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WO2010097977A1 - Vaccin polyvalent par voie orale contre le rouget/la pneumonie à mycoplasme du porc - Google Patents

Vaccin polyvalent par voie orale contre le rouget/la pneumonie à mycoplasme du porc Download PDF

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Publication number
WO2010097977A1
WO2010097977A1 PCT/JP2009/065502 JP2009065502W WO2010097977A1 WO 2010097977 A1 WO2010097977 A1 WO 2010097977A1 JP 2009065502 W JP2009065502 W JP 2009065502W WO 2010097977 A1 WO2010097977 A1 WO 2010097977A1
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Prior art keywords
vaccine
strain
gene
erysipelas
swine
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Ceased
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PCT/JP2009/065502
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English (en)
Japanese (ja)
Inventor
善弘 下地
吉広 宗田
洋介 小川
英司 大石
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National Agriculture and Food Research Organization
Kyoto Biken Laboratories Inc
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National Agriculture and Food Research Organization
Kyoto Biken Laboratories Inc
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Publication of WO2010097977A1 publication Critical patent/WO2010097977A1/fr
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    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • 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
    • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
    • 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
    • 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
    • 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
    • C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6006—Cells

Definitions

  • the present invention relates to an invention of swine erysipelas / pig mycoplasma pneumonia oral administration type mycoplasma vaccine.
  • the present invention relates to a mycoplasma hyopneumoniae adhesin protein (P97) and a polypeptide containing a repetitive sequence in its paralog, a gene encoding the polypeptide, and use of the gene as a vaccine for protection against swine mycoplasma pneumonia infection.
  • P97 mycoplasma hyopneumoniae adhesin protein
  • Mycoplasma hyopneumoniae one of the major virulence factors of swine complex respiratory disease, is a causative agent of swine mycoplasma pneumonia and affects the gain and feed efficiency of pigs during the fattening period, resulting in an economic loss Is big. It is also considered one of the first pathogens to trigger complex respiratory disease. Therefore, in Japan, vaccine control is performed, and the penetration rate is high. These vaccines are obtained by inactivating mycoplasma hyopneumoniae cells or culture supernatants and adding an oil-based adjuvant or an aluminum gel adjuvant as an adjuvant. Inoculate once or twice.
  • swine erysipelas vaccines There are two types of swine erysipelas vaccines, live vaccines and inactivated vaccines, both of which are highly evaluated for their effectiveness.
  • the live vaccine in Japan uses the Koganei strain of swine venom that has been managed as a seed lot for a long time, and its safety is high. Since the damage caused by swine erysipelas is primarily a sudden death due to bacteremia with systemic rhombus, the main component of the vaccine effect is systemic immunity.
  • an object of the present invention is to provide a safe and effective vaccine that protects against the development of swine erysipelas and Mycoplasma hyopneumoniae by oral administration in order to reduce the stress on pigs caused by vaccine administration.
  • the insertion of a foreign gene is a single crossover, it is genetically unstable, and the selective pressure for drug resistance. If the culture is performed without applying the antibiotic, that is, if the antibiotic is not added to the medium, there is a possibility of returning to the original strain. In addition, since the entire gene on the plasmid including the antibiotic resistance gene remains on the chromosome, it is not suitable as a genetically modified vaccine. Therefore, the development of genetically engineered vaccines involves the use of genetically stable recombinant bacteria that do not contain antibiotic resistance genes or vector-derived genes by double crossover, and only introduce the desired gene (p97 gene). Must be made.
  • Non-Patent Document 1 After introducing a gene by transformation, the probability of homologous recombination occurring at a single crossover is very low, and the probability of homologous recombination occurring at a double crossover is lower. Therefore, it is extremely difficult to select a clone in which homologous recombination has occurred in double crossover (see Non-Patent Document 1).
  • a part of Mycoplasma hyopneumoniae P97 adhesin gene was introduced into this bacterium by homologous recombination as described above, and a porcine erysipelas koganei strain expressing a part of Mycoplasma hyopneumoniae P97 adhesin was produced.
  • a porcine erysipelas koganei strain expressing a part of Mycoplasma hyopneumoniae P97 adhesin was produced.
  • the vaccine effect of this strain was confirmed by an infection and onset prevention test in pigs, a good vaccine effect was confirmed against highly virulent swine erysipelas and virulent Mycoplasma hyopneumoniae.
  • the present invention relates to a porcine syphilis in which a part of the P97 adhesin protein of Mycoplasma hyopneumoniae is expressed on the surface of attenuated porcine erysipelas having the ability to impart a vaccine effect by oral administration.
  • the part of the P97 adhesin protein may be a region called repeat R1 or repeat R2 on the C-terminal side, and includes a protein that is expressed from the base sequence shown in SEQ ID NO: 1.
  • porcine erysipelas of the present invention those in which a foreign gene is inserted into the host DNA by double crossing are preferred.
  • a gene having the base sequence shown in SEQ ID NO: 1 can be mentioned.
  • the present invention also relates to a vaccine using the porcine erysipelas.
  • live cells can be used.
  • the vaccine can include stabilizers and / or adjuvants.
  • the stabilizer include skim milk, lactose, sorbitol, trehalose, and polyvinylpyrrolidone.
  • adjuvants include Freund's complete or incomplete adjuvant, aluminum compounds such as aluminum hydroxide and aluminum phosphate, muramyl dipeptide, potassium alum, saponin, mineral oil or vegetable oil, mannitol oleate added oil, polyoxy Ethylene sorbitan fatty acid-added oil, carboxyl polymer, fatty acid, squalane and the like can be mentioned.
  • the vaccine of this invention is manufactured by mixing the said swine venom, and if necessary, a stabilizer and / or an adjuvant.
  • the present invention also relates to a method for inducing immunity by orally administering the vaccine.
  • Examples of subjects to be administered orally include pigs.
  • Plasmid constructs for transformation into swine erysipelas The insert having the above sequence was cleaved at the restriction enzyme site shown below, pGA14, which is a shuttle vector between Gram-positive bacteria and Escherichia coli, and the p97 gene sandwiched between the spaA.1 genes was inserted. This was introduced into porcine erysipelas (attenuated swine venom koganei strain, animal health research institute stock) by electroporation, and the strain where the spaA.1 upstream / downstream region was replaced with the introduced gene by double crossover was selected. did.
  • the structure of the pGA14 shuttle vector incorporating the p97 gene is shown in FIG.
  • mice ErMhKo-A, B, C and D culture broth (10 8 CFU / ml) addressed to 0.1 ml subcutaneously in the inner thigh of 4-week-old ddy mice (Japan SLC)
  • the mice were inoculated and observed for the existence of life and death of mice and arthritis.
  • Mouse of the LD 100 of swine erysipelas bacteria strong Dokukabu is 10 3, the mouse will die if the toxicity return.
  • the incidence of mouse arthritis was used as an indicator of clones that did not recover toxicity and retained immunogenicity.
  • Mycoplasma hyopneumoniae culture solution Hanks solution (Nissui), Brucella broth (Gibco), lactalbumin hydrolyzate, swine serum, yeast extract, methicillin, phenol red)
  • infected lung emulsion homemade made from sterile pigs
  • Necropsy was performed 4 weeks after administration, and the rate of lung lesion formation was compared.
  • respiratory symptoms such as coughing and sneezing and fever were not observed in both groups.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Communicable Diseases (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Pulmonology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un vaccin qui peut protéger efficacement contre l'infection par Erysipelothrix rhusiopathiae ou Mycoplasma hyopneumoniae lorsqu'il est administré par voie orale, et qui est sûr et économiquement avantageux. La souche d'Erysipelothrix rhusiopathiae Koganei, qui a été utilisée comme souche pour un vaccin vivant par injection pendant des années au Japon, est choisie comme souche d'Erysipelothrix rhusiopathiae atténuée pour être utilisée comme vecteur d'un vaccin contre le mycoplasme par voie orale. Une partie du gène codant pour l'adhésine P97 de Mycoplasma hyopneumoniae est introduite dans une cellule de la souche par recombinaison homologue pour produire la souche d'Erysipelothrix rhusiopathiae Koganei qui peut exprimer une partie d'une adhésine P97 de Mycoplasma hyopneumoniae. La souche présente un bon effet de vaccination contre le très virulent Erysipelothrix rhusiopathiae et le très virulent Mycoplasma hyopneumoniae.
PCT/JP2009/065502 2009-02-24 2009-09-04 Vaccin polyvalent par voie orale contre le rouget/la pneumonie à mycoplasme du porc Ceased WO2010097977A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2009-040970 2009-02-24
JP2009040970A JP5257939B2 (ja) 2009-02-24 2009-02-24 豚丹毒・豚マイコプラズマ肺炎経口投与型多価ワクチン

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108623663A (zh) * 2018-05-14 2018-10-09 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 猪肺炎支原体p97蛋白的免疫原性功能多肽及其编码基因和应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2684959A1 (fr) * 2012-07-10 2014-01-15 Laboratorios Hipra, S.A. Vecteurs pour la transformation de Mycoplasma hyopneumoniae, souches transformantes de M.hyopneumoniae, et leur utilisation.
KR102228308B1 (ko) * 2018-12-19 2021-03-16 주식회사 이노백 마이코플라즈마 폐렴 및 흉막폐렴 예방용 백신 조성물

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2992980B2 (ja) * 1997-11-19 1999-12-20 農林水産省家畜衛生試験場長 豚丹毒菌の莢膜欠損変異株 ｙｓ−１株
JP2004501979A (ja) * 2000-06-30 2004-01-22 ワイス 経口ワクチン接種のための方法および組成物
JP3793889B2 (ja) * 2000-10-13 2006-07-05 独立行政法人農業・食品産業技術総合研究機構 マイコプラズマ・ハイオニューモニエ抗原を発現する豚丹毒菌および該菌による免疫化方法
JP2006311824A (ja) * 2005-05-09 2006-11-16 National Agriculture & Food Research Organization 豚マイコプラズマ肺炎ワクチン

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2992980B2 (ja) * 1997-11-19 1999-12-20 農林水産省家畜衛生試験場長 豚丹毒菌の莢膜欠損変異株 ｙｓ−１株
JP2004501979A (ja) * 2000-06-30 2004-01-22 ワイス 経口ワクチン接種のための方法および組成物
JP3793889B2 (ja) * 2000-10-13 2006-07-05 独立行政法人農業・食品産業技術総合研究機構 マイコプラズマ・ハイオニューモニエ抗原を発現する豚丹毒菌および該菌による免疫化方法
JP2006311824A (ja) * 2005-05-09 2006-11-16 National Agriculture & Food Research Organization 豚マイコプラズマ肺炎ワクチン

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
HIROKAZU MAKIYAMA ET AL.: "Nomu Vaccine no Yuyosei", GEKKAN YOTONKAI, vol. 41, no. 10, 1 October 2006 (2006-10-01), pages 44 - 45 *
OGAWA Y ET AL.: "Oral vaccination against mycoplasmal pneumonia of swine using a live Erysipelothrix rhusiopathiae vaccine strain as a vector", VACCINE, vol. 27, no. 33, 16 July 2009 (2009-07-16), pages 4543 - 4550, XP026238180, DOI: doi:10.1016/j.vaccine.2009.04.081 *
SAWADA T ET AL.: "Cross protection of mice and swine given live-organism vaccine against challenge exposure with strains of Erysipelothrix rhusiopathiae representing ten serovars", AM. J. VET. RES., vol. 48, no. 1, 1987, pages 81 - 84 *
SHIMOJI Y ET AL.: "Erysipelothrix rhusiopathiae YS-1 as a live vaccine vehicle for heterologous protein expression and intranasal immunization of pigs", INFECT. IMMUN., vol. 70, no. 1, 2002, pages 226 - 232 *
YOSHIHIRO SHIMOJI ET AL.: "Jisedai Tontandoku Vaccine no Shorai Tenbo", PROCEEDINGS OF THE JAPANESE PIG VETERINARY SOCIETY, no. 34, 1 February 1999 (1999-02-01), pages 8 - 11 *
YOSUKE OGAWA ET AL.: "Mycoplasma hyopnemoniae P97 Adhesin o Hatsugen suru Tontandoku Kin no Keiko Toyo ni yoru Bogyo Koka", DAI 146 KAI NIPPON JUI GAKKAI GAKUJUTSU SHUKAI KOEN YOSHISU, vol. 146, 5 September 2008 (2008-09-05), pages 208 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108623663A (zh) * 2018-05-14 2018-10-09 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 猪肺炎支原体p97蛋白的免疫原性功能多肽及其编码基因和应用
CN108623663B (zh) * 2018-05-14 2021-09-21 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 猪肺炎支原体p97蛋白的免疫原性功能多肽及其编码基因和应用

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JP5257939B2 (ja) 2013-08-07

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