DK154749B - PROCEDURE FOR PREPARING AN INFLUENZA VIRUS VACCINE IN A LIQUID CELL CULTURE - Google Patents

Description

in

DK 154749 B

The present invention relates to a novel method of producing a influenza vaccine in which cells are infected with an influenza virus, the virus is propagated in the cells, after which the virus is isolated, and from the isolated virus a vaccine is produced.

Influenza vaccines have been used since the early 1940s for human vaccination and since the end of I960 for horse vaccination. All of the influenza vaccines currently in use are prepared by growing the vaccine virus strains 10 in the embryonic stage of chicken eggs. The resulting virus strains are then used to prepare live virus vaccines or further processed to produce killed virus vaccines.

It is well known by virologists that influenza-15 zaviruses grow to a very limited extent in cell cultures. Growth is referred to as the "one-step growth cycle"; i.e. only replication of the virus types in the initially infected cells. This phenomenon is described e.g. by Davis et al. MICROBIOLOGY, Harper and Row Publishers, Chapter 44, pp. 1138-39 (1968). Because the viruses of the initially infected cells are unable to infect subsequent numbers of cells in the same cell culture, the resulting yields are too low to be used in the preparation of virus vaccines. Thus, liquid cell cultures have not been used for commercial production of influenza virus vaccines.

Fetal stage chicken eggs are used to produce viruses with titers sufficiently high to be used in the preparation of vaccines. Unfortunately, embryo-cultured viruses usually require concentration and, in the case of human vaccines, also require some sort of purification to reduce toxic reactions due to unwanted egg proteins. The use of the eggs for vaccine production is time consuming, requires a lot of work and relatively high material costs, and the yield from an egg is generally only enough to produce vaccine for approx. 1 to 1.5 doses. Thus, the preparation of millions of doses requires inoculation.

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2 laying and harvesting millions of eggs at the fetal stage.

It has recently been found that many different types of influenza A virus, including human, equine, porcine and avian strains, grow productively in an established line of 5 canine kidney cells under a superimposed trypsin-containing agent and form well-defined spots regardless of their previous passage history. See the article by K. Tobita et al, "Plaque Assay and Primary Isolation of Influenza A Viruses in an Established Line of Canine Kidney Cells (MDCK) 10 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 emphasized that the effects of trypsin on influenza virus propagation in the above-mentioned reports have been found in semi-solid cultures by stain analysis and isolation methods, none of which relate to liquid cell cultures or the large scale or commercial propagation of influenza viruses for vaccine production. The term liquid cell culture used in the present text describes the in vitro growth of cells and the propagation of viruses in a chemically defined liquid agent.

Surprisingly, it has now been found that proteolytic enzymes can also be used in liquid cell cultures to promote the infection of subsequent numbers of cells in the same cell culture. Thus, by overcoming the limitations of the "one-step growth cycle" of prior liquid cell culture techniques, it is possible to obtain a flu virus yield which is in the range of about 1,000 to 10,000 times greater than non-protease treated cultures. This enables the application of liquid cell culture technique to the commercial preparation of influenza vaccines, thereby avoiding the disadvantages of using chicken eggs at the fetal stage. Details of the culture medium of the present invention, the virus propagation technique and vaccine preparation and methods of use are described below.

In Chem. Abstr. 84 (1976), 71361p and 71362q, are (equiv.

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3 as described in the above publications) the use of trypsin to increase the infectivity of influenza viruses in staining. However, this cannot be compared to the use of trypsin (or other proteases) for the propagation of 5 influenza viruses in liquid cell cultures and for the preparation of vaccines. In the two literature sites, nothing about vaccine preparation is stated. They, on the other hand, are concerned with understanding the mechanism of viral infectiousness and by increasing this infectiousness by enzymatic cleavage of 10 specific groups on these viruses. This phenomenon is followed by staining.

The staining indicates that the viruses studied have a notable infectiousness. Such spots are examined in a semi-solid medium, usually agar.

In contrast, according to the present invention, no semi-solid media is used, nor are any stains formed. On the contrary, liquid cell cultures are used for virus propagation. According to the prior art, such liquid cell cultures have not been able to be used for the preparation of influenza virus vaccines, since the virus does not exhibit any significant propagation rate in such cultures. The present application of proteolytic enzymes for the first time allows the use of liquid cell cultures to produce influenza virus vaccines at the above scale.

Accordingly, the present invention relates to a method of the aforementioned kind for producing an influenza virus vaccine, which is characterized in that a portion of the cells of a liquid cell culture are infected with an influenza virus and the infected culture incubated in the presence of an influenza virus. proteolytic enzyme, the concentration of which is sufficient to cause growth in several cycles without causing a detachment of cells in cases where a confluent monolayer is used as a cell culture.

The method used in the present invention

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The 4 influenza virus propagation medium comprises a cell culture which can be infected with an influenza virus, influenza virus and a protein hydrolyzing enzyme, the amount of enzyme being sufficient to exclude the one-step growth cycle of the virus.

The present process comprises inoculating or infecting a fluid influenza cell culture with the influenza virus types, incubating the inoculum in the presence of a protein hydrolyzing enzyme under conditions sufficient to ensure the viral growth shown in the Examples (or a corresponding cytopathic effect), and harvesting the virus. Infection of cells with the virus can occur before or after cell monolayer formation or, alternatively, by simply infecting a liquid suspension of the cells. In a subsequent step, the harvested virus is killed or attenuated for vaccine use by further cell culture passage. Particularly preferred embodiments employ protease trypsin in conjunction with a dog kidney cell line to propagate any of the different types of influenza viruses.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be further illustrated below with reference to the drawings.

FIG. 1-5 are bar graphs illustrating the increases in titre obtained when the indicated virus strains are incubated in a liquid cell culture in the presence of various amounts of a typical protease, trypsin. Results are given as geometric mean titers. Particular embodiments of the invention are described below.

The present vaccine preparation method involves the use of influenza vaccine viruses. As used in the present text, the term influenza virus includes any type of virus that is capable of causing a febrile disease state in animals and humans, which is manifested by respiratory symptoms, inflammation of mucous membranes, and often systemic consequences. The present process is particularly useful in the cultivation of various influenza viruses, including human, equine, swine and avian strains. examples

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5 on the production of typical A and B influenza virus types is described below.

Vaccine preparations prepared comprise any preparation of killed, live, attenuated or fully virulent influenza viruses that can be administered to produce or artificially enhance immunity to any influenza disease. In preferred embodiments, the vaccine comprises an aqueous suspension of the virus particles in ready-to-use form.

The cell cultures considered to be useful in carrying out the method of the present invention comprise any animal cell line or cell strain that can be infected by allowing replication of one or more given influenza virus strains. Although a number of 15 such cells are known and considered useful for the present process, particularly good results have been obtained with a particular cell line, namely a canine kidney cell line known as the Cutter Laboratories Dog Kidney (CLDK) cell line. The CLDK cell line is recognized by U.S. Pat.

20 Department of Agriculture for use in the manufacture of veterinary vaccines and is similar to the Madin Darby Dog Kidney Cell Line (ATCC NO. CCL 34) and the canine kidney cell line disclosed in U.S. Patent No. 3,616,203. A brief history and description of the particular master cell strain used for the cell line of the Examples follows, although it is to be understood that the present invention is applicable to any influenza virus susceptible cell culture.

30 CUTTER LABORATORIES DOG KIDNEY (CLDK) CELL LINE History

The pedigree of CLDK is established at Cutter Laborato- ries, Inc., Berkeley, California, from the kidney of a seemingly normal beagle dog from the University of California at Davis.

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The line is maintained on 0.5% lactalbumin hydrolyzate and 0.2% yeast extract in Earle's balanced salt solution plus 5% calf, lamb or horse serum and antibiotics and grown according to methods of J.S. Younger (Proc. Of Exp. Biol, and 5 Med., Vol. 85, 202 (1963)).

A frozen ampoule of the 142nd passage of this cell line is then planted in a 75 cm 2 (250 ml) Falcon flask, in tissue culture medium consisting of Earle's balanced saline Minimum Essential Medium (MEM) and 10% bovine-10 serum. The cells are grown as a subculture in the same manner and medium to produce the frozen master cell strain at passage 148.

An ampule of the master cell strain is thawed, planted and cultured in series as a subculture 20 times to obtain bottle cultures of the 168th passage. These cells are then frozen.

DESCRIPTION OF MASTER CELL STEMS fMCSl Number of serial subcultures from original tissue: 148 20 Freezing medium: Minimum Essential Medium (Eagle) in Earle's BSS with reduced bicarbonate (1.65 gm / liter) 80%; bovine fetal serum 10%, dimethyl sulfoxide 10%.

Viability: Approx. 75% (color exclusion).

Culture Medium: Minimum Essential Medium (Eagle) in Earle's 25 BSS with reduced bicarbonate (1.65 gm / liter) 90%; bovine serum 2-10%; antibiotics penicillin and streptomycin 100 U. or Tr / ml.

Thawed Growth Characteristics: A seed of 3 x 10 6 viable cells / ml grown in the above culture medium at 37 ° C in a closed system is multiplied 6-8 fold in 5 days.

Morphology: Epithelium-like.

Karyology: Chromosome frequency distribution 100 cells: 2N = 72.

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Cells 112469 69 53

Chromosomes 60 65 68 69 70 71 72 73 74 5 No marker chromosomes.

Sterility test: Free of mycoplasma, bacteria and fungi. species; Confirmed as being of the dog type by immunofluorescence experiments.

Virusmodtaqeliahed; Susceptible to influenza viruses, rabies virus, infectious bovine hepatitis, infectious canine hepatitis, canine virus, and possibly other viruses.

Protein hydrolyzing enzymes (proteolytic enzyme or protease) which have been found useful for the purposes of the invention include well-known proteases such as trypsin, chymotrypsin, pepsin, pancreatin, papain, pronase and carboxypeptidase, of which trypsin is a particularly preferred enzyme. The exact mechanism (s) by which a protease such as trypsin enhances influenza virus infectivity is not completely known. A possible mechanism is suggested in the above article by Klenk et al.

As described below, the amount of active protease required to enhance subsequent infectivity must be at least as large as to exclude the limitations of the one-step growth cycle, but in the case of confluent 25 monolayer cultures not sufficient to disassociate confluent cells from the surfaces of the tissue culture vessel (e.g., the inner surface of a rotating bottle).

In the case of the specific enzyme used in the following examples, it is preferred that the amount of the enzyme be in the range of 4 to 25 µg per day. in liquid tissue culture medium, preferably approx. 10 µg / ml.

VIRUSPROPAGERINGSMETODER

The present method of influenza virus propagation comprises the three general steps of infecting a portion of the cells in a liquid cell culture with influenza virus.

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8, the incubates incubate the cells in the presence of a proteolytic enzyme under conditions sufficient to ensure maximum cytopathic (CP) action and harvest the viruses from the culture. For vaccine preparation, there is associated a subsequent 5 step for modifying the harvested virus by known methods which result in a live, virulent or attenuated virus vaccine or a (inactive) virus vaccine. The vaccine may be available in dry form which can be mixed with a diluent, or in liquid, ready-to-use form. Suitable adjuvants may be added, as described below, to increase immunogenicity.

The protease may be added to an aqueous suspension cell culture or after the formation of a confluent monolayer culture. Examples of some of the different propagation methods are described below.

METHOD A - Infecting pre-prepared monolayers 1. Cells are grown for confluence in culture containers, e.g.

20 rotary bottles, Povitsky flasks, or Roux bottles using known cell culture growth media.

2. Prior to infection, the growth medium is removed from the cell monolayers.

3. The influenza virus working graft is dissolved in a growth medium having a pH of 6.6 to 6.8 and containing additional vitamins, non-essential amino acids, L-glutamine, dextrose and antibiotics.

4. An amount of diluent containing the virus is added to the cell monolayer in amounts of from 10% to 100% of the final harvest volume.

5. The infected monolayers are incubated at 34 ° C to 37 ° C for 1 35 to 72 hours.

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6. Protease, alone or in combination with the viral propagator, is added at a concentration that stimulates multiple cycle growth without producing detached cells. For trypsin, this optimal concentration is between 8 and 15 μ ^ / τύ.

5 7. The virus growth vessels are incubated again at 34 ° C to 37 ° C until maximum cytopathic effects are found. At this stage, the virus fluids are harvested.

8. Harvesting involves vigorously shaking the vials to remove the cells and transferring the fluids and cells to a sterile vial for further processing.

METHOD B - Infecting liquid cell suspension prior to formation of monolayer 1. Cells are removed from growth vessels using conventional methods.

2. Cells are concentrated by centrifugation, then resuspended in an amount 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 to 37 ° C) in a sterile closed container (e.g., an Erlenmeyer flask with 30 screw capsule) while mixed on a shaker with magnetic stirrer or rotated for 10 minutes to 4 hours. hours.

5. Aliquots of the cell virus suspension are placed in growth vessels (rotary bottles, Roux bottles, Povitsky flasks) 35 with the full volume of medium containing ingredients indicated in B-2 plus 5% fetal calf serum.

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6. Growth vessels are incubated at 34 ° C to 37 ° C until confluent monolayers are formed (2 to 4 days).

7. After monolayer formation, protease is added at a concentration which will stimulate multiple cycle growth without producing cell slurry. For trypsin, this optimal concentration is between 10 and 25 μg / ml.

8. Growth vessels are incubated at 34 ° C to 37 ° C until maximum cytopathic effects are found. Virus is then harvested.

9. Harvesting causes containers to be shaken vigorously to remove cells and transfer cells and fluids to a sterile container for further processing.

METHOD C - Infecting Liquid Suspension Culture 1. Cells adapted to a suspension culture are grown to an optimal point in a growth medium in suspension growth vessels.

2. Cells are centrifuged and resuspended in an amount of fresh medium containing additional vitamins, non-essential amino acids, L-glutamine, dextrose and antibiotics.

3. Influenza virus is then added and the culture incubated at 34 ° C to 37 ° C for 1 to 72 hours.

4. Calf fetal serum can be added and the culture suspension 30 is further incubated at 34 ° C to 37 ° C for 1 to 72 hours.

5. Protease is added at a concentration that will allow multiple cycle growth without producing a deleterious effect on the cells.

6. Incubation at 34 ° C to 37 ° C is continued until

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11 states maximum cytopathic effects at which time the fluids are harvested.

7. Harvesting involves transferring liquids to a sterile container 5 for further processing.

Specific examples of the use of the methods and media at issue here propagate selected strains of influenza viruses. Unless otherwise stated, 10 common tissue culture methods are used. As the production of both cells and media is known, it is not described in detail here.

Example 1 The A2 horse flu virus, designated the Miami strain, was originally isolated from a horse at the University of Miami. This virus is obtained from the University of Pennsylvania Medical School, where six passages are made in chicken embryos. The seventh passage is made and obtained from Lederle Laboratories. The strain is subjected to additional chicken fetal passage and 20 is used at passages 11-16.

The preferred method of tissue culture propagation of A2 strains herein involves infecting a young confluent monolayer of CLDK cells. Cells are planted in cylinders using Hank's Minimum Essential 25 Medium (MEMH) containing the following ingredients:

Bovine fetal serum 5-10%

Non-essential amino acids, 10 ml / liter (Gibco) L-glutamine, 10 ml / liter (Gibco) 30 Neomycin sulfate, 30,000 / xg / liter

Polymyxin B, 30,000 units / liter Mycostatin, 25,000 units / liter

The cells are usually confluent within 35 72 hours, at which time the media is poured out and the cells are infected. The inoculation medium contains A2 virus diluted

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12 for an Egg Infective Dose (EID50) titer of approx. 103 ° / hlL in MEMH supplemented with the following ingredients: 50% dextrose, 2.6 ml / liter 5 MEH vitamins, 30 ml / liter (Gibco)

Non-essential amino acids, 10 ml / liter (Gibco) L-Glutamine, 10 ml / liter (Gibco)

Neomycin sulphate, 30,000 jug / liter Polymyxin B, 30,000 units / liter 10 Mycostatin, 25,000 units / liter

Inoculation medium corresponding to 14% of the final volume is added to each cylinder and the vessels are incubated at 34 ° C to 35 ° C for 72 hours. At this point, the remaining 15 medium (86%) containing 12 µg / ml sterile trypsin (Sigma, 1: 250) solution (0.1 g / 100 ml) is added to each cylinder. The vessels are again incubated at 34 ° C to 35 ° C until maximum cytopathic effect (48-72 hours) is found, at which time the fluids are harvested. Harvesting vigorously shakes 20 all cylinders to remove all attached cells and transfer cells and virus fluids to a sterile charging container for further processing.

Harvest EID5o titers of A2 influenza virus grown using this technique are shown in Table 25 I. See also FIG. 1. A comparison is made with A2 virus grown by the same method, but without the addition of trypsin.

30 35

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Table I eid50 of

Equine influenza virus of the Miami strain grown in CLDK cells with and without trypsin 5 Amount of trypsin Titre (EID5o / ml) Fold increase (/ xg / ml) Feed Harvest with trypsin 10

No 102'6 106'2 ----

None ίο3'2 io6'2 ---- 5 102'9 108'3 126 5 ΙΟ2'9 108'1 19 15 5 102'6> 109'2> 1.00.0 10 102'9 109'2 1,000 10 ΙΟ2 '9 108'5 200 10 103'2 108'2 100 10 ίο3'2 1010'0 6.310 20 10 103/1 1014'5 199,526,232 15 ΙΟ3'1 108/9 50: 1

Incorporating trypsin into the growth medium gives a geometric mean increase of 3.2 log or 1711 times as many 25 virus particles / ml during the production of the Miami strain.

Example 2

A sample of a virulent type of Al equine influenza virus is obtained from the University of Pennsylvania Medical School.

30 The tribe, designated Pennsylvania (Al), is isolated from a horse and passed through chicken fetuses six times. The strain is subjected to further chicken fetal passage and used for tissue culture preparation at passages 12-17.

The preferred method for tissue culture propagation of the 35 Al strain is to infect a suspension of CLDK cells before monolayer formation. CLDK cells at a concentration of ca. 105'5 / ml, Pennsylvania virus strain at an EIB50 titer of 103'0 of 10 3 '° / ml to 105' ° / ml and Hank's Minimum

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Essential Medium (MEMH) supplemented with the ingredients listed below is incubated at 25 ° C while mixing on a magnetic stirrer in a closed sterile Erlenmeyer flask. The pH is maintained at 6.7-6.8 with 1N HCl during the 2-3 hour incubation period.

Supplemented MEMH

50% dextrose, 2.6 ml / liter MEM vitamins, 30 ml / liter (gibco) 10 Non-essential amino acids, 10 ml / liter (Gibco) L-glutamine, 10 ml / liter (Gibco)

Neomycin Sulfate, 30,000 / xg / liter Polymyxin B, 30,000 units / liter Mycostatin, 25,000 units / liter 15

Following this suspension incubation, 10 ml aliquots of the cell virus suspension are added to cylinders containing 1 liter of MEMH supplemented as mentioned above and containing 5% fetal calf serum. The cylinders are incubated at 34 ° C to 35 ° C, 20 until the monolayer is confluent (about 48 to 72 hours), then 20 ml of a sterile 1 mg / ml trypsin solution (Sigma 1: 250) is added to each cylinder. The cylinders are again incubated at 34 ° C to 35 ° C until maximum cytopathic effect is established (3-5 days). The virus fluids are harvested by shaking vigorously all the cylinders to remove remaining cells and transfer cells and fluids to a sterile container for further processing.

Harvest EIDsg titers of Al influenza virus grown using the technique of this invention are shown in Table II.

30 See also FIG. 2. A comparison is made with Al virus grown by the same method, excluding trypsin.

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Table II EID lake titers of

Horse influenza virus of the Pennsylvania strain grown in CLDK cells with and without trypsin 5 Amount of trypsin Titre (EIDgg / ml) Fold increase (pg / ml) Feed Harvest with trypsin 10

None 105'3 105'9 ----

None ΙΟ4'9 105'4 ---- 10 105'3 107'4 50 10 104'9 106.8 13 15 10 105/1 108'0 200 20 IO5 / 3 108'7 1,000 20 ΙΟ4'9 108 ' 5 631 20 105'0 107'1 25 20 ΙΟ4'9 IO8 / 3 398 20 20 103'2 109'2 3,162 20 ΙΟ3'2 107'5 63 20 103'2 108'2 316

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

Example 3 A human influenza virus strain designated B / Hong

King / 5/72 (BX-1) is obtained from The Center for Disease Control in Atlanta, Georgia. This is passed once through chicken eggs at the fetal stage and is frozen at -70 ° C as a graft virus.

The preferred method for tissue culture propagation 35 of the B / Hong Kong / 5/72 strain involves infecting a young confluent monolayer of CLDK cells similar to the monolayer of Example 1. The cells are grown as described in Example 1. The growth medium is removed from the cells and discarded. . The cells in =

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16 is then infected with virus dissolved in the inoculated medium set forth in Example 1 to an EID 103 '° "5' ° / ml. The inoculum consists of a volume equivalent to 33.3% of the final harvest volume. Containers are incubated at 34 ° C to 35 ° C for 40 to 5 48 hours, after which the remaining medium (66.7%) in containing 12 Mg / ml trypsin solution (0.1 g / 100 ml) is added to each vessel. Incubation at 34 ° C - 35 ° C is continued until maximum cytopathic effect (48-72 hours) is established, at which time the virus fluids are harvested. Harvest 10 causes vigorous shaking of all containers to remove cells and transfer cells and fluids to a sterile container for further processing.

Harvest EID5Q titers of B / Hong Kong / 5/72 influenza virus grown using this technique are shown in Table III.

15 See also FIG. 3. A comparison is made with this strain grown by the same method, but without the addition of trypsin.

Table III

Human influenza EID50 titers of B / Hong 20 Kong / 5/72 strain grown in CLDK cells with and without trypsin Amount of trypsin Titre (EID 50 / ml) Fold Increase 25 (gout / ml) Delivery Harvest with trypsin

No 103'2 106'0 ----

None ΙΟ5'7 106'4 ---- 30 8 ΙΟ3'0 108'7 316 8 ΙΟ2'0 109'0 631 8 ΙΟ4'5 109'5 1.995 8 103'5> 1010'2> 10,000 35 Incorporation of trypsin in the growth medium gives a geo-metric mean increase of 3.1 log or 1412 times as many virus particles during the production of the B / Hong Kong strain.

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Example 4

A strain of human influenza virus designated A / Texas / -1 / 77 is obtained from The Center for Disease Control in Atlanta, Georgia. This is passed once through fetal-stage chicken eggs and frozen at -70 ° C as a graft virus.

The preferred method for tissue culture propagation of the A / Texas / l / 77 strain is that described in Example 3.

Harvest EID50 titers of A / Texas / l / 77 human influenza virus grown using this technique are shown in Table IV.

A comparison is made with this strain grown by the same method, but without the addition of trypsin.

Table IV

15 EIDgg titers of human influenza virus of the A / Texas / 1/77 strain grown in CLDK cells with and without trypsin Amount of trypsin Titre (EID50 / 111I) Fold Increase 20 (Mg / ml) Delivery Harvest with trypsin

None ΙΟ5'2 105'2 ---- 8 105'1 108'9 5,012 25 8 104'1> 1010'2> 100,000 10 ΙΟ3'0 108'3 1,259 10 102'0 109'8 39,811

Incorporating trypsin with the growth medium provides a 30 geometric mean increase of 4.1 log or 12590 times as many virus particles / ml during A / Texas strain production.

Example 5 A human influenza virus strain designated A / USSR / 9Q / - 77 is obtained from The Center for Disease Control in Atlanta, Georgia. This is passed once through chicken eggs at the fetal stage and frozen at -70 ° C as a graft virus.

ICE

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The preferred method of tissue culture propagation of the A / USSR / 90/77 strain is the same as described in Example 3.

Harvest EID50 titers of A / USSR / 90/77 human influenza virus 5 grown using this technique are shown in Table V. See also Figs. 5. A comparison is made with this strain grown by the same method, but without the addition of trypsin.

Table V

10 Human influenza virus EIDgQ titers of the A / USSR / 90/77 strain grown in CLDK cells with and without trypsin Amount of trypsin Titre (EIDsg / ml) Fold Increase 15 (µq / τal) Feed Harvest with trypsin

None ΙΟ4'0 106'3 ---- 10 ΙΟ4'0 108'7 251 20 10 104'0 109'0 501 10 ΙΟ4'5 108'4 126

Incorporation of trypsin into the growth medium gives a geometric mean increase of 2.4 log or 251 times as many 25 virus particles / ml during the preparation of the A / USSR strain.

VACCINE PRODUCTION

Example 6 - Virus attenuation

Attenuation of the virus from harvested fluids of Example 1 is achieved chemically or by standard series passages including final dilution passage methods, using a sufficient number of passages in a susceptible cell culture until the virus is rendered non-pathogenic without loss of immunogenicity. A vaccine prepared in this way will stimulate an immune response in animals susceptible to disease without noticeably eliciting clinical symptoms.

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19 inches usually caused by the virulent substance. The propagation can be done in tissues which are the same or different from the tissues used in the preceding passage.

Example 7 - Virus Inactivation

The method is the same as described in Examples 1 and 2, but the harvested virus-saturated fluids are further worked up by inactivating with 0.1% concentration of formaldehyde (ranging from 0.05 to 0.2%) and the treated material in-10. cubed at 4 ° C for 10 to 14 days. Testing of the final inactivated virus preparation shows that it does not contain live virus. Known excipients, e.g. aluminum hydroxide, alum, aluminum phosphate, Freund's, or the adjuvants disclosed in U.S. Patent Nos. 3,790,665 and 3,919,411 may be added. The preferred adjuvant of the present invention and that used in the present vaccine is an acrylic acid polymer cross-linked with a polyallylsaccharide ("Carbopol® 934 P") which is the same as that described in the above patent writings.

Example 8 - Inactivated Virus Vaccine Preparation and Use

A 0.1 ml horse dose consists of 0.45 ml of the Pennsylvania (Al) strain, 0.45 ml of the Miami (A2) strain and 0.10 ml of the "Carbopol®" adjuvant. Equal portions of the inactivated vaccine strains obtained from Example 7 are mixed and 1 ml ali-quota is administered intramuscularly to 19 horses. No clinical disease or influenza symptoms were observed in 30 of the horses after vaccination. Antibody titers against both Al equine influenza and A2 equine influenza are obtained on blood sera in all animals at 2-, 4- and 8-week subsequent inoculation.

These are compared to pre-inoculation levels (Table VI) using standard haemagglutination inhibition 35 assays (DIAGNOSTIC PROCEDURES for Viral and Rickettsial Infections, 4th Edition; Lennette and Schmidt ^ pages 665-66

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20 (1969). American Public Health Association, N.Y., N.Y.

10019).

Table VI 5

Antibody Reactions (Haemagglutination Inhibition)

All-Horse Flu A2 Horse Flu 10__

Horse No. Pre. 2 weeks 4 weeks *) 8 weeks Pre. 2 weeks 4 weeks *) 8 weeks 15 2 <8 8.192 8.192 8.192 <8 512 128 256 11 <8 128 256 128 <8 64 1.024 64 13 <8 128 256 2.048 <8 256 256 1.024 19 <8 256 64 64 <8 128 128 64 20 21 <8 128 64 512 <8 64 64 512 23 <8 256 128 128 <8 512 512 128 29 <8 128 32 1,024 <8 256 64 128 32 <8 1,024 256 2,048 <8 64 64 128 37 < 8 128 32 512 <8 64 32 64 25 38 <8 1,024 512 1,024 <8 512 256 64 40 <8 256 128 1,024 <8 128 128 128 55 <8 512 512 8.192 <8 512 256 256 61 <8 512 64 256 < 8 1,024 128 128 68 <8 128 32 128 <8 512 256 256 30 79 <8 64 128 2,048 <8 256 128 128 121 <8 <8 32 512 <8 1,024 256 64 125 <8 256 32 128 <8 256 128 128 128 <8 512 128 512 <8 256 128 256 129 <8 256 128 2,048 <8 512 512 256 35 *) Renewed administration to increase power.

As Table VI shows, antibodies against both viral antigens are developed in horses receiving inoculations.

40 These vaccinated animals are immune to equine influenza, with antibody titers greater than 1:20 to A2 and 1:60 to Al being recognized by the National Veterinary Services Laboratories of the U.S. Department of Agriculture as being protective.

45 Clinical tests in 420 horses of different breed and age show that the vaccine is safe and does not cause it

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21 adverse reactions following intramuscular inoculation.

Example 9 - Use of attenuated virus vaccine 5 ml of the live attenuated A2 equine influenza vaccine strain of Example 6 is administered intranasally to three horses. No clinical disease or flu symptoms were observed in any of the horses after vaccination. Vaccine strain antibody titers obtained on blood sera in all animals by inoculation after 1, 2 and 4 weeks and compared 10 with the pre-inoculation level using standard haemagglutination inhibition (HAI) assay.

Table VII

ANTISTOFREACTION fHAIi - A2 Horse Flu 15

Horse No. Pre 1 uae 2 hrs 4 hrs 19 <8 128 256 128 23 8 64 128 128 20 61 <8 128 256 128

Once again, the antibody titers obtained are greater than necessary for protection.

It should be noted that the present invention relates to both a novel influenza culture system and the use of the medium for the preparation of a novel influenza vaccine. The liquid cell culture system used in the present invention involves the use of susceptible cells, influenza viruses, a nutrient medium and a proteolytic enzyme, but unlike known systems (e.g., Tobita et al), it does not require the use of agar, which reduces the system to a semi-solid state. Thus, the exclusion of agar allows for large-scale production of viruses in a generally known manner and results in the preparation of virus fluids with sufficiently high titers to produce vaccines.

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The influenza vaccine preparation itself comprises effective amounts of one or more strains of given influenza virus particles and a pharmaceutically acceptable carrier, the final product, preferably in aqueous form, being substantially free of reactive proteins, e.g., egg proteins. As used herein, the term substantially free of egg protein means that the only possible source of egg protein in the vaccine preparations is the graft virus, which is diluted> 1: 100,000.

The vaccines are administered to animals in various ways, e.g.

intramuscular, intravenous, subcutaneous, intratracheal, intranasally or by aerosol spray, and the vaccines are intended for beneficial use in various animals, including human horse, worm and bird groups.

The virus preparations prepared according to the present invention can be diluted with water to control their potency, and they may be added with stabilizers, e.g. sucrose, dextrose, lactose, or other non-toxic constituents. The virus preparations may be dried by freeze-drying for storage purposes or for subsequent formulation in attenuated vaccines, or they may be chemically inactivated to produce killed virus vaccines.

Claims (5)

A method for producing an influenza virus vaccine, wherein method infects cells with an influenza virus, the virus is propagated in the cells, then the virus is isolated, and from the isolated virus a vaccine is produced, characterized in that a portion of the cells in a liquid cell culture is infected with an influenza virus and the infected culture is incubated in the presence of a proteolytic enzyme whose concentration is sufficient to cause growth for several cycles without causing detachment of cells in cases where a confluent cell culture is used. monolayers. Process according to claim 1, characterized in that trypsin, chymotrypsin, pepsin, pancreatin, papain, pronase and carboxy-peptidase are used as proteolytic enzyme. Process according to claims 1 and 2, characterized in that as a proteolytic enzyme, trypsin is added in an amount of from 4 to 25 µg per day. of liquid cell culture. Method according to claims 1-3, characterized in that a horse influenza virus or human influenza virus is used as virus. 5. A method according to claims 1-4, characterized in that dog kidney cells are used as cells in the liquid cell culture.