Classifications

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  • 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
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
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  • 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
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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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