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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
  • A61K39/025—Enterobacteriales, e.g. Enterobacter
  • A61K39/0275—Salmonella
• • 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
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
• • 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 live attenuated bacteria for use in a medicament, to vaccines based upon such bacteria useful for the prevention of microbial pathogenesis, to the use of such bacteria for the manufacture of a vaccine and to methods for the preparation of such vaccines.
• Immunity to microbial pathogenesis is one means by which a warm blooded animal avoids pathogenesis, or suffers a less intense pathogenic state. Incomplete immunity to a given pathogen results in morbidity and mortality in a population exposed to a pathogen. It is generally agreed that vaccines based on live but attenuated micro-organisms (live attenuated vaccines) induce a highly effective type of immune response. Such vaccines have the advantage that, once the animal host has been vaccinated, entry of the microbial pathogen into the host induces an accelerated recall of earlier, cell-mediated or humoral immunity which is able to control further growth of the organism before the infection can assume clinically significant proportions.
• Vaccines based on a killed pathogen are generally conceded to be unable to achieve this type of response.
• vaccines that contain a live pathogen present, depending on the level of attenuation, the danger that the vaccinated host upon vaccination may contract the disease against which protection is being sought.
• Vaccines against bacteria belonging to e.g. the closely related families of Escherichia and Salmonella follow the general rules given above. Many members of these families of bacteria are pathogenic due to the fact that they infect the digestive tract and/or the bladder. The pathogenic effect of these bacteria is closely related to their ability to colonise the mucosal layers of the digestive tract and/or the bladder. It is the phenomenon of colonisation that leads to the prolonged presence of the pathogen in the digestive tract and to a very close contact of the pathogen to the mucosal layers, which can also lead to invasion of other tissues.
• a first prerequisite for such a vaccine would seem to be its ability to colonise the digestive tract. It is the phenomenon of colonisation that provides a firm trigger of the immune system.
• one embodiment of the present invention relates to a live attenuated bacterium that is not capable to express a functional Eda protein as a result of a mutation in the eda gene, for use in a vaccine.
• Eda-mutants as described in the invention show a very low pathogenicity, which makes them attractive as vaccine strains.
• the eda gene, encoding the Eda protein plays a key role in what is known as the Entner-Doudoroff Pathway.
• the Eda-protein is an enzyme, also known as KDPG-aldolase. This enzyme is used in the metabolism of glucuronate, galacturonate and gluconate.
• Fraenkel, D. G. has described the Entner-Doudoroff pathway in e.g. E.coli and Salmonella at p. 142-150 in F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington D.C. (1987).
• Egan et al. have analysed the Entner-Doudoroff pathway in E. coli and they have analysed the sequence and localized promoters for the edd-eda operon (Egan et al., J. Bacteriology 174: 4638-4646 (1992)).
• SEQ ID NO: 1 The sequence of the DNA encoding Eda of Salmonella typhimurium is depicted in SEQ ID NO: 1.
• the sequence of the Eda protein itself is given in SEQ ID NO: 2.
• Eda protein is a highly conserved protein. It can be found in e.g. Escherichia coli, in Salmonella species, more specifically Salmonella enterica species, such as serotype Typhimurium, Enteritidis and Dublin and in Yersinia species such as Y. pestis.
• the mutation can be an insertion, a deletion, a substitution or a combination thereof, provided that the mutation leads to the failure to express a functional Eda protein.
• a functional Eda protein is understood to be a protein having the regulating characteristics of the wild-type protein.
• an Eda protein that is defective in at least one of its functions is considered to be a non-functional Eda protein.
• a non-functional Eda protein would not or to a lesser extend, when compared to its wild-type counterpart, be able to mediate the synthesis of KDPG from pyruvate and glyceraldehyde-3-phosphate and vice versa.
• a strain having a non-functional Eda-protein would not or to a lesser extent, when compared to its wild-type counterpart, be able to utilise glucuronate, galacturonate and gluconate.
• Live attenuated bacteria for use according to the invention can be obtained in several ways.
• One possible way of obtaining such bacteria is by means of classical methods such as the treatment of wild-type bacteria having the eda gene with mutagenic agents such as base analogues, treatment with ultraviolet light or temperature treatment.
• transposon mutagenesis is also a mutagenesis-technique well-known in the art. This is a mutation accomplished at a localised site in the chromosome. Transposon-insertions can not be targeted to a specific gene. It is however very easy to pick up eda-mutants since they do not grow in vitro without nutrient compensation for lack of Eda activity. Therefore, they can easily be selected from a pool of randomly transposon-mutated bacteria.
• Such a mutation may again be an insertion, a deletion, a replacement of one nucleotide by another one or a combination thereof, with the only proviso that the mutated gene no longer encodes functional Eda.
• Such a mutation can e.g. be made by deletion of a number of base pairs. Even very small deletions such a stretches of 10 base pairs can already render Eda nonfunctional. Even the deletion of one single base pair may already lead to a non-functional Eda, since as a result of such a mutation, the other base pairs are no longer in the correct reading frame.
• Each deletion of insertion of a number of base pairs indivisible by three causes such a frame shift. More preferably, a longer stretch is removed e.g. 100 base pairs. Even more preferably, the whole eda gene is deleted.
• Eda-negative mutants All techniques for the construction of Eda-negative mutants are well-known standard techniques. They relate to cloning of the Eda-gene, modification of the gene sequence by site-directed mutagenesis, restriction enzyme digestion followed by re-ligation or PCR-approaches and to subsequent replacement of the wild type eda gene with the mutant gene (allelic exchange or allelic replacement). Standard recombinant DNA techniques such as cloning the eda gene in a plasmid, digestion of the gene with a restriction enzyme, followed by endonuclease treatment, re-ligation and homologous recombination in the host strain, are all known in the art and described i.a. in Maniatis/Sambrook (Sambrook, J. et al.
• Site-directed mutations can e.g. be made by means of in vitro site directed mutagenesis using the Transformer® kit sold by Clontech. PCR-techniques are extensively described in (Dieffenbach & Dreksler; PCR primers, a laboratory manual. ISBN 0-87969-447-3 and ISBN 0-87969-447-5).
• the eda gene comprises not only the coding sequence encoding the Eda protein, but also regulatory sequences such as the promoter.
• the gene also comprises sites essential for correct translation of the Eda mRNA, such as the ribosome binding site.
• the invention relates to live attenuated bacteria of the genera Escherichia, Salmonella and Yersinia for use in a vaccine.
• the live attenuated bacterium according to the invention is selected from the group consisting of S. enterica serotype typhimurium, enteritidis, choleraesuis, dublin, typhi, gallinarum, abortusovi, abortus - equi, pullorum, E. coli or Y. pestis.
• S. enterica serotype typhimurium enteritidis
• choleraesuis choleraesuis
• typhi gallinarum
• abortusovi abortus - equi
• pullorum E. coli or Y. pestis.
• the live attenuated bacterium according to the invention is selected from the group consisting of S. enterica serotype typhimurium, enteritidis, gallinarum, pullorum, E. coli or Y. pestis.
• this embodiment of the invention refers to live attenuated bacteria in which the eda gene comprises an insertion and/or a deletion.
• live attenuated bacteria as a recombinant carrier for heterologous genes, encoding antigens selected from other pathogenic micro-organisms or viruses.
• Administration of such a recombinant carrier has the advantage that immunity is induced against two or more diseases at the same time.
• the live attenuated bacteria for use in a vaccine, according to the present invention provide very suitable carriers for heterologous genes, since the gene encoding the Eda protein can be used as an insertion site for such heterologous genes.
• eda gene as an insertion site has the advantage that at the same time the eda gene is inactivated and the newly introduced heterologous gene can be expressed (in concert with the homologous bacterial genes).
• the construction of such recombinant carriers can be done routinely, using standard molecular biology techniques such as allelic exchange.
• another embodiment of the invention relates to live attenuated recombinant bacteria, preferably of the genera Escherichia, Salmonella and Yersinia that do not produce a functional Eda protein and in which a heterologous gene is inserted, for use in a vaccine.
• a heterologous gene can, as mentioned above, e.g. be a gene encoding an antigen selected from other pathogenic micro-organisms or viruses.
• Such genes can e.g. be derived from pathogenic herpesviruses (e.g. the genes encoding the structural proteins of herpesviruses), retroviruses (e.g. the gp160 envelope protein), adenoviruses and the like.
• a heterologous gene can be obtained from pathogenic bacteria.
• genes encoding bacterial toxins such as Actnobacillus pleuropneumoniae toxins, Clostridium toxins, outer membrane proteins and the like are very suitable bacterial heterologous genes.
• Another possibility is to insert a gene encoding a protein involved in triggering the immune system, such as an interleukin or an interferon, or another gene involved in immune-regulation.
• Insertion of the heterologous gene in the eda gene is advantageous, since in that case there is no need to find a new suitable insertion site for the heterologous gene, and at the same time the eda gene is knocked out.
• the heterologous gene is inserted in the eda gene.
• the heterologous gene can be inserted somewhere in the eda gene or it can be inserted at the site of the eda gene while this gene has been partially or completely deleted.
• the bacteria for use in a vaccine are very suitable as a basis for live attenuated vaccines.
• still another embodiment of the invention relates to live attenuated vaccines for the protection of animals and humans against infection with a bacterium of which the wild type form comprises a eda gene.
• Such vaccines comprise an immunogenically effective amount of a live attenuated bacterium for use in a vaccine, according to the invention or a live recombinant carrier bacterium according to the invention, and a pharmaceutically acceptable carrier.
• the vaccine comprises a live attenuated bacterium according to the invention, selected from the group of Escherichia, Salmonella and Yersinia.
• Immunogenically effective means that the amount of live attenuated bacteria administered at vaccination is sufficient to induce in the host an effective immune response against virulent forms of the bacterium.
• a vaccine according to the present invention also contains a pharmaceutically acceptable carrier.
• a carrier may be as simple as water, but it may e.g. also comprise culture fluid in which the bacteria were cultured.
• Another suitable carrier is e.g. a solution of physiological salt concentration.
• the useful dosage to be administered will vary depending on the age, weight and animal vaccinated, the mode of administration and the type of pathogen against which vaccination is sought.
• the vaccine may comprise any dose of bacteria, sufficient to evoke an immune response. Doses ranging between 10 3 and 10 10 bacteria are e.g. very suitable doses.
• one or more compounds having adjuvant activity may be added to the vaccine.
• Adjuvants are non-specific stimulators of the immune system. They enhance the immune response of the host to the vaccine. Examples of adjuvants known in the art are Freunds Complete and Incomplete adjuvant, vitamin E, non-ionic block polymers, muramyldipeptides, ISCOMs (immune stimulating complexes, cf. for instance European Patent EP 109942), Saponins, mineral oil, vegetable oil, and Carbopol.
• Adjuvants specially suitable for mucosal application are e.g. the E. coli heat-labile toxin (LT) or Cholera toxin (CT).
• LT heat-labile toxin
• CT Cholera toxin
• Suitable adjuvants are for example aluminium hydroxide, aluminium phosphate or aluminium oxide, oil-emulsions (e.g. of Bayol F® or Marcol 52®), saponins or vitamin-E solubilisate.
• the vaccines according to the present invention comprise an adjuvant.
• compositions e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran
• proteins such as albumin or casein
• protein containing agents such as bovine serum or skimmed milk
• buffers e.g. phosphate buffer
• the vaccine is very suitable for freeze-drying. Therefore, in a more preferred form, the vaccine is in a freeze-dried form.
• the vaccine according to the present invention can be given inter alia intranasally, intradermally, subcutaneously, orally, by aerosol or intramuscularly.
• aerosol or intramuscularly for application to poultry, wing web and eye-drop administration are very suitable.
• a vaccine according to the invention especially when it comprises bacteria belonging to the family of E. coli, Salmonella or Yersinia would preferably be given orally.
• Still another embodiment relates to the use of a bacterium or a recombinant bacterium according to the invention for the manufacture of a vaccine for the protection of animals and humans against infection with a wild type bacterium or the pathogenic effects of infection.
• Still another embodiment of the invention relates to methods for the preparation of a vaccine according to the invention.
• Such methods comprise the admixing of a live attenuated bacterium according to the invention or a live recombinant carrier bacterium according to the invention, and a pharmaceutically acceptable carrier.
• the eda deletions were made using a modified Wanner method (PNAS Jun. 6, 2000. 97(12):6640-45).
• Primers to the 5′ and 3′ eda flanking sequences of S. enteritidis SE5609 were designed with Pstl sites (Pstl eda 5′: cta gct gca ggt gct aag cgg taa tct ggg and Pstl eda 3′: cta gct gca gaa gag att gct cgt cat gtg g) and the PCR product was cloned into pBluescript SKII+ (pBSeda).
• BgIII containing primers, to the eda promoter region (ctag aga tct ctcgcctgattacta gtgtg) and the 3′ end (ctag agatct aag ccg ttaaatgcccgatgg) were used to reverse PCR amplify the flanking and vector sequences from pBSeda.
• BgIII digestion and subsequent ligation created an eda deletion clone called pBSedaA.
• a 1.2 kb BamHI digested chloramphenicol resistant gene was inserted into the BgIII site of pBSedaA to produce pBSeda ⁇ cam.
• pBSeda ⁇ cam was digested with Pstl and the insert was separated from vector sequences and used as the template for PCR. Eight 100 ⁇ l reactions were pooled, 5 ⁇ l was checked on a gel, and the linear PCR product was ethanol precipitated and resuspended in 2-4 ⁇ l of water.
• S. enteritidis SE5609 cells were electroporated with the temperature sensitive plasmid pKD46. When these cells are grown at 30° C. in the presence of arabinose the plasmid expresses the lambda Red recombinase.
• the recombinase acts to replace the wild type eda gene with the deleted eda gene containing the chloramphenicol cassette.
• SE5609 clones containing the deleted eda gene containing the chloramphenicol cassette were selected by overnight growth at 37° C. on Luria agar plates containing chloramphenicol (30 ⁇ g/ml), which also resulted in loss of the temperature sensitive pKD46 plasmid.
• flanking eda 5′ (ctagctgca gcc tca tat tcc gga cct gag c) and eda 3′ (cta gct gca ggt gac ggt aaa agg cta atg cg) primers were used to verify one of the SE5609-eda ( ⁇ ) eda mutants.
• An amplified fragment containing the wild type SE5609 eda gene produced a 927 bp band as expected, while the eda delebon/chloramphenicol mutant gave the expected 1677 bp product.
• chickens were orally inoculated at 6 and 14 weeks of age with respectively 1.1 ⁇ 10 8 CFU and 4.1 ⁇ 10 8 CFU of SE5609-eda ( ⁇ ) .
• the vaccinated chickens and unvaccinated controls received an oral challenge infection with 1.3 ⁇ 10 8 CFU of a naladixic acid resistant wild-type S.e. strain at 16 weeks of age.
• Cloaca swabs were taken 3, 7 and 14 days after challenge to determine the rate of colonization by the challenge strain.
• Swabs were inoculated on BGA containing naladixic acid (BGAnal) directly and also after incubation in an enrichment medium (buffered peptone water containing nal).
• the SE5609-eda ( ⁇ ) strain was not cultured from cloacal swabs of the vaccinated animals on days 7 and 14, indicating that the strain was impaired in its ability to colonize the digestive tract of chickens.
• a vaccine based upon a Salmonella enteritidis eda ( ⁇ ) strain which is a wild-type strain having eda ( ⁇ ) as a single attenuation is safe. Moreover, vaccination with this Salmonella enteritidis eda ( ⁇ ) strain resulted in complete prevention of colonization of the digestive tract by both the vaccine strain and the challenge strain.

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