination.
  • the invention provides an antigen(s) of the present invention or an antigenic fragment(s) thereof in a live recombinant expression and delivery system such as a recombinant virus, bacterium or parasite.
  • the antigenic molecules of the present invention are found in the outer membrane of D.nodosus.
  • the predicted size of the mature proteins, based on the nucleotide sequence data range from 70,120 to 74,779 Dalton.
  • Peptide sequence data determined directly from peptides derived from this 35-40 kDa fraction correlate with the predicted amino acid sequence of the genes described in this invention.
  • Other peptide sequences from this fraction correlate with the predicted amino acid sequence of the novel basic protease sequence described in Australian Patent Application No. 18030/88.
  • This invention also describes the expression of recombinant OMP proteins (rOMPS) in E. coli and their subsequent use as vaccine antigens.
  • rOMPS recombinant OMP proteins
  • Table 1 shows the results of a pen trial experiment designed to test the efficacy of the cloned antigens as a vaccine.
  • E, B and H genes were cloned into the IPTG inducible expression vector pMMB66EH in order to express the antigens in E. coli .
  • a hybrid gene encoding 448 amino acids from the NH 2 -terminus of the OMP C protein fused to the region encoding 221 amino acids from the COOH terminus of the OMP B protein was also constructed in pMMB66EH.
  • the region encoding 335 amino acids from COOH-terminus of the OMP C gene was cloned into pGEX-3X to express a fusion protein with Schistosona iaponicum glutathione-S-transferase.
  • the E. coli were grown in bulk liquid cultures and harvested by centrifugation. The E. coli were lysed with the detergent SDS and the proteins solubilized by boiling prior to fractionation by SDS-PAGE. Gel slices containing the recombinant B, C and E proteins were extracted with SDS and the solubilized proteins were then precipitated by methanol or acetone. The precipitate

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Vaccin vétérinaire dans lequel l'antigène est un polypeptide recombinant produit par la technique de génie génétique possédant une séquence d'acides aminés qui est homologue à des protéines de membranes externes sécrétées de D. nodosus.
PCT/AU1991/000366 1990-08-17 1991-08-15 Vaccin destine a lutter contre la maladie de pietin Ceased WO1992003553A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
AUPK1816 1990-08-17
AUPK181690 1990-08-17
AUPK2653 1990-10-05
AUPK265390 1990-10-05
AUPK5557 1991-04-11
AUPK555791 1991-04-11

Publications (1)

Publication Number Publication Date
WO1992003553A1 true WO1992003553A1 (fr) 1992-03-05

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ID=27157577

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1991/000366 Ceased WO1992003553A1 (fr) 1990-08-17 1991-08-15 Vaccin destine a lutter contre la maladie de pietin

Country Status (1)

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WO (1) WO1992003553A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2105980A1 (es) * 1995-11-13 1997-10-16 Univ Extremadura Una vacuna para el pedero, procedimiento para su preparacion y aplicaciones.
WO1999012564A1 (fr) * 1997-09-08 1999-03-18 University Technologies International, Inc. Traitement et prevention du panaris des bovins
US6241992B1 (en) 1997-09-08 2001-06-05 University Technologies International, Inc. Bovine footrot treatment and prevention
US6667040B2 (en) 1997-09-08 2003-12-23 University Technologies International, Inc. Bovine footrot treatment and prevention
WO2008074079A1 (fr) * 2006-12-21 2008-06-26 Monash University Identification d'antigènes de vaccins candidats à partir de dichelobacter nodosus

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3497984A (en) * 1983-11-04 1985-05-16 Commonwealth Scientific And Industrial Research Organisation Antigenic preparations derived from bacteroides nodosus
AU5015485A (en) * 1984-10-31 1986-05-15 Commonwealth Scientific And Industrial Research Organisation Improved antigenic preparation
AU1704988A (en) * 1987-04-27 1988-12-02 Commonwealth Scientific And Industrial Research Organisation Peptide production by protein engineering
AU1803088A (en) * 1987-06-05 1989-01-04 Commonwealth Scientific And Industrial Research Organisation Treatment and diagnosis of footrot using the basic protease of b.nodosus.

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3497984A (en) * 1983-11-04 1985-05-16 Commonwealth Scientific And Industrial Research Organisation Antigenic preparations derived from bacteroides nodosus
AU5015485A (en) * 1984-10-31 1986-05-15 Commonwealth Scientific And Industrial Research Organisation Improved antigenic preparation
AU1704988A (en) * 1987-04-27 1988-12-02 Commonwealth Scientific And Industrial Research Organisation Peptide production by protein engineering
AU1803088A (en) * 1987-06-05 1989-01-04 Commonwealth Scientific And Industrial Research Organisation Treatment and diagnosis of footrot using the basic protease of b.nodosus.

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
INFECTION AND IMMUNITY, Vol. 56, No. 3, March 1988, pages 595-600, ELLEMAN T.C. and STEWART D.J., "Efficacy against footrot of a Bacteroides nodosus 265 (serogroup H) pilus vaccine expressed in Pseudomonas aeruginosa". *
INFECTION AND IMMUNITY, Volume 51, No. 1, January 1986, pages 187-192, ELLEMAN T.C. et al., "Expression of the Pilin Gene from Bacteroides nodosus in Escherichia coli". *
JOURNAL OF BACTERIOLOGY, Vol. 160, No. 2, November 1984, pages 748-754, ANDERSON B.J. et al., "Cloning and expression of Escherichia coli of the gene encoding the structural subunit of Bacteroides nodosus fimbriae". *
JOURNAL OF BACTERIOLOGY, Vol. 168, No. 2, November 1986, pages 574-580, ELLEMAN T.C. et al., "Expression of pili from Bacteroides nodosus in Pseudomonas aeruginosa". *
JOURNAL OF BACTERIOLOGY, Volume 169, No. 1, pages 33-41, MATTICK J.S. et al., "Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa". *
VETERINARY MICROBIOLOGY, Vol. 9, 1984, pages 453-466, EMERY D.L., "Reactivity of sheep immunised with individual outer membrane proteins of Bacteroides nodosus against heterologous bacterial strains". *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2105980A1 (es) * 1995-11-13 1997-10-16 Univ Extremadura Una vacuna para el pedero, procedimiento para su preparacion y aplicaciones.
WO1999012564A1 (fr) * 1997-09-08 1999-03-18 University Technologies International, Inc. Traitement et prevention du panaris des bovins
US6241992B1 (en) 1997-09-08 2001-06-05 University Technologies International, Inc. Bovine footrot treatment and prevention
US6667040B2 (en) 1997-09-08 2003-12-23 University Technologies International, Inc. Bovine footrot treatment and prevention
WO2008074079A1 (fr) * 2006-12-21 2008-06-26 Monash University Identification d'antigènes de vaccins candidats à partir de dichelobacter nodosus
EP2069394A4 (fr) * 2006-12-21 2011-11-02 Univ Monash Identification d'antigenes de vaccins candidats a partir de dichelobacter nodosus

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