WO2005056586A1 - Lawsonia intracellularis 26 kd subunit vaccine - Google Patents

Lawsonia intracellularis 26 kd subunit vaccine Download PDF

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WO2005056586A1
WO2005056586A1 PCT/EP2004/053342 EP2004053342W WO2005056586A1 WO 2005056586 A1 WO2005056586 A1 WO 2005056586A1 EP 2004053342 W EP2004053342 W EP 2004053342W WO 2005056586 A1 WO2005056586 A1 WO 2005056586A1
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protein
lawsonia intracellularis
nucleic acid
vaccine
fragment
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PCT/EP2004/053342
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English (en)
French (fr)
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Paul Vermeij
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Intervet International B.V
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Priority to US10/580,709 priority Critical patent/US20070212373A1/en
Priority to AU2004297018A priority patent/AU2004297018A1/en
Priority to CA002548750A priority patent/CA2548750A1/en
Priority to EP04820075A priority patent/EP1694698A1/en
Priority to JP2006543544A priority patent/JP2007537715A/ja
Priority to BRPI0417440-2A priority patent/BRPI0417440A/pt
Publication of WO2005056586A1 publication Critical patent/WO2005056586A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (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

Definitions

  • the present invention relates i.a. to nucleic acids encoding novel Lawsonia intracellularis proteins, to DNA fragments, recombinant DNA molecules and live recombinant carriers comprising these sequences, to host cells comprising such nucleic acids, DNA fragments, recombinant DNA molecules and live recombinant carriers, to proteins encoded by these nucleotide sequences and to their use for the manufacturing of vaccines, to vaccines for combating Lawsonia intracellularis infections and methods for the preparation thereof and to diagnostic tests for the detection of Lawsonia intracellularis antigens and for the detection of antibodies against Lawsonia intracellularis.
  • Porcine proliferative enteropathy has become an important disease of the modern pig industry world- wide. The disease affects 15% to 50% of the growing herds and up to 30% of the individual animals in established problem herds. Today annual economical losses have been estimated US$ 5-10 in extra feed and facility time costs per affected pig.
  • PPE is a group of chronic and acute conditions of widely differing clinical signs (death, pale and anaemic animals, watery, dark or bright red diarrhoea, depression, reduced appetite and reluctance to move, retarded growth and increased FCR).
  • the first a pathological change only visible at necropsy, is a thickening of the small intestine and colon mucosa.
  • the second is the occurrence of intracytoplasmatic small-curved bacteria in the enterocytes of the affected intestine. These bacteria have now been established as the etiological agent of PPE and have been name Lawsonia intracellularis.
  • Lawsonia intracellularis has been found to affect a large group of animals including monkeys, rabbits, ferrets, hamsters, fox, horses, and other animals as diverse as ostrich and emoe.
  • Lawsonia intracellularis is a gram-negative, flagellated bacterium that multiplies in eukaryotic enterocytes only and no cell-free culture has been described.
  • Lawsonia intracellularis In order to persist and multiply in the cell Lawsonia intracellularis must penetrate dividing crypt cells. The bacterium associates with the cell membrane and quickly enters the enterocyte via an entry vacuole. This then rapidly breaks down (within 3 hours) and the bacteria flourish and multiply freely in the cytoplasm. The mechanisms by which the bacteria cause infected cells to fail to mature, continue to undergo mitosis and form hypoplastic crypt cells is not yet understood.
  • Lawsonia intracellularis infection, treatment and control of the disease has been hampered by the fact that Lawsonia intracellularis can not be cultivated in cell-free media. Although there are reports of successful co-culturing
  • Lawsonia intracellularis in rat enterocytes this has not lead to the development of inactivated vaccines for combating Lawsonia intracellularis, although there clearly is a need for such vaccines.
  • Lawsonia intracellularis produces a novel protein that is capable of inducing protective immunity against Lawsonia intracellularis.
  • the novel protein will be referred to as the 26 kD protein.
  • the amino acid sequence of the novel protein is presented in sequence identifier SEQ ID NO: 2.
  • the gene encoding this protein has been sequenced and its nucleic acid sequence is shown in sequence identifier SEQ ID NO: 1.
  • the gene will also be referred to in the Examples as "gene 5608".
  • nucleic acid sequences can encode one and the same protein. This phenomenon is commonly known as wobble in the second and especially the third base of each triplet encoding an amino acid. This phenomenon can result in a heterology of about 30% for two nucleic acid sequences still encoding the same protein. Therefore, two nucleic acid sequences having a sequence homology of about 70 % can still encode one and the same protein.
  • one embodiment relates to nucleic acids encoding a Lawsonia intracellularis protein and to parts of that nucleic acid that encode an immunogenic fragment of that protein, wherein those nucleic acids or parts thereof have a level of homology with the nucleic acid of which the sequence is given in SEQ ID NO: 1 of at least 90 %.
  • the nucleic acid encoding this Lawsonia intracellularis protein or the part of said nucleic acid has at least 92 %, preferably 94 %, more preferably 95 % and even more preferably 96% homology with the nucleic acid having the sequence given in SEQ ID NO: 1. Even more preferred is a homology level of 98 % or even 100 %.
  • the level of nucleotide homology can be determined with the computer program "BLAST 2 SEQUENCES” by selecting sub-program: “BLASTN” that can be found at www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.htinl.
  • Another approach for deciding if a certain nucleic acid is or is not a nucleic acid according to the invention relates to the question if that certain nucleic acid does hybridise under stringent conditions to nucleic acids having the nucleotide sequence as depicted in SEQ ID NO: 1.
  • nucleic acid hybridises under stringent conditions to the nucleotide sequence as depicted in SEQ ED NO: 1, it is considered to be a nucleic acid according to the invention.
  • Tm [81.5°C + 16.6(log M) + 0.41(%GC) - 0.61(%formamide) - 500 L] - l°C/l%mismatch
  • M molarity of monovalent cations
  • %GC percentage of guanosine and cytosine nucleotides in the DNA
  • L is the length of the hybrid in base pairs.
  • Stringent conditions are those conditions under which nucleic acids or fragments thereof still hybridise, if they have a mismatch of 10% at the most, to the nucleic acid having the sequence depicted in SEQ ED NO: 1.
  • the invention relates to DNA fragments comprising a nucleic acid according to the invention.
  • DNA fragments can e.g. be plasmids, into which a nucleic acid according to the invention is cloned.
  • DNA fragments are e.g. useful for enhancing the amount of DNA for use as a primer, as described below.
  • nucleic acid An essential requirement for the expression of the nucleic acid is an adequate promoter functionally linked to the nucleic acid, so that the nucleic acid is under the control of the promoter. It is obvious to those skilled in the art that the choice of a promoter extends to any eukaryotic, prokaryotic or viral promoter capable of directing gene transcription in cells used as host cells for protein expression. Therefore, an even more preferred form of this embodiment relates to a recombinant
  • DNA molecule comprising a DNA fragment or a nucleic acid according to the invention that is placed under the control of a functionally linked promoter.
  • a functionally linked promoter This can be acompli shed by means of e.g. standard molecular biology techniques. (Sambrook, J. and Russell, D.W., Molecular cloning: a laboratory manual, 2001. ISBN 0-87969-577-3).
  • Functionally linked promoters are promoters that are capable of controlling the transcription of the nucleic acids to which they are linked.
  • Such a promoter can be a Lawsonia promoter e.g. the promoter involved in in vivo expression of the gene encoding the 26 kD gene, provided that that promoter is functional in the cell used for expression.
  • useful expression control sequences which maybe used include the Tip promoter and operator (Goeddel, et al., Nucl. Acids Res., 8, 4057, 1980); the lac promoter and operator (Chang, et al., Nature, 275, 615, 1978); the outer membrane protein promoter (Nakamura, K. and Inouge, M., EMBO J., 1, 771-775, 1982); the bacteriophage lambda promoters and operators (Remaut, E. et al., Nucl. Acids Res., 11, 4677-4688, 1983); the ⁇ -amylase (B. subtilis) promoter and operator, termination sequences and other expression enhancement and control sequences compatible with the selected host cell.
  • Tip promoter and operator Goeddel, et al., Nucl. Acids Res., 8, 4057, 1980
  • the lac promoter and operator Chang, et al., Nature, 275, 615, 1978
  • useful expression control sequences include, e.g., ⁇ -mating factor.
  • the polyhedrin or plO promoters of baculoviruses can be used (Smith, G.E. et al., Mol. Cell. Biol. 3, 2156-65, 1983).
  • useful expression control sequences include the SV-40 promoter (Berman, P.W. et al., Science, 222, 524-527, 1983) or the metallothionein promoter (Brinster, R.L., Nature, 296, 39-42, 1982) or a heat shock promoter (Voellmy et al., Proc. Natl. Acad. Sci. USA, 82, 4949-53, 1985).
  • Bacterial, yeast, fungal, insect and mammalian cell expression systems are very frequently used systems. Such systems are well-known in the art and generally available, e.g. commercially through Invitrogen ( " www.invitrogen.com). Novagen (www.merckbiosciences.de) or Clontech Laboratories, Inc. 4030 Fabian Way, Palo Alto, California 94303-4607, USA. Next to these expression systems, parasite-based expression systems are very attractive expression systems. Such systems are e.g. described in the French Patent Application with Publication number 2 714 074, and in US NTIS Publication No US 08/043109 (Hoffman, S. and Rogers, W.: Public. Date 1 December 1993).
  • LRCs Recombinant Carriers
  • Such carriers are e.g. bacteria and viruses.
  • LRCs are micro-organisms or viruses in which additional genetic mformation, in this case a nucleic acid encoding the 26 kD protein or an immunogenic fragment thereof according to the invention has been cloned.
  • LRCs Animals infected with such LRCs will produce an immunogenic response not only against the immunogens of the carrier, but also against the immunogenic parts of the protein(s) for which the genetic code is additionally cloned into the LRC, e.g. the 26 kD protein.
  • the genetic code is additionally cloned into the LRC, e.g. the 26 kD protein.
  • attenuated Salmonella strains known in the art can attractively be used.
  • Live recombinant carrier parasites have i.a. been described by Vermeulen, A. N. (Int. Journ. Parasitol. 28: 1121-1130 (1998))
  • LRC viruses may be used as a way of transporting the nucleic acid into a target cell.
  • Live recombinant carrier viruses are also called vector viruses.
  • Viruses often used as vectors are Vaccinia viruses (Panicali-et al; Proc. Natl. Acad. Sci. USA, 79: 4927 (1982), Herpesviruses (E.P.A. 0473210A2), and Retroviruses (Valerio, D. et al; in Baum, S.J., Dicke, K. A., Lotzova, E. and Pluznik, D.H. (Eds.), Experimental Haematology today - 1988. Springer Verlag, New York: pp. 92-99 (1989)).
  • the technique of in vivo homologous recombination can be used to introduce a recombinant nucleic acid into the genome of a bacterium, parasite or virus of choice, capable of inducing expression of the inserted nucleic acid according to the invention in the host animal.
  • a host cell comprising a nucleic acid encoding a protein according to the invention, a DNA fragment comprising such a nucleic acid or a recombinant DNA molecule comprising such a nucleic acid under the control of a functionally linked promoter.
  • This form also relates to a host cell contaming a live recombinant carrier containing a nucleic acid molecule encoding a 26 kD protein or a fragment thereof according to the invention.
  • a host cell may be a cell of bacterial origin, e.g.
  • Escherichia coli, Bacillus subtilis and Lactobacillus species in combination with bacteria-based plasmids as pBR322, or bacterial expression vectors as pGEX, or with bacteriophages.
  • the host cell may also be of eukaryotic origin, e.g. yeast-cells in combination with yeast-specific vector molecules, or higher eukaryotic cells like insect cells (Luckow et al; Bio-technology 6: 47-55 (1988)) in combination with vectors or recombinant baculoviruses, plant cells in combination with e.g. Ti-plasmid based vectors or plant viral vectors (Barton, K.A. et al; Cell 32: 1033 (1983), mammalian cells like Hela cells, Chinese Hamster Ovary cells (CHO) or Crandell Feline Kidney-cells, also with appropriate vectors or recombinant viruses.
  • yeast-cells in combination with yeast-specific vector molecules
  • Another embodiment of the invention relates to the novel proteins and to irnmunogenic fragments thereof according to the invention.
  • One form of this embodiment relates i.a. to Lawsonia intracellularis proteins that have an amino acid sequence that is at least 90 % homologous to the amino acid sequence as depicted in SEQ ID NO: 2 and to immunogenic fragments of said protein.
  • the embodiment relates to such Lawsonia intracellularis proteins that have a sequence homology of at least 92 %, preferably 94 %, more preferably 96 % homology to the amino acid sequence as depicted in SEQ ED NO: 2 and to immunogenic fragments of such proteins. Even more preferred is a homology level of 98 % or even 100 %.
  • the level of protein homology can be determined with the computer program "BLAST 2 SEQUENCES” by selecting sub-program: “BLASTP”, that can be found at www.ncbi.nlm.nih.gov blast bl2seq/bl2.html.
  • Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M.D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3).
  • Other amino acid substitutions include Asp/Glu, Thr/Ser, Ala Gly, Ala/Thr, Ser/Asn, Ala/Val, Thr/Phe, Ala Pro, Lys/Arg, Leu He, Leu Val and Ala/Glu.
  • immunogenic fragment is understood to be a fragment of the full-length protein that still has retained its capability to induce an immune response in the host, i.e. comprises a
  • PEPSCAN method is an easy to perform, quick and well- established method for the detection of epitopes; the immunologically important regions of the protein.
  • the method is used world- wide and as such well-known to man skilled in the art. This (empirical) method is especially suitable for the detection of B-cell epitopes.
  • T-cell epitopes can likewise be predicted from the sequence by computer with the aid of Berzof sky's amphiphilicity criterion (Science 235, 1059-1062 (1987) and US Patent application NTIS US 07/005,885).
  • a condensed overview is found in: Shan Lu on common principles: Tibtech 9: 238-242 (1991), Good et al on Malaria epitopes; Science 235: 1059-1062 (1987), Lu for a review; Vaccine 10: 3-7 (1992), Berzowsky for HTV-epitopes; The FASEB Journal 5:2412-2418 (1991).
  • one form of still another embodiment of the invention relates to vaccines capable of protecting pigs against Lawsonia intracellularis infection, that comprise a protein or an immunogenic fragment thereof, according to the invention as described above together with a pharmaceutically acceptable carrier.
  • Still another embodiment of the present invention relates to the proteins according to the invention for use in a vaccine.
  • Still another embodiment relates to the use of a protein according to the invention for the manufacturing of a vaccine for combating Lawsonia intracellularis infections.
  • One way of making a vaccine according to the invention is by biochemical purification of the proteins or immunogenic fragments thereof according to the invention from bacteria obtained through mucosal scrapings taken from the infected intestine wall. This is however a very time-consuming way of making the vaccine. It is therefore much more convenient to use the expression products of the genes
  • a vaccine according to the invention can comprise live recombinant carriers as described above, capable of expressing the proteins according to the invention or immunogenic fragments thereof according to the invention.
  • Such vaccines e.g. based upon a Salmonella carrier or a viral carrier infecting the enteric epithelium, or e.g. the respiratory epithelium have the advantage over subunit vaccines that they better mimic the natural way of infection of Lawsonia intracellularis.
  • their self-propagation is an advantage since only low amounts of the recombinant carrier are necessary for immunisation.
  • Vaccines described above all contribute to active vaccination, i.e.
  • the host's immune system is triggered by a protein according to the invention or an immunogenic fragment thereof, to make antibodies against these proteins.
  • a protein according to the invention or an immunogenic fragment thereof can be raised in e.g. rabbits or can be obtained from antibody-producing cell lines as described below.
  • Such antibodies can then be administered to the host animal.
  • This method of vaccination passive vaccination, is the vaccination of choice when an animal is already infected, and there is no time to allow the natural immune response to be triggered. It is also the preferred method for vaccinating immune-compromised animals.
  • Administered antibodies against Lawsonia intracellularis can in these cases bind directly to the bacteria. This has the advantage that it immediately decreases or stops Lawsonia intracellularis growth. Therefore, one other form of this embodiment of the invention relates to vaccines comprising antibodies against the 26 kD Lawsonia intracellularis proteins according to the invention.
  • Vaccines can also be based upon host cells as described above, that comprise the proteins or immunogenic fragments thereof according to the invention.
  • this embodiment of the invention relate to vaccines comprising nucleic acids encoding a protein according to the invention or immunogenic fragments thereof according to the invention, and to vaccines comprising DNA fragments that comprise such nucleic acids. Still other forms of this embodiment relate to vaccines comprising recombinant DNA molecules according to the invention.
  • DNA vaccines can easily be administered through intradermal application e.g. using a needle-less injector. This way of administration delivers the DNA directly into the cells of the animal to be vaccinated. Amounts of DNA in the microgram range between 1 and 100 ⁇ g provide very good results.
  • the vaccine according to the present invention additionally comprises one or more antigens derived from other pig pathogenic organisms and viruses, or genetic information encoding such antigens.
  • Such organisms and viruses are preferably selected from the group of Pseudorabies virus, Porcine influenza virus, Porcine parvo virus, Transmissible gastro-enteritis virus,
  • Rotavirus Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Salmonella cholerasuis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae, Brachyspira hyodysenteriae and Actinobacillus pleuropneumoniae.
  • All vaccines according to the present invention comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier can be e.g. sterile water or a sterile physiological salt solution.
  • the carrier can e.g. be a buffer.
  • Methods for the preparation of a vaccine comprise the admixing of a protein according to the invention, or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.
  • Vaccines according to the present invention may in a preferred presentation also contain an adjuvant.
  • Adjuvants in general comprise substances that boost the immune response of the host in a non-specific manner.
  • a number of different adjuvants are known in the art. Examples of adjuvants are Frevmds Complete and Incomplete adjuvant, vitamin E, non- ionic block polymers, muramyldipeptides, Quill A ⁇ ), mineral oil e.g. BayolOv or
  • the vaccine may also comprise a so-called "vehicle".
  • a vehicle is a compound to which the polypeptide adheres, without being covalently bound to it. Often used vehicle compounds are e.g. aluminium hydroxide, -phosphate or -oxide, silica, Kaolin, and
  • the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span or Tween.
  • the vaccine is mixed with stabitisers, e.g. to protect degradation-prone polypeptides from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze-drying efficiency.
  • stabitisers e.g. to protect degradation-prone polypeptides from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze-drying efficiency.
  • Useful stabilisers are i.a. SPGA (Bovarmk et al; J. Bacteriology 59: 509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates.
  • the vaccine may be suspended in a physiologically acceptable diluent.
  • Vaccines according to the invention can very suitably be administered in amounts ranging between 1 and 100 micrograms, although smaller doses can in principle be used. A dose exceeding 100 micrograms will, although immunologically very suitable, be less attractive for commercial reasons.
  • Vaccines based upon live attenuated recombinant carriers, such as the LRC-viruses and bacteria described above can be administered in much lower doses, because they multiply themselves during the infection. Therefore, very suitable amounts would range between 10 3 and 10 9 CFU PFU for respectively bacteria and viruses.
  • Oral application is a very attractive way of administration, because the infection is an infection of the digestive tract.
  • a preferred way of oral administration is the packaging of the vaccine in capsules, known and frequently used in the art, that only disintegrate after they have passed the highly acidic environment of the stomach.
  • the vaccine could be mixed with compounds known in the art for temporarily enhancing the pH of the stomach.
  • Systemic application is also suitable, e.g. by intramuscular application of the vaccine. If this route is followed, standard procedures known in the art for systemic application are well-suited.
  • a diagnostic test for the detection of Lawsonia intracellularis antibodies in sera can be e.g. a simple standard sandwich-ELISA-test in which 26 kD protein or antigenic fragments thereof according to the invention are coated to the wall of the wells of an ELISA-plate.
  • a method for the detection of such antibodies is e.g. incubation of 26 kD protein or antigenic fragments thereof with serum from mammals to be tested, followed by e.g. incubation with a labelled antibody against the relevant mammalian antibody.
  • a colour reaction can then reveal the presence or absence of antibodies against Lawsonia intracellularis.
  • Another example of a diagnostic test system is e.g. the incubation of a Western blot comprising the 26 kD protein or an antigenic fragment tibtereof according to the invention, with serum of mammals to be tested, followed by analysis of the blot.
  • Another embodiment of the present invention relates to diagnostic tests for the detection of antibodies against Lawsonia intracellularis.
  • Such tests comprise a protein or a fragment thereof according to the invention.
  • a diagnostic test based upon the detection of antigenic material of the specific 26 kD protein of Lawsonia intracellularis antigens and therefore suitable for the detection of Lawsonia intracellularis infection can e.g. also be a standard ELISA test.
  • the walls of the wells of an ELISA plate are coated with antibodies directed against the 26 kD protein. After incubation with the material to be tested, labelled anti- Lawsonia intracellularis antibodies are added to the wells. A colour reaction then reveals the presence of antigenic material from Lawsonia intracellularis.
  • Still another embodiment of the present invention relates to diagnostic tests for the detection of antigenic material of Lawsonia intracellularis.
  • Such tests comprise antibodies against a protein or a fragment thereof according to the invention.
  • polypeptides or immunogenic fragments thereof according to the invention expressed as characterised above can be used to produce antibodies, which may be polyclonal, monospecific or monoclonal (or derivatives thereof). If polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are well-known in the art (e.g. Mayer and Walter, eds. Irnmunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987).
  • Monoclonal antibodies reactive against the polypeptide according to the invention (or variants or fragments thereof) according to the present invention, can be prepared by immunising inbred mice by techniques also known in the art (Kohler and Milstein, Nature, 256, 495-497, 1975).
  • L. intracellularis infected ilea confirmed by histopathology and acid-fast Ziehl-Neelsen staining, were collected from pigs died with PE, and stored at -80°C. After thawing L. intracellularis bacteria were isolated from mucosal scrapings taken from the infected intestinal wall. The ileal scrapings were homogemzed repeatedly in PBS in an onmimixer to release the intracellular bacteria as described by Lawson et al. (Vet. Microbiol. 10: 303-323 (1985)). Supernatant obtained after low-speed centrifugation to remove cell debris was filtered through 5.0, 3.0, 1.2, and 0.8 ⁇ m filters (Millipore).
  • the filtrate was subsequently centrifuged at 8000 g for 30 min, giving a small pellet of L. intracellularis bacteria. These bacteria were further purified using a Percoll gradient. The identity of the purified bacteria was assessed by PCR (Jones et al., J. Clin. Microbiol. 31 : 2611-2615 (1993)) whereas purity of the isolated bacteria (>95%) was assessed by phase contrast microscopy to reveal any contaminating bacteria or gut debris present.
  • L. intracellularis cells were isolated from infected ileal material as described above.
  • Escherichia coli host strain BL21star(DE3) containing vector pLysSrare and plasmid pET22b were purchased from Novagen (Madison, Wisconsin, USA.
  • TOP10F' was purchased from Invitrogen (Groningen, the Netherlands). Stocks of all bacterial strains, containing 30% glycerol, were stored at — 70°C.
  • Luria Bertani broth (LB) and LB plates were prepared according to standard procedures.
  • DNA was prepared from bacterial cells using a Biorad chromosomal DNA isolation kit (Biorad, Veenendaal, the Netherlands). Plasmid DNA was isolated using Qiagen products.
  • PCR amplification PCR amplification was performed using a PCR mixture containing 52 U/ml Expand High
  • the PCR mixture contained 20 U/ml Supertaq and Supertaq buffer (HT Biotechnology Ltd, Cambridge, UK), containing 8 mM dNTPs
  • Ligation and transformation Ligations were performed in a 1 x ligation buffer with 1 unit of ligation enzyme (Gibco BRL Life Technologies Inc., USA) at 16 °C overnight. 1 ⁇ l of the ligation reaction was transformed to E. coli competent cells by heat shock. The BL21star(DE3) E. coli competent cells and the TOP10F' E .coli competent cells were made competent using standard methods.
  • the DNA sequence of the expression vector was confirmed before the expression vector was transformed to BL21star(DE3) containing pLysSrare.
  • the resulting strain was grown overnight at 37°C at 200 rpm in 5 ml LB with 100 ⁇ g/ml ampicillin.
  • the overnight culture was diluted 1 : 100 in 50 ml LB with 100 ⁇ g/ml ampicillin. This culture was grown under the same conditions until the ODeoo reached 0.5.
  • the culture was induced with EPTG to a final concentration of ImM and continued to grow for a subsequent 3 hours. 100 ⁇ l samples were taken for analysis.
  • E. coli strain BL21star(DE3) containing pLysSrare was grown and induced under the same conditions and samples were taken as a negative control. The samples were analyzed by SDS page.
  • Plasmid pET5608 was transformed to BL21Star(DE3)pLysSrare. The resulting strain was tested for recombinant protein production as described above. Samples of the induced culture and control samples were analysed by SDS-PAGE gel electrophoresis (Fig. 1 A). A clear protein band of approximately 26 kDa was observed in sample that had been taken after 3 hours of induction (Fig. 1A, lane 3) in comparison with the uninduced sample (Fig. 1 A, lane 2). The same samples were also analysed by western blot using the pig and chicken serum. A reaction with protein 5608 was observed using the serum from the pig that had been orally challenged with purified L. intracellularis cells (Fig IB, lane 3). and with the chicken anti-X. intracellularis serum (Fig 1C, lane 3).

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PCT/EP2004/053342 2003-12-09 2004-12-08 Lawsonia intracellularis 26 kd subunit vaccine WO2005056586A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US10/580,709 US20070212373A1 (en) 2004-12-08 2004-12-08 Lawsonia Intracellularis 26 Kd Subunit Vaccine
AU2004297018A AU2004297018A1 (en) 2003-12-09 2004-12-08 Lawsonia intracellularis 26 KD subunit vaccine
CA002548750A CA2548750A1 (en) 2003-12-09 2004-12-08 Lawsonia intracellularis 26 kd subunit vaccine
EP04820075A EP1694698A1 (en) 2003-12-09 2004-12-08 Lawsonia intracellularis 26 kd subunit vaccine
JP2006543544A JP2007537715A (ja) 2003-12-09 2004-12-08 ローソニア・イントラセルラーリスの２６ｋＤサブユニットワクチン
BRPI0417440-2A BRPI0417440A (pt) 2003-12-09 2004-12-08 ácido nucleico, fragmento e molécula de dna, veìculo recombinante vivo, célula hospedeira, proteìna de lawsonia intracellularis, vacina para combater as infecções com lawsonia intracelularis, método para a preparação de uma vacina, e, testes de diagnóstico para aldetecção de anticorpos contra lawsonia intracellularis e material antigênico de lawsonia intracellularis

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CN103157100A (zh) * 2011-12-08 2013-06-19 普莱柯生物工程股份有限公司 副猪嗜血杆菌病、猪链球菌病二联灭活疫苗及其制备方法

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WO2020172757A1 (en) * 2019-02-28 2020-09-03 University Of Saskatchewan Lawsonia intracellularis compositions and methods of using the same
CN110029079B (zh) * 2019-03-27 2022-10-11 南京农业大学 表达分泌猪伪狂犬病毒蛋白优势抗原区的重组枯草芽孢杆菌及应用

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EP1094070A2 (en) * 1999-10-22 2001-04-25 Pfizer Products Inc. Lawsonia intracellularis proteins, and related methods and materials
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CN101124241A (zh) 2008-02-13
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CA2548750A1 (en) 2005-06-23
KR20060112674A (ko) 2006-11-01
AU2004297018A1 (en) 2005-06-23

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