Lung cells of cotton rats for cultivation of viruses

FIELD: medicine.

SUBSTANCE: method is suggested for production of virus on lung cells of cotton rats, and also line of lung cells of cotton rats ATCC PTA-3930 for growth, reproduction and cultivation of viruses.

EFFECT: improved efficiency of method.

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Relevant applications/enable for information

This application claims the priority of provisional patent application U.S. No. 60/366014 filed March 20, 2002, incorporated here by reference. The above-mentioned application and all documents cited here or during its execution ("attached cited documents")and all documents cited or referred to in the attached cited documents, and all documents cited or referenced herein (the"cited here documents")and all documents cited or referred to in the cited here documents, along with instructions, descriptions, specifications of products and inserts for any products manufacturers mentioned herein or in any document included here for the information that is included here for information and can be used in the practice of the invention.

The scope of the invention

This invention relates to a new cell line and its applications, including a new method of producing organisms, or pathogens, such as viruses, for example, the virus that causes the disease of pigs, known as reproductive and respiratory syndrome swine (RRS).

In a more General sense, the invention relates to the reproduction of microorganisms or pathogens, such as viruses, for example, virulent or attenuated viruses, using cells which agcih cotton rats or cells, or cell line according to the invention (for example, deposited cells or cell lines, or cells or cell lines, with all their distinctive properties) to obtain microorganisms or pathogens, such as viruses, for example, virulent or attenuated viruses, such as microorganisms or pathogens, such as viruses, which are usually rodents or rats, or cotton rats, or cells of the lungs of cotton rats are not natural hosts, or RNA viruses, for example, odnozadachnaya with "plus"-chain RNA viruses, for example, viruses of the order Nidovirales, for example antivirus or viruses of the family Arteriviridae, for example, a virus that increases the activity of lactate dehydrogenase (LDV), the virus of infectious Takayasu horses (EAV), haemorrhagic fever monkeys (SHFV) and PRRS virus (vector which, as a rule, are arthropods), viruses of the family Coronavirida, for example, coronaviruses, such as infectious bronchitis virus, coronavirus dogs, coronavirus cats, coronavirus human 229F, virus epidemic diarrhoea of pigs, the virus of infectious gastroenteritis virus, infectious gastroenteritis of swine respiratory virus swine coronavirus bovine coronavirus human OS, hepatitis mice, hemagglutinine virus encephalomyelitis pigs, rat coronavirus, virus salad is cryogenica, the virus infectious bronchitis of poultry, coronavirus turkeys, coronavirus rabbits (viruses, vectors which, as a rule, are arthropods) and torovirus, such as torovirus horses, torovirus pigs, torovirus person, torovirus cattle.

Viruses, mainly breeding using cells of the lungs of cotton rats or cells or cell lines of the invention include parainfluenza virus dogs (CPI), for example, the CPI type 2 (CPI-2), adenovirus, such as adenovirus dogs (CAV), for example, adenovirus dogs type 2 (CAV-2), adenovirus pigs (PAV), for example, adenovirus pigs type 3 (PAV-3) and type 5, herpesvirus cattle, for example, herpesvirus bovine type 1 (causative agent of infectious bovine rhinotracheitis large cattle (IBR)), herpesvirus horses (EHV), for example, type 1 (EHV-1) or type 4 (EHV-4), rotavirus cattle (BRV), parainfluenza virus bovine type 3 (PI-3), intestinal and respiratory coronavirus bovine (BCV), the virus reproductive and respiratory syndrome swine (PRRSV).

More preferably the virus is a virus PRRS.

In more preferred embodiments the cells of the lungs of cotton rats or cell line are such deposited in ADS as MOUTH-3930, or lung cells, or kletocna the line, with all the distinctive properties of ATSS MOUTH-3930, or cells of the lungs of cotton rats, or cell line, obtained by culturing cells in the lungs of cotton rats, at least, for 10, for example, at least within 21 or 76, or 100 passages, for example, 10 or more, or 21 or more, or 76, or more passages, for example, at least for 10 or 21, or passages 76 and preferably up to (and including) 100 passages and the receipt of such cell lines, in which cells of the lungs of cotton rats are mainly epithelial cells, and morphological characteristics are retained when the passages.

Consequently, the invention relates to such cells or cell lines, as well as all of their applications.

Viruses, which are multiplied using the cells of the lungs of cotton rats or cell or cell line according to the invention, suitable for the production of immunogenic compositions and vaccines against diseases caused by viruses such as PRRSV. Similarly, bacteria or other pathogens cultured using cells of the lungs of cotton rats or cell or cell line according to the invention, suitable for receiving the immunogenic and vaccine compositions against other pathogens or microorganisms.

The invention relates to the use of viruses grown passages in KL is located according to the invention, for example, to obtain attenuated, inactivated and subunit immunogenic compositions and vaccines; and thus, the invention relates to such attenuated, inaktivirovannye and subunit immunogenic compositions and vaccines.

The level of technology

Reproductive and respiratory syndrome swine (PRRS) was first described in North Carolina (USA) in 1987, At that time, the disease of pigs called the "mysterious" disease of pigs or MSD, later it became known as "the syndrome of infertility and respiratory disorders in pigs" or SIRS. Then, in 1990, it appeared in Central Europe. In the beginning it was called in Europe "syndrome epidemic abortion and respiratory disorders or PEARS, and finally "reproductive and respiratory syndrome swine" or PRRS, which has become a common name in the world.

The PRRS virus is a coated virus with single-stranded RNA was first isolated in the Netherlands and called Lelystad virus. He was carried to the family Arterivirida. Viruses of the family Arterivirida included in the order Nidovirales. Other viruses in the order Nidovirales are viruses of the family Coronaviridae, such as coronaviruses and toroviruses.

The PRRS virus was described in the application WO 92/21375. The isolate was deposited in the National collection of microorganism cultures (CNCM) of the Institut Pasteur, Paris, France is, under inventory number I-1102. Was also selected North American type (WO 93/03760), and the virus was deposited in the American type culture collection (ATS) under inventory number VR-2332.

In pigs, the disease is characterized PRRS violations by the reproductive system, and respiratory symptoms. Target cells for virus PRRS are macrophages.

One of the problems impeding the receipt of immunological products against PRRS virus, is the limited availability of stable substrates for viral replication.

The PRRS virus can multiply only in alveolar macrophages (PAM) pigs (Wensvoort G. et al., in The Vet. Quart. 13:121-130, 1991). The necessity of using healthy pigs of a certain age to obtain data macrophages has several shortcomings. In addition, sensitivity to virus infection is not guaranteed in the selected FRAMES as cellular substrates, obtained from various animals, always very variable. This creates the main disadvantage in obtaining quantities of antigen constant and uniform quality, and each party must be evaluated to determine its sensitivity.

Research was conducted to search for other cellular substrates. Thus, in U.S. patent No. 5476778 tested 15 cell lines derived from other animal species (for example, rupnagar cattle, dogs, cats, humans, pigs, monkeys) and other tissues (e.g. liver, kidney, lung, testes) for cultivation of PRRS virus. Only one type (cells MA-104) of the 15 cell lines were growth of PRRS virus.

Cell line of monkey kidney (e.g., VERO, MA-104, MARC-145, see, respectively, of U.S. patent No. 5476778 and application WO 98/00165) is used for cultivation and attenuative of PRRS virus. But the title of PRRS virus in these cells are low and, consequently, the cost of getting high.

Despite the fact that currently inactivated and attenuated vaccines against PRRS are industrially available, it would be very important to have an alternative cell substrates to obtain PRRS virus, for example, for the production of vaccines or immunogenic compositions. And in a more General sense it is very important to have an alternative cell substrates to obtain pathogens, such as viruses, for example, RNA viruses, for example, with single-stranded plus-chain RNA viruses, such as viruses of the order Nidovirales, such as antivirus, or family viruses Arterivirida, and viruses of the family Coronaviridae, for example, to get vaccines or immunogenic compositions.

In addition, it would be preferable to provide a new line of cells in the lungs of cotton rats.

Brief description of the invention

The present invention is based on setting the AI of the fact, that despite the fact that rodents are not natural hosts of PRRS virus, PRRS virus grows or multiplies in the cells of the lungs of cotton rats. In addition, was received and deposited a new cell line produced from the tissues of the lungs of cotton rats.

The present invention is also based on the finding that other viruses grow and multiply in the cells of the lungs of cotton rats, such viruses, which are not a natural pathogen of rodents. These viruses include parainfluenza virus dogs type 2 (CPI-2), adenovirus dogs type 2 (CV-2), herpes virus horses type 1 (EHV-1), herpes virus horses type 4 (EHV-4), rotavirus cattle (BRV) and coronavirus bovine (BCV).

In a more General sense, the present invention includes the use of cells in the lungs of cotton rats or cell or cell line according to the invention for the propagation of microorganisms or pathogens, such as viruses, for example, virulent or attenuated viruses, such as viruses, which are usually rodents or rats, or cotton rats, or cells of the lungs of cotton rats are not natural hosts, or viruses of the order Nidovirales, for example, antivirus or family viruses Arterivirida, for example, a virus that increases the activity of lactate dehydrogenase (LDV), the virus of infectious Takayasu horses (EAV), the virus hemorrhagic the maternal fever monkeys (SHFV) and PRRS virus (vector which, as a rule, are arthropods), viruses of the family Coronavirida, for example, coronaviruses, such as infectious bronchitis virus, coronavirus dogs, coronavirus cats, coronavirus human 229F, virus epidemic diarrhoea of pigs, the virus of infectious gastroenteritis virus, infectious gastroenteritis of swine respiratory virus swine coronavirus bovine coronavirus human OS, hepatitis mice, hemagglutinine virus encephalomyelitis pigs, rat coronavirus, virus sialodacryoadenitis, virus, infectious bronchitis of poultry, coronavirus turkeys, coronavirus rabbits (viruses, vectors which, as a rule, are arthropods) and torovirus such as torovirus horses, torovirus pigs, torovirus person, torovirus cattle.

Viruses, mainly breeding using cell line the lungs of cotton rats or cells or cell lines of the invention include parainfluenza virus dogs (CPI), for example, the CPI type 2 (CPI-2), adenovirus, such as adenovirus dogs (CAV), for example, adenovirus dogs type 2 (CAV-2), adenovirus pigs (PAV), for example, adenovirus pigs type 3 and (PAV-3), herpes virus of cattle, for example, herpesvirus bovine type 1 (causative agent of infectious bovine rhinotracheitis cattle (IBR)), GE shall pesvirus horses (EHV), for example, type 1 (EHV-1) or type 4 (EHV-4), rotavirus cattle (BRV), parainfluenza virus bovine type 3 (PI-3 or bPI-3), coronavirus bovine (BCV) and the virus reproductive and respiratory syndrome swine (PRRSV). More preferably the virus is a virus PRRS. Consequently, the invention relates to reproduction, or cultivating, or farming of these viruses.

Some viruses can grow in the cells of the lungs of cotton rats, such as herpesvirus bovine type 1 (BHV-1) (see Papp Z. et al., J. Gen. Virol., 1997, 78, 2933-2943, for example, p.2935), parainfluenza virus bovine type 3 (bPI-3) (see Breker-Klassen M.M. et al., J. Virol., 1995, 69 (7), 4308-4315) and adenovirus pigs type 3 (PAV-3) (see PCT patent WO-A3-99/53047). However, in these documents is not described or are not offered the use of such cells and viruses cultivated in them, to receive inactivated, attenuated and subunit immunogenic compositions or vaccines and their introduction to the animals, and methods of immunization or vaccination.

The invention also includes cells in the lungs of cotton rats or cell line deposited in ADS as MOUTH-3930, or cells of the lungs of cotton rats, or cell line, with all the distinctive properties of ATSS MOUTH-3930, or cells of the lungs of cotton rats or cell line derived from the cult is the cultivation of cells in the lungs of cotton rats at least for 10 or 21, or passages 76, for example, 10 or more, or 21 or more, or 76, or more passages, for example, at least for 10 or 21, or passages 76 and preferably up to and including 100 passages and the receipt of such cell lines in which cells of the lungs of cotton rats are mainly epithelial cells, and morphological characteristics are retained when the passages.

Primarily the present invention includes the use of cells in the lungs of cotton rats (cells CRL), for example, cell lines derived from these cells for the propagation of virulent or attenuated PRRS virus.

Primarily the present invention also includes the use of cells CRL, for example, cells or cell lines derived from these cells, such as cells or cell lines of the invention (for example, the deposited cell lines or cells with their properties), for the propagation of microorganisms or pathogens, such as viruses, for example, virulent or attenuated viruses, such as CPI-2, CAV-2, EHV-1, EHV-4, BRV, BCV.

Thus, the present invention provides a method of cultivation or propagation of the virus, such as PRRSV, CPI-2, CAV-2, PAV-5, EHV-1, EHV-4, BRV, BCV, LDV, EAV, SHFV, infectious bronchitis virus, coronavirus dogs, coronavirus cats, coronavirus human 229F, virus epidemics the th diarrhoea of pigs, the virus of infectious gastroenteritis virus, infectious gastroenteritis of swine respiratory virus swine coronavirus bovine coronavirus human OS, hepatitis mice, hemagglutinine virus encephalomyelitis pigs, rat coronavirus, virus sialodacryoadenitis, virus, infectious bronchitis of poultry, coronavirus turkeys, coronavirus rabbits, torovirus horses, torovirus pigs, torovirus person, torovirus cattle, comprising the contacting of the virus with the cells of the lungs of cotton rats in conditions suitable for culturing or breeding. The method may further include collecting the obtained virus. To obtain immunogenic or immunological or vaccine composition, the virus is not necessarily possible to inactivate and/or it can include a protein(protein) or antigen(s)or epitope(s)isolated from them (for inactivated or subunit compositions), and the virus or subunit can be mixed with a suitable carrier or diluent and/or adjuvant, for example, suitable for veterinary or pharmaceutically acceptable carrier or diluent and/or adjuvant.

The terms "immunogenic composition" and "immunological composition", and "immunogenic or immunological composition" includes any composition that induces an immune response FR is in pathogen target; for example, after the introduction or injection of the animal (such as a pig) stimulated immune response against the pathogen target (for example, PRRS). The terms "vaccine composition" and "vaccine", and "vaccine composition" includes any composition that induces a protective immune response against the pathogen target or which effectively protects against pathogen; for example, after the introduction or injection of the animal (e.g. pig) is developing a protective immune response against the pathogen target or provides effective protection against a pathogen (for example, PRRS). Subunit of a pathogen such as a virus, is an antigen or immunogen or epitope selected from a pathogen such as a virus; and subunit composition comprises or consists essentially of one or more antigens, immunogens or epitopes isolated from a pathogen such as a virus.

The present invention also provides a culture of the microorganism or pathogen, such as a virus, this virus, which is typically the rodents or rats, or cotton rats, or cells of the lungs of cotton rats are not natural hosts, such as PRRSV, LDV, EAV, SHFV, infectious bronchitis virus, coronavirus dogs, coronavirus cats, coronavirus human 229F, virus epidemic diarrhoea of pigs, the virus of infectious gastroenteritis virus, infectious gastro is Narita pigs, respiratory virus swine coronavirus bovine coronavirus human OS, hepatitis mice, the virus hemagglutinins encephalomyelitis pigs, rat coronavirus, virus sialodacryoadenitis, virus, infectious bronchitis of poultry, coronavirus turkeys, coronavirus rabbits, torovirus horses, torovirus pigs, torovirus person, torovirus cattle, CPI-2, CAV-2, PAV-5, EHV-1, EHV-4, BRV or BCV, mainly the PRRS virus, CPI-2, CAV-2, EHV-1, EHV-4, BRV or BCV resulting from cultivation or breeding in the cells of the lungs of cotton rats or cells, or cell lines according to the invention, for example, inactivated, attenuated and subunit culture or drugs.

Such a culture different from previous crops because they may not contain contaminants from cultures obtained in other cells, and may have other titles in comparison with cultures obtained in other cells. For example, if you return to the PRRS virus obtained in porcine cells, these cultures contain pollutants from pig cells, while PRRSV, cultured cells CRL does not contain contaminants detected in porcine cells. This analysis can be extended to culture PRRSV and other viruses that have been cultivated in other cells, which ispolzovalis is for the propagation of PRRSV and other viruses compared with cultures of viruses obtained in cells CRL according to the present invention, which makes it obvious that the culture of the present invention differ from the cultures of the prior art.

The present invention also encompasses immunogenic compositions and vaccines against PRRS or against PRRSV, which can be obtained from the culture of PRRS virus according to the invention, primarily live attenuated immunogenic compositions and vaccines, inactivated immunogenic compositions and vaccines and subunit immunogenic compositions and vaccines.

The present invention also encompasses immunogenic compositions and vaccines against other pathogens or microorganisms that have been cultivated and propagated in cells CRL or cell lines, or cells according to the invention, for example, live, attenuated, inactivated or subunit composition or vaccine. Data immunogenic composition or vaccine can be against any microorganism or pathogen or virus, cultivated, cultured, reproduced or the like, in cells CRL or cell lines, or cells according to the invention, for example, immunogenic or vaccine composition against any of these viruses or microorganisms, or pathogens, such as CPI-2, CAV-2, HV-1, EHV-1, EHV-4, BRV, bPI-3 or BCV.

The invention includes the em sets, containing cells and virus, or microorganism or pathogen for cultivating in them, preferably in separate containers and even more preferably, when the individual containers are in one package, and a set of optional contains instructions for cultivation, cultivation, maintenance of cells and/or virus with instructions, optionally including instructions for collecting and/or virus inactivation and/or releasing the subunit antigen or immunogen or epitope.

The invention further includes a combined composition, for example, compositions containing one or more vaccines or immunogenic compositions according to the invention, or a vaccine or immunogenic composition according to the invention in combination with another vaccine or immunogenic composition, or active component (for example, inaktivirovannye or attenuated virus, or fungus, or microorganism, or subunit antigen, or immunogen, or protein, or epitope).

Thus, the invention includes immunogenic compositions and vaccines against diseases of swine, containing a mixture of cultures of PRRS viruses and PAV-3 according to the invention, such as live, attenuated immunogenic compositions and vaccines, inactivated immunogenic compositions and vaccines or subunit immunogenic compositions and vaccines. PAV-3 may is also be a recombinant PAV-3, which contains one or more nucleic acid sequence encoding and expressing one or more foreign or heterologous or exogenous immunogen(s), antigen(s) or epitope(s) (alien, heterologous or exogenous with respect to adenovirus). In applications WO 99/53047, WO 99/08706, WO 01/83737 and WO 00/47756 examples of recombinant adenoviruses pigs that can be used in the practice of the invention.

Another example is that the invention includes immunogenic compositions and vaccines against diseases of dogs containing a mixture of cultures of viruses CPI-2 and CAV-2 according to the invention, such as live attenuated immunogenic compositions and vaccines, inactivated immunogenic compositions and vaccines or subunit immunogenic compositions and vaccines. CAV-2 can also be a recombinant AV-2, which contains one or more nucleic acid sequence encoding and expressing one or more foreign or heterologous or exogenous immunogen(s), antigen(s) or epitope(s) (alien, heterologous or exogenous with respect to adenovirus). In U.S. patent No. 6090393 examples of adenoviruses dogs that can be used in the practice of the invention.

An additional example is that the invention includes immunogenic compositions and is Accini against diseases of cattle, containing a mixture of at least two or at least three, or all four viral cultures according to the invention, including BHV-1, BRV, bPI-3 and/or BCV, for example, BHV-1+BRV, BHV-1+BRV+bPI-3, BHV-1+BRV+bPI-3+BCV, BRV+bPI-3, BRV+bPI-3+BCV, bPI-3+BCV, BHV+bP-3, BHV+bPI-3+BCV, BHV-1+BCV, BRV+BCV, such as live attenuated immunogenic compositions and vaccines, inactivated immunogenic compositions and vaccines or subunit immunogenic compositions and vaccines. Immunogenic compositions and vaccines against respiratory disease in cattle, containing a mixture of at least two cultures of viruses according to the invention, including BHV-1 and bPI-3, and immunogenic compositions and vaccines against enteric diseases of cattle, containing a mixture of at least two cultures of viruses according to the invention, including BCV and BRV, are preferred.

Another example of combined compositions according to the invention are immunogenic compositions and vaccines against diseases of horses, containing a mixture of the cultures of the virus EHV-1 and EHV-4 according to the invention, such as live attenuated immunogenic compositions and vaccines, inactivated immunogenic compositions and vaccines or subunit immunogenic compositions and vaccines.

The invention includes obtaining such combination compositions, for example, by mixing the active components, the advantage is but together with a carrier or diluent, and/or adjuvant.

The invention also includes a kit for obtaining a combined immunogenic and vaccine compositions according to the invention, for example, the set containing (a) a microorganism, pathogen or virus, or its antigen or epitope (preferably the virus is referred to here) and (b) the microorganism, pathogen or virus, or its antigen or epitope (mainly virus or immunogen, antigen or epitope, but also included other microorganisms mentioned here), which is different from (a), in separate containers, optionally in one package and optional instructions for mixing and/or administration.

The invention further includes methods of inducing an immune response by the administration to the host, such as host, Prednisolonum to the disease caused by a pathogen or microorganism or virus, obtained in the cells of the lungs of cotton rats or cell line, for example, the cells of the lungs of cotton rats or cell line according to the invention, for example, virus, mentioned here, immunogenic or vaccine compositions according to the invention, containing an inactivated or attenuated pathogen or microorganism or virus, or its antigen or immunogen or epitope in an amount sufficient for the induction of an immune response.

Thus, the present invention provides methods to immunity the purpose or vaccination, or the induction of an immune response against a disease or against PRRS virus using immunogenic compositions and/or vaccines according to the invention, for example, the introduction of the immunogenic or vaccine composition of the host, for example, a pig in a quantity sufficient for the induction of an appropriate immune response. And the invention additionally provides methods of immunization or vaccination, or the induction of an immune response against the disease CPI-2, CAV-2, BHV-1, EHV-1, EHV-4, BRV, bPI-3 or BCV, or viruses, using immunogenic compositions and/or vaccines according to the invention, the introduction of the immunogenic or vaccine composition of the host, for example, the natural host of the virus, in a quantity sufficient for the induction of an appropriate immune response.

The present invention, for example, when using mixed or combined composition includes methods of immunization or vaccination, or to stimulate an immune response in an animal host against two or more different antigens, epitopes or pathogens or microorganisms, or viruses, such as pigs against PRRS and PAV-3, comprising introducing an effective amount to stimulate the immune response to the composition(s) and/or vaccine(s) according to the invention. (The plural is used because the method can also implement a coherent introduction to the of mposite or vaccines according to the invention.) In the same way as an additional example, the invention includes methods of immunization or vaccination, or stimulation of the immune response in dogs against diseases CPI-2 and CAV-2 or viruses, using, or administration of an effective amount of the immunogenic composition(s) and/or vaccine(n) according to the invention. Similarly the invention provides methods of immunization or vaccination, or stimulation of the immune response in cattle, at least against two or at least three or at least four of the diseases of cattle, or against viruses with the use or introduction of an effective amount of the immunogenic composition(s) and/or vaccine(n) according to the invention. Additionally, the invention includes methods of immunization or vaccination, or stimulation of the immune response in cattle against diseases BHV-1 and bPI-3 or viruses, using, or administration of the immunogenic composition(s) and/or vaccine(n) according to the invention. The invention also includes methods of immunization or vaccination, or stimulation of the immune response in cattle against diseases BCV and BRV or against viruses, use, or administration of an effective amount of the immunogenic composition(s) and/or vaccine(n) according to the invention. And the invention includes methods of immunization or vaccination, or stimulation of the immune response in horses against disease EHV-1 and HV-4 or anti-virus to use the drug, or administration of the immunogenic composition(s) and/or vaccine(n) according to the invention.

It should be noted that in this description and, in particular, in the claims the terms such as "contains", "contained", "contains", and the like can be set relating to such patent law of the United States, for example, they can mean "includes", "included", "including" and the like, and terms such as "mainly composed of" and "consists essentially of" have the meaning as defined in the patent law of the United States, for example, they allow you misquote the elements, but exclude elements that are available in the prior art or affect basic or new characteristics of the invention.

These and other embodiments disclosed here or are obvious from the essence of the following detailed description.

Detailed description of the invention

Applicants have developed a method of receiving or reproduction of viruses, which uses cells from the lungs of cotton rats, as well as a new cell line, and the method of reproduction or receiving, or cultivation, or growth of microorganisms, or pathogens, or viruses in these cells.

Light is extracted from cotton rats and subjected to enzymatic cleavage (e.g., under the action of trypsin and/or collagenase). Collect the supernatant containing the separated cells, and cultured, for example, in the form of a monolayer or in suspension. Suppose the equipment cells cultured as monolayer, for example, on a minimum maintenance medium (MEM) supplemented with fetal calf serum (FBS) and/or lactalbumin of hydrosylate (LAH). Mainly the culture medium may contain antibiotics, such as gentamicin, streptomycin and/or penicillin.

The PRRS virus is cultivated in the culture of cells of the lungs. Viruses PAV-3, CPI-2, CAV-2, BHV-1, EHV-1, EHV-4, BRV, bPI-3 or BCV is also possible to cultivate such a culture of cells of the lungs.

Cell culture preferably receive as a result of several passages of these cells. Mainly in the passages of these cells can be obtained cell line that is suitable for reproduction of PRRS virus. For example, cells can be subjected to at least 10 passages, for example, from 10 to 100 passages. Preferably the data cell lines long remain viable in the absence of fetal calf serum (FS), although to obtain monolayer need some FBS or other suitable growth factor cells.

Another advantage of cell culture CRL according to the present invention is that cell culture can exist, at least within the month as a viable monolayer or suspension without the need of replacing the spent medium or adding additional nutrients. This PR is durability provides the advantage of multiple receiving a virus from the same cell culture without the need in the starting period (prizhivaniyu culture) and without depletion of uterine cell culture.

Thus, it is possible to obtain a high yield of virus from the same cell culture. In one embodiment of the cultivation process includes two or more collecting virus with or without re-inoculation of the virus and/or addition of fresh culture medium. Preferably, after each collection in a cell culture was added to fresh culture medium.

Following this method, the cells of the lungs of cotton rats cultivate up to 76 passages and receive in the form of cell lines. The data cells of the lungs of cotton rats are mainly epithelial cells, and their morphological characteristics are retained during passages. The passage 21 has been deposited on 18 December 2001, under the terms of the Budapest agreement in the American type culture collection, 10801 University Blvd., Manassas, VA 20110-2209, under the inventory number of ATSS MOUTH-3930.

In the embodiment of the present invention includes the use of epithelial cells in the lungs of cotton rats, lines of epithelial cells derived from them, such as the cell line of the MOUTH-3930, or cell line, with its distinctive properties, to obtain virulent or attenuated PRRS virus. In additional embodiments of the present invention includes the use of epithelial cells in the lungs of cotton rats, lines of epithelial cells derived from them, such as cleto is the first line of the MOUTH-3930 or cell line, with its distinctive properties, to obtain virulent or attenuated virus CPI-2, CAV-2, EHV-1, EHV-4, BRV or BCV. In additional embodiments of the present invention includes the use of cell lines of the MOUTH-3930 or cell line, with its distinctive properties, to obtain virulent or attenuated virus BHV-1 or bPI-3. Cell line or cell line, with its distinctive properties, can also be used for other mentioned here viruses.

Thus, another aspect of the invention is a method of producing virus PRRS, including the cultivation of PRRS virus in the culture of cells in the lungs of cotton rats, containing epithelial cells, preferably cell lines the lungs of cotton rats. For example, the virus get into the cell line of the MOUTH-3930 or cell lines that have distinctive properties. This method of obtaining viruses can also be used against viruses CPI-2, CAV-2, BHV-1, EHV-1, EHV-4, BRV, bPI-3 or BCV, as well as other these viruses.

The virus can be virulent virus or an attenuated virus.

Getting the virus involves the stages of planting and cultivation of the virus in these cells. Before seeding the cells according to the invention it is possible to grow up in a suitable culture medium, such as medium MEM with the addition of FBS or on the natives of the appropriate growth factor cells. The cultivation is preferably carried out at a temperature in the range of 35 to 39°C, preferably at about 37°C. Mainly cell culture is a monolayer. Alternative cell culture is a suspension. Basically the virus is sown when the cells in the monolayer grown to confluence. Extracellular virus can directly select from the supernatant. Intracellular virus can be isolated after the subsequent destruction of cells, for example, by freezing/thawing or sonication. In the case of cell cultures in suspension of the virus can be isolated, for example, after filtration of the filtrate. The virus can be isolated through 2-15 days, for example, through 3-7 days, more preferably 5-7 days after sowing. Virus isolation is performed by methods well known to experts in the field relating to get viruses. At this stage receive untreated viral culture.

Into the culture medium used in this invention can be added antibiotics.

If necessary, the virus, such as PRRS virus, adapted to growth in the cells according to the invention. Adaptation can be performed by the joint cultivation of the cells according to the invention and kidney cells of monkeys, such as MA-104. As is well known, the adaptation performed by serial passages in coculture PR is a gradual increase in the number of cells according to the invention. This adaptation can also be carried out for viruses CPI-2, CAV-2, BHV-1, EHV-1, EHV-4, BRV, bPI-3 or BCV, as well as other these viruses.

Crude culture can concentrate and/or purify.

The concentration can be performed in any conventional way known to the experts in this field, such as selective precipitation or ultrafiltration. Cleaning can be performed in any conventional way known to the experts in this field, for example by ultracentrifugation or chromatographic methods such as gel filtration. At this stage receive concentrated viral culture treated culture or concentrated and purified culture.

In another aspect of the invention the viral culture according to the invention (raw, concentrated, purified or concentrated and purified) can be inactivated using any conventional method, for example, thermal and/or chemical method. The predominant method is the chemical inactivation, for example, using beta propiolactone, formalin, ethylenimine or its derivative, such as a binary ethylenimine (BEI), and combinations of data inactivating compounds. At this stage receive inactivated viral culture of PRRS.

In another aspect of the invention of the virus in viral culture is according to the invention (raw, concentrated, purified or concentrated and purified, optionally inactivated), can be extracted immunogenic fractions, such as glycoproteins or proteins. Extraction immunogenic fractions of virus carried out by methods known to experts in this field. At this stage receive subunit viral drugs.

Thus, other aspects of the invention are described herein untreated viral culture, concentrated culture treated culture, concentrated and purified cultures, inactivated cultures, subunit preparations.

In the preferred embodiment of the invention virulent virus can be attenuate holding a sufficient number of passages of the cells according to the invention. The person skilled in the art can determine by ordinary experimentation the number of passages required to attenuative virus. Get the drug attenuated virus.

The purpose of this invention is to provide immunogenic compositions and vaccines to prevent infection. Data immunogenic compositions or vaccines contain at least one culture or the product described here.

The term "immunogenic composition" includes, in this case, any composition capable, upon the introduction of an animal such as a pig, trims which can regulate the immune response against the virus or antigen, or immunogen or epitope. The term "vaccine" includes in this case any composition capable, upon the introduction of an animal such as a pig, to stimulate a protective immune response against the virus, or to effectively protect the animal from the specified virus.

Immunogenic compositions or vaccines according to the invention may include viral culture, or drug, or antigen, or immunogen or epitope of the virus and at least one immunogen, antigen or epitope of another pathogenic, or other pathogen (e.g., inactivated or attenuated pathogen). Such an immunogen, antigen or epitope may be, for example, bacterial, or parasitic, or viral origin, or to submit an inactivated or attenuated form of the pathogen. The invention also includes kits for receiving data of the combined compositions, and methods of obtaining data of the combined compositions and the use of data components of the combined compositions for the preparation of the combined compositions. Therefore, the invention includes a kit for obtaining a combined immunogenic or vaccine compositions according to the invention, for example the set that contains: (a) organism, pathogen or virus, or its antigen or epitope (mainly virus mentioned here) and (b) the microorganism, pathogen, or VIR is with, or its antigen or epitope (mainly virus or immunogen, antigen, or epitope, but also included other pathogens mentioned here), which is different from (a), in separate containers, not necessarily in the same package and optional instructions for mixing and/or injection.

Immunogenic compositions and/or vaccines of the invention may include cultural or agent of PRRS virus (e.g., inactivated or attenuated PRRSV, or immunogen or antigen, or epitope) and at least one immunogen, antigen or epitope of another pathogen of swine (including, without limitation, the pathogen is in the inactivated or attenuated form). This pathogen can be selected from the group including, but not limited to, virus pseudoleskeella, virus, swine flu, swine parvovirus, a virus of infectious gastroenteritis (coronavirus), circovirus pigs, such as the swine circovirus type 2, rotavirus, adenovirus pigs type 3, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchiseptica, Clostridium spp., Salmonella spp., Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae.

Mainly immunogenic compositions and vaccines according to the invention may include cultural or agent of PRRS virus and virus-PAV-3, grown and propagated in cells or cell lines according to the invention, for example, cell lines of the MOUTH-II cell lines, with all its distinctive properties. Immunogen pathogens of pigs may include virus pseudoleskeella gB virus pseudoleskeella gC, virus pseudoleskeella gD, influenza virus swine HA, influenza virus swine NA, influenza virus swine N, ORF4 of the virus reproductive and respiratory syndrome swine, ORF7 of the virus reproductive and respiratory syndrome swine PRRSV ORF5, PRRSV ORF3, PRRSV ORF6, open reading frames PRRSV 5 (ORF5) and 6 (ORF6), open reading frames PRRSV 5 (ORF5) and 3 (ORF3), and 6 (ORF6), the virus cholera swine E1 gene of the virus of swine cholera E2, parvovirus VP2, ORF1 of swine circovirus type 2 or ORF2 of swine circovirus type 2. In respect of immunogenes swine pathogens, nucleic acid molecules encoding them, and constructs expressing them, you should contact the U.S. patent№6517843, 6497883, 6391314, 6379676, 6217883, 6207165 and the publication of U.S. patent No. 2003003112 and patent applications WO 99/53047, WO 99/08706, WO 01/83737 and WO 00/47756. Thus, the invention also includes methods of obtaining these compositions, and kits for this purpose.

Immunogenic compositions and vaccines according to the invention may include the culture or preparation viruses CPI-2 and/or CAV-2 (e.g., inactivated or attenuated CPI-2 and/or CAV-2, or immunogen or antigen, or epitope) and at least one immunogen, antigen or epitope of another pathogen of dogs (including, but not limited to, the pathogen in inacti the new or attenuated form). This pathogen can be selected from the group including, but not limited to, virus plague dogs, parvovirus dogs, coronavirus dogs, herpesvirus dogs, Lyme disease pathogen, Borrelia burgdorferi, and rabies virus. Mainly immunogenic compositions and vaccines according to the invention include the culture or preparation of virus CPI-2 and/or culture, or preparation of the virus CAV-2 according to the invention. Immunogenum CPI-2 can be CPI-2 F and/or HN. Cm. also U.S. patent No. 5616326, 6090393, 6159477, 6228846 regarding immunogenic pathogens of dogs and nukleinovokisly molecules encoding them, and constructs expressing them. Thus, the invention also includes methods of obtaining these compositions, and kits for this purpose.

Immunogenic compositions and vaccines according to the invention may include cultural or drug virus BHV-1, BRV, bPI-3 and/or BCV (e.g., inactivated or attenuated BHV-1, BRV, bPI-3 and/or BCV or immunogen or antigen, or epitope) and at least one immunogen, antigen or epitope of another pathogen of cattle (including, but not limited to, the pathogen is in the inactivated or attenuated form). This pathogen can be selected from the group including, but not limited to, respiratory syncytial virus bovine virus diarrhoea in cattle. Mainly immunogenic HDMI is tion and vaccines according to the invention include, at least two viral culture or preparation according to the invention, including BHV-1, BRV, bPI-3 and/or BCV. Immunogenum BRSV can be BRSV F or G, or N, such as BRSV F and/or G, or N, and/or G. Immunogenum BHV-1 can be gB and/or gC and/or gD. Immunogenum BVDV can be protein E0 (gp48) and/or protein E2 (gp53). BVDV can be type 1 and/or type 2. Immunogen bPI-3 may represent a bPI-3 F and/or HN. In respect of immunogenes pathogens of cattle and nucleic acid molecules encoding them, and constructs expressing them, see U.S. patent No. 6451770, 6376473, 6224878. Thus, the invention includes methods for obtaining these compositions, and kits for this purpose.

Immunogenic compositions and vaccines according to the invention may include cultural or drug virus EHV-1 and/or EHV-4 (e.g., inactivated or attenuated EHV-1 and/or EHV-4, or immunogen, antigen or epitope), and at least one immunogen, antigen or epitope of another pathogen of horses (including, but not limited to, the pathogen is in the inactivated or attenuated form). This pathogen can be selected from the group including, but not limited to, virus equine influenza virus in Eastern encephalomyelitis (EEV), the virus Western encephalomyelitis (WEV), the virus Venezuelan encephalomyelitis (VEV), the causative agent of Lyme disease, Borrelia burgdorferi, Clostridium tetani, the virus Takayasu horses (EAV) and the Rus rabies. Preferably immunogenic compositions and vaccines according to the invention may include the culture or preparation of virus EHV-1 and culture or preparation of virus EHV-4 according to the invention. The glycoproteins of EHV can be gB, gD, gB+gD, gC and de. In respect of immunogenes pathogens of horses and nucleic acid molecules encoding them, and constructs expressing them, please refer to U.S. patent No. 6207166 and 6368603. Thus, the invention also includes methods of obtaining these compositions, and kits for this purpose.

Immunogenic composition or vaccine according to the invention, which also includes such additional immunogenic component (additional immunogen, antigen or epitope), has the advantage that it induces an immune response or protection against several infections or diseases, or their agents at the same time. This additional immunogenic component can be an attenuated or inactivated microorganism, the recombinant construct or subunits (e.g., proteins, glycoproteins, polypeptides or epitope). In the practice of the invention can be used ways to determine epitopes, such as receiving overlapping libraries of peptides (Hemmer B. et al., Immunology Today, 1998, 19 (4), 163-168; Pepscan (H.M. Geysen et al., Proc. Nat. Acad. Sci. USA, 1984, 81 (13), 3998-4002; H.M. Geysen et al., Proc. Nat. Acad. Sci. USA, 1985, 82 (1), 178-182; Van der Zee R. et al., Eur. J. Immunol., 198, 19 (1), 43-47; H.M. Geysen, Southeast Asian J. Trop.Med. Public. Health, 1990, 21 (4), 523-533; Multipin Peptide Synthesis Kits de Chiron) and algorithms (De Groot, A. et al., Nature Biotechnology, 1999, 17, 533-561) to determine the epitopes of immunogens, antigens, polypeptides, glycoproteins, and the like, without undue experimentation. Using this information, you can construct nukleinovokisly molecule encoding this epitope and using these specific data and information in this field can be constructed vectors or constructs, such as recombinant viruses or vectors, or plasmids expressing immunogenic, epitopes or antigens, without undue experimentation.

Immunogenic compositions or vaccine compositions optionally include pharmaceutically acceptable or acceptable for veterinary use excipient, the diluent or solvent and optionally a stabilizer and/or adjuvant. Suitable dosage forms well known to specialists in this field. Dosage forms can be developed for any appropriate route of administration.

Pharmaceutically acceptable solvent may be water or saline solution.

Inactivated immunogenic composition or inactivated vaccines preferably include at least one adjuvant.

Live attenuated viruses can be liofilizirovannami preferably with a stabilizer. The lyophilization can be a well-known conventional methods of freeze drying. Pharmaceutically acceptable stabilizers can be SPGA (sucrose-phosphate-glutamate-albumin) (Bovarnik et al., J. Bacteriology, 1950, 59: 509), hydrocarbons (for example, sorbitol, mannitol, lactose, sucrose, glucose, dextran, trehalose), MSG (Tsvetkov, T. et al., Cryobiology 1983, 20 (3): 318-23; Israeli E. et al., Cryobiology 1993, 30 (5): 519-23), proteins such as peptone, albumin or casein, protein containing agents such as separated milk (Mills C.K. et al., Cryobiology 1988, 25 (2): 148-52; E. Wolff et al., Cryobiology 1990, 27 (5): 569-75) and buffers (e.g. phosphate buffer, the buffer based on a phosphate of an alkali metal).

Adjuvant can be used to make soluble lyophilized preparations.

Examples of adjuvants are emulsions of oil-in-water, water-in-oil-in-water-based mineral oils and/or vegetable oils and nonionic surfactants such as block copolymers, Tween®, Span®. Other suitable adjuvants are vitamin E, saponins, Carbool®, aluminum hydroxide, aluminum phosphate or oxide of ammonium (“Vaccine Design, The subunit and adjuvant approach”, Pharmaceutical Biotechnology, vol. 6, Edited by Michael F. Powell and Mark J. Newman, 1995, Plenum Press New York). In the documents cited here, you can also get information on adjuvants, and excipients, diluents, carriers and solvents.

Other TSA the drive of the present invention is a method of immunization or method of vaccination using respectively immunogenic compositions or vaccine compositions according to the invention.

The method includes at least one introduction to the animal an effective amount of the immunogenic composition or vaccine according to the invention. The animal can be male, female, pregnant female and newborn. This introduction can especially hold intramuscular (I/m), intradermal () or subcutaneous (s/C) injection or intranasally, or orally. Immunogenic composition or vaccine of the present invention can be introduced from a syringe or needleless injector (such as, for example, Pigjet or Biojector (Bioject, Oregon, USA)).

For attenuated compositions dose of the virus or microorganism or pathogen, received in the new cell culture can be in the range from approximately

103up to about 107CCID50(average infectious dose for cell culture), preferably within about 104up to about 106CCID50and more preferably, they can be 105CCID50. Volumes for injection of 0.2-2.0 ml, preferably about 2,0 ml Can be one or more inputs, for example, two injections with an interval of 2-4 weeks, and preferably booster immunization after about 3 weeks after the first injection.

For inactivated compositions of the virus or microorganism or pathogen obtained on a new cell culture, animal WMS is about to enter about 10 4-109equivalent CCID50(titer before inactivation), preferably about 105-108equivalent CCID50in a single dosage form. The volume of a single dosage form can be in the range of 0.2 to 5.0 ml, and preferably is in the range from 0.5 to 2.0 ml, and more preferably is about 2,0 ml Can be one or more inputs, for example, two injections with an interval of 2-4 weeks, and preferably booster immunization after about 3 weeks after the first injection.

For the subunit compositions, for example, from a virus or pathogen or microorganism obtained on a new cell culture, animal can be entered from 5 to 500 μg, preferably from 20 to 50 mcg. The volume of injection is from 0.2 to 2.0 ml, preferably approximately 2,0 ml Can be one or more inputs, for example, two injections with an interval of 2-4 weeks, and preferably booster immunization after about 3 weeks after the first injection.

The composition of the invention can also enter other mammals, such as mice or laboratory animals, for example, to obtain polyclonal antibodies or to obtain hybridomas for monoclonal antibodies.

The invention will be described with further non-limiting examples.

When the career

Example 1: obtaining cells of the lungs of cotton rats (CRL)

Light, after extraction of the subject of animal euthanasia, were placed in a medium for cell culture containing the minimum supporting environment F-15 (MEM F-15, Hyclone, cat#SH30024.02) and gentamicin at a concentration of 30 µg/ml of this medium were added industrial available antibiotics (penicillin and streptomycin) and fungicide (amphotericin) at a concentration of 2% vol./about. The lungs were treated with enzymes at 37°C in a conical centrifuge tube with a capacity of 50 ml, containing 25 ml of collagenase (Sigma, cat#L-0130) and trypsin (Sigma, cat#T-8003) in concentrations of 1 mg/ml and stiffness 1X (corresponds to a concentration of 2.5 mg/ml) in MEM F-15, containing gentamicin at a concentration of 30 μg/ml After 30 min treatment was added fetal calf serum (FBS) (Bio Whittaker) to a final concentration of 10% vol./about. After dispersion of the cells using a pipette and clarification collected supernatant containing diisocyanate cells, and were sown in mattresses size 25 cm2environment MEME F-15, containing gentamicin at a concentration of 30 μg/ml and with the addition of FBS to a final concentration of 10% vol./about.

Example 2: cell cultivation CRL

To remove residual amounts of FBS with fused monolayer cells CRL obtained in example 1 was used trypsin solution. Cells coated with 0.1% (1X) mixture St. the Noah trypsin-ethylenediaminetetraacetic acid (EDTA) (JRH Biosciences, cat#62244-79P) (3 ml per 75 cm2, 5 ml mattress size 150 cm2) was placed in a thermostat at 37°C and carefully observed until then, until it was ended by attaching cells. The cells were dispersively using a pipette and collected 3 ml of cell suspension. At this time of 1:4 of the collected cells were cultured in fresh medium for cell cultures containing MEM F-15, gentamicin at a concentration of 30 μg/ml) and FBS at a concentration of 10% vol./about.

Cells CRL were cultured at 37°C in an incubator with 5% CO2. The proportion of 1:4 is fused monolayer was blended for approximately 4 days, and the proportion of 1:8 gave flushed monolayer within approximately 7 days. This is the passage.

Then cells were propagated from passage 2 to passage 76.

After addition of the cryoprotectant dimethyl sulfoxide (DMSO), a small portion was frozen at all passages up to 76. Portions were stored in liquid nitrogen.

Cells CRL from 21 passage deposited in the American type culture collection under the inventory number of ATSS MOUTH-3930.

Example 3: reproduction of PRRS virus in cells CRL

Used attenuated PRRS virus, which is known that it multiplies in the cells of the kidneys of monkeys.

The cultivation of PRRS virus were performed on monolayer cells CRL mattress size 75 cm2(mattress Kzt75) in 50 ml of culture medium containing MEM F-15, gentamicin at a concentration of 30 μg/ml) and FBS concentration is then 10% vol./about. Before inoculation of the virus, the cells were incubated at 37°C for 24 h until they were merged. After inoculation of 1 ml of PRRS virus infected cells were incubated at 37°C for 5-7 days. The growth of the virus was assessed by indirect reaction immunofluorescence assay (IFA) and by titration of the supernatant. After freezing/thawing collected supernatant. This material is a crude culture of PRRS virus.

Indirect reaction immunofluorescence assay (IFA)

To conduct IFA cells were trypsinization, dispersible, collected and resuspendable in fresh medium MEM F-15, containing gentamicin (at a concentration of 30 μg/ml) and with the addition of FBS to a concentration of 10% vol./about. Cells were planted in 96-well plates to the cultivation of tissues at the rate of 100 µl/well and cultured overnight prior to the merger. Prepared fourfold serial dilution PRRS. Then 100 µl or 200 µl of the diluted virus was introduced into each well in row 96-well plate. The tablet was placed at 37°C in a thermostat with 5% CO2for 7 days. Wells containing infected cells CRL was determined by indirect reaction immunofluorescence assay (IFA) with monoclonal antibodies against PRRS virus (SDOW 17, obtained from USDA; Magar R. et al., Can j vet Res., 1995, 59 (3): 232-4).

Characteristics of the PRRS virus, grown in culture CRL

The PRRS virus was titrated in 96-well tablet containing cells CRL. The titer was calculated the method of Spearman-Cerberus to determine the 50% endpoint and expressed in terms of ml The results were expressed in log10(FAID50)/ml Titer was $ 4.3 log10(FAID50)/ml.

Example 4: adaptation of PRRS virus to growth in the CRL and reproduction

Virus NADC 8 PRRS adapted to growth in cell CRL. NADC 8 were obtained from the National animal disease center (USDA).

The virus was consistently replicated in joint cell cultures CRL cells and MA-104 (the kidney cells of the African green monkey, the original cell line MARC-145), containing a gradually decreasing number of cells MA-104. Initially cells CRL were sown in the ratio of 1:9 cells MA-104 (20000 cells CRL/ml: 180000 cells MA-104/ml) at 37°C, MEM F-15 with hydrosilation lactalbumin (LAH in a concentration of 0.1%.vol.), gentamicin at a concentration of 30 μg/ml) and FBS at a concentration of 10% vol./about. When the data collaborative cultures mingled, were sown the PRRS virus (1 ml mattress Kzt75) without any substitution environment. The growth of the virus was assessed by indirect reaction immunofluorescence assay (IFA, as described in example 3) and by titration of the supernatant. Inoculated cells were incubated at 37°C for 5-7 days. The passage includes the freezing/thawing, collect supernatant and subsequent inoculation of 1 ml of viable joint culture in the mattress Kzt75, i.e. about 10% of the collected supernatant.

This procedure was repeated by passirovannym seed at the joint cultures CRL:MA-104 in the ratio of eniah 2:8, 3:7, 4:6, 5:5, 6:4; 7:3, 8:2 and 9:1.

At the end of the virus was cultured in the mattress Kzt75 in the monolayer, consisting only of cells CRL, 50 ml of culture medium containing MEM F-15, gentamicin at a concentration of 30 μg/ml) and FBS at a concentration of 10% vol./about. (no LAH). Infected cells CRL incubated at 37°C for 5-7 days. After freezing/thawing collected supernatant. This material is a crude culture of PRRS virus.

Data IFA showed that NADC 8 infected cells CRL.

Characteristics of the PRRS virus, grown in culture CRL

Strain NADC 8 PRRS virus was titrated in 96-well tablet containing cells CRL. Cultivation was performed as described above. The titer was calculated using the method of Spearman-Cerberus when determining the 50% endpoint and expressed in terms of ml Results were expressed in log10(FAID50)/ml.

The titer was 4,12 log in10(FAID50)/ml.

Other viruses, such as other viruses of the order Nidovirales, such as antivirus or viruses of the family Arteriviridae, for example a virus that increases the activity of lactate dehydrogenase (LDV), the virus Takayasu horses (EAV), haemorrhagic fever monkeys (SHFV), viruses of the family Coronaviridae, such as coronaviruses, such as infectious bronchitis virus, coronavirus dogs, coronavirus cats, coronavirus human 229F, virus epidemic diarrhoea of pigs, infectious virus is about gastroenteritis, the virus of infectious gastroenteritis of swine respiratory virus swine coronavirus bovine coronavirus human OS, hepatitis mice, hemagglutinine virus encephalomyelitis pigs, rat coronavirus, virus sialodacryoadenitis, virus, infectious bronchitis of poultry, coronavirus turkeys, coronavirus rabbits, torovirus, such as torovirus horses, torovirus pigs, torovirus person, torovirus cattle and other viruses for which the rodents or rats, or cotton rats are not natural hosts, can be adapted to growth in the cells of the lungs of cotton rats, preferably cells of the lungs of cotton rats or cell line according to the invention (for example, deposited using methods described herein or methods similar to those described here for PRRS.

Example 5: the method of inactivation

Collect the PRRS virus grown on CRL (example 3 or 4). The virus suspension treated with ultrasound at a temperature of about 5°C. the Virus suspension was filtered through a membrane with pore size of 50-100 microns at about 5°C.

In viral suspension make beta propiolactone at a final concentration of 1/3000 (about./vol.). After homogenization by stirring, the suspension is transferred into another sterile mattress.

Inactivation carried out under stirring for 24 hours at about 5°C. Zn is an increase in the pH was adjusted to about 7.2 by the addition of 1M NaOH.

Inactivated viral suspension is concentrated by ultrafiltration to about 50 times. Concentrated viral suspension stored at -40°C.

Example 6: receiving inactivated vaccine in the form of an emulsion in mineral oil

The vaccine is prepared with inaktivirovannye PRRS virus obtained in example 5 (after thawing and dilution), with the following composition:

suspension of inactivated PRRS virus167 ml
the oil phase83 ml

The oil phase consists of 7% by weight (wt./about.) anhydromannitol oleate, 8% (wt./about.) ethoxylated oleic acid (11 ethylenoxide) and 85% (vol./about.) light liquid paraffin oil (European Pharmacopoeia).

The aqueous phase and the oil phase are sterilized separately by filtration. The emulsion is prepared by mixing and homogenization of the ingredients with the help of turbine Silverson emulsifier.

One dose of the vaccine contains approximately 107,5CCID50(titer before inactivation). The volume of one dose of vaccine is 2.0 ml for intramuscular injection path.

Example 7: the replication of viruses in cells CRL

The strains of virus used as a standard (reference) virus, for which there are fluorescers the antibody (FA), was titrated using cells CRL. Because the data standard viruses usually was titrated using standard cell lines, each has a known titer with different degrees of variation. Used a ten - or fourfold dilution data standard viruses for seeding cells CRL in 96-well tablets using conventional titration method for each virus. After 7 days incubation, the plates were fixed with acetone and stained the corresponding FA. Titles fluorescent-positive cultures were compared to titers obtained in standard cell cultures. The results are presented below (see table).

Standard virusTitle/CRLTitle/standard cell line
Parainfluenza virus dogs type 2 (CPI-2)4,965,6 in cells D
Adenovirus dogs type 2 (CAV-2)1,725.8 cells D
Herpesvirus bovine type 1 (BHV-1)of 3.647.1 in cells D
Herpesvirus horses t is 1 (EHV-1) 5,74The title is not defined
Herpesvirus horses type 4 (EHV-4)5,446,13 in Vero cells
Rotavirus cattle (BRV)5,326.0 in cells MA-104
Parainfluenza virus bovine type 3 (bPI-3)6,467.0-cells D
Coronavirus bovine (BCV)3,524,79 in cells D
The virus reproductive and respiratory syndrome swine (PRRSV)5,225,34 in cells RC-145

Viral titres are expressed in log1050% infectious dose for cell cultures per milliliter (log10CCID50/ml).

Despite the fact that in each case, the titres obtained at CRL, were lower compared to the standard cell lines, it must be emphasized that these viruses were not cellular adaptation when using cells CRL that was standard for virus using standard cell lines. In this case zaljubila only to detect viral replication. In other words, with some effort, the number of live virus grown in cell CRL, you can increase to such an extent that it will be equal to or higher than the amount of virus grown in cell lines to which it is adapted. It shows data on the adaptation of PRRS virus to the CRL (example 4), when the titles are the same serial dilution of the virus was, respectively, 5,22 and 5,34 (log10CCID50/ml) using, respectively, cells CRL cells and MARC-145 (sensitive subclone cells MA-104). The difference in titer values were insignificant.

The invention additionally describes the subsequent numbered provisions:

1. The method of producing PRRS virus where to get the culture of cells in the lungs of cotton rats and cultured PRRS virus in this cell culture.

2. The method according to claim 1, where the cell culture comprises epithelial cells.

3. The method of producing PRRS virus where to get the cell culture of cell line the lungs of cotton rats and cultured PRRS virus in the culture.

4. The method according to claim 3, where culture consists of epithelial cells.

5. The method of producing PRRS virus where to get the culture of epithelial cells of the lungs of cotton rats and cultured PRRS virus in this cell culture.

6. The method of producing PRRS virus where to get the culture of cells from the epithelial line to etok the lungs of cotton rats and cultured PRRS virus in this cell line.

7. The method according to 3 or 6, where the cell line is the cell line deposited in ATSS under inventory number of MOUTH-3930 or a line of cells in the lungs of cotton rats with all the distinctive properties of the cell line deposited in ATSS under inventory number of MOUTH-3930.

8. The method according to claim 7, where the cell line is the cell line deposited in ATSS under inventory number of MOUTH-3930.

9. The method according to any of items 1 to 8, where the PRRS virus is virulent PRRS virus.

10. The method according to any of items 1 to 8, where the PRRS virus is attenuated PRRS virus.

11. The method according to any of items 1 to 8, where the PRRS virus is grown in cell and get a crude culture of PRRS virus.

12. The method according to any of items 1 to 8, where the PRRS virus is cultivated in the cells that receive the virus, giving a crude culture of PRRS virus, and expose this crude culture cleaned with obtaining a purified culture of PRRS virus.

13. The method according to any of items 1 to 8, where the PRRS virus is cultivated in the cells that receive the virus, giving a crude culture of PRRS virus, and expose this crude culture concentration of obtaining a concentrated culture of PRRS virus.

14. The method according to any of items 1 to 8, where the PRRS virus is cultivated in the cells that receive the virus, giving a crude culture of PRRS virus, and expose this crude culture concentration is the key and clean with getting concentrated and purified culture of PRRS virus.

15. The method according to claim 11, where the crude culture inactivate.

16. The method according to item 12, where the purified culture inactivate.

17. The method according to item 13, where a concentrated culture inactivate.

19. The method according to 14, where concentrated and purified culture inactivate.

20. The method according to PP, 16, 17 or 18, where the culture inactivate chemical agent.

21. The method according to claim 19, where the chemical agent is selected from the group consisting of beta-propiolactone, formalin, ethylenimine and binary ethylenimine.

22. The method according to PP, 12, 13, 14, 15, 16, 17, 18, 19 or 20, where the culture is treated for separation of subunits PRRS.

23. The method according to claim 9, where the PRRS virus cultured cells and produce attenuated PRRS virus.

24. The PRRS virus or culture of PRRS virus obtained after cultivation of PRRS virus in the cells of the lungs of cotton rats.

25. The PRRS virus or culture of PRRS virus obtained after cultivation of PRRS virus in the cell line the lungs of cotton rats.

26. The PRRS virus or culture of PRRS virus on PP or 24, where the cells are epithelial cells.

27. The PRRS virus or culture of PRRS virus on PP or 24, where the cells include epithelial cells.

28. The PRRS virus or culture of PRRS virus on A.25, where the cell line is a cell line deposited in ATSS under inventory number of MOUTH-3930 or a line of cells in the lungs of cotton rats, obladaushimi distinctive properties of the cell line, deposited in ATSS under inventory number of MOUTH-3930.

29. The PRRS virus or culture of PRRS virus on A.25, where the cell line is a cell line deposited in ATSS under inventory number of MOUTH-3930.

30. The PRRS virus or culture of PRRS virus obtained by the method according to any one of items 1 to 20, or 22.

31. The PRRS virus or culture of PRRS virus according to any one of PP-28, which is inactivated.

32. The PRRS virus or culture of PRRS virus according to any one of PP-28, which is attenuated.

33. Subunit preparation of PRRS virus, obtained by implementing the method according to item 21.

34. Immunogenic composition comprising a PRRS virus or culture of PRRS virus according to any one of PP-31, and acceptable for veterinary use excipient, the diluent or solvent.

35. Immunogenic composition containing subunit preparation of PRRS virus on p and acceptable to the veterinary excipient, the diluent or solvent.

36. Immunogenic composition according p additionally contains a stabilizer.

37. Immunogenic composition according PP or 34 or 35, further containing adjuvant.

38. Immunogenic composition comprising a culture of PRRS virus obtained by the method according to claim 1.

39. Immunogenic composition comprising a culture of PRRS virus obtained by the method according to claim 3.

40. Immunogenic composition comprising a culture of PRRS virus, p is obtained by the method according to any of items 1 to 22.

41. Vaccine containing a culture of PRRS virus obtained by the method according to claim 1.

42. Vaccine containing a culture of PRRS virus obtained by the method according to claim 3.

43. Vaccine containing a culture of PRRS virus obtained by the method according to any of items 1 to 22.

44. Vaccine containing virus PRRS or culture of PRRS virus according to any one of PP-31 and acceptable for veterinary use excipient, the diluent or solvent.

45. Vaccine containing subunit preparation of PRRS virus on p and acceptable to the veterinary excipient, the diluent or solvent.

46. The vaccine according to item 43, additionally contains a stabilizer.

47. The vaccine PP or 44 or 45, further containing adjuvant.

48. The method of immunization of pigs, including the introduction of the pig immunogenic composition according to any one of PP-39.

49. Method of vaccination of pigs, including the introduction of the pig vaccine according to any one of PP-46.

50. Cell line the lungs of cotton rats, deposited in ATSS under inventory number of MOUTH-3930 or cell line the lungs of cotton rats with all the distinctive properties of the cell line deposited in ATSS under inventory number of MOUTH-3930.

51. Cell line the lungs of cotton rats, deposited in ATSS under inventory number of MOUTH-3930.

Thus, having described in detail preferred embodiments of the present invention, it should be understood that the invention, certain of the above provisions is not limited to specific details in the above description, since it may be obvious to numerous variations without departure from the essence or scope of the present invention.

1. The method of obtaining the virus, including:
I) culturing cells in the lungs of cotton rats for at least 10 passages with obtaining cell line cotton rats;
II) inoculation of the cell line with a virus;
III) the multiplication of the virus in the cells; and
IV) isolation of the virus.

2. A method of obtaining a virus according to claim 1, where the cells of the lungs of cotton rats cultured for at least 21 of the passage.

3. A method of obtaining a virus according to claim 2, where the cells of the lungs of cotton rats cultured for at least 76 passages.

4. A method of obtaining a virus according to claim 1, where the line of cells in the lungs of cotton rats is ATSS MOUTH-3930.

5. A method of obtaining a virus according to claim 1, where the virus is a virus that increases the activity of lactate dehydrogenase (LDV), the virus Takayasu horses (EAV), haemorrhagic fever monkeys (SHFV), the virus reproductive and respiratory syndrome swine (PRRSV), infectious bronchitis virus, coronavirus dogs, coronavirus cats, coronavirus human E, virus epidemic diarrhoea of pigs, the virus of infectious gastroenteritis virus, infectious gastro is Narita pigs, respiratory virus swine coronavirus bovine (BCV), human coronavirus OS, hepatitis mice, hemagglutinine virus encephalomyelitis pigs, rat coronavirus, virus sialodacryoadenitis, virus, infectious bronchitis of poultry, coronavirus turkeys, coronavirus rabbits, torovirus horses, torovirus pigs, torovirus person, torovirus bovine parainfluenza virus dogs (CPI), adenovirus, herpesvirus bovine (BHV), herpesvirus horses (EHV), rotavirus cattle (BRV) or parainfluenza virus bovine type 3 (bPI-3).

6. The method according to claim 5, where the virus is a PRRSV, CPI type 2, adenovirus dogs type 2, adenovirus pigs type 3, BHV-1, EHV-1, EHV-4, bPI-3, BRV or BCV.

7. The method according to claim 6, where the virus is a PRRSV.

8. Cell line the lungs of cotton rats intended to be the method of obtaining the virus according to claim 1, where the cell line is a cell line the lungs of cotton rats was ATSS MOUTH-3930.



 

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