EP0804232A1 - Para-influenza virus-containing vaccines for preventing porcine reproductive and respiratory syndrome - Google Patents

Para-influenza virus-containing vaccines for preventing porcine reproductive and respiratory syndrome

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Publication number
EP0804232A1
EP0804232A1 EP95910510A EP95910510A EP0804232A1 EP 0804232 A1 EP0804232 A1 EP 0804232A1 EP 95910510 A EP95910510 A EP 95910510A EP 95910510 A EP95910510 A EP 95910510A EP 0804232 A1 EP0804232 A1 EP 0804232A1
Authority
EP
European Patent Office
Prior art keywords
virus
leu
thr
ser
ile
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.)
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Application number
EP95910510A
Other languages
German (de)
French (fr)
Inventor
Ernst Heinen
Norbert Schmeer
Werner Herbst
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.)
Bayer AG
Original Assignee
Bayer AG
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Publication date
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Publication of EP0804232A1 publication Critical patent/EP0804232A1/en
Withdrawn legal-status Critical Current

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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • 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
    • A61P31/12—Antivirals
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18711—Rubulavirus, e.g. mumps virus, parainfluenza 2,4
    • C12N2760/18721—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18711—Rubulavirus, e.g. mumps virus, parainfluenza 2,4
    • C12N2760/18722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18711—Rubulavirus, e.g. mumps virus, parainfluenza 2,4
    • C12N2760/18734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C12N2770/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a viral agent.
  • PRRS Phorcine Reproductive and Respiratory Syndrome
  • the disease in sows is characterized by late abortions, stillbirths and the birth of mummified and poorly-performing piglets.
  • the primary infection agent is assumed to be a new RNA virus that multiplies in lung alveolar macrophages.
  • respiratory and reproductive diseases are caused or intensified by secondary or multiple infections with other viruses or viruses and bacteria. It is therefore desirable not only to protect pigs against the main pathogen of the PRRS, but also against the pathogens which are jointly responsible for respiratory and reproductive diseases.
  • the present invention relates to:
  • Vaccine against diseases of the respiratory and reproductive tract of pigs in particular in connection with the disease complex called PRRS, characterized in that they contain, as an antigenic material, parainfluenza viruses and their variants and mutants in living, attenuated or recombinant form, in whole or in part or contains fragments.
  • Antigenic material based on parainfluenza viruses cause diseases of the respiratory and reproductive tract of pigs.
  • antigenic material The following can be mentioned as antigenic material:
  • Virus antigens which are expressed via vector systems, the genome of the virus or parts thereof in genome vectors such as vaccina viruses and herpes viruses by means of recombinant genetic engineering.
  • Adenoviruses or other suitable vector systems is used.
  • Parainfluenza viruses of type 2 (PIV-2) are preferably used.
  • PIV-2 which are isolated from the respiratory or reproductive tract of pigs which show symptoms similar to PRRS.
  • PIV-2 strain with the designation SER which was released on June 12, 1993 by the Collection Nationale des Cultures et de. Microorganisms
  • the antigenic material of the parainfluenza viruses can be present in a mixture with antigenic material from other viruses or bacteria.
  • a mixture of PIV-2 and chlamydia is particularly preferred, in particular
  • the different DNA sequences are (1.) foreign DNA, which can be inserted in shuttle vectors and (2.) the purified genome of the vector virus.
  • Genome vector Living pathogens in particular viruses, which are suitable for inserting foreign DNA and which infect cells or organisms with the foreign DNA inserted in their genome and express the foreign DNA therein.
  • Immunogenic peptides or proteins that trigger an immunological reaction in a higher organism and can be expressed in vectors by foreign DNA sequences.
  • Shuttle vector bacteriophages or plasmids in particular bacterial plasmids which contain inserted foreign DNA which is flanked by DNA sequences of the vector virus.
  • Vectors plasmids, bacteriophages or viruses which carry foreign DNA sequences in their genetic information.
  • the multiplication of viruses for the production of complete living virus particles takes place in the usual way on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned, permanent pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or their descendants) or Vero (ATCC CCL81 or its descendants) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its descendants) and on the other hand in embryonated chicken eggs (eg Valo hatching eggs, Lohmann).
  • tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned, permanent pig kidney cell PK15 (ATCC CCL33
  • the multiplication in cell cultures takes place in a manner known per se in stationary roller or carrier cultures in the form of closed cell groups (monolayers) or in suspension cultures.
  • all cell culture media known per se are used, for example described in the product catalog of Gibco BRL GmbH, Dieselstrasse 5, 76344 Eggenstein, such as in particular the Minimal Essential Medium (MEM), which contains essential components of amino acids, vitamins, salts and carbohydrates punch with puff ersub such as sodium bicarbonate (NaHCO 3 ) or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and optionally animal sera such as sera from cattle, horses or their fetuses.
  • Eagles MEM with a NaHCO 3 content of 0.1-5 g / 1, preferably 0.5-3 g / l and fetal calf serum in a concentration of 1-30% by volume, preferably 2- 10 vol%.
  • the "for multiplication of the viruses serving cells and cell lawns are grown in conventional manner almost to confluence or to the optimal cell density.
  • the Zeilverioloungsmedium is preferably removed and the cells preferably with virus replication medium washed.
  • the virus replication medium employed all cell culture media known per se, in particular the above-mentioned MEM.
  • infection is carried out with a virus suspension.
  • the viruses are propagated with or without the addition of animal sera. If serum is used, it is added to the propagation medium in a concentration of 1-30% by volume, preferably 2-10% by volume.
  • Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 32 and 39 ° C., particularly preferably at 37 ° C. for several days, preferably until the infected cells are completely destroyed.
  • the virus-containing medium of the infected cells is processed further, for example by removing the cells and cell debris by means of filtration with pore sizes of, for example, 0.1-0.45 ⁇ m and / or centrifugation up to 10,000 ⁇ g.
  • the multiplication in embryonated chicken eggs takes place in a manner known per se in the Allantoic cave of hatching hatching eggs, which lasts 9-12 days, preferably 10 days, at a temperature of 37-39 ° C., preferably 38.5 ° C. and a relative atmospheric humidity of 30-90%, preferably 50-60%, are pre-incubated in a commercial incubator, preferably a motor breeder.
  • the hatching eggs used to multiply the viruses are stored vertically in the incubator for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation and then infected with 10-200 ⁇ l, preferably 75-125 ⁇ l, of a virus suspension after preparation of the injection site .
  • Infection and virus multiplication take place under the breeding conditions specified above over several days, preferably 2-5 days, particularly preferably 3 days.
  • the virus-containing allantoic fluid is obtained by suction after opening the lime shell as well as the skin of the shell and the chorioallantoic membrane and can e.g. by filtration with pore sizes of e.g. 0.1-0.45 ⁇ m and / or centrifugation up to 10,000 x g can be processed further.
  • live virus is produced in the usual way by long-term passages and / or alternating passages on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned permanent pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or its descendants) or Vero (ATCC CCL81 or its descendants) ) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC
  • animal CCL22 or its descendants or dog kidney cells like the permanent one Dog kidney cell MDCK (ATCC CCL34 or its descendants) and on the other hand in embryonated chicken, pigeon or duck eggs, preferably in embryonated chicken eggs (e.g. Valo hatching eggs, Lohmann) or in experimental animals, preferably in small laboratory animals, e.g. in guinea pigs, Rat or mouse in which the virus reproduces without causing serious symptoms of the disease.
  • embryonated chicken, pigeon or duck eggs preferably in embryonated chicken eggs (e.g. Valo hatching eggs, Lohmann) or in experimental animals, preferably in small laboratory animals, e.g. in guinea pigs, Rat or mouse in which the virus reproduces without causing serious symptoms of the disease.
  • embryonated chicken, pigeon or duck eggs preferably in embryonated chicken eggs (e.g. Valo hatching eggs, Lohmann) or in experimental animals, preferably in small laboratory animals, e.g. in guin
  • Diesel No. 5, 76344 Eggenstein such as in particular the Minimal Essential Medium (MEM), which contains amino acids, vitamins, salts and carbohydrates as essential components, completed with buffer substances such as sodium bicarbonate (NaHCO 3 ) or hydroxyethylpiperazine N-2- ethanesulfonic acid (Hepes) and optionally animal sera, such as sera from cattle, horses or their fetuses.
  • Eagles MEM with a NaHCO 3 content of 0.1-5 g / 1, preferably 0.5-3 g / l and fetal calf serum in a concentration of 1-30 vol.%, Preferably 2-10 vol. Are particularly preferably used -%.
  • the cells and cell turf used for the passage of the viruses are multiplied in the usual way almost to confluence or to the optimal cell density.
  • MOI multiplicity of infection
  • viruses are propagated with or without the addition of animal sera. In the event that serum is used, this becomes the propagation medium in a
  • the virus-containing medium of the infected cells is used to infect a fresh cell culture (subsequent passage).
  • the passage in embryonated poultry eggs takes place in a known manner in the Allantoic cave of e.g. Chicken hatching eggs that 9-12 days, preferably 10 days at a temperature of 37-39 ° C, preferably 38.5 ° C and a relative humidity of 30-90%>, preferably 50-60% in a commercial incubator cabinet, preferably pre-incubated for a motor breeder.
  • Chicken hatching eggs that 9-12 days, preferably 10 days at a temperature of 37-39 ° C, preferably 38.5 ° C and a relative humidity of 30-90%>, preferably 50-60% in a commercial incubator cabinet, preferably pre-incubated for a motor breeder.
  • the hatching eggs used to pass the viruses are stored vertically in the incubator for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation and then infected with 10-200 ⁇ l, preferably 75-125 ⁇ l, of a virus suspension after preparation of the injection site.
  • Infection and virus multiplication take place under the above-mentioned breeding conditions over several days, preferably 2-5 days, particularly preferably 3 days.
  • the virus-containing allantoic fluid is obtained by suction after opening the calcareous skin, the skin of the skin and the chorioallantoic membrane. It is used for the infection of freshly prepared, embryonic eggs (subsequent passage).
  • the test animals are infected parenterally with 0.1-2.0 ml of a virus suspension, for example by intradermal, intramuscular, intranasal, intraperitoneal, intravenous or subcutaneous administration.
  • the virus replication medium employed are all cell culture media known per se, such as especially the v o ge above-mentioned MEM.
  • Virus propagation takes place over several days, preferably 1-12 days.
  • the virus is reisolated from tissues, preferably internal organs of the test animals, in the usual way.
  • internal organs e.g. Lung, liver or spleen removed.
  • the organs or parts of the organs are mechanically comminuted, e.g. with the aid of scissors and mortar, fine suspensions are prepared in virus propagation medium, which are worked up further, e.g. by removing the cells and cell debris by means of filtration with pore sizes of e.g. 0.1-0.45 ⁇ m and / or centrifugation up to 10,000 x g.
  • All cell culture media known per se, such as in particular the MEM mentioned above, are used as the virus propagation medium.
  • the virus-containing medium obtained is used to infect new experimental animals (subsequent passages).
  • the process of the subsequent passage is repeated several times, preferably 10-20 times, in the same propagation system (homologous passages) or in different multiplication systems (heterologous passages).
  • the virus is checked for attenuation by experimental infection of fully susceptible test animals, preferably pigs, with a virus suspension which originates from the last passage of a series of subsequent passages.
  • the viruses in the last subsequent passage are increased as described above and the filtrates or centrifugation supernatants from virus-containing culture supernatants or allantoic fluid are used to produce vaccines.
  • viruses for the production of killed virus particles takes place in the usual way on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned, permanent
  • Pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary.
  • Pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or its descendants) or Vero (ATCC CCL81 or its descendants) or bovine kidneys - Cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its descendants) and on the other hand in embryonated chicken eggs (eg Valo hatching eggs, Lohmann).
  • the multiplication in cell cultures takes place in a manner known per se in stationary roller or carrier cultures in the form of closed cell groups (monolayers) or in suspension cultures. All cell culture media known per se are used as propagation media for the cells, e.g. described in
  • Sera from cattle, horses and their fetuses is Eagles MEM with a NaHC0 3 content of 0.1 -5 g / 1, preferably 0.5-3 g / 1 and fetal calf serum in a concentration of 1-30% by volume, preferably 2-10% by volume.
  • the cells and cell turf used to multiply the viruses are multiplied in the usual way almost to confluence or to the optimal cell density.
  • the cell proliferation medium is preferably removed and the cells are preferably washed with virus multiplication medium. All known cell culture media, such as in particular the MEM mentioned above, are used as the virus propagation medium.
  • the viruses are propagated with or without the addition of animal sera. In the event that serum is used, this becomes the propagation medium in a
  • Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 32 and 39 ° C., particularly preferably at 37 ° C. for several days, preferably until the infected cells are completely destroyed.
  • the virus-containing medium of the infected cells is processed further, e.g. by removing the cells and cell debris by means of filtration with pore sizes of e.g. 0.1-0.45 ⁇ m and / or centrifugation up to 10,000 x g.
  • the multiplication in embryonated chicken eggs takes place in a manner known per se in the Allantoic cavity of chicken hatching eggs, which lasts 9-12 days, preferably 10 days, at a temperature of 37-39 ° C., preferably 38.5 ° C. and a relative atmospheric humidity of 30-90%), preferably 50-60%> are pre-incubated in a commercial incubator, preferably a motor breeder.
  • the hatching eggs used for the multiplication of the viruses are standing upright for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation Incubator stored and then infected after preparation of the injection site with 10-200 ul, preferably 75-125 ul of a virus suspension.
  • Infection and virus multiplication take place under the breeding conditions specified above over several days, preferably 2-5 days, particularly preferably 3 days.
  • the virus-containing allantoic fluid is obtained by suction after opening the lime shell as well as the skin of the shell and the chorioallantoic membrane and can e.g. by filtration with pore sizes of e.g. 0.1-0.45 ⁇ m and / or centrifugation up to 10,000 x g can be processed further.
  • viruses are inactivated in the usual way by physical methods, e.g. by exposure to heat, UV or gamma radiation or preferably by chemical methods, e.g. by the action of ethanol, formaldehyde, ⁇ -propiolactone and preferably by ethylene amines.
  • the chemical inactivation takes place in a manner known per se in suitable reaction vessels which have a device for maintaining a constant reaction temperature and for constant movement of the reaction mixture (e.g. fermenter).
  • the preferred inactivating agent is ethylene amines, particularly preferably 2-bromoethylamine hydrobromide (2-BEA) in a concentration of 1-10 mmol / 1, preferably 2.5-7.5 mmol / 1.
  • a virus suspension with a concentration of 10 4 '° -10 9-0 TCID 50 / ml, preferred wise 10 5.0 -10 8' 0 KID 50 / ml, which is derived from one or more virus propagation, is prior to addition of 2- BEA solution adjusted to a pH of 8.1-8.7, preferably 8.3-8.5.
  • the inactivation takes place at 4-40 ° C, preferably 23-37 ° C, particularly preferably at 36-37 ° C for 6-48 hours, preferably 16-20 hours
  • Excess 2-BEA is neutralized after the inactivation has been completed by adding hydrolyzing agents.
  • Sodium thiosulfate is particularly suitable for this, that a final concentration of 40-80 mmol / 1, preferably 50 mmol / 1 is added.
  • the neutralization takes place at 4-40 ° C, preferably at 2-8 ° C for 2-16 hours, preferably 4-8 hours.
  • the multiplication of the viruses for the production of subunits takes place in the usual way on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned, permanent pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or their descendants) or Vero (ATCC CCL81 or their descendants) or bovine kidney cells like the permanent one
  • tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned, permanent pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or their descendants) or Vero (ATCC
  • the multiplication in cell cultures takes place in a manner known per se in stationary roller or carrier cultures in the form of closed cell groups (monolayers) or in suspension cultures.
  • the propagation media used for the cells are all cell culture media known per se, for example described in the product catalog of Gibco BRL GmbH, Dieselstrasse 5, 76344 Eggenstein, such as in particular the Minimal Essential Medium (MEM), which contains amino acids, vitamins as essential components Contains salts and carbohydrates, supplemented with buffer substances such as sodium bicarbonate (NaHC ⁇ 3 ) or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and optionally animal sera such as serums from cattle, horses or their fetuses.
  • buffer substances such as sodium bicarbonate (NaHC ⁇ 3 ) or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and optionally animal sera such as serums from cattle, horses or their fetuses.
  • the cell growth medium Before being infected with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus growth medium. All cell culture media known per se, such as in particular the MEM mentioned above, are used as the virus propagation medium. Then it is infected with a virus suspension.
  • MOI multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present
  • the "multiplication of the viruses is carried out with or without addition of animal sera.
  • serum it is preferably 2-10% by volume added to the replication medium in a concentration of 1-30% by volume.
  • Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 32 and 39 ° C., particularly preferably at 37 ° C. for several days, preferably until the infected cells are completely destroyed.
  • the virus-containing medium of the infected cells is processed further, e.g. by removing the cells and cell debris by means of filtration with pore sizes of e.g. 0.1-0.45 ⁇ m and / or centrifugation on up to 10,000 x g.
  • the multiplication in embryonated chicken eggs takes place in a manner known per se in the Allantoic cave of chicken hatching eggs, which lasts 9-12 days, preferably 10 days, at a temperature of 37-39 ° C., preferably 38.5 ° C. and a relative humidity of 30 -90%, preferably 50-60% in a commercial incubator, preferably an engine breeder.
  • the hatching eggs used to multiply the viruses are stored vertically in the incubator for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation and then infected with 10-200 ⁇ l, preferably 75-125 ⁇ l, of a virus suspension after preparation of the injection site.
  • Infection and virus multiplication take place under the above-mentioned breeding conditions over several days, preferably 2-5 days, particularly preferably 3 days.
  • the virus-containing allantoic fluid is obtained by suction after opening the lime shell as well as the shell skin and the chorioallantoic membrane and can, for example: by means of filtration with pore sizes of e.g. 0.1-0.45 ⁇ m and / or centrifugation up to 10,000 x g can be worked up further.
  • Virus isolation is achieved by isopycnic or zonal centrifugation in e.g. Sucrose density gradients.
  • the virus-containing medium or the allantoic fluid after removal of the cell debris, is subjected to zone centrifugation at 100,000 x g until the virus particles are sedimented.
  • zone centrifugation at 100,000 x g until the virus particles are sedimented.
  • Representation of the virus particles results from zone centrifugation in an aqueous solution with a higher density than the virus-containing medium.
  • an aqueous solution e.g. serve a 30-60% w / w, preferably 35-50% w / w, buffered solution of sucrose.
  • An even higher degree of purity is achieved by centrifugation in the density gradient.
  • the virus, freed from cells and cell debris is concentrated by means of zone centrifugation by isopycnic or zonal density gradient centrifugation in a density gradient of e.g. 30 to 50% w / w sucrose in buffered aqueous solution with a centrifugal acceleration of e.g. 100,000 to 150,000 x g isolated.
  • the virus concentrates thus obtained are treated with detergents.
  • Suitable detergents are:
  • Anionic surfactants such as sodium lauryl sulfate, ready alcohol ether sulfates, mono- / dialkyl polyglycol ether orthophosphoric acid ester monoethanolamine salt, calcium alkyl aryl sulfonate, sodium deoxycholate, cationic surfactants, such as cetyltrimethylammonium chloride, ampholytic surfactants, such as diropionium sodium, such as diropyl sodium or lecithin, non-ionic surfactants, for example polyoxyethylated castor oil, polyoxyethylated sorbitan monooleate, sorbitan monostearate, glycerol monostearate, polyoxyethylene stearate, alkylphenol polyglycol ether.
  • cationic surfactants such as cetyltrimethylammonium chloride
  • ampholytic surfactants such as diropionium sodium, such as diropyl sodium or lecithin
  • non-ionic surfactants for example
  • Emulsifier such as NP 40 ® (Bayer AG), alkyl Non-ionic, water-soluble emulsifiers having an HLB (hydrophilic-lipophilic lic balance value) greater than 10, aiylpolyglykolether; preferably non-ionic detergents may be mentioned Renex 678 ® (Atlas Chemical Industries), polyoxyethylene alkyl aryl ether; Tween 20® (Atlas), polyoxyethylene sorbitan monopalmitate; Myri 53 ® (Atlas), polyoxyethylene stearate; Atlas G 3707 s , polyoxyethylene lauryl ether; Atlas G 3920 ® , polyoxyethylene oleyl ether; Atlas G 9046 T ® , polyoxyethylene mannitan monolaurate; Emulsifier 1371 B® (Bayer AG), alkyl polyglycol ether; Emulsifier 1736 ® (Bayer AG),.
  • Renex 678 ® Alkyl Non-
  • Alkyl polyglycol ether (oleyl polyglycol ether); Emulsifier OX ® (Bayer AG), alkyl polyglycol (Dodecylpolyglykolether); Ninox BM-2 ® (Ste- pan Chemical Co.) ethoxyethyliertes nonylphenol; Triton X-IOO * (Rohm to Haas Co.), isooctylphenol polyethoxyethanol; Cremophor EL & , Nonidet P 40 ® (Shell).
  • the detergents are used in the form of dilute aqueous solutions. Solutions with 0.1 to 10 percent by volume, preferably with 0.5 to 5 percent by volume, particularly preferably about 1 percent by volume of detergent content are mentioned.
  • the detergent solution is added to the virus concentrate in a volume ratio of approximately 1: 1 to approximately 10: 1.
  • the ratio of detergent solution to is preferred
  • the detergent treatment is carried out with constant movement of the mixture at temperatures between 0 and approximately 24 ° C., preferably between 2 and 8 ° C.
  • the detergent treatment lasts 15 minutes to 2 days, preferably 6 to 18 hours.
  • the mixture can additionally contain one
  • the particles not dissolved in this treatment are removed, preferably by filtration or centrifugation at, for example, 150,000 x g.
  • the filtrate or the centrifugation supernatant obtained in this way can be stored at low temperatures (0 to -70 ° C) until it is processed further.
  • the glycoproteins of the virus particles contained in the lysate are isolated by treatment with lectins.
  • Lectins are proteins or glycoproteins from plants, especially their seeds, microorganisms, vertebrates and invertebrates, which specifically bind sugar and their conjugates. Lectins are used that recognize and bind glycoproteins from paramyxoviruses. Lectins which recognize mannose and / or glucose and their conjugates are preferably used.
  • the lectins from Canavalia ensifo ⁇ nis, Lens culinaris, Lathygros odoratus, Pisum sativum, Vicia faba, Sambucus nigra, Glycine max, Ulex europaens, Helix promatia, Phytolacca americana, Lycopersicon esculentum, Datura stramonicea, bandeira should be mentioned.
  • the lectins are used in water-soluble or water-insoluble form. In the water-insoluble form, they are preferably immobilized by coupling to inert matrix such as dextrans, agaroses, celluoses as suspensions or gels.
  • inert matrix such as dextrans, agaroses, celluoses as suspensions or gels.
  • Concanavalin-A-agarose may be mentioned in detail , Concanavalin-A-Sepharose, Lentil-Lectin-Sepharose, Agarose-Wheat-
  • the lectins are used in the form of a detergent and salt-containing solution, suspension or a gel.
  • a detergent and salt-containing solution for this purpose, both the lysate and the lectin solution, lectin suspension or the lectin gel used are mixed with sufficient sodium chloride and the known lectin-stabilizing salts that a
  • Concentration of common salt of 0.5 to 2, preferably 0.7 to 1.2 mol / 1 arises.
  • concentration setting of the lysates is preferably carried out by dialysis.
  • the required concentration of the lectin-stabilizing salts is known from the prior art and is specific for the lectins to be used.
  • the lectin solution, lectin suspension or the lectin gel is also mixed with the detergent used to treat the lysates in the same concentration, so that the lysate and lectin solution have identical concentrations of salt and detergent.
  • Total protein 0.01 to 50 mg, preferably 0.1 to 20 mg, particularly preferably 0.5 to 5 mg, lectin can be used.
  • the lectin treatment takes place at 0 to approx. 24 ° C, preferably at 2 to 8 ° C for about 10 minutes to 3 days, preferably 1 hour
  • the reaction of the lectins with the glycoproteins can also be carried out by means of column chromatography, the lysate being brought into contact with the lectin immobilized on a gel-like matrix in a chromatography column.
  • glycoprotein-lectin complex is separated from the total solution or suspension in a conventional manner. This can be done by centrifugation, filtration or, in the case of chromatography, by washing.
  • the suspensions or gels obtained in these processes and containing the lectin-glycoprotein complexes can be filtered off. Centrifugation, dialysis or other washing processes, the concentration of detergent and / or salt can be changed within the physiologically acceptable range or until it is eliminated
  • suspensions or gels of the lectin-glycoprotein complexes obtained in this way can be used directly as antigenic material. They can be further concentrated or diluted depending on the content of the glycoprotein bound to lectin.
  • the suspensions or gels of the lectin-glycoprotein complexes can be stored at temperatures below 8 ° C. They can also be freeze-dried.
  • the glycoproteins can be isolated from the suspensions or gels of the lectin-glycoprotein complexes obtained.
  • suspensions or gels are treated with a saline, aqueous sugar solution.
  • the type of sugar to be used depends on the specificity of the lectins used.
  • the concentration of the sugar is 0.1 to 1 mol / l, preferably 0.1 to 0.5 mol / l, particularly preferably 0.3 to 0.5 mol / l.
  • the concentration and composition of the salt content corresponds to that of the glycoprotein lectin.
  • the sugar solution is treated at 0 to about 24 ° C., preferably at 2 to 8 ° C.
  • the treatment is about 15 minutes to 4 days, preferably 1 hour to 2 days, particularly preferably 10 to 24 hours.
  • glycoproteins eluted in this process are isolated from the lectins by centrifugation, filtration or by other customary separation processes (e.g. chromatography).
  • concentrations of detergent, salt and sugar can be changed as already described above.
  • the isolated glycoproteins obtained in this way can be used as antigenic material.
  • the glycoprotein content can be changed by concentration or dilution.
  • the preparations are stored in the form of their solutions at temperatures below 0 ° C. or in lyophilized form.
  • the virus genome is first obtained.
  • the viruses are first multiplied in the usual way on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned permanent pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent one
  • tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned permanent pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent one
  • Monkey kidney cells BGM Flow 03-240 or their descendants
  • Vero ATCC CCL81 or their descendants
  • bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or their descendants) and on the other hand in embryonated chicken eggs (e.g. Valo - hatching eggs, Lohmann).
  • Roller or carrier cultures in the form of closed cell groups (monolayers) or in suspension cultures. All known cell culture media are used as propagation media for the cells, for example described in the product catalog of Gibco BRL GmbH, Dieselstrasse 5, 76344 Eggenstein, such as in particular the Minimal Essential Medium (MEM), which contains amino acids, vitamins, salts and carbohydrates as essential components, completed with buffer substances such as sodium bicarbonate (NaHCO ? ) or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and optionally animal sera, such as sera from cattle, horses or their fetuses.
  • buffer substances such as sodium bicarbonate (NaHCO ? ) or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes)
  • animal sera such as sera from cattle, horses or their fetuses.
  • the cells and cell turf used to multiply the viruses are multiplied in the usual way almost to confluence or to the optimal cell density.
  • MOI multiplicity of infection
  • viruses are propagated with or without the addition of animal sera. In the event that serum is used, this becomes the propagation medium in a
  • Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 32 and 39 ° C., particularly preferably at 37 ° C. for several days, preferably until the infected cells are completely destroyed.
  • the virus-containing medium of the infected cells is processed further, e.g. by removing the cells and cell debris by means of filtration with pore sizes of e.g. 0.1-0.45 ⁇ m and / or centrifugation up to 10,000 x g.
  • the multiplication in embryonated chicken eggs takes place in a manner known per se in the Allantoic cave of chicken hatching eggs, which lasts 9-12 days, preferably 10 days , > ⁇
  • the hatching eggs used to multiply the viruses are stored vertically in the incubator for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation and then infected with 10 to 200 ⁇ l, preferably 75 to 125 ⁇ l, of a virus suspension after preparation of the injection site
  • the virus propagation medium used are all cell culture media known per se, in particular the MEM mentioned above
  • Infection and virus multiplication take place under the breeding conditions specified above over several days, preferably 2-5 days, particularly preferably 3 days.
  • the virus-containing allantoic fluid is obtained by suction after opening the lime shell and the skin of the shell and the choalloallantoic membrane and can be worked up further, for example by means of filtration with pore sizes of, for example, 0 1-0.45 ⁇ m and / or centrifugation up to 10,000 ⁇ g
  • Virus purification or isolation is achieved by isopycnic or zonal centrifuge in, for example, sucrose density gradients.
  • the virus-containing medium or the allantoic fluid is subjected to zone centrifugation at 100,000 xg after removal of the cell debris until sedimentation of the virus particles results in a cleaner representation of the virus particles by zone centrifugation in an aqueous solution with a higher density than the virus-containing medium.
  • a 30-60% w / w, preferably 35-50%> w / w, buffered solution of sucrose can serve as a water solution.
  • An even higher degree of purity is achieved by centrifugation in a density gradient.
  • the cells and cell debris are freed from Zone centrifugation concentrated virus by an isopycnic or zonal density gradient centrifugation in a density gradient of, for example, 30 to 50%> w / w sucrose in buffered aqueous solution with a centrifugal acceleration ⁇ jn z 100 000 to 150 000 xg isolated
  • the viral genome is first isolated from the purified virus particles.
  • the native virus RNA is preferably obtained by treating the purified viral poultry with detergent- and protease-containing, aqueous solutions
  • Ionic detergents preferably sodium dodecyl sulfate, are preferably used in a concentration of 0.1-10% by volume, preferably 0.5-3% by volume.
  • proteases used are those which act in the presence of detergents, such as pronase and, preferably, proteinase K.
  • the proteases are used in a concentration of 0.01-10 mg / ml, preferably 0.05-0.5 mg / ml used
  • Aqueous, buffered solutions with the addition of RNase inhibitors are preferably used
  • Salts of weak acids with strong bases such as, for example, tris (hydroxymethyl) aminomethane
  • salts of strong acids with weak bases such as, for example, primary phosphates or mixtures thereof, are preferably used as tris (hydroxymethyl) aminomethane used, which ensure a pH value at which the RNA does not denature.
  • Preferred pH values are 6-8.5, particularly preferably 7-8
  • RNase inhibitors such serve B ribonucleoside vanadyl complexes, Protem inhibitors (eg RNAguard ® / Pharmac ⁇ a) or preferably diethyl pyrocarbonate
  • the lipophilic substances of the virus lysate are then extracted using solvents such as phenol, chloroform or mixtures thereof. The extraction takes place in one or more stages.
  • the RNA is precipitated using aqueous solutions containing alcohols such as e.g. Ethanol or isopropanol and monovalent chloride or acetate salts such as e.g. Contain sodium chloride, sodium acetate or potassium acetate.
  • alcohols such as e.g. Ethanol or isopropanol
  • monovalent chloride or acetate salts such as e.g. Contain sodium chloride, sodium acetate or potassium acetate.
  • the concentration of the alcohols is between 40 and 100% by volume, preferably 60 and 80% by volume, and that of the chloride or acetate salts is between 0.01 and 1 mol / l, preferably 0.1 to 0.8 mol / l.
  • the precipitated RNA is extracted from the aqueous solution e.g. obtained by centrifugation and in an aqueous solution e.g. DEPC water dissolved again.
  • This aqueous solution preferably contains buffer substances such as e.g. Tris (hydroxymethyl) aminomethane in concentrations of 1-100 mmol / l, preferably 10-50 mmol / l, possibly with the addition of ethylenediaminetetraacetate (EDTA) in concentrations of 0.1-10 mmol / l, preferably 1 -10 mmol / l or dithiothreitol (DTT) in concentrations of 0.1-10 mmol / l, preferably 1-10 mmol / l.
  • buffer substances such as e.g. Tris (hydroxymethyl) aminomethane in concentrations of 1-100 mmol / l, preferably 10-50 mmol / l, possibly with the addition of ethylenediaminetetraacetate (EDTA) in
  • the isolated RNS is stored at temperatures below -65 ° C.
  • RNA isolation is e.g. RNA extraction with guanidinium thiocyanate and subsequent cesium chloride density gradient centrifugation of the virus lysate.
  • Suitable genes are identified using the isolated virus
  • Genome e.g. through: * ⁇ -
  • RNA / DNA hybridization of the genome using known gene probes DNA probes with nucleotide sequences of known genes for immunogens of related virus strains, such as e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
  • cDNA complementary DNA
  • bacterial plasmids such as pBR322
  • gene probes are DNA samples with nucleotide sequences of known genes for immunogens of related virus strains, e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
  • cDNA complementary DNA
  • plasmid expression vectors such as e.g. pUC18 / 19 or pUC 118/119 or in ⁇ bacteriophage expression vectors such as e.g. ⁇ gtl 1 and its descendants or ⁇ ZAP or ⁇ ORF8.
  • the genes are identified by detecting their expressed immunogens with the help of
  • Antibodies that directly or indirectly e.g. by means of immunofluorescence or immunoprecipitation. Suitable antibodies are those which react with immunogens of related virus strains, e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
  • cDNA complementary DNA
  • bacterial plasmids for the enrichment of viral DNA.
  • the viral DNA of the clones is sequenced and examined for sequence homologies with known genes from related virus strains, e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
  • Genes are selected in which a nucleotide sequence which codes for one or more immunogens can be detected using the methods mentioned above.
  • sequence listing shows the nucleotide sequences with the corresponding amino acid
  • the Simian Virus 40 (SV40) and plasmid expression vectors are suitable which are suitable for being selected and propagated in prokaryotes (e.g. E. coli) and which have regulatory elements for the expression of the foreign DNA in higher cells.
  • prokaryotes e.g. E. coli
  • Suitable plasmid expression vectors are e.g. plasmid vectors such as pMSG, pSVT7 or pMT2 based on the SV40, or plasmid vectors such as pHEBo or p205 based on the Ebbstein-Barr virus.
  • Suitable cells are animal cells, in particular permanent cell lines, such as the pig kidney cell PK15 (ATCC CCL33 or its descendants), the monkey kidney cell BGM (Flow 03-240 or its descendants) or Vero (ATCC CCL81 or its descendants) , the bovine kidney cell MDBK (ATCC CCL22 or its descendants), the dog kidney cell MDCK (ATCC CCL34 or its descendants) or the rabbit kidney cell RK-13 (ATCC CCL37).
  • the transfection takes place e.g. in the form of calcium phosphate-DNA coprecipitates or by the DEAE / dextran method, the liposome method or by electroporation.
  • Suitable antibodies are those which react with immunogens of related virus strains, e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
  • Suitable are e.g. (i) bacterial plasmid expression vectors, (ii) viral expression vectors for bacteria or (iii) viral expression vectors for higher ones
  • Suitable bacterial plasmid expression vectors are e.g. pUC18 / 19 or pUC
  • Cells preferably bacteria, used and multiplied. Suitable is e.g. Escherichia coli K12 and its descendants.
  • plasmid e.g. calcium phosphate-DNA coprecipitation or electroporation.
  • Suitable viral expression vectors for bacteria are ⁇ bacteriophage vectors such as e.g. ⁇ gtl l and descendants, ⁇ ZAP or ⁇ ORF8.
  • the multiplication of the ⁇ bacteriophage vectors takes place in particular in Escherichia coli e.g. E. coli Kl 2 and its descendants.
  • Suitable viral expression vectors for higher cells are e.g. the Simian Virus 40, adenoviruses, herpes simplex virus or baculoviruses.
  • the viral vectors are propagated in appropriate cell systems.
  • the expressed immunogens are used either directly in the form of the expression systems (culture substrate and / or cells) or after preparation and purification by means of biochemical and / or immunological methods and, if appropriate, after concentration or dilution as an antigenic material.
  • Suitable for cleaning are e.g. Affinity or gel chromatographic processes in which the immunogens are separated or isolated from the expression system, if appropriate after its disruption by detergent treatment.
  • the virus genome is first obtained.
  • the viruses are first multiplied in the usual way in tissue cultures of animal cells as primary cells or permanent cell lines, for example in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as, for example cloned, permanent pig kidney cell PK15 (ATCC CCL33 or its derivatives) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or its derivatives) or Vero (ATCC CCL81 or its derivatives) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its derivatives) and, on the other hand, in embryonic chicken eggs (eg Valo hatching eggs, Lohmann).
  • embryonic chicken eggs eg Valo hatching eggs, Lohmann.
  • the multiplication in cell cultures takes place in a manner known per se in stationary roller or carrier cultures in the form of closed cell groups (monolayers) or in suspension cultures. All cell culture media known per se are used as propagation media for the cells, for example described in the product catalog of Gibco BRL GmbH, Dieselstrasse 5, 76344 Eggenstein, such as in particular the Minimal Essential Medium (MEM). which contains amino acids, vitamins, salts and carbohydrates as essential components, supplemented with buffer substances such as sodium bicarbonate (NaHCO 3 ) or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and optionally animal sera such as sera from Cattle, horses and their fetuses. Eagles MEM with a NaHC ⁇ 3 content of 0.1-5 g / 1, preferably 0.5-3 g / 1 and fetal calf serum in a concentration of 1-30% by volume, preferably 2-10, are particularly preferably used Vol%.
  • MEM Minimal Essential Medium
  • the cells and cell turf used to multiply the viruses are multiplied in the usual way almost to confluence or to the optimal cell density.
  • the cell growth medium Before being infected with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus growth medium. All known cell culture media, such as in particular the MEM mentioned above, are used as the virus propagation medium.
  • the viruses are propagated with or without the addition of animal sera. If serum is used, it is added to the propagation medium in a concentration of 1-30% by volume, preferably 2-10% by volume.
  • Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 32 and 39 ° C., particularly preferably at 37 ° C. for several days, preferably until the infected cells are completely destroyed.
  • the virus-containing medium of the infected cells is processed further, e.g. by removing the cells and cell debris by means of filtration with pore sizes of e.g. 0.1-0.45 ⁇ m and / or centrifugation up to 10,000 x g.
  • the multiplication in embryonated chicken eggs takes place in a manner known per se in the Allantoic cavity of chicken hatching eggs, which lasts 9-12 days, preferably 10 days, at a temperature of 37-39 ° C., preferably 38.5 ° C. and a relative atmospheric humidity 30-90%, preferably 50-60%, in a commercial incubator, preferably a motor breeder.
  • the hatching eggs used to multiply the viruses are stored vertically in the incubator for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation and then infected with 10-200 ⁇ l, preferably 75-125 ⁇ l, of a virus suspension after preparation of the injection site .
  • All known cell culture media, such as in particular the MEM mentioned above, are used as virus propagation medium.
  • Infection and virus multiplication take place under the breeding conditions specified above over several days, preferably 2-5 days, particularly preferably 3 days.
  • the virus-containing allantoic fluid is obtained by suctioning off after opening the lime shell as well as the skin of the shell and the chorioallantoic membrane and can eg by filtration with pore sizes of, for example, 0.1-0.45 ⁇ m and / or centrifugation up to 10,000 ⁇ g.
  • Virus cleaning or isolation is calibrated by isopycnic or zonal centrifugation in e.g. Sucrose density gradients.
  • the virus-containing medium or the allantoic fluid is removed after removing the cell debris
  • Zone centrifugation at 100,000 x g subjected to sedimentation of the virus particles A purer representation of the virus particles results from zone centrifugation in an aqueous solution with a higher density than the virus-containing medium.
  • an aqueous solution e.g. serve a 30-60% w / w, preferably 35-50% w / w, buffered solution of sucrose. An even higher one
  • the degree of purity is calibrated by centrifugation in the density gradient.
  • the virus, freed from cells and cell debris is concentrated by means of zone centrifugation by isopycnic or zonal density gradient centrifugation in a density gradient of, for example, 30 to 50% w / w * sucrose in buffered aqueous solution with a centrifugal acceleration of, for example, 100,000 to
  • the virus genome is first isolated from the purified virus particles.
  • the native virus RNA is preferably obtained by treating the purified virus particles with aqueous solutions containing detergent and proteases.
  • Ionic detergents preferably sodium dodecyl sulfate, are preferably used in a concentration of 0.1-10% by volume, preferably 0.5-3% by volume.
  • Proteases used are those which act in the presence of detergents, such as e.g. Pronase and, preferably used Proteinase K.
  • the proteases are used in a concentration of 0.01-10 mg / ml, preferably 0.05-0.5 mg / ml.
  • Aqueous, buffered solutions with the addition of RNase inhibitors are preferably used.
  • Salts of weak acids with strong bases such as, for example, tris (hydroxymethyl) aminomethane, salts of strong acids with weak bases such as, for example, primary phosphates or mixtures thereof are used as buffer substances.
  • Tris (hydroxymethyl) aminomethane is preferably used.
  • the buffer substances are used in concentrations that ensure a pH value at which the RNA does not denature. PH values of 6-8.5 are preferred, particularly preferably 7-8.
  • RNase inhibitors are used, for example, ribonucleoside-vanadyl complexes, protein inhibitors (eg RNAguard ® / Pharmacia) or preferably diethyl pyrocarbonate (DEPC) in concentrations of 0.01-2% by volume, preferably 0.1-0.5 vol- %.
  • protein inhibitors eg RNAguard ® / Pharmacia
  • DEPC diethyl pyrocarbonate
  • the ipophilic substances of the virus lysate are then extracted using solvents such as e.g. Phenol, chloroform or mixtures thereof.
  • solvents such as e.g. Phenol, chloroform or mixtures thereof.
  • the extraction takes place in one or more stages.
  • the RNA is precipitated using aqueous solutions containing alcohols such as e.g. Ethanol or isopropanol and monovalent chloride or acetate salts such as e.g. Contain sodium chloride, sodium acetate or potassium acetate.
  • alcohols such as e.g. Ethanol or isopropanol
  • monovalent chloride or acetate salts such as e.g. Contain sodium chloride, sodium acetate or potassium acetate.
  • the concentration of the alcohols is between 40 and 100% by volume, preferably 60 and 80% by volume, and that of the chloride or acetate salts is between 0.01 and 1 mol / l, preferably 0.1 to 0.8 mol / l .
  • the precipitated RNA is extracted from the aqueous solution e.g. obtained by centrifugation and in an aqueous solution e.g. DEPC water dissolved again.
  • This aqueous solution preferably contains buffer substances such as e.g. Tris (hydroxymethyl) aminomethane in concentrations of 1-100 mmol / l, preferably 10-50 mmol / l, possibly with the addition of ethylenediaminetetraacetate (EDTA) in
  • DTT dithiothreitol
  • RNA isolation is, for example, RNA extraction with guanidinium thiocvanate and subsequent casium chloride density gradient centrifugation of the virus lysate
  • Suitable genes are identified using the isolated virus genome, for example by:
  • RNA / DNA hybridization of the genome using known gene probes DNA probes with nucleotide sequences of known genes for immunogens of related ⁇ uß strains, such as the Simian virus 5 or the canine paramfluenza virus 2, serve as suitable gene probes
  • cDNA complementary DNA
  • DNA probes with nucleotide sequences of known genes for immunogens of related virus strains such as, for example, the Simian virus 5 or the canine parainfluenza virus 2, serve as suitable gene probes
  • cDNA complementary DNA
  • plasmid expression vectors such as e.g. pUC18 / 19 or pUC 118/119 or in ⁇ -bacteriophage expression vectors such as ⁇ gtl l and its descendants or ⁇ ZAP or ⁇ ORF8.
  • the genes are identified by detecting their exposed immunogens with the help of antibodies which are directly or indirectly, for example, by means of immunofluorescence or Immunoprecipitation can be detected.
  • Suitable antibodies are those which react with immunogens of related virus strains, such as the Simian virus 5 or the canine parainfluenza virus 2
  • cDNA complementary DNA
  • bacterial plasmids for the enrichment of viral DNA DNA of the clones is sequenced and examined for sequence homologies with known genes from related virus strains, such as the Simian virus 5 or the canine parainfluenza virus 2.
  • Genes are selected in which a nucleotide sequence can be detected using the methods mentioned above, which sequence is suitable for one or more
  • the sequence listing shows the nucleotide sequences with the corresponding amino acid sequences of the hemagglutinin neuraminidase and the fusion protein gene of the Parainfluenza virus 2 deposited with CNCM under number 1-1331.
  • genes which code for one or more immunogens (foreign DNA) are inserted into a genome vector which expresses the foreign gene when a cell or an organism is infected.
  • Vector viruses and vector bacteria are suitable for this.
  • apathogenic DNA viruses are used which have a stable genome with known insertion sites for the uptake of 0.1 to - .->->
  • Suitable shuttle vectors are plasmid or bacteriophage vectors.
  • Examples of useful plasmid vectors are pBR322, pUC18 / 19, pAT153, ⁇ ACYC184 or pSP64 / 65 and for bacteriophage vectors ⁇ gtlO / 11, ⁇ ZAP or M13mpl8 / 19
  • the DNA fragment carrying the vector virus insertion site is inserted into the shuttle vector DNA.
  • the shuttle vector DNA prepared in this way is mixed with an excess of the DNA fragment to be inserted, for example in a ratio of 1 5 Das DNA mixture is treated with DNA ligases in order to covalently bind the DNA fragment in the vector.
  • shuttle plasmid is used in pro- or eukaryotic cells, preferably bacteria, and is increasingly suitable.
  • pro- or eukaryotic cells preferably bacteria
  • Escherichia coli K12 and its derivatives are suitable
  • Bacteria carrying plasmids containing DNA fragments are selected
  • Polylinkers are DNA sequences with at least two defined restriction enzyme interfaces in a row.
  • the DNA fragment carrying the insertion site is treated with a restriction enzyme such that the fragment is opened (cut) only at one point.
  • the fragment thus prepared is incubated together with the polylinker and DNA ligase for the targeted insertion of defined restriction-erizyme sites.
  • the polylinker can be inserted into the isolated or the insertion site-carrying DNA fragment cloned in shuttle vectors.
  • polylinker is used in isolated DNA fragments, these must then be inserted into a shuttle vector. If a shuttle plasmid is used, it is used and propagated in pro- or eukaryotic cells, preferably bacteria. Suitable is e.g. Esche ⁇ chia coli K12 and its descendants. Bacteria containing plasmids containing DNA fragments are selected.
  • polylinker is inserted into the DNA fragment cloned into shuttle vectors, these are multiplied and selected.
  • Genes that code for one or more immunogens are inserted into the insertion sites.
  • partial sequences of the DNA fragment carrying the insertion site are removed beforehand.
  • the DNA fragment is treated with restriction enzymes, and the resulting DNA fragments are separated.
  • the isolated or the DNA fragment cloned in shuttle vectors which carries the insertion site, is first inserted with or treated several restriction enzymes and opened the fragment at the insertion site or at the polylinker used.
  • the foreign DNA is inserted into the insertion site prepared in this way, for example with the aid of DNA ligases.
  • Shuttle plasmids are used and propagated in pro- or eukaryotic cells, preferably bacteria. Suitable is e.g. Escherichia coli K12 and its descendants. Bacteria which contain plasmids containing foreign DNA are selected.
  • Method (i) which is carried out in the form of the calcium phosphate DNA precipitation technique, is preferably used. The following steps are necessary:
  • the shuttle vector is multiplied, isolated and further purified.
  • the purification of the shuttle vector DNA takes place e.g. by means of isopycnic centrifugation in density gradient, e.g. a cesium chloride density gradient.
  • the vector virus is multiplied and purified.
  • the viral genome is isolated and further purified.
  • the vector virus DNA is cleaned e.g. by means of isopycnical centrifugation in density gradient, e.g. a cesium chloride density gradient.
  • Circular or preferably linearized shuttle vector DNA is used for cotransfection.
  • the linearized shuttle vector DNA is obtained e.g. by treating the purified DNA with restriction enzymes. Restriction enzymes are preferred which have no recognition site (interface) in the inserted foreign DNA, i.e. the foreign DNA sequence is not broken up.
  • the vector virus DNA and the shuttle vector DNA are mixed, for example, in a ratio of 0.01 to 0.1 ⁇ 10 12 mol l vector virus DNA to 1 ⁇ 3 ⁇ 10 12 mol / l shuttle Vector DNS.
  • the DNA mixture is coprecipitated with e.g. Calcium phosphate and transferred to suitable cells.
  • Suitable cells are animal cells, in particular permanent cell lines, such as e.g. the pig kidney cell PK15 (ATCC CCL33 or its
  • Bovine kidney cell MDBK (ATCC CCL22 or its descendants), the dog kidney cell MDCK (ATCC CCL34 or its descendants) or the rabbit kidney cell RK-13 (ATCC CCL37).
  • the cotransfection can also be carried out using other methods. As such, e.g. called the DEAE / dextran method, the liposomes
  • the cells are cultured, e.g. according to the methods described above. If a cythopathogenetic effect occurs, clones of the vector virus are isolated using the single plaque purification methods and further multiplied.
  • Recombinant vector viruses are selected (i) by detecting the expression of the foreign gene or (ii) by detecting the inserted foreign DNA in the vector virus genome, e.g. through DNA / DNA hybridization.
  • the expression of the foreign DNA is detected, for example, with the aid of antibodies.
  • Suitable antibodies are those which react with immunogens of related virus strains, e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
  • Foreign DNS can e.g. by means of immunofluorescence or immunoprecipitation.
  • the inserted foreign DNA is detected by hybridization with gene probes, the corresponding foreign gene.
  • Stable recombinant vector viruses are used in known, customary processes, as described above, isolated and further processed, as antigenic material.
  • the antigenic material is present as such or in a mixture with the usual formulation auxiliaries. These include pharmacologically acceptable solvents or diluents, adjuvants, preservatives, suspending agents or solubilizers such as emulsifiers.
  • the antigenic material is used as a biologically active substance in the
  • the antigenic material is used in the form of living virus particles, to which additives and, if necessary, defoamers and preservatives are added.
  • the live vaccine is freeze-dried for better durability.
  • the lyophilized product is vaccinated with a solvent such as e.g. Reconstituted aqua dest., Aqua purificata or 0.9% saline.
  • a solvent such as e.g. Reconstituted aqua dest., Aqua purificata or 0.9% saline.
  • the virus particles freed from the cell substrate are at a concentration of at least 10 6 KED 50 / ml together with protective colloids or stabilizers, such as celluloses, dextrans, gelatins, collidones or stearates and optionally with the addition of defoamers, such as tributyl phosphate, isopropanol or Silicone oil and preservatives such as merthiolate or thimerosal mixed in an aqueous pH buffered solution, filled into appropriate containers and freeze-dried.
  • protective colloids or stabilizers such as celluloses, dextrans, gelatins, collidones or stearates
  • defoamers such as tributyl phosphate, isopropanol or Silicone oil
  • preservatives such as merthiolate or thimerosal mixed in an aqueous pH buffered solution, filled into appropriate containers and freeze-dried.
  • Material uses complete, killed virus particles in a concentration of 10 '° -10 9 - 0 KID 50 / ml, preferably 10 5 * ° -10 so KID 50 / ml before inactivation or parts (subunits) of the virus particles in such a concentration that 10-250 mg protein, preferably 10-100 mg protein are contained per vaccine dose.
  • the antigenic material is present in the vaccine in a mixture with the usual ones
  • Formulation auxiliaries such as solvents and diluents, adjuvants, preservatives, suspending agents or solubilizers, pH regulators and optionally defoamers.
  • Solvents and diluents which may be mentioned are aqua dest., Aqua purificata, physiologically compatible salt solutions and cell culture media. Find in particular Use the above-mentioned E-MEM and phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • Mineral salts such as aluminum hydroxide, aluminum phosphate, calcium phosphate, kaolin or silicon. 10-50% by volume, preferably 25-35% by volume, of an aluminum hydroxide gel with a proportion of 1-5% (w / v), preferably 2-3% (w / v), of aluminum hydroxide are preferably used.
  • Oily adjuvants such as non-toxic mineral oils (e.g. Draceol ® , paraffin oil), vegetable oils (e.g. lecithins, peanut oils) or animal oils - "(Squalane, Squalene), which are in a concentration of 1-40 vol% , preferably 1-15% by volume are used.
  • mineral oils e.g. Draceol ® , paraffin oil
  • vegetable oils e.g. lecithins, peanut oils
  • animal oils - "(Squalane, Squalene) which are in a concentration of 1-40 vol% , preferably 1-15% by volume are used.
  • Hydrophilic and hydrophobic polymers such as polyoxyethylene and polyoxypropylene.
  • Synthetically produced block polymers eg Pluronic ® L101, Pluronic ® L121, Pluronic ® L122, Tetronic ® 1501 are preferably used in a concentration of 1-10% by volume.
  • Adjuvants of bacterial origin such as pertussis toxin (Bordetella pertus- sis), Salmonella typhimurium mltogen or bacterial endotoxins such as lipopolysaccharides (LPS, e.g. from Mycobacteria or Salmonella) as well as LPS analogues or derivatives such as e.g. Lipid-A, Monophosphoryl-Lipid-A (MPL), Diphosphoryl-Lipid-A, (DPL), Trehalose-Dimycoiat (TDM),
  • LPS lipopolysaccharides
  • MPL Monophosphoryl-Lipid-A
  • DPL Diphosphoryl-Lipid-A
  • TDM Trehalose-Dimycoiat
  • MDP Muramyl dipeptide
  • AdDP adamantyl dipeptide
  • MDP derivatives or AdDP are preferably used in a concentration of 0.0001-10%) (w / v).
  • Organic water-dispersible adjuvants such as cholesterol, gelatin, phosphatidylcholine, polysaccharides (e.g. zymosan, agar), aliphatic
  • Amines e.g. dimethyldioctadecylamine / DDA, N, N.diotadecyl-N ', N'-bis (2-hydroxyethyl) propanediamine Avridin ® ), DEAE-dextrans or saponin (from the
  • formalin in concentrations of up to 1%, phenol and benzyl alcohol in concentrations of up to 0.5%, sorbic acid, benzoic acid, sodium benzoate, and their derivatives such as e.g. the sodium salt of 2- (ethylmercurio-thio) -benzoic acid (merthiolate, thimerosal, thiomersal) or the sodium salt of 4- (ethylmercurio-thio) -benzenesulfonic acid (thiomerfonate).
  • Merthiolate is preferably used in concentrations of 0.01% to 0.5%.
  • Suspensions and solubilizers which may be mentioned are non-toxic surface-active substances such as vegetable proteins, alginates, celluloses, phospholipids and in particular substances based on glycol ethers such as polyethylene glycols and their derivatives.
  • Polyethylene glycol (PEG) 200, 300, 400, 600 and 900 and PEG derivatives are preferably used, particularly preferably Tween ® 80 in a concentration of 0.05- 5 vol%, preferably 0.2-1 vol%.
  • substances that regulate pH are e.g. Sodium and potassium hydroxide, sodium and potassium carbonate, acetic, tartaric and citric acid or hydroxyethy lpiperazi n-N-2-ethanesulfonic acid (HEPES).
  • HEPES hydroxyethy lpiperazi n-N-2-ethanesulfonic acid
  • Defoamers include tributyl phosphate, isopropanol, silicone oil, Antifoam ® or Baysilon ® defoamers EBZ.
  • Parainfluenza viruses according to the invention which can cause diseases of the respiratory and reproductive tract of pigs can be e.g. received as follows:
  • Organs are removed from pigs suffering from PRRS-like symptoms and subjected to a virus isolation test. Weak or sick piglets from infected herds are particularly suitable.
  • the internal organs in particular the lungs, liver, kidney and spleen, are removed from a suitable animal. Parts of these organs or organ mixtures homogenized with physiologically compatible aqueous solutions to form suspensions, the proportion of organ parts accounting for approx. 10% (w / v).
  • the Eagles Minimum Essential Medium (E-MEM) described above is particularly suitable as a suspending agent. The suspensions are separated by centrifugation approx. 1500 xg freed of cells and cell drums. Another cleaning of the E-MEM
  • Centrifugation supernatant can be done by filtration. Filters with a pore size of 0.2-5 ⁇ m, preferably 0.2-0.45 ⁇ m, are suitable for this.
  • a primary cell culture can be created from the organs removed, preferably the lungs, which is examined for the appearance of a cythopathogenic effect (CPE).
  • CPE cythopathogenic effect
  • the tissue is roughly crushed and subjected to enzymatic digestion by proteases. Trypsin in a concentration of 0.1-0.5% (w / v), preferably 0.125-0.25% (w / v) in a physiological, watery solution.
  • the trypsin digestion takes place at 20-37 ° C., preferably at room temperature in 2-8 hours Undigested tissue components are separated by coarse filtration.
  • the trypsinized cells are obtained by centrifugation at 500-1500 xg.
  • the cell sediment is resuspended in a suitable growth medium, such as the E-MEM described, and in a concentration of l ⁇ ⁇ cells / ml medium sown in culture vessels Depending on the growth rate, the growth medium is replaced every 3-7 days.
  • a suitable growth medium such as the E-MEM described
  • the cell culture supernatant can be checked for hemagglutinating properties at fixed time intervals of 2-7 days
  • the centrifugation supernatants or filtrates of the organ homogenates and the cell culture supernatants of the primary organ cultures applied are applied in a dilution of 1 1 to 1 1000, preferably 1 10 to 1 100, to primary or permanent mammalian cell cultures and at 32-39 ° C, preferably 37 ° C, incubated over several days.
  • cell turf that has grown to 20-100%, preferably 80-100%) confluency is used.
  • the cell cultures are examined daily for the occurrence of a CPE
  • the supernatant can be checked for hemagglutinating properties at fixed intervals of 2-7 days. If there are no signs of virus multiplication, the cell culture supernatants in the dilutions mentioned are passed on to fresh cell cultures. This process can be repeated several times.
  • the virus is adapted to the cell culture used by further passages.
  • a cythopathogenic agent was isolated from the lungs of this piglet by applying a primary lung cell culture and by passing the resulting culture supernatant onto animal cell lines. It was characterized as a enveloped, hemagglutinating, approx. 200 nm large, single-stranded RNA virus which has the morphology of a paramyxovirus by electron microscopy. Proteins of this virus were recognized in the Western blot from an antiserum against a parainfluenza virus type 2 (Pl-2). In the same test system, an antiserum produced against the isolated virus recognized a Pl-2 strain "SV5", which shows the serological relationship of the isolated
  • Virus to parainfluenza virus type 2 is secured.
  • the isolated virus can be used on a large scale using animal
  • Purified antigen preparations can be produced from virus suspensions produced in this way using suitable technical processes (centrifugation, tangential filtration). These can be used as starting material for the diagnosis and prevention of respiratory and reproductive diseases in pigs, in particular the PRRS. example 1
  • the parainfluenza isolate "SER" could be isolated from the lungs of a piglet, which was removed from the uterus during the sectioning of a euthanized, aborting sow, which came from a herd with PRRS-like symptoms.
  • PK15 cells (cloned pig kidney cell, ATCC No. CCL 33)
  • E-MEM Eagles Minimum Essential Medium with Earle 's Salts
  • E-MEM - powder with phenol red e.g. Gibco BRL 072-01 10
  • 100 1 non-essential amino acids stock solution 100 x 1000 ml
  • trypsin solution e.g. Gibco BRL 043-05050
  • PBS buffer Phosphate Buffered Saline
  • Tissue culture bottle 80 cm “(Roux bottle, e.g. Greiner 658 170)
  • CPE cytopathogenic effect
  • the culture was subjected to a freeze-thaw process and the supernatant was diluted 1:10 and inoculated into a fresh culture, the supernatant of which was tested after 6-7 days in the hemagglutination test using chicken erythrocytes. In the 4th passage, the cultures showed almost 100% cytopathogenic effect after 6 days of incubation. Culture supernatants positive in the hemagglutination test were stored at -70 ° C.
  • SER Parainfluenza isolate "SER"
  • basic seed material - PK-15 cells (cloned pig kidneys - cell, ATCC No. CCL 33)
  • E-MEM Eagles Minimum Essential Medium with Earle 's salts
  • FCS Fetal calf serum
  • FCS Fetal calf serum
  • Growth medium E-MEM with 2.0 g / 1 sodium bicarbonate and 2%
  • FCS Maintenance medium E-MEM with 0.85 g / 1 sodium bicarbonate and 5% FCS - tissue culture bottle, 80 cm (Roux bottle, e.g. Greiner 658 170)
  • Tub stack 6000 cm 2 (e.g. Nunc 164 327)
  • the growth medium of a tissue culture bottle confluently overgrown with PK-15 cells is decanted and this is charged with 40 ml of the basic virus seed material diluted 1:50 in maintenance medium. After 7 days incubation with
  • the contents of the tissue culture flask subjected to a freeze-thaw process and suspended by ultrasound are filled with maintenance medium to a volume of 3000 ml and inoculated with this a stack of tubs grown confluently with PK-15 cells. After 7 days of incubation at 37 ° C, the culture supernatant is harvested and stored at -70 ° C until further processing.
  • Aluminum hydroxide suspension 3% (e.g. Superfos)
  • PBS buffer Phosphate Buffered Saline
  • the supernatant of the virus propagated on cell cultures is freed from cells and cell debris by centrifugation at 10,000 xg.
  • a virus suspension purified in this way with a concentration of virus particles of 10 6.0 KID 50 / ml, which originates from one or more virus harvests, is transferred into a sterile vessel.
  • the pH is adjusted to 8.4 with sodium hydroxide solution (2 N NaOH).
  • Such an amount of 0.5 M 2-bromoethylamine hydrobromide solution (2-BEA) is added with constant stirring until a final concentration of 5 mmol / l 2-BEA is calibrated.
  • the virus is inactivated within 18 hours 37 ° C.
  • the inactivating agent is then neutralized by adding a 2.5 M sodium thiosulfate solution to a final concentration of 50 mmol / l at 4 ° C.
  • 62 ml of the inactivated virus suspension are added to 31 ml of a sterile aluminum hydroxide suspension (3% Al (OH) 3 , pH 7.3) and stirred at 4 ° C. for 2 hours. After adding 1.25 ml of Quil A (2% solution) and 0.1 ml of thimerosal (2% solution), make up to 100 ml with PBS buffer and stir at 4 ° C. for a further 20 hours . The finished vaccine is filled into multiple withdrawal containers and stored at 4 ° C.
  • Pigs of all ages are vaccinated by subcutaneous application of 2 ml of this vaccine
  • Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal prteins P and V of the paramyxovirus SV5. Cell, 54, 891-902.

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Abstract

A viral agent based on para-influenza virus is useful as a vaccine component for protecting pigs against the porcine reproductive and respiratory syndrome.

Description

VAKZINE ZUR PRÄVENTION VON RESPIRATIONS- UND REPRODUKTIONSERKRANKUNGEN DE SCHWEINES , DIE PARAINFLUENZAVIREN ENTHÄLT. VACCINE FOR THE PREVENTION OF REPIRATIONAL AND REPRODUCTION DISEASES OF THE PIGS, CONTAINING PARAIN INFLUENCE VIRUSES.
Die "vorliegende Erfindung betrifft ein virales Agens. Verfahren zur Anzucht und Vermehrung dieses Agens sowie die Nutzung dieses Agens alleine oder in Kom¬ bination mit anderen bakteriellen oder viralen Erregern als Impfstoffkomponente zum Schutz von Schweinen vor Erkrankungen des Respirations- und Reproduk- tionsstraktes.The present invention relates to a viral agent. A method for cultivating and multiplying this agent and using this agent alone or in combination with other bacterial or viral pathogens as a vaccine component for protecting pigs against diseases of the respiratory and reproductive tract.
Ende der 80er bis Anfang der 90er Jahre trat in Nordamerika bzw. Europa eine neue, sich seuchenhaft ausbreitende und mit hohen wirtschaftlichen Verlusten einhergehende Schweinekrankheit auf. Diese wird mittlerweile offiziell "Porcine Reproductive and Respiratory Syndrome" (PRRS) genannt.From the late 80s to the early 90s, a new, epidemic spreading pig disease with high economic losses appeared in North America and Europe. This is now officially called "Porcine Reproductive and Respiratory Syndrome" (PRRS).
Die klinischen Hauptsymptome dieser seuchenhaften Erkrankungen sind Fruchtbar¬ keitsstörungen bei Sauen und Atemwegserkrankungen bei Ferkeln und Mast¬ schweinen.The main clinical symptoms of these epidemic diseases are fertility disorders in sows and respiratory diseases in piglets and fattening pigs.
Neben den unregelmäßig auftretenden unspezifischen Symptomen wie Inappetenz, Apathie und Fieber, ist die Erkrankung bei Sauen durch Spätaborte, Totgeburten und durch die Geburt mumifizierter und lebensschwacher Ferkel gekennzeichnet.In addition to the irregular non-specific symptoms such as anorexia, apathy and fever, the disease in sows is characterized by late abortions, stillbirths and the birth of mummified and poorly-performing piglets.
Infolge der Seuche kommt es gehäuft zu Symptomen aus dem Mastitis-Metritis- Agalaktie (MMA)-Komplex und zu Umrauschen.As a result of the epidemic, symptoms from the mastitis-metritis-agalactic (MMA) complex and noise come up.
Sind in einer endemischen Region zu Beginn der Seuche hauptsächlich Saug- undAre in an endemic region at the beginning of the epidemic mainly suction and
Läuferferkel betroffen, so erkranken im weiteren Verlauf zunehmend Mast- schweine. Hierbei können, neben den hauptsächlich vorkommenden Erkrankungen des Respirationstraktes, begleitend auch vermehπ andere klassische Schweine¬ erkrankungen beobachtet werden. Die Seuche verursacht erhebliche wirtschaftliche Verluste, die sich neben den direkten Tierverlusten aus der Verringerung der Pro¬ duktionskennzahlen ergibt (Abferkel- und Absetzergebnisse, Trächtigkeitsrate, Lebendgewichtzunahme).As piglet piglets are affected, fattening pigs become increasingly ill in the further course. Here, in addition to the mainly occurring diseases of the respiratory tract, accompanied by an increasing number of other classic swine diseases. The epidemic causes considerable economic losses which, in addition to direct animal losses, result from the reduction in the production figures (farrowing and weaning results, pregnancy rate, increase in live weight).
Als primäres Infektionsagens nimmt man ein neues, sich in Lungenalveolarmakro- phagen vermehrendes RNS-Virus an. Andererseits deuten weitergehende epi¬ demiologische Untersuchungen darauf hin, daß Respirations- und Reproduk¬ tionserkrankungen durch Sekundär- bzw. Mehrfachinfektionen mit anderen Viren bzw. Viren und Bakterien mit hervorgerufen oder verstärkt werden. Es ist daher wünschenswert Schweine nicht nur gegen den Haupterreger der PRRS zu schüt¬ zen, sondern auch gegen die Erreger die mitverantwortlich sind für Respirations¬ und Reproduktionserkrankungen.The primary infection agent is assumed to be a new RNA virus that multiplies in lung alveolar macrophages. On the other hand, further epidemiological studies indicate that respiratory and reproductive diseases are caused or intensified by secondary or multiple infections with other viruses or viruses and bacteria. It is therefore desirable not only to protect pigs against the main pathogen of the PRRS, but also against the pathogens which are jointly responsible for respiratory and reproductive diseases.
Gegenstand der vorliegenden Erfindung sind:The present invention relates to:
1. Vakzine gegen Erkrankungen des Respirations- und Reproduktionstrakts von Schweinen, insbesondere im Zusammenhang mit dem PRRS genannten Krankheitskomplex, dadurch gekennzeichnet, daß sie als antigenes Material Parainfluenzaviren sowie deren Varianten und Mutanten in lebender, attenuierter oder über rekombinante Technologie hergestellter Form ganz oder in Teilen oder Bruchstücken enthält.1. Vaccine against diseases of the respiratory and reproductive tract of pigs, in particular in connection with the disease complex called PRRS, characterized in that they contain, as an antigenic material, parainfluenza viruses and their variants and mutants in living, attenuated or recombinant form, in whole or in part or contains fragments.
2. Antigenes Material auf Basis von Parainfluenzaviren die Erkrankungen des Respirations- und Reproduktionstrakts von Schweinen hervorrufen.2. Antigenic material based on parainfluenza viruses cause diseases of the respiratory and reproductive tract of pigs.
3. Verfahren zur Herstellung von antigenem Material auf Basis von Para¬ influenzaviren die Erkrankungen des Respirations- und Reproduktionstrakts von Schweinen hervorrufen, dadurch gekennzeichnet, daß man Parain¬ fluenzaviren vermehrt und aus den so erhaltenen Virussuspensionen in übli¬ cher Weise das antigene Material isoliert. 4 Verwendung von antigenem Material auf Basis \ on Parainfluenzaviren die Erkrankungen des Respirations- und Reproduktionstrakts von Schweinen hervorrufen zur Diagnose und/oder Prävention dieser Erkrankungen3. Process for the production of antigenic material based on para influenza viruses which cause diseases of the respiratory and reproductive tract of pigs, characterized in that parainfluenza viruses are propagated and the antigenic material is isolated from the virus suspensions thus obtained in a conventional manner. 4 Use of antigenic material based on parainfluenza viruses which cause diseases of the respiratory and reproductive tract of pigs for the diagnosis and / or prevention of these diseases
5 Verwendung von antigenem Material auf Basis von Parainfluenzaviren, die Erkrankungen des Respirations- und Reprodukuonstrakts von Schweinen hervorrufen zur Herstellung von Diagnosemitteln zur Feststellung dieser Erkrankungen und zur Herstellung von Vakzinen zur Prävenüon dieser Erkrankungen.5 Use of antigenic material based on parainfluenza viruses which cause diseases of the respiratory and reproductive tract of pigs for the production of diagnostic tools for the detection of these diseases and for the production of vaccines for the prevention of these diseases.
Als antigenes Material seien genannt:The following can be mentioned as antigenic material:
1 Komplette, lebende Viruspartikel, gewonnen durch Vermehrung des Virus in Zellkulturen oder embryonierten Hühnereiern1 Complete, live virus particles, obtained by multiplying the virus in cell cultures or embryonated chicken eggs
2 Komplette, lebende, attenuierte Viruspartikel, gewonnen durch Dauer¬ passagen des Virus in primären Zellkulturen, permanenten Zellinien, embryonierten Geflügeleiern oder Versuchstieren mit anschließender Ver- mehrung in Zellkulturen oder embryonierten Hühnereiern.2 Complete, live, attenuated virus particles obtained by continuous passage of the virus in primary cell cultures, permanent cell lines, embryonated poultry eggs or experimental animals with subsequent multiplication in cell cultures or embryonated chicken eggs.
3 Komplette, abgetötete Viruspartikel, die mittels herkömmlicher Verfahren, wie chemischer oder physikalischer Inaktivierung. hergestellt werden.3 Complete, killed virus particles by conventional methods such as chemical or physical inactivation. getting produced.
4 Teilstucke (subunits) der Viruspartikel, hergestellt aus Virus, das in Zell¬ kulturen oder embryonierten Hühnereiern vermehrt wird.4 subunits of the virus particles, produced from virus that is propagated in cell cultures or embryonic chicken eggs.
5 Teilstucke (subunits) der Viruspartikel, die aufgrund rekombinanter Gen¬ techniken von Zellsystemen exprimiert werden und sich gegebenfalls von diesen abtrennen oder aus diesen isolieren lassen5 subunits of the virus particles, which are expressed by cell systems on the basis of recombinant genetic engineering and, if appropriate, can be separated from them or isolated from them
6. Virusantigene, die über Vektorsysteme exprimiert werden, wobei mittels rekombinanter Gentechniken das Genom des Virus oder Teile davon in Genom- Vektoren wie Vaccinaviren, Herpesviren. Adenoviren oder andere geeignete Vektorsysteme eingesetzt wird. Bevorzugt verwendet werden Parainfluenzaviren vom Typ 2 (PIV-2).6. Virus antigens which are expressed via vector systems, the genome of the virus or parts thereof in genome vectors such as vaccina viruses and herpes viruses by means of recombinant genetic engineering. Adenoviruses or other suitable vector systems is used. Parainfluenza viruses of type 2 (PIV-2) are preferably used.
Besonders bevorzugt sind PIV-2, die aus dem Respirations- oder Reproduktions¬ trakt von Schweinen isolsiert werden, die eine PRRS ähnliche Symptomatik zeigen. Besonders geeignet ist der PIV-2 Stamm mit der Bezeichnung SER, der am 12.6.1993 bei der Collection Nationale des Cultures et de. MicroorganismesParticularly preferred are PIV-2, which are isolated from the respiratory or reproductive tract of pigs which show symptoms similar to PRRS. Particularly suitable is the PIV-2 strain with the designation SER, which was released on June 12, 1993 by the Collection Nationale des Cultures et de. Microorganisms
(Institut Pasteur, Paris, Frankreich) unter der Nummer 1-1331 gemäß Budapester Vertrag hinterlegt wurde.(Institut Pasteur, Paris, France) under number 1-1331 in accordance with the Budapest Treaty.
In den erfindungsgemäßen Vakzinen kann das antigene Material der Parain¬ fluenzaviren in Mischung mit antigenem Material aus anderen Viren oder Bak-terien vorliegen. Als solche seien genannt: Chlamydien, insbesondereIn the vaccines according to the invention, the antigenic material of the parainfluenza viruses can be present in a mixture with antigenic material from other viruses or bacteria. The following may be mentioned: chlamydia, in particular
Chlamydia psittaci und Chlamydia pecorum in Konzentrationen von 10 010 EBE/Dosis, Erysipelothrix rhusiopathiae in Konzentrationen von 10 -10 KBE/Dosis, PRRS-Viren in Konzentrationen von 104-109 KID50/Dosis, Porcines Parvovirus in Konzentrationen von 104- 109 KED50/Dosis.Chlamydia psittaci and Chlamydia pecorum in concentrations of 10 0 10 EBE / dose, Erysipelothrix rhusiopathiae in concentrations of 10 -10 CFU / dose, PRRS viruses in concentrations of 10 4 -10 9 KID 50 / dose, Porcines Parvovirus in concentrations of 10 4 - 10 9 KED 50 / dose.
Besonders bevorzugt ist eine Mischung aus PIV-2 und Chlamydia, insbesondereA mixture of PIV-2 and chlamydia is particularly preferred, in particular
Chlamydia psittaci oder Ch. pecorum.Chlamydia psittaci or Ch. Pecorum.
In den folgenden Ausführungen werden die folgenden Begriffe verwendet:The following terms are used in the following explanations:
Cotransfektion Gleichzeitige Übertragung zweier verschiedener DNS-Se¬ quenzen in Zellen, in denen Viren vermehrt werden können, mit dem Ziel, Virus-Rekombinationen zu induzieren, dieCotransfection Simultaneous transmission of two different DNA sequences in cells in which viruses can be propagated with the aim of inducing virus recombinations that
Fremd-DNS-Sequenzen enthalten. Die verschiedenen DNS- Sequenzen sind (1.) Fremd-DNS, die in Shuttle- Vektoren inseriert sein kann und (2.) das gereinigte Genom des Vektor- Virus.Foreign DNA sequences included. The different DNA sequences are (1.) foreign DNA, which can be inserted in shuttle vectors and (2.) the purified genome of the vector virus.
Genom- Vektor Lebende Erreger, insbesondere Viren, die geeignet zur Inser- tion von Fremd-DNS sind und mit der in ihrem Genom inserierten Fremd-DNS Zellen oder Organismen infizieren und hierin die Fremd-DNS exprimieren. Immunogene Peptide oder Proteine, die in einem höheren Organismus eine immunologische Reaktion auslösen und durch fremde DNS- Sequenzen in Vektoren exprimiert werden können.Genome vector Living pathogens, in particular viruses, which are suitable for inserting foreign DNA and which infect cells or organisms with the foreign DNA inserted in their genome and express the foreign DNA therein. Immunogenic peptides or proteins that trigger an immunological reaction in a higher organism and can be expressed in vectors by foreign DNA sequences.
Klonierung Einführung von fremden DNS-Sequenzen in Vektoren.Cloning Introduction of foreign DNA sequences in vectors.
Plas id Extrachromosomale, ringförmige DNS-Sequenzen, die in nie¬ deren oder höheren Zellen repliziert werden.Plas id extrachromosomal, circular DNA sequences that are replicated in their cells or higher cells.
Shuttle-Vektor Bakteriophagen oder Plasmide. insbesondere bakterielle Plasmide, die inserierte Fremd-DNS enthalten, die flankiert ist von DNS-Sequenzen des Vektor- Virus.Shuttle vector bacteriophages or plasmids. in particular bacterial plasmids which contain inserted foreign DNA which is flanked by DNA sequences of the vector virus.
Transfektion Übertragung von DNS-Sequenzen in niedere oder höhere Zellen mit dem Ziel, Rekombinationen des Zeil-Genoms mit den eingeführten DNS-Sequenzen zu induzierenTransfection Transfer of DNA sequences into lower or higher cells with the aim of inducing recombination of the Zeil genome with the introduced DNA sequences
Vektoren Plasmide, Bakteriophagen oder Viren, die in ihrer geneti¬ schen Information fremde DNS-Sequenzen tragen.Vectors plasmids, bacteriophages or viruses which carry foreign DNA sequences in their genetic information.
Die Vermehrung der Viren zur Herstellung kompletter lebender Viruspartikel er¬ folgt in üblicher Weise einerseits in Gewebekulturen animaler Zellen als Primärzellen oder permanenten Zell-Linien, z.B. in Schweine-Zellen, Affen-Zellen oder Rinder-Zellen, bevorzugt in Schweinenieren-Zellen wie z.B. der geklonten, permanenten Schweinenieren-Zelle PK15 (ATCC CCL33 oder deren Abkömm- linge) oder der primären Schweinenieren-Zelle EPK oder Affennieren-Zellen wie den permanenten Affennieren-Zellen BGM (Flow 03-240 oder deren Abkömm¬ linge) oder Vero (ATCC CCL81 oder deren Abkömmlinge) oder Rindernieren- Zellen wie der permanenten Rindernieren-Zelle MDBK (ATCC CCL22 oder deren Abkömmlinge) und andererseits in embryonierten Hühnereiern (z.B. Valo-Bruteier, Fa. Lohmann).The multiplication of viruses for the production of complete living virus particles takes place in the usual way on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned, permanent pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or their descendants) or Vero (ATCC CCL81 or its descendants) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its descendants) and on the other hand in embryonated chicken eggs (eg Valo hatching eggs, Lohmann).
Die Vermehrung in Zellkulturen erfolgt in an sich bekannter Weise in stationären Roller- oder Carrier-Kulturen in Form von geschlossenen Zellverbänden (Mono- layern) oder in Suspensions-Kulturen. Als Vermehrungsmedien für die Zellen werden eingesetzt alle an sich bekannten Zellkulturmedien z.B. beschrieben im Produktkatalog der Fa. Gibco BRL GmbH, Dieselstraße 5, 76344 Eggenstein, wie insbesondere das Minimal Essential Medium (MEM), das als wesentliche Bestandteile Aminosäuren, Vitamine, Salze und Kohlenhydrate enthält, kom- plettiert mit Puff ersub stanzen wie z.B. Natrium-Bicarbonat (NaHCO3) oder Hy- droxyethylpiperazin-N-2-ethansulfonsäure (Hepes) und gegebenenfalls Tierseren, wie z.B. Seren von Rindern, Pferden bzw. deren Föten. Besonders bevorzugt eingesetzt wird Eagles MEM mit einem Gehalt an NaHCO3 von 0,1-5 g/1, vor¬ zugsweise 0,5-3 g/l sowie fötales Kälberserum in einer Konzentration von 1-30 Vol-%, vorzugsweise 2-10 Vol-%.The multiplication in cell cultures takes place in a manner known per se in stationary roller or carrier cultures in the form of closed cell groups (monolayers) or in suspension cultures. As propagation media for the cells all cell culture media known per se are used, for example described in the product catalog of Gibco BRL GmbH, Dieselstrasse 5, 76344 Eggenstein, such as in particular the Minimal Essential Medium (MEM), which contains essential components of amino acids, vitamins, salts and carbohydrates punch with puff ersub such as sodium bicarbonate (NaHCO 3 ) or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and optionally animal sera such as sera from cattle, horses or their fetuses. Eagles MEM with a NaHCO 3 content of 0.1-5 g / 1, preferably 0.5-3 g / l and fetal calf serum in a concentration of 1-30% by volume, preferably 2- 10 vol%.
Die" zur Vermehrung der Viren dienenden Zellen und Zellrasen werden in üblicher Weise nahezu bis zur Konfluenz oder bis zur optimaler Zelldichte vermehrt. Vor ihrer Infektion mit Viren wird bevorzugt das Zeilvermehrungsmedium entfernt und die Zellen bevorzugt mit Virusvermehrungsmedium gewaschen. Als Virusvermeh- rungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben genannte MEM. Danach wird mit einer Virussuspension infiziert. In der Virussuspension liegt das Virus im Virusvermehrungsmedium derart verdünnt vor, daß mit einer MOI (= multiplicity of infection, entspricht dem Verhältnis der Anzahl der infektiösen Viruspartikel zur Anzahl der vorhandenen Zellen) von 0,01-50, bevorzugt 0,1-10 infiziert wird.The "for multiplication of the viruses serving cells and cell lawns are grown in conventional manner almost to confluence or to the optimal cell density. Before their infection with viruses, the Zeilvermehrungsmedium is preferably removed and the cells preferably with virus replication medium washed. The virus replication medium employed all cell culture media known per se, in particular the above-mentioned MEM. After that, infection is carried out with a virus suspension. In the virus suspension, the virus is diluted in the virus propagation medium in such a way that with an MOI (= multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) from 0.01-50, preferably 0.1-10.
Die Vermehrung der Viren erfolgt mit oder ohne Zusatz von Tierseren. Für den Fall, daß Serum eingesetzt wird, wird dieses zum Vermehrungsmedium in einer Konzentration von 1-30 Vol-%, vorzugsweise 2-10 Vol-% zugegeben.The viruses are propagated with or without the addition of animal sera. If serum is used, it is added to the propagation medium in a concentration of 1-30% by volume, preferably 2-10% by volume.
Infektion und Virusvermehrung erfolgen bei Temperaturen zwischen Raumtem- peratur und 40°C, bevorzugt zwischen 32 und 39°C, besonders bevorzugt bei 37°C über mehrere Tage, bevorzugt bis zur vollständigen Zerstörung der infizierten Zellen.Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 32 and 39 ° C., particularly preferably at 37 ° C. for several days, preferably until the infected cells are completely destroyed.
Das virushaltige Medium der infizierten Zellen wird weiter aufgearbeitet, z.B. durch Entfernung der Zellen und Zelltrümmer mittels Filtration mit Porengrößen von z.B. 0,1-0,45 μm und/oder Zentrifugation bis zu 10.000 x g. Die Vermehrung in embryonierten Hühnereiern erfolgt in an sich bekannter Weise in der Allantoishöhle von Huhner-Bruteieren, die 9- 12 Tage, bevorzugt 10 Tage bei einer Temperatur von 37-39°C, bevorzugt 38,5°C und einer relativen Luft¬ feuchtigkeit von 30-90%, bevorzugt 50-60% in einem handelsüblichen Brut- schrank, bevorzugt einem Motorbrüter vorbebrütet werden.The virus-containing medium of the infected cells is processed further, for example by removing the cells and cell debris by means of filtration with pore sizes of, for example, 0.1-0.45 μm and / or centrifugation up to 10,000 × g. The multiplication in embryonated chicken eggs takes place in a manner known per se in the Allantoic cave of hatching hatching eggs, which lasts 9-12 days, preferably 10 days, at a temperature of 37-39 ° C., preferably 38.5 ° C. and a relative atmospheric humidity of 30-90%, preferably 50-60%, are pre-incubated in a commercial incubator, preferably a motor breeder.
Die zur Vermehrung der Viren dienenden Bruteier werden vor der Beimpfung 1-3 Stunden, bevorzugt 2 Stunden auf dem spitzen Eipol senkrecht stehend im Brut¬ schrank gelagert und anschließend nach Vorbereitung der Injektionstelle mit 10- 200 μl, bevorzugt 75-125 μl einer Virussuspension infiziert. In der Virus- Suspension liegt das Virus im Virusvermehrungsmedium in einer Konzentration von- 101 - 107 KTD50/ml (50%-Kultur-infektiöse Dosis pro ml Suspension = die Verdünnungsstufe, bei der noch 50%) der eingesetzten Zellkulturen infiziert würden), bevorzugt lO^lO3 KJD50/ml vor. Als Virusvermehrungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben genannte MEM.The hatching eggs used to multiply the viruses are stored vertically in the incubator for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation and then infected with 10-200 μl, preferably 75-125 μl, of a virus suspension after preparation of the injection site . In the virus suspension, the virus is in the virus propagation medium in a concentration of - 10 1 - 10 7 KTD 50 / ml (50% culture-infectious dose per ml suspension = the dilution level at which 50%) of the cell cultures used would be infected ), preferably 10 ^ 10 3 KJD 50 / ml. All known cell culture media, such as in particular the MEM mentioned above, are used as virus propagation medium.
Infektion und Virusvermehrung erfolgen unter den oben angegebenen Brut¬ bedingungen über mehrere Tage, bevorzugt 2-5 Tage, besonders bevorzugt 3 Tage.Infection and virus multiplication take place under the breeding conditions specified above over several days, preferably 2-5 days, particularly preferably 3 days.
Die virushaltige Allantoisflüssigkeit wird gewonnen durch Absaugen nach Öffnung der Kalkschale sowie der Schalenhaut und der Chorioallantoismembran und kann z.B. mittels Filtration mit Porengrößen von z.B. 0,1-0,45 μm und/oder Zentri- fugation bis zu 10.000 x g weiter aufgearbeitet werden.The virus-containing allantoic fluid is obtained by suction after opening the lime shell as well as the skin of the shell and the chorioallantoic membrane and can e.g. by filtration with pore sizes of e.g. 0.1-0.45 μm and / or centrifugation up to 10,000 x g can be processed further.
Die Herstellung von attenuiertem, lebendem Virus erfolgt in üblicher Weise durch Dauerpassagen und/oder Wechselpassagen einerseits in Gewebekulturen animaler Zellen als Primärzellen oder permanenten Zell-Linien, z.B. in Schweine-Zellen, Affen-Zellen oder Rinder-Zellen, bevorzugt in Schweinenieren-Zellen wie z.B. der geklonten, permanenten Schweinenieren-Zelle PK15 (ATCC CCL33 oder deren Abkömmlinge) oder der primären Schweinenieren-Zelle EPK oder Affennieren- Zellen wie den permanenten Affennieren-Zellen BGM (Flow 03-240 oder deren Abkömmlinge) oder Vero (ATCC CCL81 oder deren Abkömmlinge) oder Rindernieren-Zellen wie der permanenten Rindernieren-Zelle MDBK (ATCCAttenuated, live virus is produced in the usual way by long-term passages and / or alternating passages on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned permanent pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or its descendants) or Vero (ATCC CCL81 or its descendants) ) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC
CCL22 oder deren Abkömmlinge) oder Hundennieren-Zellen wie der permanenten Hundenieren-Zelle MDCK (ATCC CCL34 oder deren Abkömmlinge) und andererseits in embryonierten Hühner- , Tauben- oder Enteneiern, bevorzugt in embryonierten Hühnereiern (z.B. Valo-Bruteier, Fa. Lohmann) oder in Versuchstieren, bevorzugt in kleinen Labortieren, z.B. in Meerschweinchen, Ratte oder Maus, in denen sich das Virus vermehrt, ohne ernsthafte Krankheitssymptome hervorzurufen.CCL22 or its descendants) or dog kidney cells like the permanent one Dog kidney cell MDCK (ATCC CCL34 or its descendants) and on the other hand in embryonated chicken, pigeon or duck eggs, preferably in embryonated chicken eggs (e.g. Valo hatching eggs, Lohmann) or in experimental animals, preferably in small laboratory animals, e.g. in guinea pigs, Rat or mouse in which the virus reproduces without causing serious symptoms of the disease.
Die Passagierung in Zellkulturen erfolgt in an sich bekannter Weise in stationären Kulturen in Form von geschlossenen Zellverbänden (Monolayern). Als Vermeh¬ rungsmedien für die Zellen werden eingesetzt alle an sich bekannten Zell- kulturmedien z.B. beschrieben im Produktkatalog der Fa. Gibco BRL GmbH,Passenging in cell cultures takes place in a manner known per se in stationary cultures in the form of closed cell groups (monolayers). All cell culture media known per se are used as propagation media for the cells, e.g. described in the Gibco BRL GmbH product catalog,
Dieselstraße 5, 76344 Eggenstein, wie insbesondere das Minimal Essential Medium (MEM), das als wesentliche Bestandteile Aminosäuren, Vitamine, Salze und Kohlenhydrate enthält, komplettiert mit Puff ersub stanzen wie z.B. Natrium- Bicarbonat (NaHCO3) oder Hydroxyethylpiperazin-N-2-ethansulfonsäure (Hepes) und gegebenenfalls Tierseren, wie z.B. Seren von Rindern, Pferden bzw. deren Föten. Besonders bevorzugt eingesetzt wird Eagles MEM mit einem Gehalt an NaHCO3 von 0,1-5 g/1, vorzugsweise 0,5-3 g/l sowie fötales Kälberserum in einer Konzentration von 1-30 Vol-%, vorzugsweise 2-10 Vol-%.Dieselstraße 5, 76344 Eggenstein, such as in particular the Minimal Essential Medium (MEM), which contains amino acids, vitamins, salts and carbohydrates as essential components, completed with buffer substances such as sodium bicarbonate (NaHCO 3 ) or hydroxyethylpiperazine N-2- ethanesulfonic acid (Hepes) and optionally animal sera, such as sera from cattle, horses or their fetuses. Eagles MEM with a NaHCO 3 content of 0.1-5 g / 1, preferably 0.5-3 g / l and fetal calf serum in a concentration of 1-30 vol.%, Preferably 2-10 vol. Are particularly preferably used -%.
Die zur Passagierung der Viren dienenden Zellen und Zellrasen werden in üblicher Weise nahezu bis zur Konfluenz oder bis zur optimaler Zelldichte vermehrt. Vor ihrer Infektion mit Viren wird bevorzugt das Zellvermehrungsmedium entfernt und die Zellen bevorzugt mit Virusvermehrungsmedium gewaschen. Als Virusvermeh¬ rungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben genannte MEM. Danach wird mit einer Virussuspension infiziert. In der Virussuspension liegt das Virus im Virusvermehrungsmedium derart verdünnt vor, daß mit einer MOI (= multiplicity of infection, entspricht dem Verhältnis der Anzahl der infektiösen Viruspartikel zur Anzahl der vorhandenen Zellen) von 0,01-50, bevorzugt 0, 1-10 infiziert wird.The cells and cell turf used for the passage of the viruses are multiplied in the usual way almost to confluence or to the optimal cell density. Before being infected with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus growth medium. All known cell culture media, such as in particular the MEM mentioned above, are used as the virus propagation medium. Then it is infected with a virus suspension. In the virus suspension, the virus is diluted in the virus propagation medium in such a way that it is infected with an MOI (= multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) of 0.01-50, preferably 0.1-10 becomes.
Die Vermehrung der Viren erfolgt mit oder ohne Zusatz von Tierseren. Für den Fall, daß Serum eingesetzt wird, wird dieses zum Vermehrungsmedium in einerThe viruses are propagated with or without the addition of animal sera. In the event that serum is used, this becomes the propagation medium in a
Konzentration von 1-30 Vol-%, vorzugsweise 2-10 Vol-% zugegeben. Infektion und Virusvermehrung erfolgen bei Temperaturen zwischen Raumtem¬ peratur und 40°C, bevorzugt zwischen 30 und 39°C über mehrere Tage, bevorzugt bis zur vollständigen Zerstörung der infizierten ZellenConcentration of 1-30 vol%, preferably 2-10 vol% added. Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 30 and 39 ° C. over several days, preferably until the infected cells are completely destroyed
Das virushaltige Medium der infizierten Zellen wird zur Infektion einer frischen Zellkultur verwendet (Folgepassage).The virus-containing medium of the infected cells is used to infect a fresh cell culture (subsequent passage).
Die Passagierung in embryonierten Geflügeleiern erfolgt in an sich bekannter Weise in der Allantoishöhle von z.B. Hühner-Bruteieren, die 9-12 Tage, bevorzugt 10 Tage bei einer Temperatur von 37-39°C, bevorzugt 38,5°C und einer relativen Luftfeuchtigkeit von 30-90%>, bevorzugt 50-60% in einem handelsüblichen Brut- schränk, bevorzugt einem Motorbrüter vorbebrütet werden.The passage in embryonated poultry eggs takes place in a known manner in the Allantoic cave of e.g. Chicken hatching eggs that 9-12 days, preferably 10 days at a temperature of 37-39 ° C, preferably 38.5 ° C and a relative humidity of 30-90%>, preferably 50-60% in a commercial incubator cabinet, preferably pre-incubated for a motor breeder.
Die zur Passagierung der Viren dienenden Bruteier werden vor der Beimpfung 1-3 Stunden, bevorzugt 2 Stunden auf dem spitzen Eipol senkrecht stehend im Brutschrank gelagert und anschließend nach Vorbereitung der Injektionstelle mit 10-200 μl, bevorzugt 75-125 μl einer Virussuspension infiziert. In der Virus- Suspension liegt das Virus im Virusvermehrungsmedium in einer Konzentration von 10 x - 107 KID50/ml (50%-Kultur-infektiöse Dosis pro ml Suspension = die Verdünnungsstufe, bei der noch 50% der eingesetzten Zellkulturen infiziert würden), bevorzugt lo o5 KID50/ml vor. Als Virusvermehrungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das obenThe hatching eggs used to pass the viruses are stored vertically in the incubator for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation and then infected with 10-200 μl, preferably 75-125 μl, of a virus suspension after preparation of the injection site. In the virus suspension, the virus is in the virus propagation medium in a concentration of 10 x 10 7 KID 50 / ml (50% culture-infectious dose per ml suspension = the dilution level at which 50% of the cell cultures used would still be infected), preferably lo o 5 KID 50 / ml. All known cell culture media, in particular the one above, are used as the virus propagation medium
C genannte MEM.C called MEM.
Infektion und Virusvermehrung erfolgen unter den oben angegebenen Brutbe¬ dingungen über mehrere Tage, bevorzugt 2-5 Tage, besonders bevorzugt 3 Tage.Infection and virus multiplication take place under the above-mentioned breeding conditions over several days, preferably 2-5 days, particularly preferably 3 days.
Die virushaltige Allantoisflüssigkeit wird durch Absaugen nach Öffnung der Kalk¬ schale sowie der Schalenhaut und der Chorioallantoismembran gewonnen. Sie wird -zur Infektion von frischen vorberüteten, embryonierten Eiern verwendet (Folge¬ passage).The virus-containing allantoic fluid is obtained by suction after opening the calcareous skin, the skin of the skin and the chorioallantoic membrane. It is used for the infection of freshly prepared, embryonic eggs (subsequent passage).
Die Passagierung in Versuchstieren erfolgt in an sich bekannter Weise durch parenterale Applikation einer Virussupension und Reisolierung des Virus aus Organen und Geweben der Versuchstiere. Zur Passagierung in Versuchstieren werden bevorzugt juvenile, kleine Labortiere eingesetzt, die SPF(spezifιziert pathogen-freien)-Zuchten entstammen. z.B. Meer¬ schweinchen (Hsd/Win:DH, Fa. Harlan-Winkelmann GmbH, Borchen), Ratte (Hsd/Win:WU, Fa. Harlan-Winkelmann GmbH, Borchen) oder Maus (Hsd/Win:NMRI, Fa. Harlan-Winkelmann GmbH, Borchen; .Balb/C/JICO, IffaPassenging in experimental animals is carried out in a manner known per se by parenteral application of virus suspension and rice isolation of the virus from organs and tissues of the experimental animals. Juvenile, small laboratory animals which originate from SPF (specified pathogen-free) breeds are preferably used for the passage in experimental animals. e.g. guinea pigs (Hsd / Win: DH, Harlan-Winkelmann GmbH, Borchen), rats (Hsd / Win: WU, Harlan-Winkelmann GmbH, Borchen) or mice (Hsd / Win: NMRI, Harlan -Winkelmann GmbH, Borchen;. BALB / c / jico, Iffa
Credo Belgium). Die Versuchstiere werden mit 0,1-2,0 ml einer Virussupension parenteral infiziert, z.B. durch intradermale, intramuskuläre, intranasale, intraperi- toneale, intravenöse oder subkutane Applikation. In der Virussupension liegt das Virus im Virusvermehrungsmedium derart vor, daß die Versuchstiere jeweils eine Virusdosis von lO o7 KID50, bevorzugt 10 05 KED50 erhalten (50%-Kultur- infektiöse Dosis pro ml Suspension = die Verdünnungsstufe, bei der noch 50% der eingesetzten Zellkulturen infiziert würden). Als Virusvermehrungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben o gevnannte MEM.Credo Belgium). The test animals are infected parenterally with 0.1-2.0 ml of a virus suspension, for example by intradermal, intramuscular, intranasal, intraperitoneal, intravenous or subcutaneous administration. In the virus suspension, the virus is present in the virus propagation medium in such a way that the test animals each receive a virus dose of 10 o 7 KID 50 , preferably 10 0 5 KED 50 (50% culture-infectious dose per ml suspension = the dilution level at which 50 % of the cell cultures used would be infected). The virus replication medium employed are all cell culture media known per se, such as especially the v o ge above-mentioned MEM.
Die Virusvermehrung erfolgt über mehrere Tage bevorzugt 1-12 Tage.Virus propagation takes place over several days, preferably 1-12 days.
Das Virus wird aus Geweben, bevorzugt inneren Organen der Versuchstiere in üblicher Weise reisoliert. Hierzu werden den Versuchstieren innere Organe, z.B. Lunge, Leber oder Milz entnommen. Aus den Organen oder Teilen der Organe werden durch mechanische Zerkleinerung, z.B. mit Hilfe von Schere und Mörser in Virusvermehrungsmedium feine Suspensionen hergestellt, die weiter aufge¬ arbeitet werden, z.B. durch Entfernung der Zellen und Zelltrümmer mittels Filtration mit Porengrößen von z.B. 0,1-0,45 μm und/oder Zentrifugation bis zu 10.000 x g. Als Virusvermehrungsmedium werden eingesetzt, alle an sich be¬ kannten Zellkulturmedien, wie insbesondere das oben genannte MEM.The virus is reisolated from tissues, preferably internal organs of the test animals, in the usual way. For this purpose, internal organs, e.g. Lung, liver or spleen removed. The organs or parts of the organs are mechanically comminuted, e.g. with the aid of scissors and mortar, fine suspensions are prepared in virus propagation medium, which are worked up further, e.g. by removing the cells and cell debris by means of filtration with pore sizes of e.g. 0.1-0.45 μm and / or centrifugation up to 10,000 x g. All cell culture media known per se, such as in particular the MEM mentioned above, are used as the virus propagation medium.
Das gewonnene virushaltige Medium wird zur Infektion von neuen Versuchstieren verwendet (Folgepassagen).The virus-containing medium obtained is used to infect new experimental animals (subsequent passages).
Der Vorgang der Folgepassage wird mehrmals, bevorzugt 10-20mal im gleichen Vermehrungssystem (homologe Passagen) oder in verschiedenen Yermehrungs- systemen (heterologe Passagen) wiederholt. Die Überprüfung des Virus auf Attenuierung, erfolgt durch experimentelle Infek¬ tion von voll empfänglichen Versuchstieren, bevorzugt Schweinen, mit einer Virussupension, die der letzten Passage einer Serien von Folgepassagen entstammt.The process of the subsequent passage is repeated several times, preferably 10-20 times, in the same propagation system (homologous passages) or in different multiplication systems (heterologous passages). The virus is checked for attenuation by experimental infection of fully susceptible test animals, preferably pigs, with a virus suspension which originates from the last passage of a series of subsequent passages.
Treten noch typische Krankheitssymptome auf, z.B. Aborte und Totgeburten bei trächtigen Sauen oder respiratorische Erkrankungen, so wird ausgehend von denAre there still typical symptoms of the disease, e.g. Abortions and stillbirths in pregnant sows or respiratory diseases, it is based on the
Viren der letzten Folgepassagen weitere homologe oder hetereologe Dauerpassagen durchgeführt.Viruses of the last following passages carried out further homologous or heterologous long-term passages.
Treten keine typischen Krankheitssymptome mehr auf, so werden die Viren der letzten Folgepassage wie oben beschrieben vermehrt und die Filtrate oder Zentri- fügätionsüberstände von virushaltigen Kulturüberständen oder Allantoisflüssigkeit zur Herstellung von Vakzinen verwendet.If there are no longer any typical symptoms of the disease, the viruses in the last subsequent passage are increased as described above and the filtrates or centrifugation supernatants from virus-containing culture supernatants or allantoic fluid are used to produce vaccines.
Die Vermehrung der Viren zur Herstellung von abgetöteten Viruspartikeln erfolgt in üblicher Weise einerseits in Gewebekulturen animaler Zellen als Primärzellen oder permanenten Zell-Linien, z.B. in Schweine-Zellen, Affen-Zellen oder Rinder- Zellen, bevorzugt in Schweinenieren-Zellen wie z.B. der geklonten, permanentenThe multiplication of viruses for the production of killed virus particles takes place in the usual way on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned, permanent
Schweinenieren-Zelle PK15 (ATCC CCL33 oder deren Abkömmlinge) oder der primären .Schweinenieren-Zelle EPK oder Affennieren-Zellen wie den permanenten Affennieren-Zellen BGM (Flow 03-240 oder deren Abkömmlinge) oder Vero (ATCC CCL81 oder deren Abkömmlinge) oder Rindernieren-Zellen wie der permanenten Rindernieren-Zelle MDBK (ATCC CCL22 oder deren Abkömmlinge) und andererseits in embryonierten Hühnereiern (z.B. Valo-Bruteier, Fa. Lohmann).Pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary. Pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or its descendants) or Vero (ATCC CCL81 or its descendants) or bovine kidneys - Cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its descendants) and on the other hand in embryonated chicken eggs (eg Valo hatching eggs, Lohmann).
Die Vermehrung in Zellkulturen erfolgt in an sich bekannter Weise in stationären Roller- oder Carrier-Kulturen in Form von geschlossenen Zellverbänden (Mono- layern) oder in Suspensions-Kulturen. Als Vermehrungsmedien für die Zellen werden eingesetzt alle an sich bekannten Zellkulturmedien z.B. beschrieben imThe multiplication in cell cultures takes place in a manner known per se in stationary roller or carrier cultures in the form of closed cell groups (monolayers) or in suspension cultures. All cell culture media known per se are used as propagation media for the cells, e.g. described in
Produktkatalog der Fa. Gibco BRL GmbH. Dieselstraße 5, 76344 Eggenstein, wie insbesondere das Minimal Essential Medium (MEM), das als wesentliche Be¬ standteile Aminosäuren, Vitamine, Salze und Kohlenhydrate enthält, komplettiert mit Puffersubstanzen wie z.B. Natrium-Bicarbonat (NaHCO3) oder Hydroxyethyl- piperazin-N-2-ethansulfonsäure (Hepes) und gegebenenfalls Tierseren, wie z.B.Gibco BRL GmbH product catalog. Dieselstraße 5, 76344 Eggenstein, such as in particular the Minimal Essential Medium (MEM), which contains amino acids, vitamins, salts and carbohydrates as essential components, completed with buffer substances such as sodium bicarbonate (NaHCO 3 ) or hydroxyethylpiperazine-N- 2-ethanesulfonic acid (Hepes) and optionally animal sera, such as, for example
Seren von Rindern, Pferden bzw. deren Föten. Besonders bevorzugt eingesetzt wird Eagles MEM mit einem Gehalt an NaHC03 von 0, 1 -5 g/1, vorzugsweise 0,5- 3 g/1 sowie fötales Kälberserum in einer Konzentration von 1 -30 Vol-%, vorzugsweise 2-10 Vol-%.Sera from cattle, horses and their fetuses. Particularly preferably used is Eagles MEM with a NaHC0 3 content of 0.1 -5 g / 1, preferably 0.5-3 g / 1 and fetal calf serum in a concentration of 1-30% by volume, preferably 2-10% by volume.
Die zur Vermehrung der Viren dienenden Zellen und Zellrasen werden in üblicher Weise nahezu bis zur Konfluenz oder bis zur optimaler Zelldichte vermehrt. Vor ihrer Infektion mit Viren wird bevorzugt das Zeilvermehrungsmedium entfernt und die Zellen bevorzugt mit Virusvermehrungsmedium gewaschen. Als Virusvermeh¬ rungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben genannte MEM. Danach wird mit einer Virussuspension infiziert. In der Virussuspension liegt das Virus im Virusvermehrungsmedium derart verdünnt vor, daß mit einer MOI (= multiplicity of infection, entspricht dem Verhältnis der Anzahl der infektiösen Viruspartikel zur Anzahl der vorhandenen Zellen) von 0,01-50, bevorzugt 0,1-10 infiziert wird. Die Vermehrung der Viren erfolgt mit oder ohne Zusatz von Tierseren. Für den Fall, daß Serum eingesetzt wird, wird dieses zum Vermehrungsmedium in einerThe cells and cell turf used to multiply the viruses are multiplied in the usual way almost to confluence or to the optimal cell density. Before they are infected with viruses, the cell proliferation medium is preferably removed and the cells are preferably washed with virus multiplication medium. All known cell culture media, such as in particular the MEM mentioned above, are used as the virus propagation medium. Then it is infected with a virus suspension. In the virus suspension, the virus is diluted in the virus propagation medium in such a way that an MOI (= multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) of 0.01-50, preferably 0.1-10, is infected becomes. The viruses are propagated with or without the addition of animal sera. In the event that serum is used, this becomes the propagation medium in a
Konzentration von 1-30 Vol-%, vorzugsweise 2-10 Vol-% zugegeben.Concentration of 1-30 vol%, preferably 2-10 vol% added.
Infektion und Virusvermehrung erfolgen bei Temperaturen zwischen Raumtempe¬ ratur und 40°C, bevorzugt zwischen 32 und 39°C, besonders bevorzugt bei 37°C über mehrere Tage, bevorzugt bis zur vollständigen Zerstörung der infizierten Zellen.Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 32 and 39 ° C., particularly preferably at 37 ° C. for several days, preferably until the infected cells are completely destroyed.
Das virushaltige Medium der infizierten Zellen wird weiter aufgearbeitet, z.B. durch Entfernung der Zellen und Zelltrümmer mittels Filtration mit Porengrößen von z.B. 0,1-0,45 μm und/oder Zentrifugation bis zu 10.000 x g.The virus-containing medium of the infected cells is processed further, e.g. by removing the cells and cell debris by means of filtration with pore sizes of e.g. 0.1-0.45 μm and / or centrifugation up to 10,000 x g.
Die Vermehrung in embryonierten Hühnereiern erfolgt in an sich bekannter Weise in der Allantoishöhle von Hühner-Bruteieren, die 9-12 Tage, bevorzugt 10 Tage bei einer Temperatur von 37-39°C, bevorzugt 38,5°C und einer relativen Luft¬ feuchtigkeit von 30-90%), bevorzugt 50-60%> in einem handelsüblichen Brut¬ schrank, bevorzugt einem Motorbrüter vorbebrütet werden.The multiplication in embryonated chicken eggs takes place in a manner known per se in the Allantoic cavity of chicken hatching eggs, which lasts 9-12 days, preferably 10 days, at a temperature of 37-39 ° C., preferably 38.5 ° C. and a relative atmospheric humidity of 30-90%), preferably 50-60%> are pre-incubated in a commercial incubator, preferably a motor breeder.
Die zur Vermehrung der Viren dienenden Bruteier werden vor der Beimpfung 1-3 Stunden, bevorzugt 2 Stunden auf dem spitzen Eipol senkrecht stehend im Brutschrank gelagert und anschließend nach Vorbereitung der Injektionstelle mit 10-200 μl, bevorzugt 75- 125 μl einer Virussuspension infiziert. In der Virus¬ suspension liegt das Virus im Virusvermehrungsmedium in einer Konzentration von lO^lO7 KID50/ml (50%-Kultur-infektiöse Dosis pro ml Suspension = die Verdünnungsstufe, bei der noch 50%> der eingesetzten Zellkulturen infiziert würden), bevorzugt lO4-^3 KED50/ml vor. Als Virusvermehrungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben genannte MEM.The hatching eggs used for the multiplication of the viruses are standing upright for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation Incubator stored and then infected after preparation of the injection site with 10-200 ul, preferably 75-125 ul of a virus suspension. In the virus suspension, the virus is in the virus propagation medium in a concentration of 10 ^ 10 7 KID 50 / ml (50% culture-infectious dose per ml suspension = the dilution level at which 50% of the cell cultures used would still be infected), preferably 10 4 - ^ 3 KED 50 / ml before. All known cell culture media, such as in particular the MEM mentioned above, are used as virus propagation medium.
Infektion und Virusvermehrung erfolgen unter den oben angegebenen Brutbe- dingungen über mehrere Tage, bevorzugt 2-5 Tage, besonders bevorzugt 3 Tage.Infection and virus multiplication take place under the breeding conditions specified above over several days, preferably 2-5 days, particularly preferably 3 days.
Die virushaltige Allantoisflüssigkeit wird gewonnen durch Absaugen nach Öffnung der Kalkschale sowie der Schalenhaut und der Chorioallantoismembran und kann z.B. mittels Filtration mit Porengrößen von z.B. 0,1-0,45 μm und/oder Zentri- fugation bis zu 10.000 x g weiter aufgearbeitet werden.The virus-containing allantoic fluid is obtained by suction after opening the lime shell as well as the skin of the shell and the chorioallantoic membrane and can e.g. by filtration with pore sizes of e.g. 0.1-0.45 μm and / or centrifugation up to 10,000 x g can be processed further.
Die Inaktivierung der Viren erfolgt in üblicher Weise durch physikalische Ver¬ fahren, z.B. durch Einwirkung von Hitze, UV- oder Gamma-Bestrahlung oder bevorzugt durch chemische Verfahren, z.B. durch Einwirkung von Ethanol, Form¬ aldehyd, ß-Propiolakton und bevorzugt durch Ethylenamine.The viruses are inactivated in the usual way by physical methods, e.g. by exposure to heat, UV or gamma radiation or preferably by chemical methods, e.g. by the action of ethanol, formaldehyde, β-propiolactone and preferably by ethylene amines.
Die chemische Inaktivierung erfolgt in an sich bekannter Weise in geeigneten Re- aktionsgefäßen, die eine Einrichtung zur Haltung einer konstanten Reaktions¬ temperatur sowie zur ständigen Bewegung des Reaktionsgemisches besitzen (z.B. Fermenter). Als Inaktivierungsmittel werden bevorzugt Ethylenamine, besonders bevorzugt 2-Bromoethylamin-Hydrobromid (2-BEA) in einer Konzentration von 1- 10 mmol/1, vorzugsweise 2,5-7,5 mmol/1, eingesetzt.The chemical inactivation takes place in a manner known per se in suitable reaction vessels which have a device for maintaining a constant reaction temperature and for constant movement of the reaction mixture (e.g. fermenter). The preferred inactivating agent is ethylene amines, particularly preferably 2-bromoethylamine hydrobromide (2-BEA) in a concentration of 1-10 mmol / 1, preferably 2.5-7.5 mmol / 1.
Eine Virussuspension mit einer Konzentration von 104'°-109-0 KID50/ml, vorzugs¬ weise 105,0-108'0 KID50/ml, die einer oder mehr Virusvermehrungen entstammt, wird vor Zugabe der 2-BEA-Lösung auf einen pH-Wert von 8,1-8,7, bevorzugt 8,3-8,5 eingestellt. Die Inaktivierung erfolgt bei 4-40°C, bevorzugt 23-37"C, besonders bevorzugt bei 36-37°C über 6-48 Stunden, bevorzugt 16-20 StundenA virus suspension with a concentration of 10 4 '° -10 9-0 TCID 50 / ml, preferred wise 10 5.0 -10 8' 0 KID 50 / ml, which is derived from one or more virus propagation, is prior to addition of 2- BEA solution adjusted to a pH of 8.1-8.7, preferably 8.3-8.5. The inactivation takes place at 4-40 ° C, preferably 23-37 ° C, particularly preferably at 36-37 ° C for 6-48 hours, preferably 16-20 hours
Überschüssiges 2-BEA wird nach Abschluß der Inaktivierung durch Zugabe von hydrolisierenden Agentien neutralisiert. Hierzu eignet sich insbesondere Natrium- Thiosulfat, daß in einer Endkonzentration von 40-80 mmol/1, vorzugsweise 50 mmol/1 zugegeben wird. Die Neutralisation erfolgt bei 4-40°C, bevorzugt bei 2- 8°C über 2-16 Stunden, bevorzugt 4-8 Stunden.Excess 2-BEA is neutralized after the inactivation has been completed by adding hydrolyzing agents. Sodium thiosulfate is particularly suitable for this, that a final concentration of 40-80 mmol / 1, preferably 50 mmol / 1 is added. The neutralization takes place at 4-40 ° C, preferably at 2-8 ° C for 2-16 hours, preferably 4-8 hours.
Die Vermehrung der Viren zur Herstellung von subunits erfolgt in üblicher Weise einerseits in Gewebekulturen animaler Zellen als Primarzellen oder permanenten Zell-Linien, z.B. in Schweine-Zellen, Affen-Zellen oder Rinder-Zellen, bevorzugt in Schweinenieren-Zellen wie z.B. der geklonten, permanenten Schweinenieren- Zelle PK15 (ATCC CCL33 oder deren Abkömmlinge) oder der primären Schweinenieren-Zelle EPK oder Affennieren-Zellen wie den permanenten Affen- nieren-Zellen BGM (Flow 03-240 oder deren Abkömmlinge) oder Vero (ATCC CCL81 oder deren Abkömmlinge) oder Rindernieren-Zellen wie der permanentenThe multiplication of the viruses for the production of subunits takes place in the usual way on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned, permanent pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or their descendants) or Vero (ATCC CCL81 or their descendants) or bovine kidney cells like the permanent one
Rindernieren-Zelle MDBK (ATCC CCL22 oder deren Abkömmlinge) und andererseits in embryonierten Hühnereiern (z.B. Valo-Bruteier, Fa. Lohmann).Beef kidney cell MDBK (ATCC CCL22 or its descendants) and on the other hand in embryonated chicken eggs (e.g. Valo hatching eggs, Lohmann).
Die Vermehrung in Zellkulturen erfolgt in an sich bekannter Weise in stationären Roller- oder Carrier-Kulturen in Form von geschlossenen Zellverbänden (Mono- layern) oder in Suspensions-Kulturen. Als Vermehrungsmedien für die Zellen wer¬ den eingesetzt alle an sich bekannten Zellkulturmedien z.B. beschrieben im Produktkatalog der Fa. Gibco BRL GmbH, Dieselstraße 5, 76344 Eggenstein, wie insbesondere das Minimal Essential Medium (MEM), das als wesentliche Be¬ standteile Aminosäuren, Vitamine, Salze und Kohlenhydrate enthält, komplettiert mit Puffersubstanzen wie z.B. Natrium-Bicarbonat (NaHCÖ3) oder Hydroxyethyl- piperazin-N-2-ethansulfonsäure (Hepes) und gegebenenfalls Tierseren, wie z.B. Seren von Rindern, Pferden bzw. deren Föten. Besonders bevorzugt eingesetzt wird Eagles MEM mit einem Gehalt an NaHCO3 von 0,1-5 g/1, vorzugsweise 0,5- 3 g/1 sowie fötales Kälberserum in einer Konzentration von 1-30 Vol-%, vor- zugsweise 2-10 Vol-%. Die zur Vermehrung der Viren dienenden Zellen und Zellrasen werden in üblicher Weise nahezu bis zur Konfluenz oder bis zur optimaler Zelldichte vermehrt. Vor ihrer Infektion mit Viren wird bevorzugt das Zellvermehrungsmedium entfernt und die Zellen bevorzugt mit Virusvermehrungsmedium gewaschen. Als Virusver- mehrungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben genannte MEM. Danach wird mit einer Virussuspension infiziert. In der Virussuspension liegt das Virus im Virusvermehrungsmedium derart verdünnt vor, daß mit einer MOI (= multiplicity of infection, entspricht dem Verhältnis der Anzahl der infektiösen Viruspartikel zur Anzahl der vorhandenen Zellen) von 0,01-50, bevorzugt 0,1-10 infiziert wird.The multiplication in cell cultures takes place in a manner known per se in stationary roller or carrier cultures in the form of closed cell groups (monolayers) or in suspension cultures. The propagation media used for the cells are all cell culture media known per se, for example described in the product catalog of Gibco BRL GmbH, Dieselstrasse 5, 76344 Eggenstein, such as in particular the Minimal Essential Medium (MEM), which contains amino acids, vitamins as essential components Contains salts and carbohydrates, supplemented with buffer substances such as sodium bicarbonate (NaHCÖ 3 ) or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and optionally animal sera such as serums from cattle, horses or their fetuses. Eagles MEM with a NaHCO 3 content of 0.1-5 g / 1, preferably 0.5-3 g / 1 and fetal calf serum in a concentration of 1-30% by volume, preferably 2- 10 vol%. The cells and cell turf used to multiply the viruses are multiplied in the usual way almost to confluence or to the optimal cell density. Before being infected with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus growth medium. All cell culture media known per se, such as in particular the MEM mentioned above, are used as the virus propagation medium. Then it is infected with a virus suspension. In the virus suspension, the virus is diluted in the virus propagation medium in such a way that it is infected with an MOI (= multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) of 0.01-50, preferably 0.1-10 becomes.
Die" Vermehrung der Viren erfolgt mit oder ohne Zusatz von Tierseren. Für den Fall, daß Serum eingesetzt wird, wird dieses zum Vermehrungsmedium in einer Konzentration von 1-30 Vol-%, vorzugsweise 2-10 Vol-% zugegeben.The "multiplication of the viruses is carried out with or without addition of animal sera. In the case where serum is employed, it is preferably 2-10% by volume added to the replication medium in a concentration of 1-30% by volume.
Infektion und Virusvermehrung erfolgen bei Temperaturen zwischen Raumtem- peratur und 40°C, bevorzugt zwischen 32 und 39°C, besonders bevorzugt bei 37°C über mehrere Tage, bevorzugt bis zur vollständigen Zerstörung der infizierten Zellen.Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 32 and 39 ° C., particularly preferably at 37 ° C. for several days, preferably until the infected cells are completely destroyed.
Das virushaltige Medium der infizierten Zellen wird weiter aufgearbeitet, z.B. durch Entfernung der Zellen und Zelltrümmer mittels Filtration mit Porengrößen von z.B. 0,1-0,45 μm und/oder Zentrifugati on bis zu 10.000 x g.The virus-containing medium of the infected cells is processed further, e.g. by removing the cells and cell debris by means of filtration with pore sizes of e.g. 0.1-0.45 μm and / or centrifugation on up to 10,000 x g.
Die Vermehrung in embryonierten Hühnereiern erfolgt in an sich bekannter Weise in der Allantoishöhle von Hühner-Bruteieren, die 9-12 Tage, bevorzugt 10 Tage bei einer Temperatur von 37-39°C, bevorzugt 38,5°C und einer relativen Luftfeuchtigkeit von 30-90%, bevorzugt 50-60% in einem handelsüblichen Brutschrank, bevorzugt einem Motorbrüter vorbebrütet werden.The multiplication in embryonated chicken eggs takes place in a manner known per se in the Allantoic cave of chicken hatching eggs, which lasts 9-12 days, preferably 10 days, at a temperature of 37-39 ° C., preferably 38.5 ° C. and a relative humidity of 30 -90%, preferably 50-60% in a commercial incubator, preferably an engine breeder.
Die zur Vermehrung der Viren dienenden Bruteier werden vor der Beimpfung 1-3 Stunden, bevorzugt 2 Stunden auf dem spitzen Eipol senkrecht stehend im Brutschrank gelagert und anschließend nach Vorbereitung der Injektionstelle mit 10-200 μl, bevorzugt 75-125 μl einer Virussuspension infiziert. In der Virus- Suspension liegt das Virus im Virusvermehrungsmedium in einer Konzentration von lO'- lO' KID50/ml (50%-Kultur-infektiöse Dosis pro ml Suspension = die Ver¬ dünnungsstufe, bei der noch 50% der eingesetzten Zellkulturen infiziert würden), bevorzugt lO^lO3 KID50/ml vor. Als Virusvermehrungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben genannte MEM.The hatching eggs used to multiply the viruses are stored vertically in the incubator for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation and then infected with 10-200 μl, preferably 75-125 μl, of a virus suspension after preparation of the injection site. In the virus suspension, the virus is concentrated in the virus propagation medium from 10'- 10 'KID 50 / ml (50% culture-infectious dose per ml suspension = the dilution stage at which 50% of the cell cultures used would still be infected), preferably 10 ^ 10 3 KID 50 / ml. All known cell culture media, such as in particular the MEM mentioned above, are used as virus propagation medium.
Infektion und Virusvermehrung erfolgen unter den oben angegebenen Brutbedin¬ gungen über mehrere Tage, bevorzugt 2-5 Tage, besonders bevorzugt 3 Tage.Infection and virus multiplication take place under the above-mentioned breeding conditions over several days, preferably 2-5 days, particularly preferably 3 days.
Die virushaltige Allantoisflüssigkeit wird gewonnen durch Absaugen nach Öffnung der Kalkschale sowie der Schalenhaut und der Chorioallantoismembran und kann z:B: mittels Filtration mit Porengrößen von z.B. 0.1-0,45 μm und/oder Zentrifu¬ gation bis zu 10.000 x g weiter aufgearbeitet werden.The virus-containing allantoic fluid is obtained by suction after opening the lime shell as well as the shell skin and the chorioallantoic membrane and can, for example: by means of filtration with pore sizes of e.g. 0.1-0.45 μm and / or centrifugation up to 10,000 x g can be worked up further.
Die Virusisolierung wird erreicht durch isopyknische oder zonale Zentrifugation in z.B. Saccharose-Dichtegradienten. Hierzu wird das virushaltige Medium bzw. die Allantoisflüssigkeit nach Entfernung der Zelltrümmer einer Zonenzentrifugation bei 100.000 x g bis zur Sedimentation der Viruspartikel unterworfen. Ein reinereVirus isolation is achieved by isopycnic or zonal centrifugation in e.g. Sucrose density gradients. For this purpose, the virus-containing medium or the allantoic fluid, after removal of the cell debris, is subjected to zone centrifugation at 100,000 x g until the virus particles are sedimented. A purer one
Darstellung der Viruspartikel ergibt sich durch Zonenzentrifugation in einer wä߬ rigen Lösung mit einer höheren Dichte als das virushaltige Medium. Als wäßrige Lösung kann z.B. eine 30-60 % w/w, bevorzugt 35-50 % w/w, gepufferte Lösung von Saccharose dienen. Ein noch höherer Reinheitsgrad wird durch Zentrifugation im Dichtegradienten erreicht. Hierzu wird das von Zellen und Zelltrümmer be¬ freite, mittels Zonenzentrifugation konzentrierte Virus durch eine isopyknische oder zonale Dichtegradienten-Zentrifugation in einem Dichtegradienten von z.B. 30 bis 50 % w/w Saccharose in gepufferter wäßriger Lösung bei einer Zentrifugal¬ beschleunigung von z.B. 100.000 bis 150.000 x g isoliert.Representation of the virus particles results from zone centrifugation in an aqueous solution with a higher density than the virus-containing medium. As an aqueous solution e.g. serve a 30-60% w / w, preferably 35-50% w / w, buffered solution of sucrose. An even higher degree of purity is achieved by centrifugation in the density gradient. For this purpose, the virus, freed from cells and cell debris, is concentrated by means of zone centrifugation by isopycnic or zonal density gradient centrifugation in a density gradient of e.g. 30 to 50% w / w sucrose in buffered aqueous solution with a centrifugal acceleration of e.g. 100,000 to 150,000 x g isolated.
Die so erhaltenen Viruskonzentrate werden mit Detergentien behandelt.The virus concentrates thus obtained are treated with detergents.
Geeignete Detergentien sind:Suitable detergents are:
Anionaktive Tenside, wie Natrium-Laurylsulfat, Fertalkoholethersulfate, Mono-/Di- alkylpolyglykoletherorthophosphorsäureester-Monoethanolaminsalz, Calziumalkyl- arylsulfonat, Natriumdesoxycholat, kationaktive Tenside, wie Cetyltrimethylammo- niumchlorid, ampholytische Tenside, wie Di-Natrium-N-lauryl-iminodipropiont oder Lecithin, nicht lonogene Tenside, z.B. polyoxyethyliertes Ricinusol, polyoxy- ethyliertes Sorbitan-Monooleat, Sorbitan-Monostearat, Glycerinmonostearat, Poly- oxyethylenstearat, Alkylphenolpolyglykolether.Anionic surfactants, such as sodium lauryl sulfate, ready alcohol ether sulfates, mono- / dialkyl polyglycol ether orthophosphoric acid ester monoethanolamine salt, calcium alkyl aryl sulfonate, sodium deoxycholate, cationic surfactants, such as cetyltrimethylammonium chloride, ampholytic surfactants, such as diropionium sodium, such as diropyl sodium or lecithin, non-ionic surfactants, for example polyoxyethylated castor oil, polyoxyethylated sorbitan monooleate, sorbitan monostearate, glycerol monostearate, polyoxyethylene stearate, alkylphenol polyglycol ether.
Bevorzugt seien nicht ionogene Detergentien genannt: Nicht-ionische, wasserlösliche Emulgatoren mit einem HLB (hydrophilic-lipophi- lic-balance-Wert) größer 10, z.B. Emulgator NP 40® (Bayer AG), Alkyl- aiylpolyglykolether; Renex 678® (Atlas Chemical Industries), Polyoxyethylenalkyl- arylether; Tween 20® (Atlas), Polyoxyethylensorbitanmonopalmitat; Myri 53® (Atlas), Polyoxyethylenstearat; Atlas G 3707s, Polyoxyethylenlaurylether; Atlas G 3920®, Polyoxyethylenoleylether; Atlas G 9046 T®, Polyoxyethylenmannitan- monolaurat; Emulgator 1371 B® (Bayer AG), Alkylpolyglykolether; Emulgator 1736® (Bayer AG), .Alkylpolyglykolether (Oleylpolyglykolether); Emulgator OX® (Bayer AG), Alkylpolyglykolether (Dodecylpolyglykolether); Ninox BM-2® (Ste- pan Chemical Co.) ethoxyethyliertes Nonylphenol; Triton X-IOO* (Rohm an Haas Co.), Isooctylphenolpolyethoxyethanol; Cremophor EL&, Nonidet P 40® (Shell).Emulsifier such as NP 40 ® (Bayer AG), alkyl Non-ionic, water-soluble emulsifiers having an HLB (hydrophilic-lipophilic lic balance value) greater than 10, aiylpolyglykolether; preferably non-ionic detergents may be mentioned Renex 678 ® (Atlas Chemical Industries), polyoxyethylene alkyl aryl ether; Tween 20® (Atlas), polyoxyethylene sorbitan monopalmitate; Myri 53 ® (Atlas), polyoxyethylene stearate; Atlas G 3707 s , polyoxyethylene lauryl ether; Atlas G 3920 ® , polyoxyethylene oleyl ether; Atlas G 9046 T ® , polyoxyethylene mannitan monolaurate; Emulsifier 1371 B® (Bayer AG), alkyl polyglycol ether; Emulsifier 1736 ® (Bayer AG),. Alkyl polyglycol ether (oleyl polyglycol ether); Emulsifier OX ® (Bayer AG), alkyl polyglycol (Dodecylpolyglykolether); Ninox BM-2 ® (Ste- pan Chemical Co.) ethoxyethyliertes nonylphenol; Triton X-IOO * (Rohm to Haas Co.), isooctylphenol polyethoxyethanol; Cremophor EL & , Nonidet P 40 ® (Shell).
Die Detergentien werden in Form verdünnter wässriger Lösungen angewendet. Genannt seien Lösungen mit 0,1 bis 10 Volumenprozent, bevorzugt mit 0,5 bis 5 Volumenprozent, besonders bevorzugt ca. 1 Volumenprozent Detergensgehalt.The detergents are used in the form of dilute aqueous solutions. Solutions with 0.1 to 10 percent by volume, preferably with 0.5 to 5 percent by volume, particularly preferably about 1 percent by volume of detergent content are mentioned.
Die Detergenslösung wird im Volumenverhältnis von ca. 1 : 1 bis ca. 10: 1 zum Viruskonzentrat zugegeben. Bevorzugt ist das Verhältnis Detergenslösung zuThe detergent solution is added to the virus concentrate in a volume ratio of approximately 1: 1 to approximately 10: 1. The ratio of detergent solution to is preferred
Viruskonzentrat von ca. 3: 1.Virus concentrate of approx. 3: 1.
Die Detergensbehandlung erfolgt unter ständiger Bewegung des Gemisches bei Temperaturen zwischen 0 und ca. 24°C, bevorzugt zwischen 2 und 8°C. Die Detergensbehandlung dauert 15 Minuten bis 2 Tage, bevorzugt 6 bis 18 Stunden. Zur Verbesserung der Detergensbehandlung kann das Gemisch zusätzlich einerThe detergent treatment is carried out with constant movement of the mixture at temperatures between 0 and approximately 24 ° C., preferably between 2 and 8 ° C. The detergent treatment lasts 15 minutes to 2 days, preferably 6 to 18 hours. In order to improve the detergent treatment, the mixture can additionally contain one
Ultraschallbehandlung unterworfen werden.Be subjected to ultrasound treatment.
Die bei dieser Behandlung nicht gelösten Partikel werden entfernt, bevorzugt durch Filtration oder Zentrifugation bei z.B. 150.000 x g. Das so gewonnene Filtrat bzw. der Zentrifugationsüberstand kann bis zu seiner Weiterverarbeitung bei tiefen Temperaturen (0 bis -70°C) gelagert werden. Die im Lysat enthaltenen Glycoproteine der Viruspartikel werden durch Be¬ handlung mit Lektinen isoliert. Lektine sind Proteine oder Glycoproteine aus Pflanzen, speziell deren Samen, Mikroorganismen, Vertebraten und Invertebraten, die spezifisch Zucker und deren Konjugate binden. Verwendet werden Lektine, die Glycoproteine aus Paramyxoviren erkennen und binden. Bevorzugt verwendet werden Lektine, die Mannose und/oder Glucose sowie deren Konjugate erkennen. Im Einzelnen seien genannt die Lektine aus Canavalia ensifoπnis, Lens culinaris, Lathygros odoratus, Pisum sativum, Vicia faba, Sambucus nigra, Glycine max, Ulex europaens, Helix promatia, Phytolacca americana, Lycopersicon esculentum, Datura stramonium, Bandeiraea simplicifolia.The particles not dissolved in this treatment are removed, preferably by filtration or centrifugation at, for example, 150,000 x g. The filtrate or the centrifugation supernatant obtained in this way can be stored at low temperatures (0 to -70 ° C) until it is processed further. The glycoproteins of the virus particles contained in the lysate are isolated by treatment with lectins. Lectins are proteins or glycoproteins from plants, especially their seeds, microorganisms, vertebrates and invertebrates, which specifically bind sugar and their conjugates. Lectins are used that recognize and bind glycoproteins from paramyxoviruses. Lectins which recognize mannose and / or glucose and their conjugates are preferably used. In particular, the lectins from Canavalia ensifoπnis, Lens culinaris, Lathygros odoratus, Pisum sativum, Vicia faba, Sambucus nigra, Glycine max, Ulex europaens, Helix promatia, Phytolacca americana, Lycopersicon esculentum, Datura stramonicea, bandeira should be mentioned.
Die" Lektine werden in wasserlöslicher oder nicht wasserlöslicher Form verwendet. In der nicht wasserlöslichen Form werden sie bevorzugt immobilisiert durch Kopplung an inerte Matrize wie z.B. Dextrane, Agarosen, Celluosen als Sus¬ pensionen oder Gele eingesetzt. Im Einzelnen seien genannt Concanavalin-A- Agarose, Concanavalin-A-Sepharose, Lentil-Lectin-Sepharose, Agarose-Wheat-The lectins are used in water-soluble or water-insoluble form. In the water-insoluble form, they are preferably immobilized by coupling to inert matrix such as dextrans, agaroses, celluoses as suspensions or gels. Concanavalin-A-agarose may be mentioned in detail , Concanavalin-A-Sepharose, Lentil-Lectin-Sepharose, Agarose-Wheat-
Germ-Lectin, Helix-pomatia-Lectin-Sepharose.Germ-lectin, Helix-pomatia-lectin-Sepharose.
Die Lektine werden in Form einer Detergens- und Salz-haltigen Lösung, Suspen¬ sion oder eines Gels eingesetzt. Dazu wird sowohl das Lysat als auch die eingesetzte Lektinlösung, Lektinsuspension oder das Lektingel zuvor mit soviel Kochsalz sowie den bekannten Lektin-stabilisierenden Salzen versetzt, daß eineThe lectins are used in the form of a detergent and salt-containing solution, suspension or a gel. For this purpose, both the lysate and the lectin solution, lectin suspension or the lectin gel used are mixed with sufficient sodium chloride and the known lectin-stabilizing salts that a
Konzentration an Kochsalz von 0,5 bis 2, bevorzugt 0,7 bis 1,2 mol/1 entsteht. Die Konzentrationseinstellung der Lysate erfolgt bevorzugt durch Dialyse. Die erforderliche Konzentration der Lektin-stabilisierenden Salze ist aus dem Stand der Technik bekannt und für die einzusetzenden Lektine spezifisch. Die Lektinlösung, Lektinsuspension oder das Lektingel wird darüber hinaus mit dem zur Behandlung der Lysate eingesetzten Detergens in gleicher Konzentration versetzt, so daß Lysat und Lektinlösung identische Konzentrationen an Salz und Detergens besitzen.Concentration of common salt of 0.5 to 2, preferably 0.7 to 1.2 mol / 1 arises. The concentration setting of the lysates is preferably carried out by dialysis. The required concentration of the lectin-stabilizing salts is known from the prior art and is specific for the lectins to be used. The lectin solution, lectin suspension or the lectin gel is also mixed with the detergent used to treat the lysates in the same concentration, so that the lysate and lectin solution have identical concentrations of salt and detergent.
Verwendet werden ca. 1 bis 150 mg, bevorzugt 1 bis 50 mg, besonders bevorzugt 5 bis 20 mg, reines Lektin pro ml Lösung, Suspension oder Gel. Dem Lysat wird soviel dieser Lektinlösung, -Suspension oder dem Lektingel zugesetzt, daß pro mgAbout 1 to 150 mg, preferably 1 to 50 mg, particularly preferably 5 to 20 mg, of pure lectin are used per ml of solution, suspension or gel. So much of this lectin solution, suspension or lectin gel is added to the lysate that per mg
Gesamtprotein 0,01 bis 50 mg, bevorzugt 0,1 bis 20 mg, besonders bevorzugt 0,5 bis 5 mg, Lektin eingesetzt werden. Die Lektinbehandlung erfolgt bei 0 bis ca. 24° C, bevorzugt bei 2 bis 8° C über ca 10 Minuten bis 3 Tage, bevorzugt 1 StundeTotal protein 0.01 to 50 mg, preferably 0.1 to 20 mg, particularly preferably 0.5 to 5 mg, lectin can be used. The lectin treatment takes place at 0 to approx. 24 ° C, preferably at 2 to 8 ° C for about 10 minutes to 3 days, preferably 1 hour
Die Reaktion der Lektine mit den Glycoproteinen kann auch mittels Saulen- chromatographie erfolgen, wobei das Lysat mit dem an eine gelformige Matrix in einer Chromatographiesaule immobilisierten Lektin in Kontakt gebracht wird.The reaction of the lectins with the glycoproteins can also be carried out by means of column chromatography, the lysate being brought into contact with the lectin immobilized on a gel-like matrix in a chromatography column.
Der Glycoprotein-Lektin-Komplex wird in üblicher Weise aus der Gesamtlosung oder -Suspension abgetrennt. Dies kann durch Zentrifugation, Filtration oder im Fall der Chromatographie durch Waschen erfolgen.The glycoprotein-lectin complex is separated from the total solution or suspension in a conventional manner. This can be done by centrifugation, filtration or, in the case of chromatography, by washing.
In -den bei diesen Verfahren erhaltenen Suspensionen oder Gelen, die die Lektin- Glycoprotein-Komplexe enthalten, kann durch Filtration. Zentrifugation, Dialyse oder andere Waschvorgange die Konzentration an Detergens und/oder Salz in den physiologisch verträglichen Bereich oder bis zur Eleminierung verändert werdenThe suspensions or gels obtained in these processes and containing the lectin-glycoprotein complexes can be filtered off. Centrifugation, dialysis or other washing processes, the concentration of detergent and / or salt can be changed within the physiologically acceptable range or until it is eliminated
Die so erhaltenen Suspensionen oder Gele der Lektin-Glycoprotein-Komplexe kön¬ nen direkt als antigenes Material verwendet werden. Sie können in Abhängigkeit vom Gehalt des an Lektin gebundenen Glycoproteins weiter konzentriert oder verdünnt werden.The suspensions or gels of the lectin-glycoprotein complexes obtained in this way can be used directly as antigenic material. They can be further concentrated or diluted depending on the content of the glycoprotein bound to lectin.
Die Suspensionen oder Gele der Lektin-Glycoprotein-Komplexe können bei Tem¬ peraturen unter 8° C gelagert werde. Sie lassen sich auch gefriertrocknen.The suspensions or gels of the lectin-glycoprotein complexes can be stored at temperatures below 8 ° C. They can also be freeze-dried.
Aus den erhaltenen Suspensionen oder Gelen der Lektin-Glycoprotein-Komplexe können zur Herstellung antigenen Materials die Glycoproteine isoliert werden.To prepare antigenic material, the glycoproteins can be isolated from the suspensions or gels of the lectin-glycoprotein complexes obtained.
Dazu werden die Suspensionen oder Gele mit einer salzhaltigen, wäßrigen Zuckerlosung behandelt.For this purpose, the suspensions or gels are treated with a saline, aqueous sugar solution.
Die Art des einzusetzenden Zuckers richtet sich nach der Spezifitat der verwendeten Lektine. Die Konzentration des Zuckers betragt 0, 1 bis 1 mol/l, bevorzugt 0,1 bis 0,5 mol/l, besonders bevorzugt 0,3 bis 0,5 mol/l Konzentration und Zusammensetzung des Salzgehaltes entspricht der der Glycoprotein-Lektin- Komplex enthaltenden Suspensionen oder Gele. Die Behandlung der Zuckerlösung erfolgt bei 0 bis ca. 24° C, bevorzugt bei 2 bis 8° C. Die Behandlung beträgt ca. 15 Minuten bis 4 Tage, bevorzugt 1 Stunde bis 2 Tage, besonders bevorzugt 10 bis 24 Stunden.The type of sugar to be used depends on the specificity of the lectins used. The concentration of the sugar is 0.1 to 1 mol / l, preferably 0.1 to 0.5 mol / l, particularly preferably 0.3 to 0.5 mol / l. The concentration and composition of the salt content corresponds to that of the glycoprotein lectin. Suspensions or gels containing complexes. The sugar solution is treated at 0 to about 24 ° C., preferably at 2 to 8 ° C. The treatment is about 15 minutes to 4 days, preferably 1 hour to 2 days, particularly preferably 10 to 24 hours.
Die hierbei eluierten Glycoproteine werden durch Zentrifugation, Filtration oder durch andere übliche Trennverfahren (z.B. Chromatographie) von den Lektinen isoliert. In den resultierenden Präparationen lassen sich die Konzentrationen an Detergens, Salz und Zucker wie bereits oben beschrieben verändern.The glycoproteins eluted in this process are isolated from the lectins by centrifugation, filtration or by other customary separation processes (e.g. chromatography). In the resulting preparations, the concentrations of detergent, salt and sugar can be changed as already described above.
Die so erhaltenen isolierten Glycoproteine können als antigenes Material ver¬ wendet werden. Der Glycoproteingehalt kann durch Konzentration oder Ver- dünnung verändert werden.The isolated glycoproteins obtained in this way can be used as antigenic material. The glycoprotein content can be changed by concentration or dilution.
Die Lagerung der Präparationen erfolgt in Form ihrer Lösungen bei Temperaturen unter 0° C oder in lyophilisierter Form.The preparations are stored in the form of their solutions at temperatures below 0 ° C. or in lyophilized form.
Zur Herstellung von subunits der Viruspartikel auf rekombinantem Weg wird zuerst das Virusgenom gewonnen.In order to produce subunits of the virus particles by recombinant means, the virus genome is first obtained.
Zur Gewinnung des Virusgenoms erfolgt zunächst die Vermehrung der Viren in üblicher Weise einerseits in Gewebekulturen animaler Zellen als Primärzellen oder permanenten Zell-Linien, z.B. in Schweine-Zellen, Affen-Zellen oder Rinder- Zellen, bevorzugt in Schweinenieren-Zellen wie z.B. der geklonten, permanenten Schweinenieren-Zelle PK15 (ATCC CCL33 oder deren Abkömmlinge) oder der primären Schweinenieren-Zelle EPK oder Affennieren-Zellen wie den permanentenTo obtain the virus genome, the viruses are first multiplied in the usual way on the one hand in tissue cultures of animal cells as primary cells or permanent cell lines, e.g. in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as e.g. the cloned permanent pig kidney cell PK15 (ATCC CCL33 or its descendants) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent one
Affennieren-Zellen BGM (Flow 03-240 oder deren Abkömmlinge) oder Vero (ATCC CCL81 oder deren Abkömmlinge) oder Rindernieren-Zellen wie der per¬ manenten Rindernieren-Zelle MDBK (ATCC CCL22 oder deren Abkömmlinge) und andererseits in embryonierten Hühnereiern (z.B. Valo-Bruteier, Fa. Lohmann).Monkey kidney cells BGM (Flow 03-240 or their descendants) or Vero (ATCC CCL81 or their descendants) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or their descendants) and on the other hand in embryonated chicken eggs (e.g. Valo - hatching eggs, Lohmann).
Die Vermehrung in Zellkulturen erfolgt in an sich bekannter Weise in stationärenThe propagation in cell cultures takes place in a known manner in stationary
Roller- oder Carrier-Kulturen in Form von geschlossenen Zellverbänden (Mono- layern) oder in Suspensions-Kulturen. Als Vermehrungsmedien für die Zellen werden eingesetzt alle an sich bekannten Zellkulturmedien z.B. beschrieben im Produktkatalog der Fa. Gibco BRL GmbH, Dieselstraße 5, 76344 Eggenstein, wie insbesondere das Minimal Essential Medium (MEM), das als wesentliche Bestand¬ teile Aminosäuren, Vitamine, Salze und Kohlenhydrate enthält, komplettiert mit Puffersubstanzen wie z.B. Natrium-Bicarbonat (NaHCO?) oder Hydroxyethyl- piperazin-N-2-ethansulfonsäure (Hepes) und gegebenenfalls Tierseren, wie z.B. Seren von Rindern, Pferden bzw. deren Föten. Besonders bevorzugt eingesetzt wird Eagles MEM mit einem Gehalt an NaHCO3 von 0,1-5 g/1, vorzugsweise 0,5- 3 g/1 sowie fötales Kälberserum in einer Konzentration von 1-30 Vol-%, vorzugsweise 2-10 Vol-%.Roller or carrier cultures in the form of closed cell groups (monolayers) or in suspension cultures. All known cell culture media are used as propagation media for the cells, for example described in the product catalog of Gibco BRL GmbH, Dieselstrasse 5, 76344 Eggenstein, such as in particular the Minimal Essential Medium (MEM), which contains amino acids, vitamins, salts and carbohydrates as essential components, completed with buffer substances such as sodium bicarbonate (NaHCO ? ) or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and optionally animal sera, such as sera from cattle, horses or their fetuses. Eagles MEM with a NaHCO 3 content of 0.1-5 g / 1, preferably 0.5-3 g / 1 and fetal calf serum in a concentration of 1-30% by volume, preferably 2-10%, are particularly preferably used -%.
Die zur Vermehrung der Viren dienenden Zellen und Zellrasen werden in üblicher Weise nahezu bis zur Konfluenz oder bis zur optimaler Zelldichte vermehrt. Vor ihrer Infektion mit Viren wird bevorzugt das Zeilvermehrungsmedium entfernt und die Zellen bevorzugt mit Virusvermehrungsmedium gewaschen. Als Virusver¬ mehrungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben genannte MEM. Danach wird mit einer Virussuspension infiziert. In der Virussuspension liegt das Virus im Virusvermehrungsmedium derart verdünnt vor, daß mit einer MOI (= multiplicity of infection, entspricht dem Verhältnis der Anzahl der infektiösen Viruspartikel zur Anzahl der vorhandenen Zellen) von 0,01-50, bevorzugt 0,1-10 infiziert wird.The cells and cell turf used to multiply the viruses are multiplied in the usual way almost to confluence or to the optimal cell density. Before they are infected with viruses, the cell proliferation medium is preferably removed and the cells are preferably washed with virus multiplication medium. All known cell culture media, such as in particular the MEM mentioned above, are used as the virus propagation medium. Then it is infected with a virus suspension. In the virus suspension, the virus is diluted in the virus propagation medium in such a way that an MOI (= multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) of 0.01-50, preferably 0.1-10, is infected becomes.
Die Vermehrung der Viren erfolgt mit oder ohne Zusatz von Tierseren. Für den Fall, daß Serum eingesetzt wird, wird dieses zum Vermehrungsmedium in einerThe viruses are propagated with or without the addition of animal sera. In the event that serum is used, this becomes the propagation medium in a
Konzentration von 1-30 Vol-%, vorzugsweise 2-10 Vol-% zugegeben.Concentration of 1-30 vol%, preferably 2-10 vol% added.
Infektion und Virusvermehrung erfolgen bei Temperaturen zwischen Raumtem¬ peratur und 40°C, bevorzugt zwischen 32 und 39°C, besonders bevorzugt bei 37°C über mehrere Tage, bevorzugt bis zur vollständigen Zerstörung der infizierten Zellen.Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 32 and 39 ° C., particularly preferably at 37 ° C. for several days, preferably until the infected cells are completely destroyed.
Das virushaltige Medium der infizierten Zellen wird weiter aufgearbeitet, z.B. durch Entfernung der Zellen und Zelltrümmer mittels Filtration mit Porengrößen von z.B. 0,1-0,45 μm und/oder Zentrifugation bis zu 10.000 x g.The virus-containing medium of the infected cells is processed further, e.g. by removing the cells and cell debris by means of filtration with pore sizes of e.g. 0.1-0.45 μm and / or centrifugation up to 10,000 x g.
Die Vermehrung in embryonierten Hühnereiern erfolgt in an sich bekannter Weise in der Allantoishöhle von Hühner-Bruteieren, die 9-12 Tage, bevorzugt 10 Tage . > ιThe multiplication in embryonated chicken eggs takes place in a manner known per se in the Allantoic cave of chicken hatching eggs, which lasts 9-12 days, preferably 10 days , > ι
bei einer Temperatur von 37-39°C, bevorzugt 38 5"C und einer relativen Luft¬ feuchtigkeit von 30-90%), bevorzugt 50-60% in einem handelsüblichen Brut¬ schrank, bevorzugt einem Motorbruter vorbehaltet werdenat a temperature of 37-39 ° C, preferably 38 5 "C and a relative humidity of 30-90%), preferably 50-60% in a commercial incubator, preferably a motor breeder
Die zur Vermehrung der Viren dienenden Bruteier werden vor der Beimpfung 1-3 Stunden, bevorzugt 2 Stunden auf dem spitzen Eipol senkrecht stehend im Brut¬ schrank gelagert und anschließend nach Vorbereitung der Injeküonstelle mit 10- 200 μl, bevorzugt 75-125 μl einer Virussuspension infiziert In der Virus¬ suspension liegt das Virus im Virusvermehrungsmedium in einer Konzentration von lOl-IO7 KED50/ml (50%-Kultur-infektιose Dosis pro ml Suspension = die Verdunnungsstufe, bei der noch 50%» der eingesetzten Zellkulturen infiziert wurden), bevorzugt 104-105 KID50/ml vor Als Virusvermehrungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien wie insbesondere das oben genannte MEMThe hatching eggs used to multiply the viruses are stored vertically in the incubator for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation and then infected with 10 to 200 μl, preferably 75 to 125 μl, of a virus suspension after preparation of the injection site In the virus suspension, the virus is in the virus propagation medium in a concentration of 10 l -IO 7 KED 50 / ml (50% culture-infectious dose per ml suspension = the dilution stage at which 50% of the cell cultures used were still infected) , preferably 10 4 -10 5 KID 50 / ml before The virus propagation medium used are all cell culture media known per se, in particular the MEM mentioned above
Infektion und Virusvermehrung erfolgen unter den oben angegebenen Brutbe- dingungen über mehrere Tage, bevorzugt 2-5 Tage, besonders bevorzugt 3 Tage.Infection and virus multiplication take place under the breeding conditions specified above over several days, preferably 2-5 days, particularly preferably 3 days.
Die virushaltige Allantoisflüssigkeit wird gewonnen durch Absaugen nach Öffnung der Kalkschale sowie der Schalenhaut und der Choπoallantoismembran und kann z B mittels Filtration mit Porengroßen von z B 0 1-0,45 μm und/oder Zentri¬ fugation bis zu 10 000 x g weiter aufgearbeitet werdenThe virus-containing allantoic fluid is obtained by suction after opening the lime shell and the skin of the shell and the choalloallantoic membrane and can be worked up further, for example by means of filtration with pore sizes of, for example, 0 1-0.45 μm and / or centrifugation up to 10,000 × g
Die Virusreinigung, bzw -isolierung wird erreicht durch isopyknische oder zonale Zentπfugaüon in z.B. Saccharose-Dichtegradienten Hierzu wird das virushaltige Medium bzw. die Allantoisflüssigkeit nach Entfernung der Zelltrummer einer Zonenzentrifugation bei 100 000 x g bis zur Sedimentation der Viruspartikel unterworfen Ein reinere Darstellung der Viruspartikel ergibt sich durch Zonenzentrifugation in einer wäßrigen Losung mit einer höheren Dichte als das virushaltige Medium. Als waßπge Losung kann z B eine 30-60 % w/w, bevorzugt 35-50 %> w/w, gepufferte Losung von Saccharose dienen Ein noch höherer Reinheitsgrad wird durch Zentrifugation im Dichtegradienten erreicht Hierzu wird das von Zellen und Zelltrummer befreite, mittels Zonenzentrifugation konzentrierte Virus durch eine isopyknische oder zonale Dichtegradienten-Zentπfugation in einem Dichtegradienten von z.B. 30 bis 50 %> w/w Saccharose in gepufferter wäßriger Losung bei einer Zentrifugalbeschleunigung \ jn z B 100 000 bis 150 000 x g isoliertVirus purification or isolation is achieved by isopycnic or zonal centrifuge in, for example, sucrose density gradients. For this purpose, the virus-containing medium or the allantoic fluid is subjected to zone centrifugation at 100,000 xg after removal of the cell debris until sedimentation of the virus particles results in a cleaner representation of the virus particles by zone centrifugation in an aqueous solution with a higher density than the virus-containing medium. A 30-60% w / w, preferably 35-50%> w / w, buffered solution of sucrose can serve as a water solution. An even higher degree of purity is achieved by centrifugation in a density gradient. For this purpose, the cells and cell debris are freed from Zone centrifugation concentrated virus by an isopycnic or zonal density gradient centrifugation in a density gradient of, for example, 30 to 50%> w / w sucrose in buffered aqueous solution with a centrifugal acceleration \ jn z 100 000 to 150 000 xg isolated
Zur Gewinnung geeigneter Gene, die für immunogene Proteine codieren, wird aus den gereinigten Viruspartikeln zunächst das Virusgenom isoliert Die native Virus- RNS wird vorzugsweise gewonnen durch Behandlung der gereinigten Virusparükel mit Detergens- und Proteasen-haltigen, waßπgen LosungenTo obtain suitable genes which code for immunogenic proteins, the viral genome is first isolated from the purified virus particles. The native virus RNA is preferably obtained by treating the purified viral poultry with detergent- and protease-containing, aqueous solutions
Eingesetzt werden anionische, kationische, amphoteπsche und nicht-ionische De¬ tergentien. Bevorzugt eingesetzt werden ionische Detergentien, vorzugsweise Na- tnumdodecylsulfat, in einer Konzentration von 0,1-10 Vol-%>, vorzugsweise 0,5-3 Vol-%Anionic, cationic, amphoteric and non-ionic detergents are used. Ionic detergents, preferably sodium dodecyl sulfate, are preferably used in a concentration of 0.1-10% by volume, preferably 0.5-3% by volume.
Als Proteasen werden solche eingesetzt, die in Gegenwart von Detergentien wirken, wie z B Pronase und, vorzugsweise eingesetzt Proteinase K Die Pro¬ teasen werden in einer Konzentration 0,01-10 mg/ml, bevorzugt 0,05-0,5 mg/ml eingesetztProteases used are those which act in the presence of detergents, such as pronase and, preferably, proteinase K. The proteases are used in a concentration of 0.01-10 mg / ml, preferably 0.05-0.5 mg / ml used
Vorzugsweise werden wäßrige, gepufferte Losungen mit Zusatz von RNase-Inhi- bitoren verwendetAqueous, buffered solutions with the addition of RNase inhibitors are preferably used
Als Puffersubstanzen werden verwendet Salze schwacher Sauren mit starken Basen wie z B Tris(hydroxymethyl)-amιnomethan, Salze starker Sauren mit schwachen Basen wie z B primäre Phosphate oder Gemische hiervon Vorzugsweise verwendet wird Tris(hydroxymethyl)-amιnomethan Die Puff ersub stanzen werden in Konzentrationen eingesetzt, die einen pH-Wert sicherstellen, bei dem die RNS nicht denaturiert Bevorzugt werden pH-Werte von 6-8,5, besonders bevorzugt von 7-8Salts of weak acids with strong bases such as, for example, tris (hydroxymethyl) aminomethane, salts of strong acids with weak bases such as, for example, primary phosphates or mixtures thereof, are preferably used as tris (hydroxymethyl) aminomethane used, which ensure a pH value at which the RNA does not denature. Preferred pH values are 6-8.5, particularly preferably 7-8
Als RNase-Inhibitoren dienen z B Ribonucleosid-Vanadyl-Komplexe, Protem-Inhi- bitoren (z B. RNAguard®/Pharmacιa) oder vorzugsweise DiethylpyrocarbonatAs RNase inhibitors such serve B ribonucleoside vanadyl complexes, Protem inhibitors (eg RNAguard ® / Pharmacιa) or preferably diethyl pyrocarbonate
(DEPC) in Konzentrationen von 0,01-2 Vol-%, bevorzugt 0, 1-0,5 Vol-% Die lipophilen Substanzen des Viruslysates werden anschließend extrahiert unter Verwendung von Lösemittel wie z.B. Phenol, Chloroform oder Mischungen davon. Die Extraktion erfolgt in ein oder mehreren Stufen.(DEPC) in concentrations of 0.01-2% by volume, preferably 0.1-0.5% by volume The lipophilic substances of the virus lysate are then extracted using solvents such as phenol, chloroform or mixtures thereof. The extraction takes place in one or more stages.
In der verbleibenden wäßrigen Phase wird die RNS mittels wäßriger Lösungen gefällt, die Alkohole wie z.B. Ethanol oder Isopropanol und monovalente Chlorid¬ oder Acetat-Salze wie z.B. Natriumchlorid, Natriumacetat oder Kaliumacetat ent¬ halten.In the remaining aqueous phase, the RNA is precipitated using aqueous solutions containing alcohols such as e.g. Ethanol or isopropanol and monovalent chloride or acetate salts such as e.g. Contain sodium chloride, sodium acetate or potassium acetate.
Die Konzentration der Alkohole liegt zwischen 40 und 100 Vol-%>, bevorzugt 60 und 80 Vol-% und die der Chlorid- oder Acetat-Salze zwischen 0,01 und 1 mol/l, bevorzugt 0,1 bis 0,8 mol/l.The concentration of the alcohols is between 40 and 100% by volume, preferably 60 and 80% by volume, and that of the chloride or acetate salts is between 0.01 and 1 mol / l, preferably 0.1 to 0.8 mol / l.
Die gefällte RNS wird aus der wäßrigen Lösung z.B. durch Zentrifugation gewonnen und in einer wäßrigen Lösung z.B. DEPC-Wasser wiederum aufgelöst. Diese wäßrige Lösung enthält bevorzugt Puff ersub stanzen wie z.B. Tris(hydroxy- methyl)-aminomethan in Konzentrationen von 1-100 mmol/l, vorzugsweise 10-50 mmol/l, eventuell unter Zusatz von Ethylendiamintetraacetat (EDTA) in Konzen¬ trationen von 0,1-10 mmol/l, vorzugsweise 1-10 mmol/l oder Dithiothreit (DTT) in Konzentrationen von 0,1-10 mmol/l, vorzugsweise 1-10 mmol/l.The precipitated RNA is extracted from the aqueous solution e.g. obtained by centrifugation and in an aqueous solution e.g. DEPC water dissolved again. This aqueous solution preferably contains buffer substances such as e.g. Tris (hydroxymethyl) aminomethane in concentrations of 1-100 mmol / l, preferably 10-50 mmol / l, possibly with the addition of ethylenediaminetetraacetate (EDTA) in concentrations of 0.1-10 mmol / l, preferably 1 -10 mmol / l or dithiothreitol (DTT) in concentrations of 0.1-10 mmol / l, preferably 1-10 mmol / l.
Die isolierte RNS wird bei Temperaturen unter -65° C gelagert.The isolated RNS is stored at temperatures below -65 ° C.
Eine andere Methode zur RNS-Isolierung ist z.B. die RNS-Extraktion mit Guanidiniumthiocyanat und anschließender Cäsiumchlorid-Dichtegradienten-Zentri- fugation des Virus-Lysates.Another method for RNA isolation is e.g. RNA extraction with guanidinium thiocyanate and subsequent cesium chloride density gradient centrifugation of the virus lysate.
Methoden zur RNS-Isolierung sind beschrieben in: J. Sambrook, E.F. Fritsch and T. Maniatis (Hrsg.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989.Methods for RNA isolation are described in: J. Sambrook, E.F. Fritsch and T. Maniatis (ed.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989.
Die Identifizierung geeigneter Gene erfolgt unter Verwendung des isolierten Virus-Suitable genes are identified using the isolated virus
Genoms z.B. durch: *ι -Genome e.g. through: * ι -
a) RNS/DNS-Hybridisierung des Genoms unter Verwendung bekannter Gen¬ sonden. Als geeignete Gensonden dienen DNS-Proben mit Nucleotid-Se- quenzen von bekannten Genen für Immunogene verwandter Virusstämme, wie z.B. dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2.a) RNA / DNA hybridization of the genome using known gene probes. DNA probes with nucleotide sequences of known genes for immunogens of related virus strains, such as e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
b) Herstellung einer komplementären DNS (cDNS), Klonierung der cDNS in z.B. bakterielle Plasmide wie z.B. pBR322 zur Anreicherung von viraler DNS und Hybridisierung der klonierten DNS mittels bekannter Gensonden. Ais geeignete Gensonden dienen DNS-Proben mit Nucleotid- Sequenzen von bekannten Genen für Immunogene verwandter Virusstämme, wie z.B. dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2.b) Preparation of a complementary DNA (cDNA), cloning of the cDNA in e.g. bacterial plasmids such as pBR322 for the enrichment of viral DNA and hybridization of the cloned DNA using known gene probes. Suitable gene probes are DNA samples with nucleotide sequences of known genes for immunogens of related virus strains, e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
c) Herstellung einer komplementären DNS (cDNS) und Klonierung der cDNS in Plasmid-Expressionsvektoren wie z.B. pUC18/19 oder pUC 118/119 oder in λ-Bakteriophagen-Expressionsvektoren wie z.B. λgtl 1 und dessen Abkömmlinge oder λZAP oder λORF8. Die Identifizierung der Gene er- folgt durch Nachweis ihrer exprimierten Immunogene mit Hilfe vonc) Preparation of a complementary DNA (cDNA) and cloning of the cDNA in plasmid expression vectors such as e.g. pUC18 / 19 or pUC 118/119 or in λ bacteriophage expression vectors such as e.g. λgtl 1 and its descendants or λZAP or λORF8. The genes are identified by detecting their expressed immunogens with the help of
Antikörpern, die direkt oder indirekt z.B. mittels Immunfluoreszenz oder Immunpräzipitation nachgewiesen werden. Geeignete Antikörper sind sol¬ che, die mit Immunogenen verwandter Virusstämme reagieren, wie z.B. dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2.Antibodies that directly or indirectly e.g. by means of immunofluorescence or immunoprecipitation. Suitable antibodies are those which react with immunogens of related virus strains, e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
d) Herstellung einer komplementären DNS (cDNS) und Klonierung der cDNS in z.B. bakterielle Plasmide zur Anreicherung von viraler DNS. Die virale DNS der Klone wird sequenziert und untersucht auf Sequenzhomologien mit bekannten Genen verwandter Virusstämme, wie z.B. dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2.d) Preparation of a complementary DNA (cDNA) and cloning of the cDNA in e.g. bacterial plasmids for the enrichment of viral DNA. The viral DNA of the clones is sequenced and examined for sequence homologies with known genes from related virus strains, e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
e) Sequenzierung der cDNS bei deren Herstellung und Untersuchung auf Se¬ quenzhomologien mit bekannten Genen verwandter Virusstämme, wie z.B. dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2.e) Sequencing of the cDNA during its production and examination for sequence homologies with known genes of related virus strains, such as e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
f) Kombinationen der Methoden a) bis e). Methoden zur RNS/DNS- und DNS/DNS-Hybridisierung, Herstellung von cDNS, Klonierung von DNS in Plasmid- und Bakteriophagen-Vektoren, Sequenzierung von DNS sowie Methoden zum immunologischen Nachweis von exprimierten Immunogenen sind beschrieben in:f) combinations of methods a) to e). Methods for RNA / DNA and DNA / DNA hybridization, production of cDNA, cloning of DNA in plasmid and bacteriophage vectors, sequencing of DNA and methods for the immunological detection of expressed immunogens are described in:
- J. Sambrook, E.F. Fritsch and T. Maniatis (Hrsg.), Molecular Cloning, A- J. Sambrook, E.F. Fritsch and T. Maniatis (ed.), Molecular Cloning, A
Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989
F.M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987F.M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987
- A. Mayr, Virologische Arbeitsmethoden, Band III, Gustav Fischer Verlag,- A. Mayr, Virological Working Methods, Volume III, Gustav Fischer Verlag,
Stuttgart, 1989Stuttgart, 1989
Es werden solche Gene ausgewählt, bei denen mit den oben genannten Methoden eine Nucleotid-Sequenz nachgewiesen werden kann, die für ein oder mehrere Immunogene codiert. Als Beispiel sind im Sequenzprotokoll (unten) wiedergegeben die Nucleotid-Sequenzen mit den korrespondierenden Aminosäure-Genes are selected in which a nucleotide sequence which codes for one or more immunogens can be detected using the methods mentioned above. As an example, the sequence listing (below) shows the nucleotide sequences with the corresponding amino acid
Sequenzen des Hämagglutinin-Neuraminidase- und des Fusion-Protein-Gens des Parainfluenzavirus 2 hinterlegt bei CNCM under der Nummer 1-1331.Sequences of the hemagglutinin neuraminidase and the fusion protein gene of parainfluenza virus 2 deposited with CNCM under number 1-1331.
Die Expression der Gene zur Herstellung der Immunogene erfolgt z.B. durch:The expression of the genes for the production of the immunogens takes place e.g. by:
a) Stabile Integration der Gene in Form von komplementärer DNS in zelluläres Erbmaterial höherer Zellen. Zuvor werden die Gene in geeignetea) Stable integration of the genes in the form of complementary DNA into cellular genetic material of higher cells. Before that, the genes are put into suitable
Shuttle- Vektoren kloniert. Hierzu eignet sich beispielsweise das Simian Virus 40 (SV40) sowie Plasmid-Expressionsvektoren, die geeignet sind, in Prokaryonten (z.B. E. coli) selektiert und vermehrt zu werden sowie regulatorische Elemente zur Expression der Fremd-DNS in höheren Zellen besitzen.Cloned shuttle vectors. For this purpose, for example, the Simian Virus 40 (SV40) and plasmid expression vectors are suitable which are suitable for being selected and propagated in prokaryotes (e.g. E. coli) and which have regulatory elements for the expression of the foreign DNA in higher cells.
Geeignete Plasmid-Expressionsvektoren sind z.B. auf dem SV40 basierende Plasmid- Vektoren wie pMSG, pSVT7 oder pMT2, oder auf dem Ebbstein-Barr- Virus basierende Plasmid- Vektoren wie pHEBo oder p205.Suitable plasmid expression vectors are e.g. plasmid vectors such as pMSG, pSVT7 or pMT2 based on the SV40, or plasmid vectors such as pHEBo or p205 based on the Ebbstein-Barr virus.
Die klonierte DNS wird mittels den oben beschriebenen Methoden isoliert und gereinigt und durch Transfektion in höhere Zellen eingeführt. Geeignete Zellen sind animale Zellen, insbesondere permanente Zell-Linien, wie z.B. die Schweinenieren-Zelle PK15 (ATCC CCL33 oder deren Abkömmlinge), die Affennieren-Zelle BGM (Flow 03-240 oder deren Abkömmlinge) oder Vero (ATCC CCL81 oder deren Abkömmlinge), die Rindernieren-Zelie MDBK (ATCC CCL22 oder deren Abkömmlinge), die Hundenieren-Zelle MDCK (ATCC CCL34 oder deren Abkömmlinge) oder die Kaninchennieren-Zelle RK-13 (ATCC CCL37).The cloned DNA is isolated and purified using the methods described above and introduced into higher cells by transfection. Suitable cells are animal cells, in particular permanent cell lines, such as the pig kidney cell PK15 (ATCC CCL33 or its descendants), the monkey kidney cell BGM (Flow 03-240 or its descendants) or Vero (ATCC CCL81 or its descendants) , the bovine kidney cell MDBK (ATCC CCL22 or its descendants), the dog kidney cell MDCK (ATCC CCL34 or its descendants) or the rabbit kidney cell RK-13 (ATCC CCL37).
Die Transfektion erfolgt z.B. in Form von Calziumphosphat-DNS-Kopräzipitaten oder durch die DEAE/Dextran-Methode, die Liposomen Methode oder durch Elektroporation.The transfection takes place e.g. in the form of calcium phosphate-DNA coprecipitates or by the DEAE / dextran method, the liposome method or by electroporation.
Methoden zur Klonierung der ausgewählten Gene in geeignete Vektoren sowie zurMethods for cloning the selected genes into suitable vectors and for
Transfektion der klonierten Gene in höhere Zellen sind im Detail beschrieben in J. Sambrook, E.F. Fritsch and T. Maniatis (Hrsg.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 und F.M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.Transfection of the cloned genes into higher cells are described in detail in J. Sambrook, E.F. Fritsch and T. Maniatis (ed.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and F.M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.
Zellkulturüberstände bzw. Zelllysate von solchermaßen behandelten Zellen werden auf das Vorhandensein von exprimierten Immunogenen mit Hilfe von Antikörpern getestet, die direkt oder indirekt z.B. mittels Immunfluoreszenz oder Immun- präzipitation nachgewiesen werden. Geeignete Antikörper sind solche, die mit Immunogenen verwandter Virusstämme reagieren, wie z.B. dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2.Cell culture supernatants or cell lysates from cells treated in this way are tested for the presence of expressed immunogens with the aid of antibodies which directly or indirectly e.g. can be detected by means of immunofluorescence or immunoprecipitation. Suitable antibodies are those which react with immunogens of related virus strains, e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
b) Klonierung der Gene in Form von komplementärer DNS in geeignete Ex¬ pressionsvektoren für niedere oder höhere Zellen.b) Cloning of the genes in the form of complementary DNA into suitable expression vectors for lower or higher cells.
Geeignet sind z.B. (i) bakterielle Plasmid-Expressionsvektoren, (ii) virale Ex- pressionsvektoren für Bakterien oder (iii) virale Expressionsvektoren für höhereSuitable are e.g. (i) bacterial plasmid expression vectors, (ii) viral expression vectors for bacteria or (iii) viral expression vectors for higher ones
Zellen, in denen das klonierte Gen exprimiert wird.Cells in which the cloned gene is expressed.
ad (i):ad (i):
Geeignete bakterielle Plasmid-Expressionsvektoren sind z.B. pUC18/19 oder pUCSuitable bacterial plasmid expression vectors are e.g. pUC18 / 19 or pUC
118/119. Nach Klonierung der DNS in das Plasmid wird dieses in prokaryotische - 2S -118/119. After cloning the DNA into the plasmid, it is converted into prokaryotic - 2S -
Zellen, vorzugsweise Bakterien, eingesetzt und vermehrt. Geeignet ist z.B. Escherichia coli K12 und dessen Abkömmlinge.Cells, preferably bacteria, used and multiplied. Suitable is e.g. Escherichia coli K12 and its descendants.
Zum Einschleusen des Plasmides in die prokaryotische Zelle eignet sich z.B. die Calziumphosphat-DNS-Kopräzipitation oder die Elektroporation.For introducing the plasmid into the prokaryotic cell, e.g. calcium phosphate-DNA coprecipitation or electroporation.
ad (ii):ad (ii):
Geeignete virale Expressionsvektoren für Bakterien sind λ-Bakteriophagen- Vek¬ toren wie z.B. λgtl l und Abkömmlinge, λZAP oder λORF8. Die Vermehrung der λ-Bakteriophagen- Vektoren erfolgt insbesondere in Escherichia coli z.B. E. coli Kl 2 und dessen Abkömmlinge.Suitable viral expression vectors for bacteria are λ bacteriophage vectors such as e.g. λgtl l and descendants, λZAP or λORF8. The multiplication of the λ bacteriophage vectors takes place in particular in Escherichia coli e.g. E. coli Kl 2 and its descendants.
ad (iii):ad (iii):
Geeignete virale Expressionsvektoren für höhere Zellen sind z.B. das Simian Virus 40, Adenoviren, Herpes-Simplex Virus oder Baculoviren. Die Vermehrung der viralen Vektoren erfolgt in entsprechenden Zellsystemen.Suitable viral expression vectors for higher cells are e.g. the Simian Virus 40, adenoviruses, herpes simplex virus or baculoviruses. The viral vectors are propagated in appropriate cell systems.
Methoden zur Klonierung der ausgewählten Gene in geeignete Expressions- Vektoren sowie deren Einsatz in entsprechenden Expressionssystemen sind imMethods for cloning the selected genes into suitable expression vectors and their use in corresponding expression systems are in the
Detail beschrieben in J. Sambrook, E.F. Fritsch and T. Maniatis (Hrsg.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 und F.M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.Described in detail in J. Sambrook, E.F. Fritsch and T. Maniatis (ed.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and F.M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.
Die exprimierten Immunogene werden entweder direkt in Form der Expressions¬ systeme (Kultur Substrat und/oder Zellen) oder nach Aufbereitung und Reinigung mittels biochemischer und/oder immunologischer Methoden und gegebenenfalls nach Konzentrierung oder Verdünnung als antigenes Material verwendet.The expressed immunogens are used either directly in the form of the expression systems (culture substrate and / or cells) or after preparation and purification by means of biochemical and / or immunological methods and, if appropriate, after concentration or dilution as an antigenic material.
Geeignet zur Reinigung sind z.B. Affinitäts- oder Gel-chromatographische Ver- fahren, bei denen die Immunogene vom Expressionssystem, gegebenenfalls nach dessen Aufschluß durch Detergentien-Behandlung, abgetrennt oder isoliert werden.Suitable for cleaning are e.g. Affinity or gel chromatographic processes in which the immunogens are separated or isolated from the expression system, if appropriate after its disruption by detergent treatment.
Zur Herstellung von Virusantigenen, die von Vektorsystemen exprimiert werden, wird zunächst das Virusgenom gewonnen. Zur Gewinnung des Virusgenoms erfolgt zunächst die Vermehrung der Viren in üblicher Weise einerseits in Gewebekulturen animaler Zellen als Primarzellen oder permanenten Zell-Linien, z.B. in Schweine-Zellen, Affen-Zellen oder Rinder-Zel¬ len, bevorzugt in Schweinenieren-Zellen wie z.B. der geklonten, permanenten Schweinenieren-Zelle PK15 (ATCC CCL33 oder deren Abkömmlinge) oder der primären Schweinenieren-Zelle EPK oder Affennieren-Zellen wie den permanenten Affennieren-Zellen BGM (Flow 03-240 oder deren Abkömmlinge) oder Vero (ATCC CCL81 oder deren Abkömmlinge) oder Rindernieren-Zellen wie der permanenten Rindernieren-Zelle MDBK (ATCC CCL22 oder deren Abkömmlinge) und andererseits in embryonierten Hühnereiern (z.B. Valo-Bruteier, Fa. Lohmann). Die Vermehrung in Zellkulturen erfolgt in an sich bekannter Weise in stationären Roller- oder Carrier-Kulturen in Form von geschlossenen Zellverbänden (Mono- layern) oder in Suspensions-Kulturen. Als Vermehrungsmedien für die Zellen werden eingesetzt alle an sich bekannten Zellkulturmedien z.B. beschrieben im Produktkatalog der Fa. Gibco BRL GmbH, Dieselstraße 5, 76344 Eggenstein, wie insbesondere das Minimal Essential Medium (MEM). das als wesentliche Be¬ standteile Aminosäuren, Vitamine, Salze und Kohlenhydrate enthält, komplettiert mit Puff ersub stanzen wie z.B. Natrium-Bicarbonat (NaHCO3) oder Hydroxyethyl- piperazin-N-2-ethansulfonsäure (Hepes) und gegebenenfalls Tierseren, wie z.B. Seren von Rindern, Pferden bzw. deren Föten. Besonders bevorzugt eingesetzt wird Eagles MEM mit einem Gehalt an NaHCÖ3 von 0.1-5 g/1, vorzugsweise 0,5- 3 g/1 sowie fötales Kälberserum in einer Konzentration von 1-30 Vol-%>, vor¬ zugsweise 2-10 Vol-%.In order to produce virus antigens which are expressed by vector systems, the virus genome is first obtained. In order to obtain the virus genome, the viruses are first multiplied in the usual way in tissue cultures of animal cells as primary cells or permanent cell lines, for example in pig cells, monkey cells or bovine cells, preferably in pig kidney cells such as, for example cloned, permanent pig kidney cell PK15 (ATCC CCL33 or its derivatives) or the primary pig kidney cell EPK or monkey kidney cells such as the permanent monkey kidney cells BGM (Flow 03-240 or its derivatives) or Vero (ATCC CCL81 or its derivatives) or bovine kidney cells such as the permanent bovine kidney cell MDBK (ATCC CCL22 or its derivatives) and, on the other hand, in embryonic chicken eggs (eg Valo hatching eggs, Lohmann). The multiplication in cell cultures takes place in a manner known per se in stationary roller or carrier cultures in the form of closed cell groups (monolayers) or in suspension cultures. All cell culture media known per se are used as propagation media for the cells, for example described in the product catalog of Gibco BRL GmbH, Dieselstrasse 5, 76344 Eggenstein, such as in particular the Minimal Essential Medium (MEM). which contains amino acids, vitamins, salts and carbohydrates as essential components, supplemented with buffer substances such as sodium bicarbonate (NaHCO 3 ) or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and optionally animal sera such as sera from Cattle, horses and their fetuses. Eagles MEM with a NaHCÖ 3 content of 0.1-5 g / 1, preferably 0.5-3 g / 1 and fetal calf serum in a concentration of 1-30% by volume, preferably 2-10, are particularly preferably used Vol%.
Die zur Vermehrung der Viren dienenden Zellen und Zellrasen werden in üblicher Weise nahezu bis zur Konfluenz oder bis zur optimaler Zelldichte vermehrt. Vor ihrer Infektion mit Viren wird bevorzugt das Zellvermehrungsmedium entfernt und die Zellen bevorzugt mit Virusvermehrungsmedium gewaschen. Als Virusvermeh¬ rungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben genannte MEM. Danach wird mit einer Virussuspension in- fiziert. In der Virussuspension liegt das Virus im Virusvermehrungsmedium derart verdünnt vor, daß mit einer MOI (= multiplicity of infection, entspricht dem Ver¬ hältnis der Anzahl der infektiösen Viruspartikel zur Anzahl der vorhandenen Zellen) von 0,01-50, bevorzugt 0,1-10 infiziert wird. Die Vermehrung der Viren erfolgt mit oder ohne Zusatz von Tierseren. Für den Fall, daß Serum eingesetzt wird, wird dieses zum Vermehrungsmedium in einer Konzentration von 1-30 Vol-%, vorzugsweise 2-10 Vol-% zugegeben.The cells and cell turf used to multiply the viruses are multiplied in the usual way almost to confluence or to the optimal cell density. Before being infected with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus growth medium. All known cell culture media, such as in particular the MEM mentioned above, are used as the virus propagation medium. The virus is then infected with a virus suspension. In the virus suspension, the virus is diluted in the virus propagation medium in such a way that with an MOI (= multiplicity of infection, corresponds to the ratio of the number of infectious virus particles to the number of cells present) from 0.01-50, preferably 0.1- 10 is infected. The viruses are propagated with or without the addition of animal sera. If serum is used, it is added to the propagation medium in a concentration of 1-30% by volume, preferably 2-10% by volume.
Infektion und Virusvermehrung erfolgen bei Temperaturen zwischen Raumtempe- ratur und 40°C, bevorzugt zwischen 32 und 39°C, besonders bevorzugt bei 37°C über mehrere Tage, bevorzugt bis zur vollständigen Zerstörung der infizierten Zel¬ len.Infection and virus multiplication take place at temperatures between room temperature and 40 ° C., preferably between 32 and 39 ° C., particularly preferably at 37 ° C. for several days, preferably until the infected cells are completely destroyed.
Das virushaltige Medium der infizierten Zellen wird weiter aufgearbeitet, z.B. durch Entfernung der Zellen und Zelltrümmer mittels Filtration mit Porengrößen von-z.B. 0,1-0,45 μm und/oder Zentrifugation bis zu 10.000 x g.The virus-containing medium of the infected cells is processed further, e.g. by removing the cells and cell debris by means of filtration with pore sizes of e.g. 0.1-0.45 μm and / or centrifugation up to 10,000 x g.
Die Vermehrung in embryonierten Hühnereiern erfolgt in an sich bekannter Weise in der Allantoishöhle von Hühner-Bruteiern, die 9-12 Tage, bevorzugt 10 Tage bei einer Temperatur von 37-39°C, bevorzugt 38,5°C und einer relativen Luftfeuch¬ tigkeit von 30-90%, bevorzugt 50-60% in einem handelsüblichen Brutschrank, be- vorzugt einem Motorbrüter vorbebrütet werden.The multiplication in embryonated chicken eggs takes place in a manner known per se in the Allantoic cavity of chicken hatching eggs, which lasts 9-12 days, preferably 10 days, at a temperature of 37-39 ° C., preferably 38.5 ° C. and a relative atmospheric humidity 30-90%, preferably 50-60%, in a commercial incubator, preferably a motor breeder.
Die zur Vermehrung der Viren dienenden Bruteier werden vor der Beimpfung 1-3 Stunden, bevorzugt 2 Stunden auf dem spitzen Eipol senkrecht stehend im Brut¬ schrank gelagert und anschließend nach Vorbereitung der Injektionstelle mit 10- 200 μl, bevorzugt 75-125 μl einer Virussuspension infiziert. In der Virussus- pension liegt das Virus im Virusvermehrungsmedium in einer Konzentration von lO'-lO' KJD50/ml (50%-Kultur-infektiöse Dosis pro ml Suspension = die Ver¬ dünnungsstufe, bei der noch 50% der eingesetzten Zellkulturen infiziert würden), bevorzugt 104-105 KID50/ml vor. Als Virusvermehrungsmedium werden eingesetzt, alle an sich bekannten Zellkulturmedien, wie insbesondere das oben genannte MEM.The hatching eggs used to multiply the viruses are stored vertically in the incubator for 1-3 hours, preferably 2 hours, on the pointed egg pole before inoculation and then infected with 10-200 μl, preferably 75-125 μl, of a virus suspension after preparation of the injection site . In the virus suspension, the virus lies in the virus propagation medium in a concentration of 10'-10 'KJD 50 / ml (50% culture-infectious dose per ml suspension = the dilution stage at which 50% of the cell cultures used would still be infected ), preferably 10 4 -10 5 KID 50 / ml. All known cell culture media, such as in particular the MEM mentioned above, are used as virus propagation medium.
Infektion und Virusvermehrung erfolgen unter den oben angegebenen Brut¬ bedingungen über mehrere Tage, bevorzugt 2-5 Tage, besonders bevorzugt 3 Tage.Infection and virus multiplication take place under the breeding conditions specified above over several days, preferably 2-5 days, particularly preferably 3 days.
Die virushaltige Allantoisflüssigkeit wird gewonnen durch Absaugen nach Öffnung der Kalkschale sowie der Schalenhaut und der Chorioallantoismembran und kann z.B. mittels Filtration mit Porengrößen von z.B. 0, 1 -0,45 μm und/oder Zentri¬ fugation bis zu 10.000 x g weiter aufgearbeitet werden.The virus-containing allantoic fluid is obtained by suctioning off after opening the lime shell as well as the skin of the shell and the chorioallantoic membrane and can eg by filtration with pore sizes of, for example, 0.1-0.45 μm and / or centrifugation up to 10,000 × g.
Die Virusreinigung, bzw. -isolierung wird eπeicht durch isopyknische oder zonale Zentrifugation in z.B. Saccharose-Dichtegradienten. Hierzu wird das virushaltige Medium bzw. die Allantoisflüssigkeit nach Entfernung der Zelltrümmer einerVirus cleaning or isolation is calibrated by isopycnic or zonal centrifugation in e.g. Sucrose density gradients. For this purpose, the virus-containing medium or the allantoic fluid is removed after removing the cell debris
Zonenzentrifugation bei 100.000 x g bis zur Sedimentation der Viruspartikel unterworfen. Ein reinere Darstellung der Viruspartikel ergibt sich durch Zonenzentrifugation in einer wäßrigen Lösung mit einer höheren Dichte als das virushaltige Medium. Als wäßrige Lösung kann z.B. eine 30-60 % w/w, bevorzugt 35-50 % w/w, gepufferte Lösung von Saccharose dienen. Ein noch höhererZone centrifugation at 100,000 x g subjected to sedimentation of the virus particles. A purer representation of the virus particles results from zone centrifugation in an aqueous solution with a higher density than the virus-containing medium. As an aqueous solution e.g. serve a 30-60% w / w, preferably 35-50% w / w, buffered solution of sucrose. An even higher one
Reinheitsgrad wird durch Zentrifugation im Dichtegradienten eπeicht. Hierzu wird das von Zellen und Zelltrümmer befreite, mittels Zonenzentrifugation konzentrierte Virus durch eine isopyknische oder zonale Dichtegradienten-Zentπfugation in einem Dichtegradienten von z.B. 30 bis 50 % w/w* Saccharose in gepufferter wäßriger Lösung bei einer Zentrifugalbeschleunigung von z.B. 100.000 bisThe degree of purity is calibrated by centrifugation in the density gradient. For this purpose, the virus, freed from cells and cell debris, is concentrated by means of zone centrifugation by isopycnic or zonal density gradient centrifugation in a density gradient of, for example, 30 to 50% w / w * sucrose in buffered aqueous solution with a centrifugal acceleration of, for example, 100,000 to
150.000 x g isoliert.150,000 x g isolated.
Zur Gewinnung geeigneter Gene, die für immunogene Proteine codieren, wird aus den gereinigten Viruspartikeln zunächst das Virusgenom isoliert. Die native Virus- RNS wird vorzugsweise gewonnen durch Behandlung der gereinigten Viruspartikel mit Detergens- und Proteasen-haltigen, wäßrigen Lösungen.To obtain suitable genes that code for immunogenic proteins, the virus genome is first isolated from the purified virus particles. The native virus RNA is preferably obtained by treating the purified virus particles with aqueous solutions containing detergent and proteases.
Eingesetzt werden anionische, kationische, amphoterische und nicht-ionische De¬ tergentien. Bevorzugt eingesetzt werden ionische Detergentien, vorzugsweise Natriumdodecylsulfat, in einer Konzentration von 0,1-10 Vol-%, vorzugsweise 0,5- 3 Vol-%.Anionic, cationic, amphoteric and non-ionic detergents are used. Ionic detergents, preferably sodium dodecyl sulfate, are preferably used in a concentration of 0.1-10% by volume, preferably 0.5-3% by volume.
Als Proteasen werden solche eingesetzt, die in Gegenwart von Detergentien wirken, wie z.B. Pronase und, vorzugsweise eingesetzt Proteinase K. Die Proteasen werden in einer Konzentration 0,01-10 mg/ml, bevorzugt 0,05-0,5 mg/ml eingesetzt.Proteases used are those which act in the presence of detergents, such as e.g. Pronase and, preferably used Proteinase K. The proteases are used in a concentration of 0.01-10 mg / ml, preferably 0.05-0.5 mg / ml.
Vorzugsweise werden wäßrige, gepufferte Lösungen mit Zusatz von RNase- Inhibitoren verwendet. Als Puffersubstanzen werden verwendet Salze schwacher Säuren mit starken Basen wie z.B. Tris(hydroxymethyl)-aminomethan, Salze starker Säuren mit schwachen Basen wie z.B. primäre Phosphate oder Gemische hiervon. Vorzugsweise verwendet wird Tris(hydroxymethyl)-aminomethan. Die Puffersubstanzen werden in Konzentrationen eingesetzt, die einen pH-Wert sicherstellen, bei dem die RNS nicht denaturiert. Bevorzugt werden pH-Werte von 6-8,5, besonders bevorzugt von 7-8.Aqueous, buffered solutions with the addition of RNase inhibitors are preferably used. Salts of weak acids with strong bases such as, for example, tris (hydroxymethyl) aminomethane, salts of strong acids with weak bases such as, for example, primary phosphates or mixtures thereof are used as buffer substances. Tris (hydroxymethyl) aminomethane is preferably used. The buffer substances are used in concentrations that ensure a pH value at which the RNA does not denature. PH values of 6-8.5 are preferred, particularly preferably 7-8.
Als RNase-Inhibitoren dienen z.B. Ribonucleosid-Vanadyl-Komplexe, Protein- Inhibitoren (z.B. RNAguard®/Pharmacia) oder vorzugsweise Diethylpyrocarbonat (DEPC) in Konzentrationen von 0,01-2 Vol-%, bevorzugt 0,1-0,5 Vol-%.As RNase inhibitors are used, for example, ribonucleoside-vanadyl complexes, protein inhibitors (eg RNAguard ® / Pharmacia) or preferably diethyl pyrocarbonate (DEPC) in concentrations of 0.01-2% by volume, preferably 0.1-0.5 vol- %.
Die ipophilen Substanzen des Viruslysates werden anschließend extrahiert unter Verwendung von Lösemittel wie z.B. Phenol, Chloroform oder Mischungen davon. Die Extraktion erfolgt in ein oder mehreren Stufen.The ipophilic substances of the virus lysate are then extracted using solvents such as e.g. Phenol, chloroform or mixtures thereof. The extraction takes place in one or more stages.
In der verbleibenden wäßrigen Phase wird die RNS mittels wäßriger Lösungen gefällt, die Alkohole wie z.B. Ethanol oder Isopropanol und monovalente Chlorid¬ oder Acetat-Salze wie z.B. Natriumchlorid, Natriumacetat oder Kaliumacetat enthalten.In the remaining aqueous phase, the RNA is precipitated using aqueous solutions containing alcohols such as e.g. Ethanol or isopropanol and monovalent chloride or acetate salts such as e.g. Contain sodium chloride, sodium acetate or potassium acetate.
Die Konzentration der Alkohole liegt zwischen 40 und 100 Vol-%, bevorzugt 60 und 80 Vol-% und die der Chlorid- oder Acetat-Salze zwischen 0,01 und 1 mol/l, bevorzugt 0,1 bis 0,8 mol/l.The concentration of the alcohols is between 40 and 100% by volume, preferably 60 and 80% by volume, and that of the chloride or acetate salts is between 0.01 and 1 mol / l, preferably 0.1 to 0.8 mol / l .
Die gefällte RNS wird aus der wäßrigen Lösung z.B. durch Zentrifugation gewonnen und in einer wäßrigen Lösung z.B. DEPC-Wasser wiederum aufgelöst. Diese wäßrige Lösung enthält bevorzugt Puff ersub stanzen wie z.B. Tris(hy- droxymethyl)-aminomethan in Konzentrationen von 1-100 mmol/l, vorzugsweise 10-50 mmol/l, eventuell unter Zusatz von Ethylendiamintetraacetat (EDTA) inThe precipitated RNA is extracted from the aqueous solution e.g. obtained by centrifugation and in an aqueous solution e.g. DEPC water dissolved again. This aqueous solution preferably contains buffer substances such as e.g. Tris (hydroxymethyl) aminomethane in concentrations of 1-100 mmol / l, preferably 10-50 mmol / l, possibly with the addition of ethylenediaminetetraacetate (EDTA) in
Konzentrationen von 0,1-10 mmol/l, vorzugsweise 1-10 mmol/l oder Dithiothreit (DTT) in Konzentrationen von 0,1-10 mmol/l, vorzugsweise 1-10 mmol/l.Concentrations of 0.1-10 mmol / l, preferably 1-10 mmol / l or dithiothreitol (DTT) in concentrations of 0.1-10 mmol / l, preferably 1-10 mmol / l.
Die isolierte RNS wird bei Temperaturen unter -65° C gelagert. Eine andere Methode zur RNS-Isolierung ist z B die RNS-Extraktion mit Guanidiniumthiocvanat und anschließender Casiumchloπd-Dichtegradienten-Zentπ- fugation des Virus-LysatesThe isolated RNS is stored at temperatures below -65 ° C. Another method for RNA isolation is, for example, RNA extraction with guanidinium thiocvanate and subsequent casium chloride density gradient centrifugation of the virus lysate
Methoden zur RNS-Isolierung sind beschrieben in J Sambrook, E F Fπtsch and T Maniatis (Hrsg ), Molecular Cloning, A Laboratory Manual, 2nd edition, ColdMethods for RNA isolation are described in J Sambrook, E F Ftsch and T Maniatis (ed.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold
Spring Harbor Laboratory Press, 1989.Spring Harbor Laboratory Press, 1989.
Die Identifizierung geeigneter Gene erfolgt unter Verwendung des isolierten Virus- Genoms z B durch:Suitable genes are identified using the isolated virus genome, for example by:
a) " RNS/DNS-Hybridisierung des Genoms unter Verwendung bekannter Gen- sonden Als geeignete Gensonden dienen DNS-Proben mit Nucleotid-Se- quenzen von bekannten Genen für Immunogene verwandter λ ιrusstamme, wie z B dem Simian Virus 5 oder dem Caninen Paramfluenzavirus 2a) " RNA / DNA hybridization of the genome using known gene probes. DNA probes with nucleotide sequences of known genes for immunogens of related λ uß strains, such as the Simian virus 5 or the canine paramfluenza virus 2, serve as suitable gene probes
b) Herstellung einer komplementären DNS (cDNS), Klonierung der cDNS in z B bakterielle Plasmide wie z B. pBR322 zur Anreicherung von viraler DNS und Hybridisierung der klonierten DNS mittels bekannter Gensondenb) Production of a complementary DNA (cDNA), cloning of the cDNA in, for example, bacterial plasmids such as, for example, pBR322 for the enrichment of viral DNA and hybridization of the cloned DNA by means of known gene probes
Als geeignete Gensonden dienen DNS-Proben mit Nucleotid-Sequenzen von bekannten Genen für Immunogene verwandter Virusstamme, wie z B dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2DNA probes with nucleotide sequences of known genes for immunogens of related virus strains, such as, for example, the Simian virus 5 or the canine parainfluenza virus 2, serve as suitable gene probes
c) Herstellung einer komplementären DNS (cDNS) und Klonierung der cDNS in Plasmid-Expressionsvektoren wie z.B. pUC18/19 oder pUC 118/119 oder in λ-Bakteriophagen-Expressionsvektoren wie z.B λgtl l und dessen Abkömmlinge oder λZAP oder λORF8 Die Identifizierung der Gene erfolgt durch Nachweis ihrer expnmierten Immunogene mit Hilfe von Antikörpern, die direkt oder indirekt z B mittels Immunfluoreszenz oder Immunprazipitation nachgewiesen werden Geeignete Antikörper sind sol¬ che, die mit Immunogenen verwandter Virusstamme reagieren, wie z.B dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2c) Preparation of a complementary DNA (cDNA) and cloning of the cDNA in plasmid expression vectors such as e.g. pUC18 / 19 or pUC 118/119 or in λ-bacteriophage expression vectors such as λgtl l and its descendants or λZAP or λORF8. The genes are identified by detecting their exposed immunogens with the help of antibodies which are directly or indirectly, for example, by means of immunofluorescence or Immunoprecipitation can be detected. Suitable antibodies are those which react with immunogens of related virus strains, such as the Simian virus 5 or the canine parainfluenza virus 2
d) Hei Stellung einer komplementären DNS (cDNS) und Klonierung der cDNS in z.B bakterielle Plasmide zur Anreicherung von viraler DNS Die virale DNS der Klone wird sequenziert und untersucht auf Sequenzhomologien mit bekannten Genen verwandter Virusstämme, wie z.B. dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2.d) Position of a complementary DNA (cDNA) and cloning of the cDNA in, for example, bacterial plasmids for the enrichment of viral DNA DNA of the clones is sequenced and examined for sequence homologies with known genes from related virus strains, such as the Simian virus 5 or the canine parainfluenza virus 2.
e) Sequenzierung der cDNS bei deren Herstellung und Untersuchung auf Sequenzhomologien mit bekannten Genen verwandter Virusstämme, wie z.B. dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2.e) Sequencing the cDNA during its production and examination for sequence homologies with known genes of related virus strains, such as e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2.
f) Kombinationen der Methoden a) bis e).f) combinations of methods a) to e).
Methoden zur RNS/DNS- und DNS/DNS-Hybridisierung, Herstellung von cDNS, Klorrierung von DNS in Plasmid- und Bakteriophagen-Vektoren, Sequenzierung von DNS sowie Methoden zum immunologischen Nachweis von exprimiertenMethods for RNA / DNA and DNA / DNA hybridization, production of cDNA, cloning of DNA in plasmid and bacteriophage vectors, sequencing of DNA as well as methods for the immunological detection of expressed
Immunogenen sind beschrieben in:Immunogens are described in:
J. Sambrook, E.F. Fritsch and T. Maniatis (Hrsg.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989J. Sambrook, E.F. Fritsch and T. Maniatis (ed.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989
- F.M. Ausubel, Current protocols in molecular biology 1987-1988, John- F.M. Ausubel, Current protocols in molecular biology 1987-1988, John
Wiley & Sons, New York, 1987Wiley & Sons, New York, 1987
A. Mayr, Virologische Arbeitsmethoden, Band III, Gustav Fischer Verlag, Stuttgart, 1989A. Mayr, Virological Working Methods, Volume III, Gustav Fischer Verlag, Stuttgart, 1989
Es werden solche Gene ausgewählt, bei denen mit den oben genannten Methoden eine Nucleotid-Sequenz nachgewiesen werden kann, die für ein oder mehrereGenes are selected in which a nucleotide sequence can be detected using the methods mentioned above, which sequence is suitable for one or more
Immunogene codiert. Als Beispiel sind im Sequenzprotokoll wiedergegeben die Nucleotid-Sequenzen mit den korrespondierenden Aminosäuren-Sequenzen des Hämagglutinin-Neuraminidase- und des Fusion-Protein-Gens des Parainfluenza¬ virus 2 hinterlegt bei CNCM unter der Nummer 1-1331.Immunogenic encoded. As an example, the sequence listing shows the nucleotide sequences with the corresponding amino acid sequences of the hemagglutinin neuraminidase and the fusion protein gene of the Parainfluenza virus 2 deposited with CNCM under number 1-1331.
Diese Gene, die für ein oder mehrere Immunogene codieren (Fremd-DNS), werden in einen Genom-Vektor inseriert, der bei der Infektion einer Zelle oder eines Organismus das Fremd-Gen exprimiert. Hierzu geeignet sind Vektor-Viren und Vektor-Bakterien. Verwendung finden beispielsweise apathogene DNS-Viren, die ein stabiles Genom mit bekannten Insertionsstellen für die Aufnahme von 0,1 bis - .-> ->These genes, which code for one or more immunogens (foreign DNA), are inserted into a genome vector which expresses the foreign gene when a cell or an organism is infected. Vector viruses and vector bacteria are suitable for this. For example, apathogenic DNA viruses are used which have a stable genome with known insertion sites for the uptake of 0.1 to - .->->
zu 20 kB ( 1000 Basenpaare) Fremd-DNS besitzen, wie z B Vacciniaviren, Herpesviren oder Adenovirento 20 kB (1000 base pairs) have foreign DNA, such as vaccinia viruses, herpes viruses or adenoviruses
Hierzu ist es notig (α) das oder die Gene zunächst in einen Shuttle-Vektor zu inserieren, der die Fremd-DNS flankiert von DNS-Sequenzen des Vektor- Virus enthalt Anschließend wird (ß) das oder die Gene in das Vektor- Virus-Genom eingesetzt z.B mittels Cotransfektion des Shuttle- Vektors und des Vektor- Virus.For this purpose, it is necessary (α) to insert the gene or genes first into a shuttle vector which contains the foreign DNA flanked by DNA sequences of the vector virus. Genome used, for example, by means of cotransfection of the shuttle vector and the vector virus.
Geeignete Shuttle- Vektoren sind Plasmid- oder Bakteπophagen- Vektoren.Suitable shuttle vectors are plasmid or bacteriophage vectors.
Beispiele für gebrauchliche Plasmid-Vektoren sind pBR322, pUC18/19, pAT153, ρACYC184 oder pSP64/65 und für Bakteriophagen-Vektoren λgtlO/11, λZAP oder M13mpl8/19Examples of useful plasmid vectors are pBR322, pUC18 / 19, pAT153, ρACYC184 or pSP64 / 65 and for bacteriophage vectors λgtlO / 11, λZAP or M13mpl8 / 19
(α) Inserierung des oder der Gene in einen Shuttle- Vektor(α) Insertion of the gene or genes in a shuttle vector
Zunächst wird das DNS-Fragment, das die Insertionsstelle des Vektor- Virus tragt, in die Shuttle- Vektor-DNS eingesetzt Hierzu werden sowohl die DNS-Sequenzen bekannter Insertionsstellen des Genoms des Vektor- Virus als auch die DNS des Shuttle- Vektor mit Restriktions-Endonucleasen (Restπktions-Enzyme) behandelt, um passende Sequenz-Enden zur Insertion zu schaffen Die solchermaßen vorbe¬ reitete Shuttle- Vektor-DNS wird mit einem Überschuß des zu inserierenden DNS- Fragmentes vermischt, z B. ungefähr in einem Verhältnis von 1 5 Das DNS-Ge¬ misch wird mit DNS-Ligasen behandelt, um das DNS-Fragment kovalent im Vektor zu binden.First the DNA fragment carrying the vector virus insertion site is inserted into the shuttle vector DNA. For this purpose, both the DNA sequences of known insertion sites of the vector virus genome and the DNA of the shuttle vector with restriction Endonucleases (residual enzymes) treated to create suitable sequence ends for insertion. The shuttle vector DNA prepared in this way is mixed with an excess of the DNA fragment to be inserted, for example in a ratio of 1 5 Das DNA mixture is treated with DNA ligases in order to covalently bind the DNA fragment in the vector.
Bei Verwendung eines Shuttle-Plasmids wird dieses in pro- oder eukaryotische Zellen, vorzugsweise Bakterien, eingesetzt und vermehrt Geeignet ist z B Escherichia coli K12 und dessen AbkömmlingeIf a shuttle plasmid is used, it is used in pro- or eukaryotic cells, preferably bacteria, and is increasingly suitable. For example, Escherichia coli K12 and its derivatives are suitable
Bakterien, die DNS-Fragment-haltige Plasmide tragen, werden selektiertBacteria carrying plasmids containing DNA fragments are selected
Die Klonierung der Insertionsstelle in das Shuttle-Plasmid-Genom, dessen Inser¬ tion in pro- oder eukaryotische Zellen, Vermehrung und Selektion der transfor¬ mierten Bakterien sind im Detail beschrieben in J Sambrook, E F. Fritsch and T Maniatis (Hrsg.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 und F.M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.The cloning of the insertion site into the shuttle plasmid genome, its insertion into pro- or eukaryotic cells, the multiplication and selection of the transformed bacteria are described in detail in J Sambrook, E F. Fritsch and T Maniatis (ed.) , Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and FM Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.
Falls notwendig werden sogenannte "Polylinker" in die Insertionsstellen des Vektor- Virus eingesetzt. Polylinker sind DNS-Sequenzen mit mindestens zwei definierten Restriktions-Enzym-Schnittstellen in Folge.If necessary, so-called "polylinkers" are inserted into the insertion sites of the vector virus. Polylinkers are DNA sequences with at least two defined restriction enzyme interfaces in a row.
Hierzu wird das DNS-Fragment, das die Insertionstelle trägt mit einem solchen Restriktions-Enzym behandelt, daß das Fragment nur an einer Stelle geöffnet (geschnitten) wird. Das so vorbereitete Fragment wird zusammen mit dem Polylinker und DNS-Ligase zur gezielten Insertion von definierten Restriktions- Erizym-Schnittstellen inkubiert.For this purpose, the DNA fragment carrying the insertion site is treated with a restriction enzyme such that the fragment is opened (cut) only at one point. The fragment thus prepared is incubated together with the polylinker and DNA ligase for the targeted insertion of defined restriction-erizyme sites.
Der Polylinker kann in das isolierte oder das in Shuttle-Vektoren klonierte Insertionstellen-tragende DNS-Fragment inseriert werden.The polylinker can be inserted into the isolated or the insertion site-carrying DNA fragment cloned in shuttle vectors.
Wird der Polylinker in isolierte DNS-Fragmente eingesetzt, müssen diese an¬ schließend in einen Shuttle- Vektor inseriert werden. Bei Verwendung eines Shuttle-Plasmids wird dieses in pro- oder eukaryotische Zellen, vorzugsweise Bak¬ terien, eingesetzt und vermehrt. Geeignet ist z.B. Escheήchia coli K12 und dessen Abkömmlinge. Bakterien, die DNS-Fragment-haltige Plasmide enthalten, werden selektiert.If the polylinker is used in isolated DNA fragments, these must then be inserted into a shuttle vector. If a shuttle plasmid is used, it is used and propagated in pro- or eukaryotic cells, preferably bacteria. Suitable is e.g. Escheήchia coli K12 and its descendants. Bacteria containing plasmids containing DNA fragments are selected.
Wird der Polylinker in das in Shuttle- Vektoren klonierte DNS-Fragment eingesetzt, werden diese vermehrt und selektiert.If the polylinker is inserted into the DNA fragment cloned into shuttle vectors, these are multiplied and selected.
Gene, die für ein oder mehrere Immunogene codieren (Fremd-DNS) werden in die Insertionsstellen eingesetzt.Genes that code for one or more immunogens (foreign DNA) are inserted into the insertion sites.
Falls notwendig, werden zuvor Teilsequenzen des DNS-Fragments entfernt, das die Insertionsstelle trägt. Hierzu wird das DNS-Fragment mit Restriktions-Enzymen behandelt, und die resultierenden DNS-Fragmente aufgetrennt.If necessary, partial sequences of the DNA fragment carrying the insertion site are removed beforehand. For this, the DNA fragment is treated with restriction enzymes, and the resulting DNA fragments are separated.
Zum Einsetzen der Fremd-DNS wird zunächst das isolierte oder das in Shuttle- Vektoren klonierte DNS-Fragment, das die Insertionsstelle trägt, mit ein oder mehreren Restriktions- Enzymen behandelt und das Fragment an der Insertionsstelle bzw. am eingesetzten Polylinker geöffnet. Die Fremd-DNS wird in die so vorbereitete Insertionsstelle beispielsweise mit Hilfe von DNS-Ligasen eingefügt.To insert the foreign DNA, the isolated or the DNA fragment cloned in shuttle vectors, which carries the insertion site, is first inserted with or treated several restriction enzymes and opened the fragment at the insertion site or at the polylinker used. The foreign DNA is inserted into the insertion site prepared in this way, for example with the aid of DNA ligases.
Wird die Fremd-DNS in isolierte DNS-Fragmente eingesetzt, müssen diese an- schließend in einen Shuttle-Vektor eingesetzt werden. Bei Verwendung einesIf the foreign DNA is used in isolated DNA fragments, these must then be inserted in a shuttle vector. When using a
Shuttle-Plasmids wird dieses in pro- oder eukaryotische Zellen, vorzugsweise Bakterien, eingesetzt und vermehrt. Geeignet ist z.B. Escherichia coli K12 und dessen Abkömmlinge. Bakterien, die Fremd-DNS-haltige Plasmide enthalten, werden selektiert.Shuttle plasmids are used and propagated in pro- or eukaryotic cells, preferably bacteria. Suitable is e.g. Escherichia coli K12 and its descendants. Bacteria which contain plasmids containing foreign DNA are selected.
Wird die Fremd-DNS in das in Shuttle- Vektoren klonierte DNS-Fragment einge¬ setzt, werden diese vermehrt und selektiert.If the foreign DNA is inserted into the DNA fragment cloned into shuttle vectors, these are multiplied and selected.
Methoden zur Herstellung von Shuttle- Vektoren sind im Detail beschrieben in J. Sambrook, E.F. Fritsch and T. Maniatis (Hrsg.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 und F.M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons,Methods for producing shuttle vectors are described in detail in J. Sambrook, E.F. Fritsch and T. Maniatis (ed.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and F.M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons,
New York, 1987.New York, 1987.
(ß) Einsetzen der Fremd-DNS in das Vektor-Virus-Genom:(ß) Inserting the foreign DNA into the vector virus genome:
Folgende Methoden zur Insertion der Fremd-DNS in das Vektor-Virus-Genom können verwendet werden:The following methods for inserting the foreign DNA into the vector virus genome can be used:
(i) Cotransfektion geeigneter Zellen mit der Shuttle-Vektor-DNS und der iso¬ lierten, nativen Vektor-Virus-DNS,(i) cotransfection of suitable cells with the shuttle vector DNA and the isolated, native vector virus DNA,
(ii) Transfektion geeigneter Zellen mit der Shuttle-Vektor-DNS und Infektion mit dem Vektor-Virus,(ii) transfection of suitable cells with the shuttle vector DNA and infection with the vector virus,
(iii) Infektion geeigneter Zellen mit dem Vektor-Virus und Transfektion mit der Shuttle-Vektor-DNS.(iii) Infection of suitable cells with the vector virus and transfection with the shuttle vector DNA.
Hierzu geeignete Methoden sind im Detail beschrieben in J. Sambrook, E.F. Fritsch and T. Maniatis (Hrsg.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 und F.M. Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.Suitable methods for this are described in detail in J. Sambrook, EF Fritsch and T. Maniatis (ed.), Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989 and FM Ausubel, Current protocols in molecular biology 1987-1988, John Wiley & Sons, New York, 1987.
Bevorzugt eingesetzt wird die Methode (i), die in Form der Calziumphosphat- DNS-Präzipitations-Technik durchgeführt wird. Hierzu sind folgende Schritte not- wendig:Method (i), which is carried out in the form of the calcium phosphate DNA precipitation technique, is preferably used. The following steps are necessary:
(1) Der Shuttle- Vektor wird vermehrt, isoliert und weiter gereinigt. Die Reini¬ gung der Shuttle-Vektor-DNS erfolgt z.B. mittels isopyknischer Zentri¬ fugation im Dichtegradienten, z.B. einem Cäsiumchlorid-Dichtegradienten.(1) The shuttle vector is multiplied, isolated and further purified. The purification of the shuttle vector DNA takes place e.g. by means of isopycnic centrifugation in density gradient, e.g. a cesium chloride density gradient.
Das Vektor- Virus wird vermehrt und gereinigt. Das virale Genom wird isoliert und weiter gereinigt. Die Reinigung der Vektor- Virus-DNS erfolgt z.B. mittels isopyknischer Zentrifugation im Dichtegradienten, z.B. einem Cäsiumchlorid-Dichtegradienten.The vector virus is multiplied and purified. The viral genome is isolated and further purified. The vector virus DNA is cleaned e.g. by means of isopycnical centrifugation in density gradient, e.g. a cesium chloride density gradient.
(2) Zur Cotransfektion wird zirkuläre oder vorzugsweise linearisierte Shuttle- Vektor-DNS verwendet.(2) Circular or preferably linearized shuttle vector DNA is used for cotransfection.
Die linearisierte Shuttle- Vektor-DNS erhält man z.B. durch Behandlung der gereinigten DNS mit Restriktions-Enzymen. Bevorzugt werden Restrik¬ tions-Enzyme, die keine Erkennungsstelle (Schnittstelle) in der inserierten Fremd-DNS besitzen, d.h. die Fremd-DNS-Sequenz wird nicht zerteilt.The linearized shuttle vector DNA is obtained e.g. by treating the purified DNA with restriction enzymes. Restriction enzymes are preferred which have no recognition site (interface) in the inserted foreign DNA, i.e. the foreign DNA sequence is not broken up.
(3) Die Vektor- Virus-DNS und die Shuttle- Vektor-DNS werden vermischt z.B. in einem Verhältnis von 0,01 bis 0,lxl0'12 mol l Vektor- Virus-DNS zu 1 bis 3xl0"12 mol/l Shuttle- Vektor-DNS.(3) The vector virus DNA and the shuttle vector DNA are mixed, for example, in a ratio of 0.01 to 0.1 × 10 12 mol l vector virus DNA to 1 × 3 × 10 12 mol / l shuttle Vector DNS.
(4) Das DNS-Gemisch wird copräzipitiert mit z.B. Calziumphosphat und auf geeignete Zellen übertragen.(4) The DNA mixture is coprecipitated with e.g. Calcium phosphate and transferred to suitable cells.
Geeignete Zellen sind animale Zellen, insbesondere permanente Zell- Linien, wie z.B. die Schweinenieren-Zelle PK15 (ATCC CCL33 oder derenSuitable cells are animal cells, in particular permanent cell lines, such as e.g. the pig kidney cell PK15 (ATCC CCL33 or its
Abkömmlinge),die Affennieren-Zelle BGM (Flow 03-240 oder deren Ab¬ kömmlinge) oder Vero (ATCC CCL81 oder deren Abkömmlinge), die ?9 -Descendants), the monkey kidney cell BGM (Flow 03-240 or its descendants) or Vero (ATCC CCL81 or its descendants), the ? 9 -
Rindernieren-Zelle MDBK (ATCC CCL22 oder deren Abkömmlinge), die Hundenieren-Zelle MDCK (ATCC CCL34 oder deren Abkömmlinge) oder die Kaninchennieren-Zelle RK-13 (ATCC CCL37).Bovine kidney cell MDBK (ATCC CCL22 or its descendants), the dog kidney cell MDCK (ATCC CCL34 or its descendants) or the rabbit kidney cell RK-13 (ATCC CCL37).
Die Cotransfektion kann auch mittels anderer Methoden erfolgen. Als solche seien z.B. genannt die DEAE/Dextran-Methode, die LiposomenThe cotransfection can also be carried out using other methods. As such, e.g. called the DEAE / dextran method, the liposomes
Methode oder die Elektroporation.Method or electroporation.
(5) Die Zellen werden kultiviert, z.B. nach den weiter oben beschriebenen Methoden. Bei Auftreten eines zythopathogenetischen Effektes werden Klone des Vektor- Virus mittels der Einzel-Plaque-Reinigungsmethoden iso- liert und weiter vermehrt.(5) The cells are cultured, e.g. according to the methods described above. If a cythopathogenetic effect occurs, clones of the vector virus are isolated using the single plaque purification methods and further multiplied.
Methoden zur Einzel-Plaque-Reinung sind beschrieben in A. Mayr, Virolo- gische Arbeitsmethoden, Band I, Gustav Fischer Verlag, Stuttgart, 1974.Methods for single plaque cleaning are described in A. Mayr, Virological Working Methods, Volume I, Gustav Fischer Verlag, Stuttgart, 1974.
(6) Die Selektion rekombinanter Vektor- Viren erfolgt (i) durch Nachweis der Expression des Fremd-Gens oder (ii) durch Nachweis der inserierten Fremd-DNS im Vektor- Virus-Genom z.B. durch DNS/DNS-Hybridisierung.(6) Recombinant vector viruses are selected (i) by detecting the expression of the foreign gene or (ii) by detecting the inserted foreign DNA in the vector virus genome, e.g. through DNA / DNA hybridization.
(i)(i)
Der Nachweis der Expression der Fremd-DNS erfolgt beispielsweise mit Hilfe von Antikörpern. Geeignete Antikörper sind solche, die mit Immunogenen verwandter Virusstämme reagieren, wie z.B. dem Simian Virus 5 oder dem Caninen Parainfluenzavirus 2. Das Genprodukt derThe expression of the foreign DNA is detected, for example, with the aid of antibodies. Suitable antibodies are those which react with immunogens of related virus strains, e.g. the Simian Virus 5 or the Canine Parainfluenza Virus 2. The gene product of
Fremd-DNS kann z.B. mittels Immunfluoreszenz oder Immunpräzipitation nachgewiesen werden.Foreign DNS can e.g. by means of immunofluorescence or immunoprecipitation.
(ii):(ii):
Der Nachweis der inserierten Fremd-DNS erfolgt durch Hybridisierung mit Gensonden, des entsprechenden Fremd-Genes.The inserted foreign DNA is detected by hybridization with gene probes, the corresponding foreign gene.
Stabile rekombinante Vektor- Viren werden in bekannten, üblichen Verfahren, wie weiter oben beschrieben vermehrt, isoliert und weiter aufgearbeitet als antigenes Material verwendet. In den erfindungsgemäßen Vakzinen liegt das antigene Material als solches oder in Mischung mit den üblichen Formulierungshilfsstoffen vor. Als solche seien genannt pharmakologisch verträgliche Löse- oder Verdünnungsmittel, Adjuvantien, Konservierungsstoffe, Suspensions- oder Lösungsvermittler wie Emulgatoren.Stable recombinant vector viruses are used in known, customary processes, as described above, isolated and further processed, as antigenic material. In the vaccines according to the invention, the antigenic material is present as such or in a mixture with the usual formulation auxiliaries. These include pharmacologically acceptable solvents or diluents, adjuvants, preservatives, suspending agents or solubilizers such as emulsifiers.
Das antigene Material wird als biologisch wirksame Substanz bei derThe antigenic material is used as a biologically active substance in the
Formulierung von Vakzinen eingesetzt.Formulation of vaccines used.
Zur Herstellung einer Lebendvakzine wird das antigene Material in Form lebender Viruspartikel verwendet, dem zur Stabilisierung Zusatzstoffe und gegebenenfalls auch Entschäumer und Konservierungsstoffe zugesetzt werden. Zur besseren Halt- barkeit wird der Leb endimpf Stoff gefriergetrocknet. Vor der Verwendung dieserTo produce a live vaccine, the antigenic material is used in the form of living virus particles, to which additives and, if necessary, defoamers and preservatives are added. The live vaccine is freeze-dried for better durability. Before using this
Vakzine wird das lyophilisierte Produkt mit einem Lösungsmittel, wie z.B. Aqua dest., Aqua purificata oder 0,9%-ige Kochsalzlösung rekonstituiert.The lyophilized product is vaccinated with a solvent such as e.g. Reconstituted aqua dest., Aqua purificata or 0.9% saline.
Die vom Zellsubstrat befreiten Viruspartikel werden in einer Konzentration von mindestens 106 KED50/ml zusammen mit Schutzkolloiden bzw. Stabilisatoren, wie z.B. Cellulosen, Dextranen, Gelatinen, Kollidonen oder Stearaten und gegebenen¬ falls unter Zusatz von Entschäumern, wie z.B. Tributylphosphat, Isopropanol oder Siliconöl sowie von Konservierungsmitteln, wie z.B. Merthiolat oder Thimerosal in einer wässrigen pH-Wert-gepufferten Lösung vermischt, in entsprechende Behältnisse abgefüllt und gefriergetrocknet.The virus particles freed from the cell substrate are at a concentration of at least 10 6 KED 50 / ml together with protective colloids or stabilizers, such as celluloses, dextrans, gelatins, collidones or stearates and optionally with the addition of defoamers, such as tributyl phosphate, isopropanol or Silicone oil and preservatives such as merthiolate or thimerosal mixed in an aqueous pH buffered solution, filled into appropriate containers and freeze-dried.
Zur Herstellung von Totvakzinen (inaktivierten Vakzinen) werden als antigenesTo produce dead vaccines (inactivated vaccines) are called antigenic
Material verwendet komplette, abgetötete Viruspartikel in einer Konzentration von 10 '°-109-0 KID50/ml, vorzugsweise 105*°-10s o KID50/ml vor Inaktivierung oder Teilstücke (subunits) der Viruspartikel in einer solchen Konzentration, daß pro Impfdosis 10-250 mg Protein, bevorzugt 10-100 mg Protein enthalten sind. Das antigene Material liegt in der Vakzine vor in Mischung mit den üblichenMaterial uses complete, killed virus particles in a concentration of 10 '° -10 9 - 0 KID 50 / ml, preferably 10 5 * ° -10 so KID 50 / ml before inactivation or parts (subunits) of the virus particles in such a concentration that 10-250 mg protein, preferably 10-100 mg protein are contained per vaccine dose. The antigenic material is present in the vaccine in a mixture with the usual ones
Formulierungshilfsstoffen wie Löse- und Verdünnungsmittel, Adjuvantien, Konser¬ vierungsmittel, Suspensions- oder Lösungsvermittler, pH-Wert-regulierende Mittel und gegebenenfalls Entschäumer.Formulation auxiliaries such as solvents and diluents, adjuvants, preservatives, suspending agents or solubilizers, pH regulators and optionally defoamers.
Als Löse- und Verdünnungsmittel seien genannt Aqua dest., Aqua purificata, phy- siologisch verträgliche Salzlösungen sowie Zellkulturmedien. Insbesondere finden Verwendung das oben genannte E-MEM sowie Phosphat-gepufferte Kochsalz¬ lösung (PBS).Solvents and diluents which may be mentioned are aqua dest., Aqua purificata, physiologically compatible salt solutions and cell culture media. Find in particular Use the above-mentioned E-MEM and phosphate-buffered saline (PBS).
Als Adjuvantien seien genannt:The following may be mentioned as adjuvants:
1.) . Mineralsalze wie Aluminiumhydroxyd, Aluminiumphosphat, Calciumphos- phat, Kaolin oder Silcium. Bevorzugt eingesetzt werden 10-50 Vol-%, vorzugsweise 25-35 Vol-% eines Aluminumhydroxid-Geles mit einem An¬ teil von 1-5 % (w/v), vorzugsweise 2-3 % (w/v) Aluminiumhydroxyd.1.) . Mineral salts such as aluminum hydroxide, aluminum phosphate, calcium phosphate, kaolin or silicon. 10-50% by volume, preferably 25-35% by volume, of an aluminum hydroxide gel with a proportion of 1-5% (w / v), preferably 2-3% (w / v), of aluminum hydroxide are preferably used.
2.) Ölige Adjuvantien wie nicht-toxische Mineralöle (z.B. Draceol®, Paraf- finöl), pflanzliche Öle (z.B. Lecithine, Erdnußöle) oder tierische Öle - " (Squalane, Squalene), die in einer Konzentration von 1-40 Vol-%, vorzugs¬ weise 1-15 Vol-% eingesetzt werden.2.) Oily adjuvants such as non-toxic mineral oils (e.g. Draceol ® , paraffin oil), vegetable oils (e.g. lecithins, peanut oils) or animal oils - "(Squalane, Squalene), which are in a concentration of 1-40 vol% , preferably 1-15% by volume are used.
3.) Hydrophile und hydrophobe Polymere wie Polyoxyethylen und Polyoxy- propylen. Bevorzugt eingesetzt werden synthetisch hergestellte Block¬ polymere (z.B. Pluronic® L101, Pluronic® L121, Pluronic® L122, Tetronic® 1501) in einer Konzentration von 1-10 Vol-%.3.) Hydrophilic and hydrophobic polymers such as polyoxyethylene and polyoxypropylene. Synthetically produced block polymers (eg Pluronic ® L101, Pluronic ® L121, Pluronic ® L122, Tetronic ® 1501) are preferably used in a concentration of 1-10% by volume.
4.) Adjuvantien bakteriellen Ursprungs wie Pertussis-Toxin (Bordetella pertus- sis), Salmonella-typhimurium-Mltogen oder bakterielle Endotoxine wie Lipopolysaccharide (LPS, z.B. aus Mycobakterien oder Salmonellen) sowie LPS-Analoge oder -Derivate wie z.B. Lipid-A, Monophosphoryl-Lipid-A (MPL), Diphosphoryl-Lipid-A, (DPL), Trehalose-Dimycoiat (TDM),4.) Adjuvants of bacterial origin such as pertussis toxin (Bordetella pertus- sis), Salmonella typhimurium mltogen or bacterial endotoxins such as lipopolysaccharides (LPS, e.g. from Mycobacteria or Salmonella) as well as LPS analogues or derivatives such as e.g. Lipid-A, Monophosphoryl-Lipid-A (MPL), Diphosphoryl-Lipid-A, (DPL), Trehalose-Dimycoiat (TDM),
Muramyldipeptid (MDP) oder Adamantyldipeptid (AdDP) sowie deren Derivate. Bevorzugt eingesetzt werden MDP-Derivate oder AdDP in einer Konzentration von 0,0001-10%) (w/v).Muramyl dipeptide (MDP) or adamantyl dipeptide (AdDP) and their derivatives. MDP derivatives or AdDP are preferably used in a concentration of 0.0001-10%) (w / v).
5.) Organische in Wasser dispergi erbare Adjuvantien wie Cholesterin, Gelatine, Phosphatidylcholin, Polysaccharide (z.B. Zymosan, Agar), aliphatische5.) Organic water-dispersible adjuvants such as cholesterol, gelatin, phosphatidylcholine, polysaccharides (e.g. zymosan, agar), aliphatic
Amine (z.B. Dimethyldioctadecylamin/DDA, N,N.diotadecyl-N',N'-bis(2-hy- droxyethyl)propandiamin Avridin®), DEAE-Dextrane oder Saponin (aus derAmines (e.g. dimethyldioctadecylamine / DDA, N, N.diotadecyl-N ', N'-bis (2-hydroxyethyl) propanediamine Avridin ® ), DEAE-dextrans or saponin (from the
Rinde von Ouillaia saponaria Molina') und Saponin-Abkömmlinge (Quil A). 6.) Monokine und Lymphokine wie z.B. Interleukin- 1 , Interleukin-2 oder γ- Interferon.Bark of Ouillaia saponaria Molina ' ) and saponin descendants (Quil A). 6.) Monokines and lymphokines such as interleukin-1, interleukin-2 or γ-interferon.
7.) Mögliche Kombinationen aus 1.) bis 6.)7.) Possible combinations from 1.) to 6.)
Als Konservierungsmittel seien genannt Formalin in Konzentrationen bis 1%, Phenol und Benzylalkohol in Konzentrationen bis 0,5 %, Sorbinsäure, Benzoe- säure, Natriumbenzoat, sowie deren Derivate wie z.B. das Natriumsalz der 2- (Ethylmercurio-thio)-benzoesäure (Merthiolat, Thimerosal, Thiomersal) oder das Natriumsalz der 4-(Ethylmercurio-thio)-benzolsulfonsäure (Thiomerfonat). Bevor¬ zugt eingesetzt wird Merthiolat in Konzentrationen von 0,01 % bis 0,5 %As preservatives, formalin in concentrations of up to 1%, phenol and benzyl alcohol in concentrations of up to 0.5%, sorbic acid, benzoic acid, sodium benzoate, and their derivatives such as e.g. the sodium salt of 2- (ethylmercurio-thio) -benzoic acid (merthiolate, thimerosal, thiomersal) or the sodium salt of 4- (ethylmercurio-thio) -benzenesulfonic acid (thiomerfonate). Merthiolate is preferably used in concentrations of 0.01% to 0.5%.
Als Suspensions- und Lösungsvermittler seien genannt nicht-toxische oberflächen¬ aktive Substanzen wie pflanzliche Proteine, Alginate, Cellulosen, Phospholipide und insbesondere auf Glykolether basierende Substanzen wie Polyethylenglykole und deren Derivate. Bevorzugt eingesetzt werden Polyethylenglycol (PEG) 200, 300, 400, 600 und 900 sowie PEG-Derivate (Span®, Arlacel®, Tween®, Myri®, Brij®), besonders bevorzugt Tween® 80 in einer Konzentration von 0,05-5 Vol-%, vorzugsweise 0,2-1 Vol-%.Suspensions and solubilizers which may be mentioned are non-toxic surface-active substances such as vegetable proteins, alginates, celluloses, phospholipids and in particular substances based on glycol ethers such as polyethylene glycols and their derivatives. Polyethylene glycol (PEG) 200, 300, 400, 600 and 900 and PEG derivatives (Span ® , Arlacel ® , Tween ® , Myri ® , Brij ® ) are preferably used, particularly preferably Tween ® 80 in a concentration of 0.05- 5 vol%, preferably 0.2-1 vol%.
Als pH-Wert-regulierende Substanzen seien genannt z.b. Natrium- und Kalium¬ hydroxid, Natrium- und Kaliumcarbonat, Essig-, Wein- und Zitronensäure oder Hydroxyethy lpiperazi n-N-2-ethansulfonsäure (HEPES ) .Examples of substances that regulate pH are e.g. Sodium and potassium hydroxide, sodium and potassium carbonate, acetic, tartaric and citric acid or hydroxyethy lpiperazi n-N-2-ethanesulfonic acid (HEPES).
Als Entschäumer seien genannt Tributylphosphat, Isopropanol, Siliconöl, Anti- foam® oder Baysilon® Entschäumer EBZ.Defoamers include tributyl phosphate, isopropanol, silicone oil, Antifoam ® or Baysilon ® defoamers EBZ.
Erfindungsgemäße Parainfluenzaviren die Erkrankungen der Respirations- und Reproduktionstrakts von Schweinen hervorrufen lassen sich z.B. wie folgt erhalten:Parainfluenza viruses according to the invention which can cause diseases of the respiratory and reproductive tract of pigs can be e.g. received as follows:
Von Schweinen, die an PRRS-ähnlicher Symptomatik erkrankt sind, werden Organe entnommen und einem Virusisolationsversuch unterworfen. Besonders geeignet sind lebensschwache oder erkrankte Ferkel aus infizierten Beständen. Die inneren Organe, insbesondere Lunge, Leber, Niere und Milz werden einem geeig¬ neten Tier entnommen. Teile von diesen Organen oder Organgemischen werden mit physiologisch vertraglichen wassngen Losungen zu Suspensionen homogeni¬ siert, wobei der Anteil an Organteilen ca 10% (w/v) ausmacht Besonders geeignet als Suspensionsmittel ist das oben beschriebene Eagles Minimum Essen- tial Medium (E-MEM) Die Suspensionen werden durch Zentrifugation bei ca 1500 x g von Zellen und Zelltrummern befreit. Eine weitere Reinigung desOrgans are removed from pigs suffering from PRRS-like symptoms and subjected to a virus isolation test. Weak or sick piglets from infected herds are particularly suitable. The internal organs, in particular the lungs, liver, kidney and spleen, are removed from a suitable animal. Parts of these organs or organ mixtures homogenized with physiologically compatible aqueous solutions to form suspensions, the proportion of organ parts accounting for approx. 10% (w / v). The Eagles Minimum Essential Medium (E-MEM) described above is particularly suitable as a suspending agent. The suspensions are separated by centrifugation approx. 1500 xg freed of cells and cell drums. Another cleaning of the
Zentrifugationsüberstandes kann durch Filtrieren erfolgen. Hierzu geeignet sind Filter mit einer Porengroße von 0,2-5 μm, bevorzugt 0,2-0,45 μm.Centrifugation supernatant can be done by filtration. Filters with a pore size of 0.2-5 μm, preferably 0.2-0.45 μm, are suitable for this.
Aus den entnommen Organen, bevorzugt der Lunge kann auch eine primäre Zellkultur angelegt werden, die auf das Auftreten eines zythopathogenen Effektes (CPE) hin untersucht wird Hierzu wird das Gewebe grob zerkleinert einem enzymatischen Aufschluß durch Proteasen unterworfen Hierzu besonders geeignet ist Trypsin in einer Konzentration von 0, 1-0,5 % (w/v), bevorzugt 0,125-0,25 % (w/v) in einer physiologischen, wassngen Losung Der Trypsinverdau erfolgt bei 20-37° C, bevorzugt bei Raumtemperatur in 2-8 Stunden Nicht verdaute Gewebs- bestandteile werden durch Grob-Filtration abgetrennt Die trypsinierten Zellen wer¬ den durch Zentrifugation bei 500-1500 x g gewonnen Das Zellsediment wird in einem geeigneten Wachstumsmedium, wie z B. dem beschriebenen E-MEM, resus¬ pendiert und in einer Konzentration von lθ θ Zellen/ml Medium in Kultur- gefaße ausgesät Je nach Wachstumsgeschwindigkeit wird das Wachstumsmedium alle 3-7 Tage ausgewechselt Das Wachstum der Zellen und das Auftreten einesA primary cell culture can be created from the organs removed, preferably the lungs, which is examined for the appearance of a cythopathogenic effect (CPE). For this purpose, the tissue is roughly crushed and subjected to enzymatic digestion by proteases. Trypsin in a concentration of 0.1-0.5% (w / v), preferably 0.125-0.25% (w / v) in a physiological, watery solution. The trypsin digestion takes place at 20-37 ° C., preferably at room temperature in 2-8 hours Undigested tissue components are separated by coarse filtration. The trypsinized cells are obtained by centrifugation at 500-1500 xg. The cell sediment is resuspended in a suitable growth medium, such as the E-MEM described, and in a concentration of lθ θ cells / ml medium sown in culture vessels Depending on the growth rate, the growth medium is replaced every 3-7 days. The growth of the cells and the appearance of a s
CPE wird taglich untersucht. Zusatzlich kann der Zellkulturuberstand in festen Zeitabstanden von 2-7 Tagen auf hämagglutinierende Eigenschaften überprüft wer¬ denCPE is examined daily. In addition, the cell culture supernatant can be checked for hemagglutinating properties at fixed time intervals of 2-7 days
Die Zentrifugationsuberstande bzw. Filtrate der Organhomogenate sowie die Zell- kulturuberstande der angelegten primären Organkulturen werden in einer Verdün¬ nung von 1 1 bis 1 1000, bevorzugt 1 10 bis 1 100, auf primäre oder permanente Mammalier-Zellkulturen aufgebracht und bei 32-39° C, bevorzugt 37° C über mehrere Tage inkubiert Verwendet werden hierzu Zellrasen, die zu 20-100%), be¬ vorzugt 80-100%) Konfluenz ausgewachsen sind Die Zellkulturen werden taglich auf das Auftreten eines CPE hin untersucht Zusätzlich kann der Zellkultur¬ uberstand in festen Zeitabstanden von 2-7 Tagen auf hämagglutinierende Eigen¬ schaften überprüft werden. Treten keine Anzeichen einer Virusvermehrung auf, werden die Zellkulturüberstände in den genannten Verdünnungen auf frische Zell¬ kulturen weiterpassagiert. Dieser Vorgang kann mehrmals wiederholt werden.The centrifugation supernatants or filtrates of the organ homogenates and the cell culture supernatants of the primary organ cultures applied are applied in a dilution of 1 1 to 1 1000, preferably 1 10 to 1 100, to primary or permanent mammalian cell cultures and at 32-39 ° C, preferably 37 ° C, incubated over several days. For this purpose, cell turf that has grown to 20-100%, preferably 80-100%) confluency is used. The cell cultures are examined daily for the occurrence of a CPE The supernatant can be checked for hemagglutinating properties at fixed intervals of 2-7 days. If there are no signs of virus multiplication, the cell culture supernatants in the dilutions mentioned are passed on to fresh cell cultures. This process can be repeated several times.
Bei Auftreten von Anzeichen einer Virusvermehrung wird durch weitere Passagen das Virus an die verwendete Zellkultur adaptiert.If signs of a virus multiplication appear, the virus is adapted to the cell culture used by further passages.
Von einer abortierenden Sau, die aus einem Bestand mit PRRS-ähnlicherFrom an aborting sow from a stock similar to PRRS
Symptomatik entstammte, wurde in sich noch im Uterus befindliches Ferkel per Sektion entnommen. Aus der Lunge dieses Ferkels konnte durch Anlegen einer primären Lungenzellkultur und durch Passagieren des sich hieraus ergebenden Kulturüberstandes auf tierische Zellinien ein cythopathogenes Agens isoliert werden. Es wurde als behülltes, hämagglutinierendes, ca. 200 nm großes, ein- strängiges RNS-Virus charakterisiert, das elektronenmikroskopisch die Morpholo¬ gie eines Paramyxovirus besitzt. Proteine dieses Virus wurden im Western Blot von einem Antiserum gegen ein Parainfluenzavirus Typ 2 (Pl-2) erkannt. Im gleichen Testsystem erkannte ein gegen das isolierte Virus hergestelltes Antiserum einen Pl-2-Stamm "SV5", womit die serologische Verwandschaft des isoliertenSymptoms originated, piglets still in the uterus were removed by section. A cythopathogenic agent was isolated from the lungs of this piglet by applying a primary lung cell culture and by passing the resulting culture supernatant onto animal cell lines. It was characterized as a enveloped, hemagglutinating, approx. 200 nm large, single-stranded RNA virus which has the morphology of a paramyxovirus by electron microscopy. Proteins of this virus were recognized in the Western blot from an antiserum against a parainfluenza virus type 2 (Pl-2). In the same test system, an antiserum produced against the isolated virus recognized a Pl-2 strain "SV5", which shows the serological relationship of the isolated
Virus zu Parainfluenzavirus Typ 2 gesichert ist.Virus to parainfluenza virus type 2 is secured.
Dieses Parainfluenza-Isolat mit der Bezeichnung "SER" wurde am 12.06.1993 in der "Collection Nationale de Cultures et de Microorganismes" des "Institut Pasteur", Paris, Frankreich unter der Nummer 1-1331 hinterlegt.This parainfluenza isolate with the designation "SER" was deposited on June 12, 1993 in the "Collection Nationale de Cultures et de Microorganismes" of the "Institut Pasteur", Paris, France under the number 1-1331.
Das isolierte Virus läßt sich in großem Maßstab unter Verwendung von tierischenThe isolated virus can be used on a large scale using animal
Zellkulturen vermehren. Aus so produzierten Virus-Suspensionen können mittels geeigneter technischer Verfahren (Zentrifugation, Tangential-Filtration) gereinigte Antigenpräparationen hergestellt werden. Diese können als Ausgangsmaterial zur Diagnose und zur Prävention von Respirations- und Reproduktionserkrankungen der Schweine, insbesondere des PRRS, eingesetzt werden. Beispiel 1Multiply cell cultures. Purified antigen preparations can be produced from virus suspensions produced in this way using suitable technical processes (centrifugation, tangential filtration). These can be used as starting material for the diagnosis and prevention of respiratory and reproductive diseases in pigs, in particular the PRRS. example 1
Isolierung des Parainfluenza-Isolates "SER"Isolation of the parainfluenza isolate "SER"
Das Parainfluenza-Isolates "SER" konnte aus der Lunge eines Ferkels isoliert wer¬ den, das bei der Sektion einer euthanasierten, abortierenden Sau aus dem Uterus entnommen wurde, die aus einem Bestand mit PRRS-ähnlicher Symptomatik stammte.The parainfluenza isolate "SER" could be isolated from the lungs of a piglet, which was removed from the uterus during the sectioning of a euthanized, aborting sow, which came from a herd with PRRS-like symptoms.
PK15 Zellen (Klonierte Schweinenieren - Zelle, ATCC-Nr. CCL 33)PK15 cells (cloned pig kidney cell, ATCC No. CCL 33)
Eagles Minimum Essential Medium mit Earle's Salzen (E-MEM):Eagles Minimum Essential Medium with Earle 's Salts (E-MEM):
E-MEM - Pulver mit Phenolrot (z.B. Gibco BRL 072-01 10) für 100 1 Nicht-essentielle Aminosäuren, Stocklösung lOOx 1000 mlE-MEM - powder with phenol red (e.g. Gibco BRL 072-01 10) for 100 1 non-essential amino acids, stock solution 100 x 1000 ml
Neomycinsulfat 3 gNeomycin sulfate 3 g
Polymyxin-B-Sulfat 3 MUPolymyxin B sulfate 3 MU
Aqua purificata (EP 8) ad 100 1 Nicht-essentielle Aminosäuren, Stocklösung lOOx: Alanin (EP 752) 8,9 gAqua purificata (EP 8) ad 100 1 non-essential amino acids, stock solution 100x: alanine (EP 752) 8.9 g
Asparagin - Monohydrat (DAß 10) 15,0 gAsparagine Monohydrate (DAß 10) 15.0 g
L - Aparaginsäure 13,2 gL - Aspartic acid 13.2 g
Glycin (EP 614) 7,5 gGlycine (EP 614) 7.5 g
Glutaminsäure (EP 750) 14,7 g Prolin (EP 785) 11,5 gGlutamic acid (EP 750) 14.7 g proline (EP 785) 11.5 g
Serin (EP 788) 10,5 gSerine (EP 788) 10.5 g
Aqua purificata (EP 8) ad 10 1 Fötales Kälberserum (FKS, z.B. Gibco BRL 012-06290) Wachstumsmedium: E-MEM mit 2,0 g/1 Natriumbicarbonat und 2% FKS - Erhaltungsmedium: E-MEM mit 0,85 g/1 Natriumbicarbonat und 5% FKSAqua purificata (EP 8) ad 10 1 Fetal calf serum (FCS, e.g. Gibco BRL 012-06290) Growth medium: E-MEM with 2.0 g / 1 sodium bicarbonate and 2% FCS - Maintenance medium: E-MEM with 0.85 g / 1 sodium bicarbonate and 5% FCS
0,25% Trypsin-Lösung (z.B. Gibco BRL 043-05050) PBS-Puffer (Phosphate Buffered Saline):0.25% trypsin solution (e.g. Gibco BRL 043-05050) PBS buffer (Phosphate Buffered Saline):
NaCl 8,0 gNaCl 8.0 g
KC1 0,2 g KH:,P04 0,2 gKC1 0.2 g KH: P0 4 0.2 g
Na2HPÖ4 x 12 H20 2,9 g Aqua purificata (EP 8) ad 1000 mlNa 2 HPÖ 4 x 12 H 2 0 2.9 g Aqua purificata (EP 8) ad 1000 ml
Gewebekulturflasche, 80 cm" (Roux-Flasche, z.B. Greiner 658 170)Tissue culture bottle, 80 cm "(Roux bottle, e.g. Greiner 658 170)
Von der unter sterilen Bedingungen entnommenen Ferkellunge wurde ein Teil mit der Schere grob zerkleinert und einem enzymatischen Aufschluß in 0,25%-iger Trypsinlösung unterzogen. Die Trypsinierung erfolgte unterA portion of the piglet lung removed under sterile conditions was roughly crushed with scissors and subjected to enzymatic digestion in 0.25% trypsin solution. The trypsinization took place under
Rühren bei Raumtemperatur in 4 Stunden. Die groben Gewebsstücke wur¬ den in einem sterilen Gaze-Filter abgeschieden. Das Filtrat wurde dreimal bei niedertouriger Zentrifugation (1000 x g, 10 Minuten) in PBS gewa¬ schen. Die Zellen wurden in E-MEM und Zusatz von 5% FKS in einer Konzentration von 10 Zellen ml in SOcnr-Kulturflaschen ausgesät und beiStir at room temperature in 4 hours. The coarse pieces of tissue were separated in a sterile gauze filter. The filtrate was washed three times with low-speed centrifugation (1000 x g, 10 minutes) in PBS. The cells were seeded in E-MEM and addition of 5% FCS in a concentration of 10 cells ml in SOcnr culture bottles and at
37 °C inkubiert. Das Wachstumsmedium wurde alle 4 - 5 Tage ausge¬ wechselt. Das Wachstum dieser Primärkultur wurde täglich durch mikro¬ skopische Untersuchung beobachtet. Hierbei wurde insbesondere auf das Auftreten eines zytopathogenen Effekts (CPE) geachtet. Nach ca. 2 Wo- chen konnte ein CPE in Form von sich abrundenden, schrumpfenden Zellen mit langsamer Ausbreitungstendenz beobachtet werden. Der Kulturüber¬ stand der Primärzellen wurde 1: 10 mit Erhaltungsmedium verdünnt und auf konfluente Monolayer von PK- 15 -Zellkulturen in 80 cm2 -Kulturflaschen verimpft (Inkubationsvolumen: 40 ml). Zur Kontrolle wurde von jeder Zelle eine nicht-infizierte Kultur mitgeführt. Nach 7-tägiger Inkubation ent¬ wickelte sich ein CPE mit beginnender Lochbildung. Die Kultur wurde einem Frier-Tau-Prozeß unterworfen und der Überstand 1 :10 verdünnt auf eine frische Kultur verimpft, deren Überstand nach 6 - 7 Tagen im Hämag¬ glutinationstest unter Verwendung von Hühnererythrozyten geprüft wurde. In der 4. Passage wiesen die Kulturen nach 6-tägiger Inkubation annähernd zu 100%) einen zytopathogenen Effekt auf. Im Hämagglutinationstest positive Kulturüberstände wurden bei -70 °C eingelagert. Beispiel 2Incubated at 37 ° C. The growth medium was changed every 4-5 days. The growth of this primary culture was observed daily by microscopic examination. Particular attention was paid to the appearance of a cytopathogenic effect (CPE). After about 2 weeks, a CPE in the form of rounded, shrinking cells with a slow tendency to spread could be observed. The culture supernatant of the primary cells was diluted 1:10 with maintenance medium and inoculated onto confluent monolayers of PK-15 cell cultures in 80 cm 2 culture bottles (incubation volume: 40 ml). A non-infected culture was carried from each cell as a control. After 7 days of incubation, a CPE developed with the start of hole formation. The culture was subjected to a freeze-thaw process and the supernatant was diluted 1:10 and inoculated into a fresh culture, the supernatant of which was tested after 6-7 days in the hemagglutination test using chicken erythrocytes. In the 4th passage, the cultures showed almost 100% cytopathogenic effect after 6 days of incubation. Culture supernatants positive in the hemagglutination test were stored at -70 ° C. Example 2
Vermehrung des Parainfluenza-Isolates "SER"Propagation of the parainfluenza isolate "SER"
Material:Material:
Parainfluenza-Isolat "SER", Basissaatmaterial - PK-15 Zellen (Klonierte Schweinenieren - Zelle, ATCC-Nr. CCL 33)Parainfluenza isolate "SER", basic seed material - PK-15 cells (cloned pig kidneys - cell, ATCC No. CCL 33)
Eagles Minimum Essential Medium mit Earle's Salzen (E-MEM): E-MEM - Pulver mit Phenolrot (z.B. Gibco BRL 072-0110) für 100 1 Nicht-essentielle Aminosäuren, Stocklösung lOOx 1000 mlEagles Minimum Essential Medium with Earle 's salts (E-MEM): E-MEM - powder with phenol red (e.g. Gibco BRL 072-0110) for 100 1 non-essential amino acids, stock solution 100 x 1000 ml
Neomycinsulfat 3 g Polymyxin-B-Sulfat 3 MUNeomycin sulfate 3 g polymyxin B sulfate 3 MU
Aqua purificata (EP 8) ad 100 1Aqua purificata (EP 8) ad 100 1
Fötales Kälberserum (FKS, z.B. Gibco BRL 012-06290) Wachstumsmedium: E-MEM mit 2,0 g/1 Natriumbicarbonat und 2% FKS Erhaltungsmedium: E-MEM mit 0,85 g/1 Natriumbicarbonat und 5% FKS - Gewebekulturflasche, 80 cm (Roux-Flasche, z.B. Greiner 658 170)Fetal calf serum (FCS, e.g. Gibco BRL 012-06290) Growth medium: E-MEM with 2.0 g / 1 sodium bicarbonate and 2% FCS Maintenance medium: E-MEM with 0.85 g / 1 sodium bicarbonate and 5% FCS - tissue culture bottle, 80 cm (Roux bottle, e.g. Greiner 658 170)
Wannenstapel, 6000 cm2 (z.B. Nunc 164 327)Tub stack, 6000 cm 2 (e.g. Nunc 164 327)
Methodik:Methodology:
Das Wachstumsmedium einer mit PK-15-Zellen konfluent bewachsenen Gewebe- kulturflasche wird dekantiert und diese mit 40 ml des in Erhaltungsmedium 1:50 verdünnten Virus-Basissaatmaterials beschickt. Nach 7-tägiger Inkubation beiThe growth medium of a tissue culture bottle confluently overgrown with PK-15 cells is decanted and this is charged with 40 ml of the basic virus seed material diluted 1:50 in maintenance medium. After 7 days incubation with
37 °C wird der einem Einfrier- Auftau-Prozeß unterworfene und durch Ultraschall suspendierte Inhalt der Gewebekulturflasche mit Erhaltungsmedium auf ein Volumen von 3000 ml aufgefüllt und hiermit ein mit PK-15-Zellen konfluent bewachsener Wannenstapel beimpft. Nach 7-tägiger Inkubation bei 37 °C wird der Kulturüberstand geerntet und bis zur weiteren Verarbeitung bei -70 °C gelagert. 37 ° C., the contents of the tissue culture flask subjected to a freeze-thaw process and suspended by ultrasound are filled with maintenance medium to a volume of 3000 ml and inoculated with this a stack of tubs grown confluently with PK-15 cells. After 7 days of incubation at 37 ° C, the culture supernatant is harvested and stored at -70 ° C until further processing.
Beispiel 3Example 3
Totimpfstoffherstellung (Parainfluenza-Isolat "SER")Vaccine production (parainfluenza isolate "SER")
Material:Material:
Parainfluenza-Isolat "SER", Zellkulturuberstand aus Virusvermehrung(en) 2-Bromethylamin-Hydrobromid (2-BEA) 0,5 M Stocklösung: 2-Bromethylamin-Hydrobromid (2-BEA, BrCH2CH2NH2HBr,Parainfluenza isolate "SER", cell culture supernatant from virus propagation (s) 2-bromoethylamine hydrobromide (2-BEA) 0.5 M stock solution: 2-bromoethylamine hydrobromide (2-BEA, BrCH 2 CH 2 NH 2 HBr,
Merck 820176) 102,5 gMerck 820176) 102.5 g
Aqua purificata (EP 8) ad 1000 mlAqua purificata (EP 8) ad 1000 ml
Natrium-Thiosulfat 2,5 M Stocklösung:Sodium thiosulfate 2.5 M stock solution:
Na2S203 x 5H20 (EP 414) 620,5 gNa 2 S 2 0 3 x 5H 2 0 (EP 414) 620.5 g
Aqua purificata (EP 8) ad 1000 mlAqua purificata (EP 8) ad 1000 ml
Aluminiumhydroxid Suspension 3% (z.B. Superfos)Aluminum hydroxide suspension 3% (e.g. Superfos)
Quil A 1 %ige StocklösungQuil A 1% stock solution
Quil A (z.B. Superfos) 10,0 gQuil A (e.g. Superfos) 10.0 g
Aqua purificata (EP 8) ad 1000 mlAqua purificata (EP 8) ad 1000 ml
Thimerosal 2 %ige StocklösungThimerosal 2% stock solution
Thimerosal (CgHcJigNaO-^) 50,0 gThimerosal (CgHcJigNaO- ^) 50.0 g
Aqua purificata (EP 8) ad 1000 mlAqua purificata (EP 8) ad 1000 ml
PBS-Puffer (Phosphate Buffered Saline):PBS buffer (Phosphate Buffered Saline):
NaCl 8,0 gNaCl 8.0 g
KC1 0,2 gKC1 0.2 g
KH,P04 0,2 gKH, P0 4 0.2 g
Na2HP04 x 12 H20 2,9 gNa 2 HP0 4 x 12 H 2 0 2.9 g
Aqua purificata (EP 8) ad 1000 mlAqua purificata (EP 8) ad 1000 ml
Der Überstand des auf Zellkulturen vermehrten Virus wird durch Zentrifugation bei 10.000 x g von Zellen und Zelltrümmern befreit. Eine solchermaßen gereinigte Virussuspension mit einer Konzentration an Viruspartikeln von 106,0 KID50/ml, die aus einer oder mehr Virusernten stammt, wird in ein steriles Gefäß überführt. Der pH-Wert wird mit Natronlauge (2 N NaOH) auf 8,4 eingestellt. Es wird eine solche Menge an 0,5 M 2-Bromethylamin-Hydrobromid Lösung (2-BEA) unter ständigem Rühren zugegeben, bis eine Endkonzentration von 5 mmol/l 2-BEA eπeicht ist. Die Inaktivierung des Virus erfolgt innerhalb von 18 Stunden bei 37 °C Anschließend wird das Inaktivierungsmittel durch Zugabe einer 2,5 M Natπum-Thiosulfatlosung bis zu einer Endkonzentration • on 50 mmol/l bei 4°C neutralisiertThe supernatant of the virus propagated on cell cultures is freed from cells and cell debris by centrifugation at 10,000 xg. A virus suspension purified in this way with a concentration of virus particles of 10 6.0 KID 50 / ml, which originates from one or more virus harvests, is transferred into a sterile vessel. The pH is adjusted to 8.4 with sodium hydroxide solution (2 N NaOH). Such an amount of 0.5 M 2-bromoethylamine hydrobromide solution (2-BEA) is added with constant stirring until a final concentration of 5 mmol / l 2-BEA is calibrated. The virus is inactivated within 18 hours 37 ° C. The inactivating agent is then neutralized by adding a 2.5 M sodium thiosulfate solution to a final concentration of 50 mmol / l at 4 ° C.
62 ml der inaktivierten Virussuspension werden zu 31 ml einer sterilen Alumi- niumhydroxid-Suspension (3% Al(OH)3, pH 7,3) gegeben und 2 Stunden bei 4 °C gerührt. Nach Zugabe von 1,25 ml Quil A (2%-ige Losung) und 0,1 ml Thimero¬ sal (2%-ige Losung) wird mit PBS-Puffer auf 100 ml aufgefüllt und für weitere 20 Stunden bei 4 °C gerührt. Die fertige Vakzine wird in Mehrfachentnahme- Behaltnisse abgefüllt und bei 4°C gelagert.62 ml of the inactivated virus suspension are added to 31 ml of a sterile aluminum hydroxide suspension (3% Al (OH) 3 , pH 7.3) and stirred at 4 ° C. for 2 hours. After adding 1.25 ml of Quil A (2% solution) and 0.1 ml of thimerosal (2% solution), make up to 100 ml with PBS buffer and stir at 4 ° C. for a further 20 hours . The finished vaccine is filled into multiple withdrawal containers and stored at 4 ° C.
Die- Impfung von Schweinen aller Altersklassen erfolgt durch subkutane Applika¬ tion von 2 ml dieser VakzinePigs of all ages are vaccinated by subcutaneous application of 2 ml of this vaccine
Publikationen mit veröffentlichten Nucleotid-Sequenzen. die für Immunogene des Simian Virus 5 codieren:Publications with published nucleotide sequences. which code for Simian Virus 5 immunogens:
Hiebert, S W., Paterson, R G. & Lamb, R.A (1985) Hemaggluünin-neuraminidase protein of the paramyxovirus simian virus 5. nucleotide sequence of the mRNA predicts a N-terminal membrane anchor Journal of Virology, 54, 1-6Hiebert, S W., Paterson, R G. & Lamb, R.A (1985) Hemaggluünin-neuraminidase protein of the paramyxovirus simian virus 5. nucleotide sequence of the mRNA predicts a N-terminal membrane anchor Journal of Virology, 54, 1-6
Hiebert, S W , Paterson, R.G & Lamb, R A. (1985) Identification and predicted sequence of a previously unrecognized small hydrophobic protein, SH, of the paramyxovirus simian virus 5 Journal of Virology, 55. 744-751Hiebert, S W, Paterson, R.G & Lamb, R A. (1985) Identification and predicted sequence of a previously unrecognized small hydrophobic protein, SH, of the paramyxovirus simian virus 5 Journal of Virology, 55,744-751
Paterson, R G , Harris, T J R & Lamb, R.A (1984) Analysis and gene assignment of mRNAs of a paramyxovirus, simian virus 5 Virology, 138, 310-323Paterson, R G, Harris, T J R & Lamb, R.A (1984) Analysis and gene assignment of mRNAs of a paramyxovirus, simian virus 5 Virology, 138, 310-323
Paterson, R G., Harris, T J.R & Lamb, R A ( 1984) Fusion protein of the paramyxovirus simian virus 5 nucleotide sequence of mRNA predicts a highly hydrophobic glycoprotein Proc Natl. Acad Sei USA. 81, 6706-6710Paterson, R.G., Harris, T.J.R & Lamb, RA (1984) Fusion protein of the paramyxovirus simian virus 5 nucleotide sequence of mRNA predicts a highly hydrophobic glycoprotein Proc Natl. Acad Sei USA. 81, 6706-6710
Paterson, R.G., Hiebert, S W & Lamb, RA (1985) Expression at the cell surface of biologically active fusion and hemagglutinin/neuraminidase proteins of the paramyxovirus simian virus 5 from cloned cDNA. Proc. Natl. Acad. Sei. USA, 82, 7520-7524.Paterson, RG, Hiebert, SW & Lamb, RA (1985) Expression at the cell surface of biologically active fusion and hemagglutinin / neuraminidase proteins of the paramyxovirus simian virus 5 from cloned cDNA. Proc. Natl. Acad. Be. USA, 82, 7520-7524.
Thomas, S., Lamb, RA. & Paterson, RG. (1988). Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal prteins P and V of the paramyxovirus SV5. Cell, 54, 891-902. Thomas, S., Lamb, RA. & Paterson, RG. (1988). Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal prteins P and V of the paramyxovirus SV5. Cell, 54, 891-902.
SEQUENZPROTOKOLLSEQUENCE LOG
(1) ALLGEMEINE ANGABEN:(1. GENERAL INFORMATION:
(i) ANMELDER:(i) APPLICANT:
(A) NAME: Bayer AG(A) NAME: Bayer AG
(B) STRASSE: Bayerwerk(B) ROAD: Bayerwerk
(C) ORT: Leverkusen(C) LOCATION: Leverkusen
(E) LAND: Deutschland(E) COUNTRY: Germany
(F) POSTLEITZAHL: D-51368(F) POSTCODE: D-51368
(G) TELEFON: 0214/30 66400 (H) TELEFAX: 0214/30 3482 (I) TELEX: 85 101-265 by d(G) TELEPHONE: 0214/30 66400 (H) TELEFAX: 0214/30 3482 (I) TELEX: 85 101-265 by d
(ii) BEZEICHNUNG DER ERFINDUNG: Vakzine zur Praevention von Respirations- und(ii) DESCRIPTION OF THE INVENTION: Vaccine for the prevention of respiratory and
Reproduktionserkrankungen des SchweinesReproductive diseases of the pig
(iii) ANZAHL DER SEQUENZEN: 4(iii) NUMBER OF SEQUENCES: 4
(iv) COMPUTER-LESBARE FASSUNG:(iv) COMPUTER READABLE VERSION:
(A) DATENTRÄGER: Floppy disk(A) DISK: Floppy disk
(B) COMPUTER: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) BETRIEBSSYSTEM: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPA)(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA)
(2) ANGABEN ZU SEQ ID NO: 1.-(2) INFORMATION ON SEQ ID NO: 1.-
(i) SEQUENZKENNZΞICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 1698 Basenpaare(A) LENGTH: 1698 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelsträng(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear (ii) ART DES MOLEKÜLS: Genom-DNA(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(iii) HYPOTHETISCH: NEIN(iii) HYPOTHETICAL: NO
(ix) MERKMAL:(ix) FEATURE:
(A) NAME/SCHLÜSSEL: CDS(A) NAME / KEY: CDS
(B) LAGE:1..1695(B) LOCATION: 1..1695
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 1:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
ATG GTT GCA GAA GAT GCC CCT GTT AGG GGC ACT TGC CGA GTA TTA TTT 48 Met Val Ala Glu Asp Ala Pro Val Arg Gly Thr Cys Arg Val Leu Phe 1 5 10 15ATG GTT GCA GAA GAT GCC CCT GTT AGG GGC ACT TGC CGA GTA TTA TTT 48 Met Val Ala Glu Asp Ala Pro Val Arg Gly Thr Cys Arg Val Leu Phe 1 5 10 15
CGA ACA ACA ACT TTA ATT TTT CTA TGC ACA CTA CTA GCA TTA AGC ATC 96 Arg Thr Thr Thr Leu Ile Phe Leu Cys Thr Leu Leu Ala Leu Ser Ile '20 25 30CGA ACA ACA ACT TTA ATT TTT CTA TGC ACA CTA CTA GCA TTA AGC ATC 96 Arg Thr Thr Thr Leu Ile Phe Leu Cys Thr Leu Leu Ala Leu Ile Ser '20 25 30
TCT ATC CTT TAT GAG AGT TTA ATA ACC CAA AAG CAA ATC ATG AGC CAC 144 Ser Ile Leu Tyr Glu Ser Leu Ile Thr Gin Lys Gin Ile Met Ser His 35 40 45TCT ATC CTT TAT GAG AGT TTA ATA ACC CAA AAG CAA ATC ATG AGC CAC 144 Ser Ile Leu Tyr Glu Ser Leu Ile Thr Gin Lys Gin Ile Met Ser His 35 40 45
GCA GGA TAC ACT CGA TCT AAT TCT AGA TTA GGA AGT ATC ACT GAT CTT 192 Ala Gly Tyr Thr Arg Ser Asn Ser Arg Leu Gly Ser Ile Thr Asp Leu 50 55 60GCA GGA TAC ACT CGA TCT AAT TCT AGA TTA GGA AGT ATC ACT GAT CTT 192 Ala Gly Tyr Thr Arg Ser Asn Ser Arg Leu Gly Ser Ile Thr Asp Leu 50 55 60
CTT AAT AAT ATT CTC TCT GTC GCA AAT CAG ATT ATA TAT AAC TCT GCA 240 Leu Asn Asn Ile Leu Ser Val Ala Asn Gin Ile Ile Tyr Asn Ser Ala 65 70 75 80CTT AAT AAT ATT CTC TCT GTC GCA AAT CAG ATT ATA TAT AAC TCT GCA 240 Leu Asn Asn Ile Leu Ser Val Ala Asn Gin Ile Ile Tyr Asn Ser Ala 65 70 75 80
GTC.GCT CTA CCT CTA CAA TTG GAC ACT CTT GAA TCA ACA CTC CTT ACA 288 Val Ala Leu Pro Leu Gin Leu Asp Thr Leu Glu Ser Thr Leu Leu Thr 85 90 95 GCC ATT AAG TCT CTT CAA ACC AGT GAC AAG CTA GAA CAG AAC TGC TCG 336 Ala Ile Lys Ser Leu Gin Thr Ser Asp Lys Leu Glu Gin Asn Cys Ser 100 105 110GTC.GCT CTA CCT CTA CAA TTG GAC ACT CTT GAA TCA ACA CTC CTT ACA 288 Val Ala Leu Pro Leu Gin Leu Asp Thr Leu Glu Ser Thr Leu Leu Thr 85 90 95 GCC ATT AAG TCT CTT CAA ACC AGT GAC AAG CTA GAA CAG AAC TGC TCG 336 Ala Ile Lys Ser Leu Gin Thr Ser Asp Lys Leu Glu Gin Asn Cys Ser 100 105 110
TGG GGT GCT GCA CTG ATT AAT AAT AAT AGA TAC ATT AAT GGC ATC AAT 384 Trp Gly Ala Ala Leu Ile Asn Asn Asn Arg Tyr Ile Asn Gly Ile Asn 115 120 125TGG GGT GCT GCA CTG ATT AAT AAT AAT AGA TAC ATT AAT GGC ATC AAT 384 Trp Gly Ala Ala Leu Ile Asn Asn Asn Arg Tyr Ile Asn Gly Ile Asn 115 120 125
CAG TTC TAT TTT TCA ATT GCT GAG GGT CGC AAT CTG ACA CTT GGC CCA 432 Gin Phe Tyr Phe Ser Ile Ala Glu Gly Arg Asn Leu Thr Leu Gly Pro 130 135 140CAG TTC TAT TTT TCA ATT GCT GAG GGT CGC AAT CTG ACA CTT GGC CCA 432 Gin Phe Tyr Phe Ser Ile Ala Glu Gly Arg Asn Leu Thr Leu Gly Pro 130 135 140
CTT CTT AAT ATA CCT AGT TTC ATT CCA ACT GCC ACG ACA CCA GAG GGC 480 Leu Leu Asn Ile Pro Ser Phe Ile Pro Thr Ala Thr Thr Pro Glu Gly 145 150 155 160CTT CTT AAT ATA CCT AGT TTC ATT CCA ACT GCC ACG ACA CCA GAG GGC 480 Leu Leu Asn Ile Pro Ser Phe Ile Pro Thr Ala Thr Thr Pro Glu Gly 145 150 155 160
TGC ACC AGG ATC CCA TCA TTC TCG CTC ACC AAG ACA CAC TGG TGT TAT 528 Cys Thr Arg Ile Pro Ser Phe Ser Leu Thr Lys Thr His Trp Cys Tyr 165 170 175TGC ACC AGG ATC CCA TCA TTC TCG CTC ACC AAG ACA CAC TGG TGT TAT 528 Cys Thr Arg Ile Pro Ser Phe Ser Leu Thr Lys Thr His Trp Cys Tyr 165 170 175
ACA CAC AAT GTT ATC CTG AAT GGA TGC CAG GAT CAT GTA TCC TCA AAT 576 Thr His Asn Val Ile Leu Asn Gly Cys Gin Asp His Val Ser Ser Asn 180 185 190ACA CAC AAT GTT ATC CTG AAT GGA TGC CAG GAT CAT GTA TCC TCA AAT 576 Thr His Asn Val Ile Leu Asn Gly Cys Gin Asp His Val Ser Ser Asn 180 185 190
CAA TTT GTT TCC ATG GGA ATC ATT GAA CCC ACT TCT GCC GGG TTT CCA 624 Gin Phe Val Ser Met Gly Ile Ile Glu Pro Thr Ser Ala Gly Phe Pro 195 200 205CAA TTT GTT TCC ATG GGA ATC ATT GAA CCC ACT TCT GCC GGG TTT CCA 624 Gin Phe Val Ser Met Gly Ile Ile Glu Pro Thr Ser Ala Gly Phe Pro 195 200 205
TCC TTT CGA ACC CTA AAG ACT CTA TAT CTC AGC GAT GGG GTC AAT CGT 672 Ser Phe Arg Thr Leu Lys Thr Leu Tyr Leu Ser Asp Gly Val Asn Arg 210 215 220TCC TTT CGA ACC CTA AAG ACT CTA TAT CTC AGC GAT GGG GTC AAT CGT 672 Ser Phe Arg Thr Leu Lys Thr Leu Tyr Leu Ser Asp Gly Val Asn Arg 210 215 220
AAG AGC TGC TCT ATC AGT ACA GTT CCG GGG GGT TGT ATG ATG TAC TGT 720 Lys Ser Cys Ser Ile Ser Thr Val Pro Gly Gly Cys Met Met Tyr Cys 225 230 235 240 TTT GTC TCT ACT CAA CCA GAG AGG GAT GAC TAC TTT TCT ACC GCT CCT 768 Phe Val Ser Thr Gin Pro Glu Arg Asp Asp Tyr Phe Ser Thr Ala Pro 245 250 255AAG AGC TGC TCT ATC AGT ACA GTT CCG GGG GGT TGT ATG ATG TAC TGT 720 Lys Ser Cys Ser Ile Ser Thr Val Pro Gly Gly Cys Met Met Tyr Cys 225 230 235 240 TTT GTC TCT ACT CAA CCA GAG AGG GAT GAC TAC TTT TCT ACC GCT CCT 768 Phe Val Ser Thr Gin Pro Glu Arg Asp Asp Tyr Phe Ser Thr Ala Pro 245 250 255
CCA GAA CAA CGA ATT ATT ATA ATG TAC TAT AAT GAT ACA ATC GTG GAG 816 Pro Glu Gin Arg Ile Ile Ile Met Tyr Tyr Asn Asp Thr Ile Val Glu 260 265 270CCA GAA CAA CGA ATT ATT ATA ATG TAC TAT AAT GAT ACA ATC GTG GAG 816 Pro Glu Gin Arg Ile Ile Ile Met Tyr Tyr Asn Asp Thr Ile Val Glu 260 265 270
CGC ATA ATT AAT CCA CCC GGG GTA CTA GAT GTA TGG GCA ACA TTG ACC 864 Arg Ile Ile Asn Pro Pro Gly Val Leu Asp Val Trp Ala Thr Leu Thr 275 280 285CGC ATA ATT AAT CCA CCC GGG GTA CTA GAT GTA TGG GCA ACA TTG ACC 864 Arg Ile Ile Asn Pro Pro Gly Val Leu Asp Val Trp Ala Thr Leu Thr 275 280 285
CCA GGA ACA GGA AGC GGG GTA TAT TAT TTA GGT TGG GTG CTC TTT CCA 912 Pro Gly Thr Gly Ser Gly Val Tyr Tyr Leu Gly Trp Val Leu Phe Pro 290 295 300CCA GGA ACA GGA AGC GGG GTA TAT TAT TTA GGT TGG GTG CTC TTT CCA 912 Pro Gly Thr Gly Ser Gly Val Tyr Tyr Leu Gly Trp Val Leu Phe Pro 290 295 300
ATA TAT GGC GGC GTG ATT AAA GAT ACG AGT TTA TGG AAT AAT CAA GCA 960 Ile Tyr Gly Gly Val Ile Lys Asp Thr Ser Leu Trp Asn Asn Gin Ala 305 310 315 320ATA TAT GGC GGC GTG ATT AAA GAT ACG AGT TTA TGG AAT AAT CAA GCA 960 Ile Tyr Gly Gly Val Ile Lys Asp Thr Ser Leu Trp Asn Asn Gin Ala 305 310 315 320
AAT AAA TAC TTT ATC CCC CAG ATG GTT GCT GCT CTC TGC TCA CAA AAC 1008 Asn Lys Tyr Phe Ile Pro Gin Met Val Ala Ala Leu Cys Ser Gin Asn 325 330 335AAT AAA TAC TTT ATC CCC CAG ATG GTT GCT GCT CTC TGC TCA CAA AAC 1008 Asn Lys Tyr Phe Ile Pro Gin Met Val Ala Ala Leu Cys Ser Gin Asn 325 330 335
CAG GCA ACT CAA GTC CAA AAT GCT AAG TCA TCA TAC TAT AGC AGC TGG 1056 Gin Ala Thr Gin Val Gin Asn Ala Lys Ser Ser Tyr Tyr Ser Ser Trp 340 345 350CAG GCA ACT CAA GTC CAA AAT GCT AAG TCA TCA TAC TAT AGC AGC TGG 1056 Gin Ala Thr Gin Val Gin Asn Ala Lys Ser Ser Tyr Tyr Ser Ser Trp 340 345 350
TTT GGC AAT CGA ATG ATT CAG TCT GGG ATC CTG GCA TGT CCT CTT CAA 1104 Phe Gly Asn Arg Met Ile Gin Ser Gly Ile Leu Ala Cys Pro Leu Gin 355 360 365TTT GGC AAT CGA ATG ATT CAG TCT GGG ATC CTG GCA TGT CCT CTT CAA 1104 Phe Gly Asn Arg Met Ile Gin Ser Gly Ile Leu Ala Cys Pro Leu Gin 355 360 365
CAG GAT CTA ACC AAT GAG TGT TTA GTT CTG CCC TTT TCT AAT GAT CAG 1152 Gin Asp Leu Thr Asn Glu Cys Leu Val Leu Pro Phe Ser Asn Asp Gin 370 375 380 GTG CTT ATG GGT GCT GAA GGG AGA TTA TAC ATG TAT GGT GAC TCG GTG 1200 Val Leu Met Gly Ala Glu Gly Arg Leu Tyr Met Tyr Gly Asp Ser Val 385 390 395 400CAG GAT CTA ACC AAT GAG TGT TTA GTT CTG CCC TTT TCT AAT GAT CAG 1152 Gin Asp Leu Thr Asn Glu Cys Leu Val Leu Pro Phe Ser Asn Asp Gin 370 375 380 GTG CTT ATG GGT GCT GAA GGG AGA TTA TAC ATG TAT GGT GAC TCG GTG 1200 Val Leu Met Gly Ala Glu Gly Arg Leu Tyr Met Tyr Gly Asp Ser Val 385 390 395 400
TAT TAC TAC CAA AGA AGC AAT AGT TGG TGG CCT ATG ACC ATG CTG TAT 1248 Tyr Tyr Tyr Gin Arg Ser Asn Ser Trp Trp Pro Met Thr Met Leu Tyr 405 410 415TAT TAC TAC CAA AGA AGC AAT AGT TGG TGG CCT ATG ACC ATG CTG TAT 1248 Tyr Tyr Tyr Gin Arg Ser Asn Ser Trp Trp Pro Met Thr Met Leu Tyr 405 410 415
AAG GTA ACC ATA ACA TTC ACT AAT GGT CAG CCA TCT GCT ATA TCA GCT 1296 Lys Val Thr Ile Thr Phe Thr Asn Gly Gin Pro Ser Ala Ile Ser Ala 420 425 430AAG GTA ACC ATA ACA TTC ACT AAT GGT CAG CCA TCT GCT ATA TCA GCT 1296 Lys Val Thr Ile Thr Phe Thr Asn Gly Gin Pro Ser Ala Ile Ser Ala 420 425 430
CAG AAT GTG CCC ACA CAG CAG GTC CCT AGA CCT GGG ACA GGA GCC TGC 1344 Gin Asn Val Pro Thr Gin Gin Val Pro Arg Pro Gly Thr Gly Ala Cys 435 440 445CAG AAT GTG CCC ACA CAG CAG GTC CCT AGA CCT GGG ACA GGA GCC TGC 1344 Gin Asn Val Pro Thr Gin Gin Val Pro Arg Pro Gly Thr Gly Ala Cys 435 440 445
TCT GCA ACA AAT AGA TGT CCC GGT TTT TGC TTG AAA GGA GTG TAT GCT 1392 Ser Ala Thr Asn Arg Cys Pro Gly Phe Cys Leu Lys Gly Val Tyr Ala 450 ' 455 460TCT GCA ACA AAT AGA TGT CCC GGT TTT TGC TTG AAA GGA GTG TAT GCT 1392 Ser Ala Thr Asn Arg Cys Pro Gly Phe Cys Leu Lys Gly Val Tyr Ala 450 ' 455 460
GAT GCC TGG TTA CTG ACC AAC CCT TCG TCT ACC AGT ACA TTT GGA TCA 1440 Asp Ala Trp Leu Leu Thr Asn Pro Ser Ser Thr Ser Thr Phe Gly Ser 465 470 475 480GAT GCC TGG TTA CTG ACC AAC CCT TCG TCT ACC AGT ACA TTT GGA TCA 1440 Asp Ala Trp Leu Leu Thr Asn Pro Ser Ser Thr Ser Thr Phe Gly Ser 465 470 475 480
GAA GCA ACC TTC ACT GGT TCT TAT CTC AAC GCA GCA ACT CAG CGT ATC 1488 Glu Ala Thr Phe Thr Gly Ser Tyr Leu Asn Ala Ala Thr Gin Arg Ile 485 490 495GAA GCA ACC TTC ACT GGT TCT TAT CTC AAC GCA GCA ACT CAG CGT ATC 1488 Glu Ala Thr Phe Thr Gly Ser Tyr Leu Asn Ala Ala Thr Gin Arg Ile 485 490 495
AAT CCG ACG ATG TAT ATC GCG AAC AAC ACA CAG ATC ATA AGC TCA CAG 1536 Asn Pro Thr Met Tyr Ile Ala Asn Asn Thr Gin Ile Ile Ser Ser Gin 500 505 510AAT CCG ACG ATG TAT ATC GCG AAC AAC ACA CAG ATC ATA AGC TCA CAG 1536 Asn Pro Thr Met Tyr Ile Ala Asn Asn Thr Gin Ile Ile Ser Ser Gin 500 505 510
CAA TTT GGA TCA AGC GGT CAA GAA GCA GCA TAT AGC CAC ACA ACT TGT 1584 Gin Phe Gly Ser Ser Gly Gin Glu Ala Ala Tyr Ser His Thr Thr Cys 515 520 525 TTT AGG GAC ACA GGC TCT GTT ATG GTA TAC TGT CTC TAT ATT ATT GAA 1632 Phe Arg Asp Thr Gly Ser Val Met Val Tyr Cys Leu Tyr Ile Ile Glu 530 535 540CAA TTT GGA TCA AGC GGT CAA GAA GCA GCA TAT AGC CAC ACA ACT TGT 1584 Gin Phe Gly Ser Ser Gly Gin Glu Ala Ala Tyr Ser His Thr Thr Cys 515 520 525 TTT AGG GAC ACA GGC TCT GTT ATG GTA TAC TGT CTC TAT ATT ATT GAA 1632 Phe Arg Asp Thr Gly Ser Val Met Val Tyr Cys Leu Tyr Ile Ile Glu 530 535 540
TTG TCC TCA TCT CTC TTA GGA CAA TTT CAG ATT GTC CCA TTT ATC CGT 1680 Leu Ser Ser Ser Leu Leu Gly Gin Phe Gin Ile Val Pro Phe Ile Arg 545 550 555 560TTG TCC TCA TCT CTC TTA GGA CAA TTT CAG ATT GTC CCA TTT ATC CGT 1680 Leu Ser Ser Ser Leu Leu Gly Gin Phe Gin Ile Val Pro Phe Ile Arg 545 550 555 560
CAG GTG ACA CTA TCC TAA 1698CAG GTG ACA CTA TCC TAA 1698
Gin Val Thr Leu Ser 565Gin Val Thr Leu Ser 565
(2) ANGABEN ZU SEQ ID NO: 2:(2) INFORMATION ON SEQ ID NO: 2:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 565 Aminosäuren(A) LENGTH: 565 amino acids
(B) ART: Aminosäure (D)' TOPOLOGIE: linear(B) TYPE: amino acid (D) ' TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Protein(ii) MOLECULE TYPE: Protein
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 2:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Val Ala Glu Asp Ala Pro Val Arg Gly Thr Cys Arg Val Leu Phe 1 5 10 15Met Val Ala Glu Asp Ala Pro Val Arg Gly Thr Cys Arg Val Leu Phe 1 5 10 15
Arg Thr Thr Thr Leu Ile Phe Leu Cys Thr Leu Leu Ala Leu Ser Ile 20 25 30Arg Thr Thr Thr Leu Ile Phe Leu Cys Thr Leu Leu Ala Leu Ser Ile 20 25 30
Ser Ile Leu Tyr Glu Ser Leu Ile Thr Gin Lys Gin Ile Y.ez Ser His 35 40 45Ser Ile Leu Tyr Glu Ser Leu Ile Thr Gin Lys Gin Ile Y.ez Ser His 35 40 45
Ala Gly Tyr Thr Arg Ser Asn Ser Arg Leu Gly Ser Ile Thr Asp Leu 50 55 60Ala Gly Tyr Thr Arg Ser Asn Ser Arg Leu Gly Ser Ile Thr Asp Leu 50 55 60
Leu Asn Asn Ile Leu Ser Val Ala Asn Gin Ile Ile Tyr Asn Ser Ala 65 70 75 80 Val Ala Leu Pro Leu Gin Leu Asp Thr Leu Glu Ser Thr Leu Leu Thr 85 90 95Leu Asn Asn Ile Leu Ser Val Ala Asn Gin Ile Ile Tyr Asn Ser Ala 65 70 75 80 Val Ala Leu Pro Leu Gin Leu Asp Thr Leu Glu Ser Thr Leu Leu Thr 85 90 95
Ala Ile Lys Ser Leu Gin Thr Ser Asp Lys Leu Glu Gin Asn Cys Ser 100 105 110Ala Ile Lys Ser Leu Gin Thr Ser Asp Lys Leu Glu Gin Asn Cys Ser 100 105 110
Trp Gly Ala Ala Leu Ile Asn Asn Asn Arg Tyr Ile Asn Gly Ile Asn 115 120 125Trp Gly Ala Ala Leu Ile Asn Asn Asn Arg Tyr Ile Asn Gly Ile Asn 115 120 125
Gin Phe Tyr Phe Ser Ile Ala Glu Gly Arg Asn Leu Thr Leu Gly Pro 130 135 140Gin Phe Tyr Phe Ser Ile Ala Glu Gly Arg Asn Leu Thr Leu Gly Pro 130 135 140
Leu Leu Asn Ile Pro Ser Phe Ile Pro Thr Ala Thr Thr Pro Glu Gly 145 150 155 160Leu Leu Asn Ile Pro Ser Phe Ile Pro Thr Ala Thr Thr Pro Glu Gly 145 150 155 160
Cys Thr Arg Ile Pro Ser Phe Ser Leu Thr Lys Thr His Trp Cys Tyr 165 170 175Cys Thr Arg Ile Pro Ser Phe Ser Leu Thr Lys Thr His Trp Cys Tyr 165 170 175
Thr His Asn Val Ile Leu Asn Gly Cys Gin Asp His Val Ser Ser Asn 180 185 190Thr His Asn Val Ile Leu Asn Gly Cys Gin Asp His Val Ser Ser Asn 180 185 190
Gin Phe Val Ser Met Gly Ile Ile Glu Pro Thr Ser Ala Gly Phe Pro 195 200 205Gin Phe Val Ser Met Gly Ile Ile Glu Pro Thr Ser Ala Gly Phe Pro 195 200 205
Ser Phe Arg Thr Leu Lys Thr Leu Tyr Leu Ser Asp Gly Val Asn Arg 210 215 220Ser Phe Arg Thr Leu Lys Thr Leu Tyr Leu Ser Asp Gly Val Asn Arg 210 215 220
Lys Ser Cys Ser Ile Ser Thr Val Pro Gly Gly Cys Met Met Tyr Cys 225 230 235 240Lys Ser Cys Ser Ile Ser Thr Val Pro Gly Gly Cys Met Met Tyr Cys 225 230 235 240
Phe Val Ser Thr Gin Pro Glu Arg Asp Asp Tyr Phe Ser Thr Ala Pro 245 250 255Phe Val Ser Thr Gin Pro Glu Arg Asp Asp Tyr Phe Ser Thr Ala Pro 245 250 255
Pro Glu Gin Arg Ile Ile Ile Met Tyr Tyr Asn Asp Thr Ile Val Glu 260 265 270 Arg Ile Ile Asn Pro Pro Gly Val Leu Asp Val Trp Ala Thr Leu Thr 275 280 285 ' Pro Glu Gin Arg Ile Ile Ile Met Tyr Tyr Asn Asp Thr Ile Val Glu 260 265 270 Arg Ile Ile Asn Pro Pro Gly Val Leu Asp Val Trp Ala Thr Leu Thr 275 280 285 '
Pro Gly Thr Gly Ser Gly Val Tyr Tyr Leu Gly Trp Val Leu Phe Pro 290 295 300Pro Gly Thr Gly Ser Gly Val Tyr Tyr Leu Gly Trp Val Leu Phe Pro 290 295 300
Ile Tyr Gly Gly Val Ile Lys Asp Thr Ser Leu Trp Asn Asn Gin Ala 305 310 315 320Ile Tyr Gly Gly Val Ile Lys Asp Thr Ser Leu Trp Asn Asn Gin Ala 305 310 315 320
Asn Lys Tyr Phe Ile Pro Gin Met Val Ala Ala Leu Cys Ser Gin Asn 325 330 335Asn Lys Tyr Phe Ile Pro Gin Met Val Ala Ala Leu Cys Ser Gin Asn 325 330 335
Gin Ala Thr Gin Val Gin Asn Ala Lys Ser Ser Tyr Tyr Ser Ser Trp 340 345 350Gin Ala Thr Gin Val Gin Asn Ala Lys Ser Ser Tyr Tyr Ser Ser Trp 340 345 350
Phe Gly Asn Arg Met Ile Gin Ser Gly Ile Leu Ala Cys Pro Leu Gin 355 360 365Phe Gly Asn Arg Met Ile Gin Ser Gly Ile Leu Ala Cys Pro Leu Gin 355 360 365
Gin Asp Leu Thr Asn Glu Cys Leu Val Leu Pro Phe Ser Asn Asp Gin 370 375 380Gin Asp Leu Thr Asn Glu Cys Leu Val Leu Pro Phe Ser Asn Asp Gin 370 375 380
Val Leu Met Gly Ala Glu Gly Arg Leu Tyr Met Tyr Gly Asp Ser Val 385 390 395 400Val Leu Met Gly Ala Glu Gly Arg Leu Tyr Met Tyr Gly Asp Ser Val 385 390 395 400
Tyr Tyr Tyr Gin Arg Ser Asn Ser Trp Trp Pro Met Thr Met Leu Tyr 405 410 415Tyr Tyr Tyr Gin Arg Ser Asn Ser Trp Trp Pro Met Thr Met Leu Tyr 405 410 415
Lys Val Thr Ile Thr Phe Thr Asn Gly Gin Pro Ser Ala Ile Ser Ala 420 425 430Lys Val Thr Ile Thr Phe Thr Asn Gly Gin Pro Ser Ala Ile Ser Ala 420 425 430
Gin Asn Val Pro Thr Gin Gin Val Pro Arg Pro Gly Thr Gly Ala Cys 435 440 445Gin Asn Val Pro Thr Gin Gin Val Pro Arg Pro Gly Thr Gly Ala Cys 435 440 445
Ser Ala Thr Asn Arg Cys Pro Gly Phe Cys Leu Lys Gly Val Tyr Ala 450 455 460 Asp Ala Trp Leu Leu Thr Asn Pro Ser Ser Thr Ser Thr Phe Gly Ser 465 470 475 480Ser Ala Thr Asn Arg Cys Pro Gly Phe Cys Leu Lys Gly Val Tyr Ala 450 455 460 Asp Ala Trp Leu Leu Thr Asn Pro Ser Ser Thr Ser Thr Phe Gly Ser 465 470 475 480
Glu Ala Thr Phe Thr Gly Ser Tyr Leu Asn Ala Ala Thr Gin Arg Ile 485 490 495Glu Ala Thr Phe Thr Gly Ser Tyr Leu Asn Ala Ala Thr Gin Arg Ile 485 490 495
Asn Pro Thr Met Tyr Ile Ala Asn Asn Thr Gin Ile Ile Ser Ser Gin 500 505 510Asn Pro Thr Met Tyr Ile Ala Asn Asn Thr Gin Ile Ile Ser Ser Gin 500 505 510
Gin Phe Gly Ser Ser Gly Gin Glu Ala Ala Tyr Ser His Thr Thr Cys 515 520 525Gin Phe Gly Ser Ser Gly Gin Glu Ala Ala Tyr Ser His Thr Thr Cys 515 520 525
Phe Arg Asp Thr Gly Ser Val Met Val Tyr Cys Leu Tyr Ile Ile Glu 530 535 540Phe Arg Asp Thr Gly Ser Val Met Val Tyr Cys Leu Tyr Ile Ile Glu 530 535 540
Leu Ser Ser Ser Leu Leu Gly Gin Phe Gin Ile Val Pro Phe Ile Arg 545 550 555 560Leu Ser Ser Ser Leu Leu Gly Gin Phe Gin Ile Val Pro Phe Ile Arg 545 550 555 560
Gin Val Thr Leu Ser 565Gin Val Thr Leu Ser 565
(2) ANGABEN ZU SEQ ID NO: 3:(2) INFORMATION ON SEQ ID NO: 3:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 1656 Basenpaare(A) LENGTH: 1656 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Genom-DNA(ii) MOLECULE TYPE: Genomic DNA
(iii) HYPOTHETISCH: NEIN(iii) HYPOTHETICAL: NO
(ix) MERKMAL:(ix) FEATURE:
(A) NAME/SCHLÜSSEL: CDS(A) NAME / KEY: CDS
(B) LAGE:1..1653 (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 3:(B) LOCATION: 1..1653 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
ATG GGT ACT ATA ATT CAA TTT CTG GTG GTC TCC TGT CTA TTG GCA GGA 48 Met Gly Thr Ile Ile Gin Phe Leu Val Val Ser Cys Leu Leu Ala Gly 570 575 580ATG GGT ACT ATA ATT CAA TTT CTG GTG GTC TCC TGT CTA TTG GCA GGA 48 Met Gly Thr Ile Ile Gin Phe Leu Val Val Ser Cys Leu Leu Ala Gly 570 575 580
GCA GGC AGC CCT GAT CCA GCA GCC CTC ATG CAA ATC GGT GTC ATT CCA 96 Ala Gly Ser Pro Asp Pro Ala Ala Leu Met Gin Ile Gly Val Ile Pro 585 590 595GCA GGC AGC CCT GAT CCA GCA GCC CTC ATG CAA ATC GGT GTC ATT CCA 96 Ala Gly Ser Pro Asp Pro Ala Ala Leu Met Gin Ile Gly Val Ile Pro 585 590 595
ACA AAT GTC CGG CAA CTT ATG TAT TAT ACT GAG GCC TCA TCA GCA TTC 144 Thr Asn Val Arg Gin Leu Met Tyr Tyr Thr Glu Ala Ser Ser Ala Phe 600 605 610ACA AAT GTC CGG CAA CTT ATG TAT TAT ACT GAG GCC TCA TCA GCA TTC 144 Thr Asn Val Arg Gin Leu Met Tyr Tyr Thr Glu Ala Ser Ser Ala Phe 600 605 610
ATT GTT GTG AAG TTA ATG CCT ACA ATT GAC TCG CCG ATT AGT GGA TGT 192 Ile Val Val Lys Leu Met Pro Thr Ile Asp Ser Pro Ile Ser Gly Cys 615 620 625ATT GTT GTG AAG TTA ATG CCT ACA ATT GAC TCG CCG ATT AGT GGA TGT 192 Ile Val Val Lys Leu Met Pro Thr Ile Asp Ser Pro Ile Ser Gly Cys 615 620 625
AAT ATA ACA TCA ATT TCA AGC TAT AAT GCA ACA CTG ACA AAA CTC CTA 240 Asn Ile Thr Ser Ile Ser Ser Tyr Asn Ala Thr Leu Thr Lys Leu Leu 630 635 640 645AAT ATA ACA TCA ATT TCA AGC TAT AAT GCA ACA CTG ACA AAA CTC CTA 240 Asn Ile Thr Ser Ile Ser Ser Tyr Asn Ala Thr Leu Thr Lys Leu Leu 630 635 640 645
CAG CCG ATC GGT GAG AAT TTG GAA ACG ATT AGG AAC CAG TTG ATT CCA 288 Gin Pro Ile Gly Glu Asn Leu Glu Thr Ile Arg Asn Gin Leu Ile Pro 650 655 660CAG CCG ATC GGT GAG AAT TTG GAA ACG ATT AGG AAC CAG TTG ATT CCA 288 Gin Pro Ile Gly Glu Asn Leu Glu Thr Ile Arg Asn Gin Leu Ile Pro 650 655 660
ACT CGG AGG AGA CGC CGG TTT GCA GGG GTG GTG ATT GGA TTA GCT GCA 336 Thr Arg Arg Arg Arg Arg Phe Ala Gly Val Val Ile Gly Leu Ala Ala 665 670 675ACT CGG AGG AGA CGC CGG TTT GCA GGG GTG GTG ATT GGA TTA GCT GCA 336 Thr Arg Arg Arg Arg Arg Phe Ala Gly Val Val Ile Gly Leu Ala Ala 665 670 675
TTA GGA GTA GCT ACT GCC GCA CAG GTC ACT GCC GCA GTA GCA CTA GTA 384 Leu Gly Val Ala Thr Ala Ala Gin Val Thr Ala Ala Val Ala Leu Val 680 685 690TTA GGA GTA GCT ACT GCC GCA CAG GTC ACT GCC GCA GTA GCA CTA GTA 384 Leu Gly Val Ala Thr Ala Ala Gin Val Thr Ala Ala Val Ala Leu Val 680 685 690
AAG GCA AAT AAA AAT GCT GCG GCT ATA CTC AAT CTC AAA AAT GCA ATC 432 Lys Ala Asn Lys Asn Ala Ala Ala Ile Leu Asn Leu Lys Asn Ala Ile 695 700 705 CAA AAA ACA AAT ACA GCA GTT GCA GAT GTG GTC CAG GCC ACA CAA TCA 480 Gin Lys Thr Asn Thr Ala Val Ala Asp Val Val Gin Ala Thr Gin Ser 710 715 720 725AAG GCA AAT AAA AAT GCT GCG GCT ATA CTC AAT CTC AAA AAT GCA ATC 432 Lys Ala Asn Lys Asn Ala Ala Ala Ile Leu Asn Leu Lys Asn Ala Ile 695 700 705 CAA AAA ACA AAT ACA GCA GTT GCA GAT GTG GTC CAG GCC ACA CAA TCA 480 Gin Lys Thr Asn Thr Ala Val Ala Asp Val Val Gin Ala Thr Gin Ser 710 715 720 725
CTA GGA ACG GCA GTT CAA GCA GTT CAA GAT CAC ATA AAC AGT GTG GTA 528 Leu Gly Thr Ala Val Gin Ala Val Gin Asp His Ile Asn Ser Val Val 730 735 740CTA GGA ACG GCA GTT CAA GCA GTT CAA GAT CAC ATA AAC AGT GTG GTA 528 Leu Gly Thr Ala Val Gin Ala Val Gin Asp His Ile Asn Ser Val Val 730 735 740
AGT CCA GCA ATT ACA GCA GCC AAT TGT AAG GCC CAA GAT GCT ATC ATT 576 Ser Pro Ala Ile Thr Ala Ala Asn Cys Lys Ala Gin Asp Ala Ile Ile 745 750 755AGT CCA GCA ATT ACA GCA GCC AAT TGT AAG GCC CAA GAT GCT ATC ATT 576 Ser Pro Ala Ile Thr Ala Ala Asn Cys Lys Ala Gin Asp Ala Ile Ile 745 750 755
GGC TCA ATC CTC AAT CTC TAT TTG ACC GAG TTG ACA ACT ATC TTC CAC 624 Gly Ser Ile Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr Ile Phe His 760 765 770GGC TCA ATC CTC AAT CTC TAT TTG ACC GAG TTG ACA ACT ATC TTC CAC 624 Gly Ser Ile Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr Ile Phe His 760 765 770
AAT CAA ATT ACA AAC CCT GCA TTG AGT CCT ATT ACA ATT CAA GCT TTA 672 Asn Gin Ile Thr Asn Pro Ala Leu Ser Pro Ile Thr Ile Gin Ala Leu 775 780 785AAT CAA ATT ACA AAC CCT GCA TTG AGT CCT ATT ACA ATT CAA GCT TTA 672 Asn Gin Ile Thr Asn Pro Ala Leu Ser Pro Ile Thr Ile Gin Ala Leu 775 780 785
AGG ATC CTA CTG GGG AGT ACC TTG CCG ACT GTG GTC GAA AAA TCT TTC 720 Arg Ile Leu Leu Gly Ser Thr Leu Pro Thr Val Val Glu Lys Ser Phe 790 795 800 805AGG ATC CTA CTG GGG AGT ACC TTG CCG ACT GTG GTC GAA AAA TCT TTC 720 Arg Ile Leu Leu Gly Ser Thr Leu Pro Thr Val Val Glu Lys Ser Phe 790 795 800 805
AAT ACC CAG ATA AGT GCA GCT GAG CTT CTC TCA TCA GGG TTA TTG ACA 768 Asn Thr Gin Ile Ser Ala Ala Glu Leu Leu Ser Ser Gly Leu Leu Thr 810 815 820AAT ACC CAG ATA AGT GCA GCT GAG CTT CTC TCA TCA GGG TTA TTG ACA 768 Asn Thr Gin Ile Ser Ala Ala Glu Leu Leu Ser Ser Gly Leu Leu Thr 810 815 820
GGC CAG ATT GTG GGA TTA GAT TTG ACC TAT ATG CAG ATG GTC ATA AAA 816 Gly Gin Ile Val Gly Leu Asp Leu Thr Tyr Met Gin Met Val Ile Lys 825 830 835GGC CAG ATT GTG GGA TTA GAT TTG ACC TAT ATG CAG ATG GTC ATA AAA 816 Gly Gin Ile Val Gly Leu Asp Leu Thr Tyr Met Gin Met Val Ile Lys 825 830 835
ATT GAG CTG CCA ACT TTA ACT GTA CAA CCT GCA ACC CAG ATC ATA GAT 864 Ile Glu Leu Pro Thr Leu Thr Val Gin Pro Ala Thr Gin Ile Ile Asp 840 845 850 CTG GCC ACC ATT TCT GCA TTC ATT AAC AAT CAA GAA GTC ATG GCC CAA 912ATT GAG CTG CCA ACT TTA ACT GTA CAA CCT GCA ACC CAG ATC ATA GAT 864 Ile Glu Leu Pro Thr Leu Thr Val Gin Pro Ala Thr Gin Ile Ile Asp 840 845 850 CTG GCC ACC ATT TCT GCA TTC ATT AAC AAT CAA GAA GTC ATG GCC CAA 912
Leu Ala Thr Ile Ser Ala Phe Ile Asn Asn Gin Glu Val Met Ala Gin 855 860 865Leu Ala Thr Ile Ser Ala Phe Ile Asn Asn Gin Glu Val Met Ala Gin 855 860 865
TTA CCA ACA CGT GTT ATG GTG ACT GGC AGC TTG ATC CAA GCC TAT CCC 960TTA CCA ACA CGT GTT ATG GTG ACT GGC AGC TTG ATC CAA GCC TAT CCC 960
Leu Pro Thr Arg Val Met Val Thr Gly Ser Leu Ile Gin Ala Tyr ProLeu Pro Thr Arg Val Met Val Thr Gly Ser Leu Ile Gin Ala Tyr Pro
870 875 880 885870 875 880 885
GCA TCG CAA TGC ACT ATT ACA CCC AAC ACT GTG TAC TGT AGG TAT AAT 1008GCA TCG CAA TGC ACT ATT ACA CCC AAC ACT GTG TAC TGT AGG TAT AAT 1008
Ala Ser Gin Cys Thr Ile Thr Pro Asn Thr Val Tyr Cys Arg Tyr Asn 890 895 900Ala Ser Gin Cys Thr Ile Thr Pro Asn Thr Val Tyr Cys Arg Tyr Asn 890 895 900
GAT GCC CAA GTA CTC TCA GAT GAT ACG ATG GCT TGC CTC CAA GGT AAC 1056GAT GCC CAA GTA CTC TCA GAT GAT ACG ATG GCT TGC CTC CAA GGT AAC 1056
Asp Ala Gin Val Leu Ser Asp Asp Thr Met Ala Cys Leu Gin Gly Asn 905 910 915Asp Ala Gin Val Leu Ser Asp Asp Thr Met Ala Cys Leu Gin Gly Asn 905 910 915
TTG ACA AGA TGC ACC TTC TCT CCG GTG GTT GGG AGC TTT CTC ACT CGA 1104TTG ACA AGA TGC ACC TTC TCT CCG GTG GTT GGG AGC TTT CTC ACT CGA 1104
Leu Thr Arg Cys Thr Phe Ser Pro Val Val Gly Ser Phe Leu Thr Arg 920 ' 925 930Leu Thr Arg Cys Thr Phe Ser Pro Val Val Gly Ser Phe Leu Thr Arg 920 ' 925 930
TTC ATG CTG TTC GAT GGA ATA GTT TAT GCA AAT TGC AGG TCG ATG TTA 1152TTC ATG CTG TTC GAT GGA ATA GTT TAT GCA AAT TGC AGG TCG ATG TTA 1152
Phe Met Leu Phe Asp Gly Ile Val Tyr Ala Asn Cys Arg Ser Met Leu 935 940 945Phe Met Leu Phe Asp Gly Ile Val Tyr Ala Asn Cys Arg Ser Met Leu 935 940 945
TGC AAG TGC ATG CAG CCT GCT GCT GTG ATC CTA CAG CCG AGT TCA TCC 1200TGC AAG TGC ATG CAG CCT GCT GCT GTG ATC CTA CAG CCG AGT TCA TCC 1200
Cys Lys Cys Met Gin Pro Ala Ala Val Ile Leu Gin Pro Ser Ser SerCys Lys Cys Met Gin Pro Ala Ala Val Ile Leu Gin Pro Ser Ser Ser
950 955 960 965950 955 960 965
CCT GTA ACT GTC ATT GAC ATG TAC AAA TGT GTG AGT CTG CAG CTT GAC 1248CCT GTA ACT GTC ATT GAC ATG TAC AAA TGT GTG AGT CTG CAG CTT GAC 1248
Pro Val Thr Val Ile Asp Met Tyr Lys Cys Val Ser Leu Gin Leu Asp 970 975 980Pro Val Thr Val Ile Asp Met Tyr Lys Cys Val Ser Leu Gin Leu Asp 970 975 980
AAT CTC AGA TTC ACC ATC ACT CAA TTG GCC AAT GTA ACC TAC AAT AGC 1296AAT CTC AGA TTC ACC ATC ACT CAA TTG GCC AAT GTA ACC TAC AAT AGC 1296
Asn Leu Arg Phe Thr Ile Thr Gin Leu Ala Asn Val Thr Tyr Asn Ser 985 990 995 ACC ATC AAG CTT GAA ACA TCC CAG ATC TTG CCT ATT GAT CCG TTG GAT 1344 Thr Ile Lys Leu Glu Thr Ser Gin Ile Leu Pro Ile Asp Pro Leu Asp 1000 1005 1010Asn Leu Arg Phe Thr Ile Thr Gin Leu Ala Asn Val Thr Tyr Asn Ser 985 990 995 ACC ATC AAG CTT GAA ACA TCC CAG ATC TTG CCT ATT GAT CCG TTG GAT 1344 Thr Ile Lys Leu Glu Thr Ser Gin Ile Leu Pro Ile Asp Pro Leu Asp 1000 1005 1010
ATA TCC CAG AAT CTA GCT GCG GTG AAT AAG AGT CTA AGT GAT GCA CTA 1392 Ile Ser Gin Asn Leu Ala Ala Val Asn Lys Ser Leu Ser Asp Ala Leu 1015 1020 1025ATA TCC CAG AAT CTA GCT GCG GTG AAT AAG AGT CTA AGT GAT GCA CTA 1392 Ile Ser Gin Asn Leu Ala Ala Val Asn Lys Ser Leu Ser Asp Ala Leu 1015 1020 1025
CAA CAC TTA GCA CAA AGT GAC ACA TAC CTT TCT GCA ATC ACA TCA GCT 1440 Gin His Leu Ala Gin Ser Asp Thr Tyr Leu Ser Ala Ile Thr Ser Ala 1030 1035 1040 1045CAA CAC TTA GCA CAA AGT GAC ACA TAC CTT TCT GCA ATC ACA TCA GCT 1440 Gin His Leu Ala Gin Ser Asp Thr Tyr Leu Ser Ala Ile Thr Ser Ala 1030 1035 1040 1045
ACG ACT ACA AGT GTA TTA TCC ATA ATG GCA ATC TGT CTT GGA TCG TTA 1488 Thr Thr Thr Ser Val Leu Ser Ile Met Ala Ile Cys Leu Gly Ser Leu 1050 1055 1060ACG ACT ACA AGT GTA TTA TCC ATA ATG GCA ATC TGT CTT GGA TCG TTA 1488 Thr Thr Thr Ser Val Leu Ser Ile Met Ala Ile Cys Leu Gly Ser Leu 1050 1055 1060
GGT TTA ATA TTA ATA ATC TTG CTC AGT GTA GTT GTG TGG AAG TTA TTG 1536 Gly Leu Ile Leu Ile Ile Leu Leu Ser Val Val Val Trp Lys Leu Leu 1065 1070 1075GGT TTA ATA TTA ATA ATC TTG CTC AGT GTA GTT GTG TGG AAG TTA TTG 1536 Gly Leu Ile Leu Ile Ile Leu Leu Ser Val Val Val Trp Lys Leu Leu 1065 1070 1075
ACC ATT GTC ACT GCT AAT CGA AAT AGA ATG GAG AAT TTT GTT TAT CAT 1584 Thr Ile Val Thr Ala Asn Arg Asn Arg Met Glu Asn Phe Val Tyr His 1080 1085 1090ACC ATT GTC ACT GCT AAT CGA AAT AGA ATG GAG AAT TTT GTT TAT CAT 1584 Thr Ile Val Thr Ala Asn Arg Asn Arg Met Glu Asn Phe Val Tyr His 1080 1085 1090
AAT TCA GCA TTC CAC CAC TCA CGA TCT GAT CTC AGT GAG AAA AAT CAA 1632 Asn Ser Ala Phe His His Ser Arg Ser Asp Leu Ser Glu Lys Asn Gin 1095 1100 1105AAT TCA GCA TTC CAC CAC TCA CGA TCT GAT CTC AGT GAG AAA AAT CAA 1632 Asn Ser Ala Phe His His Ser Arg Ser Asp Leu Ser Glu Lys Asn Gin 1095 1100 1105
CCT GCA ACT CTT GGA ACA AGA TAA 1656CCT GCA ACT CTT GGA ACA AGA TAA 1656
Pro Ala Thr Leu Gly Thr Arg 1110 1115Pro Ala Thr Leu Gly Thr Arg 1110 1115
(2) ANGABEN ZU SEQ ID NO: 4: (i) SEQUENZKENNZEICHEN:(2) INFORMATION ON SEQ ID NO: 4: (i) SEQUENCE LABEL:
(A) LÄNGE: 551 Aminosäuren(A) LENGTH: 551 amino acids
(B) ART: Aminosäure (D) TOPOLOGIE: linear(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Protein(ii) MOLECULE TYPE: Protein
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 4:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Gly Thr Ile Ile Gin Phe Leu Val Val Ser Cys Leu Leu Ala Gly 1 5 10 15Met Gly Thr Ile Ile Gin Phe Leu Val Val Ser Cys Leu Leu Ala Gly 1 5 10 15
Ala Gly Ser Pro Asp Pro Ala Ala Leu Met Gin Ile Gly Val Ile Pro 20 25 30Ala Gly Ser Pro Asp Pro Ala Ala Leu Met Gin Ile Gly Val Ile Pro 20 25 30
Thr Asn Val Arg Gin Leu Met Tyr Tyr Thr Glu Ala Ser Ser Ala Phe 35 40 45Thr Asn Val Arg Gin Leu Met Tyr Tyr Thr Glu Ala Ser Ser Ala Phe 35 40 45
Ile Val Val Lys Leu Met Pro Thr Ile Asp Ser Pro Ile Ser Gly Cys 50 ' 55 60Ile Val Val Lys Leu Met Pro Thr Ile Asp Ser Pro Ile Ser Gly Cys 50 '55 60
Asn Ile Thr Ser Ile Ser Ser Tyr Asn Ala Thr Leu Thr Lys Leu Leu 65 70 75 80Asn Ile Thr Ser Ile Ser Ser Tyr Asn Ala Thr Leu Thr Lys Leu Leu 65 70 75 80
Gin Pro Ile Gly Glu Asn Leu Glu Thr Ile Arg Asn Gin Leu Ile Pro 85 90 95Gin Pro Ile Gly Glu Asn Leu Glu Thr Ile Arg Asn Gin Leu Ile Pro 85 90 95
Thr Arg Arg Arg Arg Arg Phe Ala Gly Val Val Ile Gly Leu Ala Ala 100 105 110Thr Arg Arg Arg Arg Arg Phe Ala Gly Val Val Ile Gly Leu Ala Ala 100 105 110
Leu Gly Val Ala Thr Ala Ala Gin Val Thr Ala Ala Val Ala Leu Val 115 120 125Leu Gly Val Ala Thr Ala Ala Gin Val Thr Ala Ala Val Ala Leu Val 115 120 125
Lys Ala Asn Lys Asn Ala Ala Ala Ile Leu Asn Leu Lys Asn Ala Ile 130 135 140 Gin Lys Thr Asn Thr Ala Val Ala Asp Val Val Gin Ala Thr Gin Ser 145 ' 150 155 160Lys Ala Asn Lys Asn Ala Ala Ala Ile Leu Asn Leu Lys Asn Ala Ile 130 135 140 Gin Lys Thr Asn Thr Ala Val Ala Asp Val Val Gin Ala Thr Gin Ser 145 ' 150 155 160
Leu Gly Thr Ala Val Gin Ala Val Gin Asp His Ile Asn Ser Val Val 165 170 175Leu Gly Thr Ala Val Gin Ala Val Gin Asp His Ile Asn Ser Val Val 165 170 175
Ser Pro Ala Ile Thr Ala Ala Asn Cys Lys Ala Gin Asp Ala Ile Ile 180 185 190Ser Pro Ala Ile Thr Ala Ala Asn Cys Lys Ala Gin Asp Ala Ile Ile 180 185 190
Gly Ser Ile Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr Ile Phe His 195 200 205Gly Ser Ile Leu Asn Leu Tyr Leu Thr Glu Leu Thr Thr Ile Phe His 195 200 205
Asn Gin Ile Thr Asn Pro Ala Leu Ser Pro Ile Thr Ile Gin Ala Leu 210 215 220Asn Gin Ile Thr Asn Pro Ala Leu Ser Pro Ile Thr Ile Gin Ala Leu 210 215 220
Arg Ile Leu Leu Gly Ser Thr Leu Pro Thr Val Val Glu Lys Ser Phe 225 230 235 240Arg Ile Leu Leu Gly Ser Thr Leu Pro Thr Val Val Glu Lys Ser Phe 225 230 235 240
Asn Thr Gin Ile Ser Ala Ala Glu Leu Leu Ser Ser Gly Leu Leu Thr 245 250 255Asn Thr Gin Ile Ser Ala Ala Glu Leu Leu Ser Ser Gly Leu Leu Thr 245 250 255
Gly Gin Ile Val Gly Leu Asp Leu Thr Tyr Met Gin Met Val Ile Lys 260 265 270Gly Gin Ile Val Gly Leu Asp Leu Thr Tyr Met Gin Met Val Ile Lys 260 265 270
Ile Glu Leu Pro Thr Leu Thr Val Gin Pro Ala Thr Gin Ile Ile Asp 275 280 285Ile Glu Leu Pro Thr Leu Thr Val Gin Pro Ala Thr Gin Ile Ile Asp 275 280 285
Leu Ala Thr Ile Ser Ala Phe Ile Asn Asn Gin Glu Val Met Ala Gin 290 295 300Leu Ala Thr Ile Ser Ala Phe Ile Asn Asn Gin Glu Val Met Ala Gin 290 295 300
Leu Pro Thr Arg Val Met Val Thr Gly Ser Leu Ile Gin Ala Tyr Pro 305 310 315 320Leu Pro Thr Arg Val Met Val Thr Gly Ser Leu Ile Gin Ala Tyr Pro 305 310 315 320
Ala Ser Gin Cys Thr Ile Thr Pro Asn Thr Val Tyr Cys Arg Tyr Asn 325 330 335 Asp Ala Gin Val Leu Ser Asp Asp Thr Met Ala Cys Leu Gin Gly Asn 340 345 350Ala Ser Gin Cys Thr Ile Thr Pro Asn Thr Val Tyr Cys Arg Tyr Asn 325 330 335 Asp Ala Gin Val Leu Ser Asp Asp Thr Met Ala Cys Leu Gin Gly Asn 340 345 350
Leu Thr Arg Cys Thr Phe Ser Pro Val Val Gly Ser Phe Leu Thr Arg 355 360 365Leu Thr Arg Cys Thr Phe Ser Pro Val Val Gly Ser Phe Leu Thr Arg 355 360 365
Phe Met Leu Phe Asp Gly Ile Val Tyr Ala Asn Cys Arg Ser Met Leu 370 375 380Phe Met Leu Phe Asp Gly Ile Val Tyr Ala Asn Cys Arg Ser Met Leu 370 375 380
Cys Lys Cys Met Gin Pro Ala Ala Val Ile Leu Gin Pro Ser Ser Ser 385 390 395 400Cys Lys Cys Met Gin Pro Ala Ala Val Ile Leu Gin Pro Ser Ser Ser 385 390 395 400
Pro Val Thr Val Ile Asp Met Tyr Lys Cys Val Ser Leu Gin Leu Asp 405 410 415Pro Val Thr Val Ile Asp Met Tyr Lys Cys Val Ser Leu Gin Leu Asp 405 410 415
Asn Leu Arg Phe Thr Ile Thr Gin Leu Ala Asn Val Thr Tyr Asn Ser 420 425 430Asn Leu Arg Phe Thr Ile Thr Gin Leu Ala Asn Val Thr Tyr Asn Ser 420 425 430
Thr Ile Lys Leu Glu Thr Ser Gin Ile Leu Pro Ile Asp Pro Leu Asp 435 440 445Thr Ile Lys Leu Glu Thr Ser Gin Ile Leu Pro Ile Asp Pro Leu Asp 435 440 445
Ile Ser Gin Asn Leu Ala Ala Val Asn Lys Ser Leu Ser Asp Ala Leu 450 455 460Ile Ser Gin Asn Leu Ala Ala Val Asn Lys Ser Leu Ser Asp Ala Leu 450 455 460
Gin His Leu Ala Gin Ser Asp Thr Tyr Leu Ser Ala Ile Thr Ser Ala 465 470 475 480Gin His Leu Ala Gin Ser Asp Thr Tyr Leu Ser Ala Ile Thr Ser Ala 465 470 475 480
Thr Thr Thr Ser Val Leu Ser Ile Met Ala Ile Cys Leu Gly Ser Leu 485 490 495Thr Thr Thr Ser Val Leu Ser Ile Met Ala Ile Cys Leu Gly Ser Leu 485 490 495
Gly Leu Ile Leu Ile Ile Leu Leu Ser Val Val Val Trp Lys Leu Leu 500 505 510Gly Leu Ile Leu Ile Ile Leu Leu Ser Val Val Val Trp Lys Leu Leu 500 505 510
Thr Ile Val Thr Ala Asn Arg Asn Arg Met Glu Asn Phe Val Tyr His 515 520 525 Asn Ser Ala Phe His His Ser Arg Ser Asp Leu Ser Glu Lys Asn Gin 530 535 540Thr Ile Val Thr Ala Asn Arg Asn Arg Met Glu Asn Phe Val Tyr His 515 520 525 Asn Ser Ala Phe His His Ser Arg Ser Asp Leu Ser Glu Lys Asn Gin 530 535 540
Pro Ala Thr Leu Gly Thr ArgPro Ala Thr Leu Gly Thr Arg
545 550 545 550

Claims

Patentansprüche claims
1. Vakzine gegen Erkrankungen des Respirations- und Reproduktionstrakts von Schweinen, dadurch gekennzeichnet, daß sie als antigenes Material Parainfluenzaviren sowie deren Varianten und Mutanten in lebender, toter, attenuierter oder über rekombinante Technologie hergestellter Form ganz oder in Teilen oder Bruchstücken enthält.1. Vaccine against diseases of the respiratory and reproductive tract of pigs, characterized in that it contains, as an antigenic material, parainfluenza viruses and their variants and mutants in whole, in part or in fragments in living, dead, attenuated or produced by recombinant technology.
2. Antigenes Material auf Basis von Parainfluenzaviren die Erkrankungen des Respirations- und Reproduktionstrakts von Schweinen hervorrufen.2. Antigenic material based on parainfluenza viruses cause diseases of the respiratory and reproductive tract of pigs.
3. Verfahren zur Herstellung von antigenem Material auf Basis von Para- influenzaviren, die Erkrankungen des Respirations- und Reproduktionstrakts von Schweinen hervorrufen, dadurch gekennzeichnet, daß man Parain¬ fluenzaviren vermehrt und aus den so erhaltenen Virussuspensionen in üblicher Weise das antigene Material isoliert.3. A process for the production of antigenic material based on para-influenza viruses which cause diseases of the respiratory and reproductive tract of pigs, characterized in that Parainfluenza viruses are propagated and the antigenic material is isolated in a conventional manner from the virus suspensions thus obtained.
4. Verwendung von antigenem Material auf Basis von Parainfluenzaviren, die Erkrankungen des Respirations- und Reproduktionstrakts von Schweinen hervorrufen zur Diagnose und/oder Prävention dieser Erkrankungen.4. Use of antigenic material based on parainfluenza viruses which cause diseases of the respiratory and reproductive tract of pigs for the diagnosis and / or prevention of these diseases.
5. Verwendung von antigenem Material auf Basis von Parainfluenzaviren, die Erkrankungen des Respirations- und Reproduktionstrakts von Schweinen hervorrufen, zur Herstellung von Diagnosemitteln zur Feststellung dieser Erkrankungen und zur Herstellung von Vakzinen zur Prävention dieser5. Use of antigenic material based on parainfluenza viruses, which cause diseases of the respiratory and reproductive tract of pigs, for the production of diagnostic agents for the detection of these diseases and for the production of vaccines for the prevention of these
Erkrankungen. Diseases.
EP95910510A 1994-03-07 1995-02-22 Para-influenza virus-containing vaccines for preventing porcine reproductive and respiratory syndrome Withdrawn EP0804232A1 (en)

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DE4407489A DE4407489A1 (en) 1994-03-07 1994-03-07 Vaccine for the prevention of respiratory and reproductive diseases of the pig
DE4407489 1994-03-07
PCT/EP1995/000642 WO1995024214A1 (en) 1994-03-07 1995-02-22 Para-influenza virus-containing vaccines for preventing porcine reproductive and respiratory syndrome

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HRP950088A2 (en) 1997-08-31
HUT74824A (en) 1997-02-28
MX9603896A (en) 1997-03-29
CA2184833A1 (en) 1995-09-14
DE4407489A1 (en) 1995-09-14
ZA951831B (en) 1995-12-12
CZ261796A3 (en) 1997-02-12
SK113996A3 (en) 1997-04-09
HU9602421D0 (en) 1996-11-28
PL316178A1 (en) 1996-12-23
RU2162710C2 (en) 2001-02-10
AU700160B2 (en) 1998-12-24
JPH09509949A (en) 1997-10-07
AU1758995A (en) 1995-09-25
CN1147769A (en) 1997-04-16
NZ281615A (en) 1997-11-24

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