NO801443L - PROCEDURE FOR MANUFACTURING INFLUENSE WASHING IN LIQUID CELL CULTURE - Google Patents

Description

This invention relates generally to a new influenza virus propagation medium and in particular to the use of that medium for influenza vaccine production.

Influenza vaccines have been used since the beginning of

late 1940s for human vaccination and since the end of the 1960s for horse vaccination. All influenza vaccines that are now used are produced by growing vaccine virus strains in embryonated chicken eggs. The virus strains obtained are then used for the production of live virus vaccines; or further processed for the production of killed virus vaccine.

It is generally known by virologists that influenza viruses grow to a very limited extent in cell cultures. The growth was termed a 'one-stage growth cycle'; i.e., only the initially infected cells produce viruses. This phenomenon is described, for example, by Davis et al, Microbiology, Harper and Row Publishers,

chapter 44, pages 1138 - 39 (1968). Then viruses. from those originally infected; cells are not suitable for infecting successive numbers of cells in the same cell culture,

the yield obtained is too low to be suitable for. production of virus vaccines. Thus, liquid cell culture has

tours have not been used for commercial production of influenza virus vaccines.

Embryoned: chicken eggs are used to produce viruses with sufficiently high titers for use in the production of vaccines. Unfortunately, honseembryd-cultured viruses generally require concentration and, in the case of human vaccines, also require some form of purification to reduce toxic reactions caused by uncoated egg proteins. The use of eggs for the production of vaccine is time-consuming, labor-intensive, requires relatively high material costs, and the yield from one egg is usually only sufficient to produce vaccine for about 1 - 1.5 doses. Thus, the production of millions of doses requires the inoculation and incubation of millions of embryonated eggs.

In recent times, it has been established that a

broad spectrum of influenza A viruses including human, horse, pig and poultry strains grow productively in an established

line of rabbit kidney cells under an overlay medium in-? retaining trypsin and forming well-defined plaques regardless of their previous passage history. See the article by K. Tobita et. al, "Plaque Assay and Primary Isolation of Influenz A Viruses'in an Established Line of Canihe Kidney Cells (MDCK) in the presence of Trypsin", Med. Microbiol. Immunol.. 162, 9-14 (1975). See also the article by Hans-Dieter Klenk et al, "Activation of Influenza A Viruses by Trypsin Treatment", Virology 68, 426 - 439 (.1975). It should be noted that in the reports cited above, the effects of trypsin were observed on influenza virus propagation in semi-solid cultures in plaque formation trials and isolation techniques, none of which relate to liquid cell cultures.

tours or large-scale'or commercial propagation of influenza viruses for vaccine production. The term liquid cell culture as used here describes in vitro growth of cells and multiplication of viruses in a chemically defined liquid medium.

Quite surprisingly, we have now found that proteolytic enzymes can also be used in liquid cell cultures to facilitate the infection of successive numbers of cells in the same cell culture. Thus, by overcoming the limitations of the "one-step growth cycle" of previous liquid cell culture techniques, it is possible to effect an influenza virus yield that is in the range of approximately 1,000 - 10,000

fold greater than non-protease-treated cultures. This enables the application of liquid cell culture techniques for the commercial production of influenza vaccine, thereby avoiding

the disadvantages associated with the use of embryonated honse eggs. Details regarding our culture medium, virus propagation technique and vaccine production and methods used are covered by the invention.

Our influenza virus propagation medium comprises a cell culture suitable for infection with an influenza virus, an influenza virus, and a protein-hydrolyzing enzyme, the amount of the enzyme being sufficient to overcome the one-step growth cycle of the virus. Virus propagation techniques include the steps of inoculating or infecting a liquid influenza virus cell culture with.1 the influenza viruses, incubating the inoculum in the presence of a protein-hydrolyzing enzyme under conditions sufficient to ensure maximum virus growth (or maximum cytopathic effect), and coughing up the virus. Infection of the cells with the virus can occur

before or after the cell monolayer formation or, alternatively, by simple infection of a liquid cell suspension. The procedure for our vaccine production includes the following steps for neutralization of coughed-up virus or weakening by further cell culture passage for vaccine use. Particularly preferred embodiments include the use of the protease trypsin in conjunction with a canine kidney cell line for propagation of any types of different influenza viruses.

Figures 1-5 show a graphical illustration of the increase in titer obtained when the indicated virus strain is incubated in a liquid cell culture in the presence of different amounts of a typical protease, trypsin. The results are given as geometric mean titers.

The influenza virus and vaccine production technique of this invention demonstrates the use of influenza vaccine viruses. As used herein, the term influenza virus includes any virus that causes a fever-like disease state in animals (including humans) characterized by respiratory symptoms, inflammation of mucous membranes and frequent systemic involvement. The medium and the methods according to the invention are particularly suitable for the production of different influenza viruses, including human, horse, pig and poultry strains. Examples for the production of typical type A and B influenza viruses are described below.

The vaccine preparations disclosed in this invention include any preparation of dead, live, attenuated or fully live malignant influenza viruses that can be administered to provide or artificially boost immunity for any influenza disease. In a preferred embodiment, the vaccine comprises an aqueous suspension of virus particles ready for use.

The cell cultures considered suitable for carrying out the principles of this invention include any animal cell line or cell strain capable of being infected by, and allowing replication of, one or more given influenza virus strains. Although a number of such cells are known and considered useful for the technique described herein, we have achieved particularly good results with an established cell line known as the Cutter Laboratories Dog Kidney (CLDK) cell line: The CLDK cell line has been approved by the U.S. Department of Agriculture for use in the production of veterinary vaccines and is similar to the Madin Darby Dog Kidney Cell Line (ATCC No. CCL 34) and the dog kidney cell line as described in U.S. Patent No. 3,616,203. A brief historical account and description of the "specific mass cell stock" used for the cell line according to the examples follows, although it should be understood that the techniques described herein are intended to be applicable to any influenza virus that is susceptible to cell cultures.

Cutter Laboratoires Canine Kidney (CLDK) cell line history

The starting line for CLDK was implemented and established

at Cutter Laboratories, Inc., Berkeley, California, from the kidney of an apparently normal rabbit dog obtained from the University of California at Davis. The line was maintained on 0.5% lactalbumin hydrolyzate and 0.2% yeast extract in Earle! balanced salt solution plus 5% calf, lamb or horse serum and antibiotics, cultured according to methods of J.S. Younger (Proe. of Éxp. Biol., and Med., v 85, 202).

A frozen vial from the 142nd passage of this cell line was then implanted in a 75 cm 2 (250 ml) falcon flask, in tissue culture medium consisting of Earle's balanced salt solution "Minimum Essential Medium" (MEM) and 10%

fetal bovine serum<*>. The cells were subcultured in the same way and in the same medium for the preparation of the frozen "Master cell stock" at the 148th passage. ;A vial of "Master cell stock" was thawed, plated and serially subcultured 20 times to obtain bottle cultures from the 168th passage. These cells were then frozen. j' ;Description of "Mater'cell stock" ( MCS) ;Number of subcultures in series from original tissue: 148 Freezing medium: Minimum.1 required medium (Eadle) in Earle's BSS with reduced bicarbonates (1.65 gm/l) 80%; fetal bovine serum 10%; dimethyl sulfoxide 10%. Viability: Approximately 75% (color exclusion). Culture medium: Minimum required medium (Eagle) in Earle'S'-BSS with reduced bicarbonates (1.65 gm/l) 90%; fetal bovine serum 2 - 10%; antibiotics penicillin and streptomycin 100 U. or Y/ml.. ;Growth characteristics of fused cells; An inoculum of '—15 • ■ • ... 3 x 10 viable cells/ml cultured in the above culture medium at 37°C in a closed system' gives about 6-8 folds in 5 days. ;Morphology; Epithelial-like. ;Karyology; Chromosome frequency distribution 100 cells: ;2N = 72. ; No marker chromosomes. Sterility test: Free from mycoplasma, bacteria and fungi. Species:. Confirmed as a dog by immunofluorensctest. Virus susceptibility: Susceptible to influenza viruses, rabies virus, infectious swine rhinotracheitis, infectious canine heptatitis, distemper virus and possibly other viruses. Protein hydrolyzing enzymes (proteolytic enzyme or protease) which are considered to be useful for use in this invention include approved proteases such as trypsin, chymotrypsin, pepsin, pancreatin, papain, pronase, carboxypeptidase and the like, with trypsin as a particularly preferred enzyme. The exact mechanism(s) by which a protease such as trypsin inhibits influenza virus infection is not fully known. A possible mechanism has been proposed in the above-mentioned article by Klenk et al. As described below, the amount of active protease required to increase successive infection should be at least sufficient to outweigh the limitations of the "one-step growth cycle", but in the case of confluent monolayer cultures, not be so high as to cause sloughing of confluent cells from the surface of the tissue culture vessel (ie inner surface of a roller bottle). In the case of the specific enzyme used in the examples set forth below, we prefer that the amount of the enzyme be in the range of about 4-25 microns. grams per ml (ug/ml) of liquid tissue culture medium, preferably about 10 ug/ml. , ;Virus propagation methods ;The influenza virus propagation method according to the invention comprises three general features of infecting part of the cells of a liquid cell culture with the influenza virus, incubating the cells in the presence of a proteolytic enzyme under conditions sufficient to ensure maximum cytopathic (CP.) effect and abortion of the viruses from the culture. Vaccine production includes the subsequent steps of modifying the coughed virus by a technique to obtain live, malignant, attenuated, or dbdt (inactive) vaccine preparation. The vaccine may be available in dry form to be mixed with a diluent, or in a liquid, ready for use. Suitable aids may be included, as described below, to increase immunogenicity. ;The protease can be added to an aqueous cell suspension culture or or after the formation of a confluent monolayer culture. Examples of some of the different propagation methods are described below. ;Method A. -. infection of pre-formed monolayers; 1. Cells are grown to confluence in culture containers such as roller bottles, Povitsky bottles or Roux bottles using cell culture growth media as previously known. 2. For infection, the growth medium is removed from the cellmo.no layers. 3. The active influenza virus seed is diluted in the growth medium containing additional vitamins, non-essential amino acids, L-glutamine, dextrose and antibiotics with the pH adjusted to 6.6-6.8. 4. A quantity of diluent containing virus is added to the cell monolayer in quantities varying from 10% - 100% of the final coughed up volume. 5. The infected monolayers are incubated at 34 - 37°C for 1-72 hours. 6. Protease, alone or in combination with virus propagation media, is added at a concentration that will stimulate multiple cycle growth without the production of dead cells. For trypsin, the optimal concentration is between about 8 and 15 µg/ml. ; 7. The virus growth containers are re-incubated at ;34 - 37°C until maximum cytopathic effect is observed...At this point the virus fluids are coughed up. 8. The coughing includes vigorous shaking of the containers to remove cells and transferring fluids and cells to a sterile container for further processing. Method B - Infection of liquid cell suspension for monblag formation 1. Cells are removed from growth containers using conventional methods. 2. Cells become; concentrated by centrifugation, then resuspended in a quantity of fresh growth medium containing additional vitamins, non-essential amino acids, L-glutamine, dextrose and antibiotics. 3. Influenza virus is then added to this concentrated cell suspension. 4. The cell virus suspension is incubated (25°C - 37°C) in a sterile, closed container (such as a screw-cap Erlenmeyer flask) while mixing on a magnetic stirrer or rotary shaker for a period of 10 minutes to .'. 4 hours.. ;5. Aliquot portions of cell virus suspension are placed in growth containers (roller bottles, Roux bottles, Povitsky bottles) with a full volume of media containing ingredients as stated in B-2, plus 5% fetal calf serum. 6. Growth containers are incubated at 34 - 37°C until confluent monolayers form (approximately 2-4 days). 7. After the monolayer is formed, protease is added at a concentration that will stimulate multiple cycle growth without the formation of dead cells. For trypsin, the optimal concentration is between 10 and 25 yug/tal.. 8. Growth containers are incubated at 34 - 37°C until maximum cytopathic effect is observed. Viruses are then hosted. 9. Harvesting involves vigorous shaking of containers to remove cells and transferring cells and fluids to a sterile container for further processing. ;Method C - Infection of liquid culture suspension ;1. Cells adapted for culture suspension are allowed to grow to an optimal number in growth medium in suspension growth containers. 2. Cells are centrifuged and resuspended in a quantity of fresh medium containing additional vitamins, non-essential amino acids, L-glutamine, dextrose and antibiotics. 3. Influenza virus is then added and the culture is incubated at 34 - 37?C from 10 minutes to several hours. 4. Fetal calf serum can be added, and the culture suspension is further incubated at 3° - 37°C for 1 - 72 hours. 5. Protease is added at a concentration that will allow multiple cycle growth without producing an adverse effect on the cells. For trypsin, the optimal concentration is between 4 and 25 yug/ml. 6. Incubation at 34 - 37°C is continued until the maximum cytopathic effect is observed during the coughing time for the liquids. 7. Coughing includes transferring liquids to a sterile container for further processing. ;Specific examples for the application of our technique and media for propagation of selected strains of influenza viruses follow. When not stated otherwise, we use conventional tissue culture techniques known for. the professional. As both the cell and the medium preparation technique are well known, it will not be described here in detail. ;i •■ ' ;Example 1 ;A2 equine influenza virus, designated Miami strain, was originally isolated from a horse at the University of Miami. This virus was obtained from the University of Pennsylvania Medical School where six passages were made in the chick embryo. The*seventh passage was made at and obtained from Lederle Laboratories. The strain was further subjected to chicken embryo assay and was used at passages 11-16.

The present preferred method of tissue culture propagation of the A2 strain involves infection of a young, confluent monolayer of CLDK cells. Cells are plated in roller bottles, using "Hahk's Minimum Essential Medium". (MEMH) containing the following ingredients:

Fetal bovine serum, 5 - 10%

Non-essential amino acids, 10 ml/Z (Gibco) L-glutamine, in 10 ml/& (Gibco)

Neomycin sulfate, 30,000 mcg/Z-

Polymyxin Bj 30,000 units/^

Mycostatin, 25,000 units/^

The cells usually become confluent after 72 hours at which time the medium is poured off and the cells infected. The inoculation medium contains A2 virus diluted to an "Egg Infective■Dose^0" (EID^q) titer of approximately 10^'°/ml in MEMH supplemented with the following ingredients:

50% dextrose, 2.6 ml/Z

MEM vitamins, 30 ml/(Gibco)

Non-essential amino acids, 10 ml/^ (Gibco) L-glutamine, 10 ml/ C (Gibco)

Neomycin sulfate, 30,000 mcg/-£

Polymyxin B;, 30,000 units/^

Mycostatin, 25,000 units//^

Inoculation medium equivalent to 14% of the final volume is added to each roller bottle and the containers are incubated at 34-35°C for 72 hours. At this time, remaining medium (86%) containing 12 µg/ml sterile trypsin (Sigma, 1:250) solution (0.1 g/100 ml) is added to each roller bottle. The containers are again incubated at 34 - 35°C until maximum cytopathic effect is observed (48 - 72 hours) at which time the liquids are coughed up. Coughing involves vigorous shaking of each roller bottle to remove accompanying cells and transfer of cells and viral fluid to a sterile batch container for further processing.

The cough EID^q titer of A2 influenza virus grown using this technique is shown in Table 1. See also Figs. 1. A comparison is made with A2 virus grown by the same method but without the addition of trypsin.

Incorporation of trypsin into the growth medium gives a geographical average increase of 3.2 logs or 1711 times as many virus particles/ml during the production of the Miami strain.

Example 2

A sample of: malignant type Al Equine influenza

virus was obtained from; University of Pennsylvania Medical School. The strain, designated Pennsylvania (Al), had been isolated from a horse and has been passaged in the chicken embryo six times. The strain has undergone further chick embryo passage and has been used for tissue culture production at passages 12 - 17.

The preferred method for tissue culture propagation

of the Al strain comprises infection of a suspension of CLDK cells for monolayer dannel.se. CLDK cells at a concentration of about 10?'^/ml, Pennsylvania virus strain at an EID50 titer of 10<3>'°/ml - 10<5>,<0>/ml, and "Hank's Minium Essential Medium " (MEMH) supplemented with the ingredients an-

fbrt below, is incubated at 25°C with mixing on a magnetic stirrer in a closed, sterile Erlenmeyer flask. The pH is maintained at 6.7 - 6.8 with IN HCl during the 2 - 3 hour incubation period.

Supplemented MEMH

50% dextrose, 2.6 ml/lb

MEM vitamins, 30 ml/Z (Gibco)

Non-essential amino acids, 10 ml/^ (Gibco)

L-glutamine, 10 ml/-^ (Gibco)

Neomycin sulfate, 30,000 meg/£

Polymyxin B, 30,000 units/£

Mycostatin, 25,000 units/^

After this suspension incubation, 10 ml aliquots of the viral cell suspension were added to roller bottles containing 1 liter of MEMH supplemented as indicated above and containing 5% fetal calf serum. The roller bottles are incubated at 34-35°C until the monolayer becomes confluent (approximately 48-72 hours) after which time 20 ml of a sterile 1 mg/ml trypsin solution (Sigma 1:250) is added to each roller bottle. The roller bottles are again incubated at 34 - 35°C until the maximum cytopathic effect is observed (3-5 days). The virus fluids are coughed up by strong shaking each roller bottle to remove adherent cells, and transferring cells and fluid to a sterile container for further processing. The cough EID^Q titer of the A1 influenza virus cultured using this technique is shown in Table 2. See also Fig 2. A comparison was made with A1 virus grown by the same method where trypsin is excluded.

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Incorporation of trypsin into the growth medium gives a geometric mean increase of 2.3 logs or 187 times as many virus particles/ml during production of the Pennsylvania strain.

Example 5

A strain of human influenza virus designated B/Hong'Kong/5/72 (BX-1) was received from The Center for Disease Control in Atlanta, Georgia. This was passed once in embryonated honse egg and frozen at -70°C as working sed virus.

The preferred method for tissue culture propagation of the B/Hong Kong/5/72 strain involves infecting a young confluent monolayer of CLDK cells similar to that in Example 1. Cells are cultured as described in Example 1. Growth medium is removed from cells and discarded. Cells are then infected with virus diluted in the inoculation medium as indicated in Example 1 1 to an EID,-n titer of approximately

3 5-5 0/

lGr'^ ^' /ml. The inoculum consists of a volume equivalent to 33.3% of the final coughed volume. Containers become in- . incubated at 34-35°C for 40-48 hours after which remaining medium (66.7%) containing 12 µg/ml trypsin solution (0.1 g/100 ml) is added to each container. Incubation at 34 - 35°C is continued until maximum cytopathic effect is observed (48 - 72 hours) at which time virus fluid is coughed up. The coughing includes vigorous shaking of each container to remove cells and transfer of cells and fluid to a sterile container for further processing.

The cough EID^Q titer of B/Hong Kong/5/72 influenza virus cultured according to this technique is shown in Table 3. See also Figs. 3> A comparison is made with this strain grown with the same method, but without the addition of. trypsin.

Incorporation of trypsin into the growth medium gives a geometric mean increase of 3.1 logs or .1412 times the number of virus particles during the production of the B/Hong Kong strain.

Example 4

A strain of human influenza virus designated A/Texas/. 1/77 was received from The Center for Disease Control in Atlanta, Georgia. This was passaged once in embryonated honse eggs and frozen at -70°C as working frb virus.

The preferred method for tissue culture propagation of the A/Texas/l/77 strain is. the one described fully in example 3•

The cough EID^Q titer of A/Texas/1/77 human influenza virus grown using this technique is shown in Table 4. See also Figs. 4. A comparison is made with this strain grown by the same method, but without the addition of trypsin.

Incorporation of trypsin into the growth medium gives a geometric mean increase of 4.1 logs or 12,590 times as many virus particles/ml during production of the A/ Texas strain.

Example 5

A strain of human influenza virus designated A/USSR/ 90/77 was received from The Center for disease.Control in

Atlanta, Georgia. This was passaged once in embryonated chicken eggs and frozen at -70°C as a working frb virus.

The preferred method of culture propagation of the A/USSR/90/77 strain is that described fully in Example 3•

The host EID50 titer of A/USSR/90/77 human influenza virus grown using this technique is shown in Table 5• A comparison is made with this strain grown by the same method but without the addition of trypsin.

Incorporation of trypsin into the growth medium gives a geometric mean increase of 2.4 logs or 251 times as many virus particles/ml during production of the A/USSR strain.

Vaccine production Example 6 - virus attenuation

Attenuation of the virus from coughed fluids from Example 1 is accomplished chemically or by standard passages in series involving the terminal dilution passage technique in which a sufficient number of passages in a susceptible cell culture are used until the virus is rendered non-pathogenic without loss of immunity. A vaccine prepared in this way will stimulate an immune response in animals susceptible to disease-like without producing clinical symptoms of an appreciable degree normally caused by a malignant pathogen. The propagation can be carried out in the same or different tissues than those used in the previous passage.

Example 7 - Virus Inactivation

The technique is similar to that described in examples 1 and 2, but: the coughed virus-containing fluids are further processed by inactivation with 0.1% concentrated formaldehyde (range 0.05 - 0.2%) and the treated material is incubated at 4° C for 10 - 14 days. Testing of the final inactivated virus preparation showed that it was free of live virus. Curing agents known to those skilled in the art, such as aluminum hydroxide, alum, aluminum phosphate, "Freund's" or such as described in U.S. Pat. patent nos. 3,790,665 and 3,919,411 can be added. The preferred adjuvant according to the invention and that used in our vaccine is an acrylic acid polymer cross-linked with a polyallyl saccharide ("Carbopol 934 P") similar to that described in the above-mentioned patents.

Example 8 - Inactivated virus vaccine

Production and application

A 1.0 ml horse dose consisting of 0.45 ml of Pennsylvania (Al) strain, :0.45 ml of Miami (A2) strain and 0.10 ml of "Carbopol" adjuvant. Equal parts of inactivated vaccine strains obtained from Example 7 were mixed and a 1 ml aliquot was administered intramuscularly to 19 horses. No clinical disease or symptoms of influenza were observed in any of the horses after vaccination. Antibody against both Equine Influenza Al and Equine Influenza A2 was obtained on blood sera from all animals after 2, 4 and 8 weeks followed by inoculation. These are compared with pre-inoculation levels (Table 6) using the standard hemagglutination inhibition test (DIAGNOSTIC PROCEDURES for Viral and Rickettsial Infections, Fourth Edition; Lennette and Schmidt, pp. 665-66 (1969). American Public Health Association, N.Y., N.Y. 10019 ).

As can be seen from Table 6, antibodies to both viral antigens developed in horses receiving the inoculations. These vaccines will be immune to Equine Influenza as antibody titers in excess of 1:20 to A2 and 1:60 for Al are accepted to be protective by the National Veterinary Services Laboratories of the U.S.' Department of Agriculture.

Clinical tests of 420 horses of different breeds and ages showed that the vaccine is safe and does not cause unfortunate reactions after intramuscular inoculation.

Example 9 - Application of weakened virus vaccine

Three horses were given intranasally 5 ml of live attenuated Equine Influenza A2 vaccine strain from Example 6. No clinical disease or symptoms of influenza were observed in any of the horses after vaccination. Antibody titers against the vaccine strain were obtained on block sera of all animals at 1, 2 and 4 weeks following inoculation and compared to pre-inoculation levels using the standard haemagglutination inhibition (HAI) test.

Once again, antibody titers obtained are stbrre

than required for protection.

This invention relates to both a new influenza culture system and the use of the medium for the production of a new influenza vaccine. The liquid cell culture system used in this invention involves the use of susceptible cells, influenza viruses, a nutrient medium and a proteolytic enzyme, but unlike previous systems (e.g. Tobita et al), the use of agar is not required, which reduces the system to a <1> semi-fixed system. Exclusion of agar enables large-scale production of viruses by conventional means known to those skilled in the art and results in the production of viruses of sufficient high titer for the manufacture of vaccines.

Influenza vaccine preparation comprises an effective amount of

those of one or more derived from given influenza virus particles and a pharmaceutically acceptable carrier, the entire preparation, preferably in liquid form, being substantially free of reactive proteins such as egg proteins. As used herein, the term substantially free of egg protein means that

the only possible source of egg protein in vaccine preparations is frovirus diluted >lrlOO,000.

The vaccines are administered to animals by various routes, including intramuscular, intravenous, subcutaneous, irritratracheal, intranasal, or by aerosol spray, and the vaccines are used for beneficial use in various animals, including humans, horses, swine, and poultry.

The virus preparation produced according to the invention can be diluted with water to adjust its strength, and stabilizers such as sucrose, dextrose, lactose or other non-toxic substances can be added. The virus preparations can be dried by freeze-drying for storage or for later formulation into attenuated vaccines or they can be chemically inactivated for the production of lethal virus vaccines.

It must be understood that the examples given above are only illustrative and that the scope of the invention is only limited by subsequent patent claims.

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Claims (39)

1. A method for producing an influenza virus vaccine comprising the following steps: (a) infusing a portion of the cells from a liquid cell culture with an influenza virus; (b) incubating the infected culture in the presence of a proteolytic enzyme under conditions sufficient to ensure further infection and propagation of the virus in cells other than the cells originally infected; , (c) withdrawal of the virus from the culture and; (d) preparing a vaccine from the host virus. 2. Method according to claim 1 where the cell culture from step (a) comprises a confluent monolayer of cells. 3. Method according to claim 1 where the cell culture from step (a) comprises a liquid suspension of cells. 4. Method according to claim 1, where the vaccine production in step (d) includes the step of inactivating the coughed virus. 5. Method according to claim 1, where the vaccine preparation in step (d) includes the step of diluting the coughed-up virus. 6. Method according to claim 1 where the proteolytic enzyme from step (b) is selected from trypsin, chymotrypsin, pepsin, pancreatin, papain, pronase and carboxypeptidase. 7. Method according to claim 1 where the virus is an equine influenza virus. 8. Method according to claim 7, wherein the virus is a strain selected from equine influenza A1 and equine influenza A2 virus strains. 9. Method according to claim wherein the virus is a human influenza virus. 10. Method according to claim 9 wherein the virus is selected from B/Hong Kong, A/Texas and A/USSR virus strains. 11. Method according to claim 1, where the cells from the cell culture are dog kidney cells. 12. Method according to claim 1 where the cell culture from step (a) is a dog kidney cell culture, the virus from step (a) is a horse influenza virus and the enzyme from step (b) is trypsin. 13. Method according to claim 1 where the cell culture from step (a) is a dog kidney cell culture, the virus from step (a) is a human influenza virus and the enzyme from step (b) is trypsin. 14. Method according to claim 1 where the enzyme from step (b) is present in an amount sufficient to ensure infection and propagation of the virus in culture cells other than the cells that comprise the part that was infected in step (a). 15. Method in, according to claim 14, where the amount of trypsin varies from approx. 4 - approx. 25 µg/ml culture media. 16. An influenza vaccine preparation comprising an effective amount of influenza virus particles and a pharmaceutically acceptable carrier, wherein the preparation is substantially free of egg proteins. 17. Preparation according to claim 16 where virus particles are weakened. 18. Preparation according to claim 16 where the virus particles are dbde..! 19. Preparation according to claim 16 where the virus is selected from the group consisting of the human, horse, poultry and pig strains. 20. Vaccine preparation according to claim 16, wherein the virus is an equine influenza virus selected from equine influenza A1 and equine influenza A2 strains. 21. Vaccine preparation according to claim 1 wherein the virus is a human virus selected from B/Hong Kong, A/Texas and A/USSR viruses. 22. Vaccine preparation according to claim 16, where the carrier is a liquid. 23. Vaccine preparation according to claim 22, wherein the liquid carrier comprises an aid selected from aluminum hydroxide, alum, aluminum fps, Freund's and an acrylic acid polymer cross-linked with a polyallyl saccharide. 24. A method for propagating influenza viruses 1 a liquid cell culture medium comprising infecting a liquid cell culture with an influenza virus, incubating the infected culture in the presence of a proteolytic enzyme under conditions sufficient to ensure virus propagation and. ? 25. Method according to claim 24, wherein the influenza virus is an equine influenza virus. 26. Method according to claim 25, wherein the virus is selected from equine influenza A1 and equine influenza A2 viruses. 27. Method according to claim 24, wherein the virus is a human influenza virus. 28. Method according to claim 27 wherein the virus is a strain selected from B/Hong Kong, A/Texas and A/USSR viruses. 29. Method according to claim 24, wherein the proteolytic enzyme is selected from trypsin, chymtrypsin, pepsin, pancreatin, papain, pronase and carboxypeptidase. 30. Method according to claim 24 where the enzyme is trypsin in an amount varying from approx. 4 - approx. 25 µg/ml liquid cell culture. 31. Method according to claim 24 where the tissue culture comprises canine kidney cells, the influenza virus is selected from horse and human virus strains and the proteolytic enzyme is trypsin. 32.. An influenza virus-inoculated liquid cell culture comprising influenza viruses, a cell culture suitable for host propagation of the viruses and a proteolytic enzyme, the amount of the enzyme being sufficient to ensure infection and propagation of the viruses in substantially all cells of the liquid cell culture. 33. Cell culture, according to claim 32, wherein the virus is an equine influenza virus. 34. Cell culture: according to claim 33 wherein the virus is a strain selected from equine influenza A1 and equine influenza A2 viruses. j 35. Cell culture 1 according to claim 32, wherein the virus is a human influenza virus. 36. Cell culture: according to claim 35 wherein the virus is a strain selected from B/Hong Kong, A/Texas and A/USSR viruses. 37. Cell culture! according to claim 32 where the proteolytic enzyme is selected from trypsin, chymotrypsin, pepsin, pancreatin, papain, prohase and carboxypeptidase. 38. Culture medium according to claim 32, where the cell-line tissue culture comprises canine kidney cells, the influenza virus is selected from equine and human influenza virus strains and the proteolytic enzyme is trypsin. 39. Culture medium according to claim 38, where the amount of trypsin varies from approx. 4 - approx. 25 yug/ml of fluid cell culture. j