CA1122527A

CA1122527A - Influenza vaccine production in liquid cell culture

Info

Publication number: CA1122527A
Application number: CA350,366A
Authority: CA
Inventors: Karen K. Brown; Richard C. Stewart
Original assignee: Cutter Laboratories Inc
Current assignee: Bayer Corp
Priority date: 1979-05-15
Filing date: 1980-04-22
Publication date: 1982-04-27
Also published as: AR220838A1; ES8104405A1; ATE16454T1; YU41923B; AU5824480A; RO79525B; DK212580A; NO801443L; EP0019218A2; RO79525A; EP0019218B1; JPH0124769B2; DK154749B; DE3071225D1; EP0019218B2; DK154749C; EP0019218A3; IL60063A0; YU126580A; AU535804B2

Abstract

ABSTRACT OF THE DISCLOSURE
Infectivity and replication of influenza viruses in successive numbers of cells of the same liquid cell culture is assured by including a protein hydrolyzing enzyme in the culture during virus incubation. Technique overcomes "one-step growth cycle" of virus and allows commercial influenza vaccine pro-duction from liquid cell cultures instead of from more costly embryonated chicken eggs. Resulting vaccine is thus substanti-ally free of egg proteins.

Description

~Z~;~7 This disclos~re is concerned generally ~ith a novel influenza virus propagation medium and specifically wit~ the use of that me~ium for influenza vaccine prcduction.
Influenza vaccines have ~een in use since the early l9a0's for human vaccination and since the late 1960's for equine vaccination. All influenza vaccines pre~ently used are made by growing the vaccine virus strains in embryonated chicken eggs. The resulting virus strains are then used for making live virus vaccines or further processed to make killed virus vaccines.
It is generally known by virologists that in~luenza viruses grow to a very limited degree in cell cultures. The growth is referred to as a "one-step growth cycle"; that is, only the originally infected cells replicate viruses. This phenomenon is descri~ed, for example, by Davis et al, rlIcRoBIo-LOGY, Harper and Rot~ Publishers, Chapter 4a, pp. 113~-39 (1968~.
Since the viruses of the originally infected cells are unable to infect successive numbers of cells in the same cell culture, the resulting yields are far too low to be useful in the prepa-ration of virus vaccines. Thus, liquid cell cultures have notbeen used for commercial production of influenza virus vaccines.
Embryonated chicken eggs are used to produce viruses with titers sufficiently high enough to be useful in the pre-paration of vaccines. Unfortunately, chick embryo-grown viruses usually require concentration, and, in the case of human vaccines, also require some form of purlfication to reduce toxic reactions due to the undesira~le egg proteins. The use of the eggs for vaccine production is time consuming, labor ~12~S~

intensive, requires relatively high material costs, and the yield from one egg is commonly only enough to produce vaccine for about one to 1.5 doses. mhus~ the manufacture of millions of doses requires innoculating and harvesting millions of embryonated eggs.
Recently, it has ~een noted that a wide variety of influenz~ A viruses comprising human, equine, porcine, and avian strains, grew productively in an established line of canine kidney cells under an overlay medium containing trypsin and formed well-defined plaques regardless of their prior passage history. See the article ~y K. To~ita et al, "Plaque Assay and Primary Isolation of Influenæe A Viruses in an ~stab-lished Line of Canine riidney Cells (~CK~ in the presence of Tr~psin", Med. Microbiol. Immunol. 162, ~-14 tl975~. See also the article by ~Tans-Dieter Klenk et al,"Activation of ~nfluenza A Viruses ~y Trypsin ~reatment", Virology 68, 426-439 (1975).
It should ~e noted that in the above reports the effects of trypsin on influenza virus propagation were observed in semi-solid cultures in plaque ormation assays and isolation tech-niques, neither of which are concerned with liquid cell cultuxesor the large scalleor commercial propagation of in1uenza viruses for vaccine production. The term liquid cell culture used herein describes the in vitro gro~th of cells and propaga-tion of virus in a chemically defined liquid medium.
Quite surprisingly, we have now found that proteolytic enzymes can also he used in liquid cell cultures to facilitate infect;on of successive num~ers of cells in the same cell culture. By thus overcoming the limitations of the "one-step ; - 2 -~, ~ 25Z7 growth cycle" of part liquid cell culture techniques, it is possible to achieve an influenza virus yield which is in the range of about 1,000 to 10,000 fold greater than non-protease treated cultures. This makes feasible the use of liquid cell culturing techni~ues for the commercial production of influenza vaccines, thereby avoiding the disadvantages associated with using embryonated chicken eggs. Details of our culturing medium, virus propagation techniques, and vaccine production and use methods are disclosed herein.
Our influenza virus propagation medium comprises a cell culture capa~le of being infected with an influen~a virus, an influenza virus, and a protein-hydrolyzing enzyme, the amount of enzyme being sufficient to overcome the one-step growth cycle of the virus. Our virus propagation technique comprises the steps of inoculating or infecting a liquid influenza virus cell culture with the influenza viruses, incuhatin~ the inocu-late in the presence of a protein-hydrolyzing enzyme under con-ditions sufficient to assure maxi~um virus growth (or maximum cytopathic effect), and harvesting the virus. Infection of the cells with the virus may occur before or after cell monolayer formation or, alternatively, by simply infecting a liquid suspension of the cells. Our vaccine production method includes the subsequent step of killing the harvested virus or attenua-ting by further cell culture passage for vaccine use. ~specially preferred embodiments involve the use of the protease trypsin in conjunction ~ith a dog kidney cell line to propagate an~ of several types of ïnfluenza viruses.
Accordingly the present invention provides a method ` ~
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~2Z~Z7 of preparing an influenza virus vacc.ine which comprises the steps of:
(a) infecting a portion of the cells of a liquid cell culture with an influenza virus;
(b~ incubating the infected culture in the presence of a pro-teolytic enzyme under conditions sufficient to assure further infection and replication of the virus in cells other than the originally infected cells;
(c) harvesting the virus from the culture, and;
(d) preparing a vaccine from the harvested virus.
The present invention also provides an influenza vaccine preparation comprising an effective amount o influenza virus particles whenever prepared by the above process and a pharmaceutically acceptable carrier, the preparation being substantially free of egg proteins.
The present invention also provides a method of pro-pagating influenza viruses in a liquid cell culture ~edium which comprises infecting a liquid cell culture with an influ-enza virus, incu~ating the infected culture in the presence of a proteolytic enzyme under conditions sufEicient to assure virus propagation, and terminating the incubation.
The present invention also provides an influen2a virus-inoculated liquid cell culture comprising influenza viruses, a cell culture capable of hosting propagation of the viruses, and a proteolytic enzyme, the amount of the enzyme being sufficient to assure infectivity and replicatïon of the viruses in substantially all the cells of t~e l;quid cell culture.

-. -- 4 25~7 Figures l-S are bar graphs illustrating the increases in titer achieved when the indicated virus strains were incu-bated in a liquïd cell culture in the presence of various amounts of a typical protease, trypsin. P~esults are reported as geometric ~ean titers.
The influenza virus and vaccine production techniques of this disclosure conte~plate the use of influenza vaccine viruses. As used herein, the term influenza virus includes any viruses capable of causing a febrile disease state in animals (including man~ marked by respiratory symptoms, inflammation of mucous membranes and often systemic involvement. The medium and methods of this disclosure are especially useful in the production of a variety of influenza viruses, including human, equine, porcine, and avian strains. Examples of the production of typical Type A and B Influenza viruses are described below.
TFle vaccine preparations contemplated by this dis-closure include any preparation of killed, livin~ attenuated, or living fully virulent influenza viruses that can he adminis-tered to produce or artificially increase immunity to any influenza disease. In preferred embodiments, the vaccine com-promises an aqueous suspension of the virus particles in ready to use form.
The cell cultures contemplated as being useful in carrying out the principles of this disclosure include any animal cell line or cell strain capable of being infected by, and which allows t~le replicatîon of, one or ~ore given influenza virus strains. Although a num~er of sucFI cells are known and thought to ~e useful for the techniques disclosed herein, we ~122~i2~

have had especially 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 United States Department of Agriculture for use in producin~ veterinary vaccines and is similar to the ~ladin Darby Dog ~idney Cell Line (ATCC No. CCL 34 I and to the dog kidney cell line descrihed in United States Patent No. 3,616,2~3 to ~. Bro~m. ~ hrief history and description of the specific master cell stock used for the cell line of t~e Examples follo~s alt~ough it should be understood that the techniques disclosed herein are thought to be useful with any influenza virus susceptible cell culture.
CUT~ER LABORATORI~S DOG KIDNEY (CLDK~ CELL LINE

_ HISTORY
The parent line of CLDK was initiated and established at Cutter Laboratories, Inc., Berkele~7, California, from the kidney of an apparently normal beagle dog obtained from the University of California at Davis. The line was maintained on 0.5% lactalbumin hydrolysate and 0.2~ yeast extract in ~arle's balanced salt solution plus 5% calf, lamb, or horse serum and antibiotics, cultivated by the methods of J. S. Younger (Proc.
of Exp. Biol., and Med., v 85, 202~.
A frozen ampoule of the 142nd passage of this cell line was subsequently planted in a 75 cm2 ~250 ml) falcon flask, in tissue culture medium consisting of ~arlels balanced salt solution Minimum Essential Medium (~ and lQ% fetal bovine serum. The cells ~ere suficultured in the sa~e manner and ~edium to prepare the frozen Master cell stock at passage 148.
An ampoule of the Master cell stock was thawed, planted - :
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25~7 and serially subcultured 2Q tïmes to o~,tain bottle cultures of the 16~th passa~e. These cells were then frozen.
DESC~IP~ION'OF ~ASTE~ CELL STOC~ (MCS) -Number of Serial Subcultures from missue of Origin: 14~
Freeze Medium: Minimum essential medium (Ea~le) in Farle's BSS with reduced bicarbonate (1.65 g~/L~ ~0~; fetal bovine seru~ 10%; dimethyl sulfoxide lOPo.
Viability: Approximately 75% (dye exclusion~.
Culture ~Sedium: Minimum essential medium ~Ea~lel in Earle's BSS with reduced bicarbonate (1.65 ~m/L 9Q%; fetal bovine serum

2-10%; antibiotics penicillin and streptomycin 100 ~. or y/ml.
Growth Characteristics-of Thawed Cells: An inoculum of 3 x 106 viable cells/ml cultured in the above culture medium at 37 C
in a closed system, multiplies approximately 6-~ fold in 5 days.
Morphology: Epithelial-like.
Karyology: Chromosome Freauency Distribution 10Q Ce]ls:
2M - 72.
Cells: l l 2 4 6 9 69 5 3 Chromosomes: 60 65 6~ 69 70 71 7?~ 73 7 No marker chromosomes.
Sterility Tests: Free of ~Sycoplasma, bacteria and fun~i.
Species: Confirmed as canine bv immunofluorescence test.
-Virus Susceptibilitv: Susceptible to influenza viruses, rabies .
virus, infectious bovine rhinotracheitis, infectious caninehepatitis, canine distenper virus, and possibly other viruses.
The protein ~ydrolyzing enzv~es ~proteol~tic enzyme or protease~ conte~plated as be;n~ useful for purposes of this disclosure include well known proteases such as trypsin, ~lZ~5Z~7 chvmotrypsin, pepsin, pancrea-tin, papain, pronase, carboxypep-tidase, and the like, with trypsin being an especially preferred enzyme. The exact mechanism(s~ ~y which a protease such as trypsin enhances the influenza virus infectivity is not fully knot~n. One possible mechanism has ~een suggested in the above cited article hy ~lenk et al.
AS described below, the amount of active protease required to enhance successive infectivity should be at least enough to overcome the limitations of the one-step grot~th cycle but, in the case of confluent ~onolayer cultures, not so much as to cause a sloughing of confluent cells from the surfaces of the tissue cultivation vessel ~i.e. t~e inner surface of a roller bottle~. In the case of the specific enzyme used in the examples below, we prefer that the amount of the enzyme be in the range of a~out ~ to 25 micro~rams per ml ~yg/ml~ of liquid tissue culture medium, preferably about 10 ~ g/ml.

VIRUS PP~OPAGATION METHOD~
The influenza virus propagation method of this disclo-sure comprises the three general steps of infecting a portion o~ the cells of a liquid cell culture with the influenza virus, incubating the cells in the presence of a proteolytic enz~e under conditions sufficient to assure maximum cytopathic (CP) effect, an~ harvesting the viruses from the culture. Vaccine preparation comprises the subsequent ste of modifying the harvested virus by knot~n techniques to result in a live virulentr attenuated, or killed cinactive~ vaccine preparation. The vaccine may ~e availa~le in dry form, to ~e mi~ed ~ith a dilu-ent, or in liquid, ready to use form. Suitable adjuvants may ~.2;Z ~;27 be included, as descri~ed ~elo~, to enhance i~munogenicity.
The protease may be added to an aqueous suspension cell culture or ~efore or after formation o~ a con1uent mono-layer culture. Examples of some of tlie various propagation methods are descri~ed ~elow.
~T~OD A - Infection o~ ~re-formed ~onolayers 1. Cells are grown to confluency in culture containers such as roller bottles, Povitsky flas~s, or Roux bottles utilizing cell culture growth media kno~m to the art.
2. Prior to infection, the growth medium is removed from the cell monolayers.

3. The influenza virus working seed is diluted in gro~th 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 the diluent containing virus is added to the cell monolayer in quantities ranging from la% to 100~ of the final harvest volume.

5. The infected monolayers are incubated at 34 - 37 C for l to 72 hours.

6. Protease, alone or in com~ination with the virus propagation media, is added at a concentration ~hich will stimulate multiple cycle ~rowth without producing cell slough. For trypsin, this optimum concentration is ~etween a~out 8 and 15 ~ g/ml.

7. The virus gro~t~ containers are once again incu~ated at 3g - 37 C untLl maximum cytopathic effects are ohserved. At this point the virus fluids are harvested.

8. ~arvest involves shaking the containers vigorously to remove _ ~ _ ~L~225:27 cells and transferring fluids and cells to a sterile container for further processing.
~ETIIOD E - ~nfection of Li~uid Cell Suspension Prior to Monolayer Formation 1. Cells are removed from growth containers usin~ conventional procedures.
2. Cells are concentrated by centrifugatîon, then resuspended in a quantity of fresh growt~ medium containing additional vitamins, non-essential amino acids, L-glutamine/ dextrose, and antibiotics.
3. Influenza virus i5 then added to this concentrated cell suspension.
4. The cell ~irus suspension is incubated C25 C - 37 C~ in a sterile, closed container Csuch as a screw-cap ~hrlenmeyer flask) while being mixed on a magnetic type stirrer or rotary shaker for 10 minutes to ~ hours.
5. Aliquots of the cell virus suspension are placed into growth containers (roller ~ottles, Roux bottles, Dovitsky flasks~ with the full volume of media containing ingredients indicated in B-2 plus 5% fetal calf serum.
6. Growth containers are incubated at 34 - 37 C until conflu-ent monolayers are formed (approximately 2-4 days~.
7. After monolayers have formed, protease is added at a concen-tration which will stimulate multiple cycle growth without produc-ing cell slough. For trypsin, this optimum concentration is between 10 and 25 ~ug/ml.
8. Growth containers are incubated at 34 - 37C until maximum cytopathic effects are observed. Virus is then harvested.

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~12Z527

9. Harvest involves shaking con-tainers vigorously to remove cells and transferring cells and fluids to a sterile container for fur-ther processing.
METHOD C - Infection of Liquid Suspension Culture 1. Cells adapted to suspension culture are grown to an optimum count 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 for 10 minutes to several hours. ~`
4. Fetal calf serum may be added and the culture suspension is further incubated at 34 - 37 C for 1 to 72 hours.
5. Protease is added at a concentration which will allow multiple cycle growth without producing a detrimental effect on the cells.
For trypsin, the optimum concentration is between 4 and 25 pg/ml.
6. Incubation at 34 - 37 C is continued until maximum cyto-pathic effects are observed at which time fluids are harvested.
7. Harvest involves transfer of fluids to a sterile container for further processing.
Specific examples of the use of our techniques and media for the propagation of selected strains of infLuenza viruses follow. Unless otherwise indicated, we used conventional tissue culturing techniques known to the art. Since, both the cell and medium preparation techniques are well known, they are not des-cribed here in detail.
_ AMPLE 1 The A2 Equine Influenza Virus, designated Miami strain, ` ~Z;~S~7 was originally isolated from a horse at the University o~ Miami.
This virus was obtained from the University of Pennsylvania Medical School where six passages were made in chick embryo. The seventh passage was made at and obtained from Lederle Labora-tories. The strain under-went further chick embryo passage and was used at passages 11 - 16.
The present preferred method of tissue culture propaga-tion of the A2 strain involves infection of a young, confluent monolayer of CLDK cells. Cells are planted in roller bottles, using Hank's Minimum Essential Medium (MEMH) containing the fol-lowing ingredients:
Fetal Bovine Serum, 5 - 10%
Non-Essential Amino Acids, 10 ml/Q (Gibco) L-Glutamine, 10 ml/Q (&ibco) Neomycin Sulfate, 30,000 mcg/Q
Polymyxin B, 30,000 units/Q
Mycostatin, 25,000 units/Q
The cells are usually confluent with 72 hours at which time the medium is poured off and the cells are infected. The inoculating medium contains A2 virus diluted to an Egg In~ective Dose50 (EID50) titer of approximately 103-/ml in MEMH supple-mented with the following ingredients:
50% Dextrose, 2.6 ml/Q
MEM Vitamins, 30 ml/Q (Gibco) Non-Essential Amino Acis, 10 ml/Q (Gibco) L-Glutamine, 10 ml/Q (Gibco) Neomycin Sulfate, 30-,000 mcg./Q
Polymyxin B, 30,000 units/Q

.. .
.

, :- ~ " . .. : : ~, :
" . ,. .. ~ , . . :

~2;~S27 Mycostatin, 25,000 units/Q
Inoculating 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, the remaining medium (86%) containing 12 ~g/ml of sterile trypsin (Sigma, 1:250) solu-tion (0.1 g/100 ml), is added to each roller bottle. Containers are again incubated at 34 - 35 C until maximum cytopathic effect is observed (48 - 72 hours) at which time the ~luids are harvest-ed. Harvest involves vigorously shaking each roller bottle to remove any attached cells and transferring cells and virus fluids to a sterile batching container for further processing.
Harvest EID50 titers of A2 Influenza Virus grown using this technique are shown in Table l. Also, see Figure 1. A
comparison is made with A2 virus grown by the same method but without adding trypsin.
TABLE l MIAMI STRAIN EQUINE INFLUENZA VIRUS
GROWN IN CLDK CELLS WITH AND WITHOUT TRYPSIN

Amt. TrypsinTiter ~EID 0/ml) Fold Increase (lug/ml) Input~arvestWith Trypsin None lo2.61o6.2 _ __ None 103.21o6.2 102-91o8.3 126 102-9 108-l 79 102-6>109-2 >1,000 lo2.9lo9.2 1,000 lo2.91o8.5 200 103.21o8.2 100 ..

~2Z5Z7 MIAMI STRAIN EQUINE INF~UENZA VIRUS
GROWN IN CLDK CELLS WITH AND WITHOUT TRYPSIN

Amt. Trypsin Titer (EIDs0/ml) Fold Increase (Jug/ml) Input HarvestWith Trypsin 103.2 1ol0.06,310 103.1 1014 5199,526,232 103.l 1o8.9 501 Incorporation of trypsin into the growth medium produc-ed a geometric mean increase of 3.2 logs or 1711 times as many virus particles/ml during production of the Miami strain.

A sample of virulen~ type Al Equine Influenza virus was obtained from the University of Pennsylvania Medical School.
The strain, designated Pennsylvania (Al), has been isolated from a horse and passaged in chick embryo six times. The strain has undergone further chick embryo passage and is being used for tissue cultureproductionat passages 12-17.
The preferred method of tissue culture propagation of the Al strain involves infection of a suspension of CLDK cells prior to monolayer formation. CLDK cells at a concentration of approximately 10 /ml, Pennsylvania strain of virus at an EID50 titer of 103-/ml to 10 /ml, and Hank's Minimum Essential Medium (MEMH) supplemented with the ingredients listed below are incubat-ed at 25 C while being mixed on a magnetic stirrer in a closed, sterile Ehrlenmeyer flask. The pH is maintained at 6.7 - 6.8 with I N HCI during the 2 - 3 hour incubation period.

, ~2~5Z7 Supplemented MEMH
50% Dextrose, 2.6 ml/Q
MEM Vitamins, 30 ml/~ (Gibco) Non-Essential Amino Acids, 10 ml/~ ~Gibco) L-Glutamine, 10 ml/Q ~Gibco) Neomycin Sulfate, 30,000 meg./Q
Polymyxin B, 30,000 units/Q
~Iycostatin, 25,000 units/Q
After this suspension incubation, 10 ml aliquots of the cell virus suspension are added to roller bottles containing 1 liter of ME~IH supplemented as listed above and containing 5% Fetal Calf Serum. The roller bottles are incubated at 34 - 35 C until the monolayer is confluent (approximately 4S -72 hours) after ~hich 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 har~ested by vigorously shaking each roller bottle to remove cells which remain attached and transferring cells and fluids to a sterile container for further processing.
~larvest EID50 titers of the Al Influen~a virus grown using this technique are shown in Table 2. Also, see Fig~lre 2. A comparison is made with Al virus gro~n by the same method e~clucling trypsin.

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~ZZ5~

PENNSYLVANIA STRAIN EQUINE INFLUENZA VIRUS
GRO~YN IN CLDK CELLS WITII AND WITHOUT TRYPSIN
Amt. TrypsinTiter (EID 0/ml) Fold Increase (~g/ml) Input HarvestWith Trypsin None 105.3 105.9 None 104.9 105.4 105.3 107~4 50 104.9 1o6.8 13 105.l 1o8.0 200 105~3 1o8.7 1,000 104~9 1o8.5 631 105. lo7.l 25 104.9 1o8.3 398 103.2 109.2 3,162 1o3.2 107~5 63 103.2 1o8.2 316 Incorporation of trypsin into the gro~th medium produced a geometric mean incrcase o 2.3 logs or 187 times ns m~ny vlrus particles/ml during production of the Pennsylvania strain.
EXAhIPLE 3 A strain of ~luman Influenza virus designated B/Hong Kong/5/72 (BX-l) 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 working 10seed virus.
The preferred method of tissue culture propagation of the B/Hong ~L~2Z~7 Kong/5/72 strain involves infection of a young confluent monolayer of CLDK
cells similar to that in Example 1. Cells are grown as described in Example 1. Growth medium is removed from cells and discarded. Cells are then infect-ed with virus diluted in the inoculating medium listed in Example 1 to an EID50 titer of approximately 103- 5'0/ml. The inoculum consists o~ a volume equivalent to 33.3% of the final harvest volume. Containers are incubated at 34 35 C for 40 - 48 hours after which the remaining mediu~ ~66.7%) con-taining 12 ~g/ml of trypsin solution ~0.1 g/100 ml) is added to each container.
Incubation at 34 - 35 C is continued until the maximum cytopathic effect is observed ~48 - 72 hours) at which time the virus fluids are harvested. Har-vest involves vigorously shaking each container to remove cells and trans-ferring cells and fluids to a sterile container for further processing.
Harvest EID50 titers of B/Hong Kong/5/72 Influenza virus gro-~n using this technique are shown in Table 3. Also, see Figure 3. A comparison is made with this strain grown ~y the same method but without adding trypsin.

HUMAN INFLUENZA VIRUS GROWN IN
CLDK CELLS WITH AND WITHOUT TRYPSIN
Amt, Trypsin Titer ~EID50/ml)Fold Increase ~g/ml) Input~larvestWith Trypsin None 103.2 1o6.0 None 105~7 1o6.4 8 103' loS.7 316 8 102' 109' 631 8 104~5 109.5 1,995 8 103.5 >1ol0.2 ~10,000 Incorporation of trypsin into the gro~th mediuml produces a geometric mean increase nf 3.1 logs or 1412 times as many virus particles during pro-.

.-, :
.

l~L22r9:27 duction of the B/Hong Kong Strain.

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 chicken eggs and frozen at -70 C as working seed virus.
The preferred method of tissue culture propagation of the A/Texas/
1/77 strain is that described in total in E~ample 3.
Harvest EID50 titers of A/Texas/1/77 Human Influenza virus grown using this technique are shown in Table 4. Also, see Figure 4. A comparison is made with this strain grown by the same method but without adding trypsin.

HUMAN INFLUENZA VIRUS GROWN IN
CLDK CELLS WITH AND WITHOUT TRYPSIN

Amt. Trypsin Titer (EID50/ml)Fold Increase ~g/ml) Input HarvestWith Trypsin None --- 105.2 8 1o5~1 1o8.9 5,012 8 1o4~1 ~1olO.2~100,000 103~ lo8.3 1,259 1o2.0 1o9.8 39,811 Incorporation of trypsin with the growth medium produced a geometric mean increase of 4.1 logs or 12590 times as many virus particles/ml during production of the A/Texas strain.

A strain of Human Influenza virus designated A/USSR/90/77 was re-ceived from The Center for Disease Control in Atlanta, Gcorgia. This was passaged once in embryonated chicken eggs and frozen at -70 C as working .. : .:, . ~ -:

%~

seed virus.
The preferred method of tissue culture propagation of the A/USSR/90/
77 strain is that described in total in Example 3.
Harvest EID50 titers of A/USSR/90/77 ~luman Influenza virus grown using this technique are shown in Table 5. Also, see Figure 5. A comparison is made with this strain grown by the same method but without adding trypsin.

HUMAN INFLUENZA VIRUS GROWN IN
CLDK CELLS WITH AND ~ITHOUT TRYPSIN
Amt. Trypsin Titer ~EID5 /ml) Fold Increase ~g/ml) Input ~arvest With Trypsin None 104' lo6.3 104~ 10~7 251 104~ 1oC~,0 501 104~5 1o8.4 126 Incorporation of trypsin into the growth medium produced a geometric mean increase of 2.4 logs or 251 times as many virus particles/ml during pro-duction of the A/USSR strain.
VACCrNE PREPARATION
EXA~lPLE 6 - VIRUS ATTENUATION
~ttcnuation of the virus from harvested fluicls of F.~ample 1 is accom-plished chemically or by standard serial passages including terminal dilution passage techniques wherein a sufficient number of passages in a susceptible cell culture is employecl until the virus is rendered non-pathogenic without loss of immunogenicity. A vaccine prepared in these manners will stimulate an immune response in animals susceptible to disease without producing the clinical symptoms normally due to the virulent agent to any significant degree.
The propagation can be done in the same or different tissues as those employed in the preceding passage.

Z~2~

The technique is similar to that described ln ~xamples 1 and 2 but the harvest viral laden fluids are -further processed by inactivation with 0.1%
concentration of formaldehyde ~range 0.05 to 0.2%) and the treated material is incubated at 4 C for 10 to 14 days. Testing of the ~inal inactivated viral preparation showed it to be free from live virus. Adjuvants known to the art, such as aluminum hydroxideJ alum, aluminum phosphate, Freund's or those described in United States Patent Nos. ~,790,665 and 3,919,411 may be added. The preferred adjuvant of this disclosure and that used in our vaccine is an acrylic acid polymer crosslinked with a polyallylsaccharide ~Carbopol 934 P*) similar to that described in the above patents.
EXAMPLE 8 - INACTIVATF.D VIRUS VACCINE
PREPARATION AND USE
A 1.0 ml equine dose consists of 0.45 ml of the Pennsylvania ~Al) strain, 0.45 ml of the kliami ~A2) strain and O.10 ml of the Carbopol adjuvant.
Equal parts of the inactivated vaccine strains obtained from Example 7 l~ere mixed and 1 ml aliquots administered to 19 horses by the intramuscular route.
No clinical disease or s)rmptoms of influenza were noted in any of the horses a~tcr vaccination. Antibody titers a~ainst both Equine In1uenza Al and 2n Equ;nc In~luenza A2 wcre obtain~d on blood sera of all animals at 2, ~, and 8 weeks following inoculation. These are compared to the pre-inoculation levels (Table 6) using the standard haemagglutination inhibition test (DIAGNOSTIC
PROC~DURES for Viral and Rickettsial Infections, Fourth Edition; Lennette and Schmidt, pp. 665-66 (1969). American Public Health Association, N.Y., N.Y.
10019) .

*Trade mark 1~22~7 . . ~
ANTIBODY RESPONSES ~HAE~AGGLUTI NATION IN~IIBITION) Equine Influenza Al Equine Influenza A2 ~lorse No. Pre. 2 wk. 4 wk.* 8 wk. Pre. 2 wk. _ 4 wk.* 8 wk.
2 <88,192 8.192 8,192 ~8 512 128 256 11 <8128 256 128 <864 1,024 64 13 ~8128 2562,048 <8256 256 1,024 19 <8256 64 64 <8128 128 64 21 <8128 64 512 <864 64 512 23 <8256 128 128 <8512 512 128 29 <8128 321,024 <8256 64 128 32 <81,0242562,048 <864 64 128 37 <8128 32 512 ~864 32 64 38 <81,0245121,024 ~8512 256 64 <8256 1281,024 <8128 128 128 <8515 5128,192 <8512 256 256 61 <8512 64 256 <81,024 128 128 68 <8128 32 128 <8512 256 256 79 <864 1282,048 <8256 128 128 121 ~8c8 32 512 ~81,024 256 64 125 <8256 32 128 <8~56 128 128 128 c8512 128 512 c8256 128 256 129 <8256 1282,048 ~8512 512 256 *Day of Booster As can be seen from Table 6, antibody developed to both viral anti-gens in horses receiving the inoculations. These vaccinates woul~ be immune to Equine Influenza since antibody titers in excess of 1:20 to A2 and 1:60 for Al are accepted as being protective by the National Veterinary Services . . .

~2S27 Laboratories of the United States Department of Agriculture.
Clinical trials in 420 horses of various breeds and ages showed the vaccine to be safe and to produce no untoward reactions after intra-muscular inoculation.

Three horses were given 5 ml of the live, attenuated Equine InEluenza A2 vaccine strain of Example 6 by the intranasal route. No clinical disease or symptoms of influenza were noted in any of the horses after vaccination.
Antibody titers against the vaccine strain were obtained on blood sera of all animals at 1, 2, and 4 weeks following inoculation and compared to the pre-inoculation level using the standard haemagglutination inhibition test ~HAI).

ANTIBODY RESPONSE ~HAI) - EQUINE INFLUENZA A2 Horse No.P _ 1 week 2 weeks 4 weeks 19 <8 128 256 128 23 8 6~ 12~ 12S
61 <8 128 25~ 12S
Once againJ the antibody titers obtained are greater than required or protection.
It should be noted that this disclosure is concerned with both a novel influenza culture system and the use of the medium to produce a novel in~luenza vaccine. The liquid cell culture system used in this invention en-tails the use of susceptible cells, influenza viruses, a nutrient medium and a proteolytic enzyme, but unlike past systems ~e.g. the Tobita et al reference), does not require the use of agar which reduces the system to a semi-solid state. The exclusion of the agar thus enables large scale production of viruses in the conventional manner known to the art and results in the produc-; ~ ~

~.2~527 tion of viral fluids of suficiently higll titers or the preparation of vaccines.
The influenza vaccine preparation itself comprises effective amounts of one or more strains of given influenza virus particles and a pharmaceutical-ly acceptable carrier, the total preparation, preferably in aqueous form, be-ing 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 the vaccine preparations is the seed virus which is diluted <1:100,000.
The vaccines are ad]ninistered to animals by various routes, includ-ing intramuscular, intravenous, subcutaneous, intratracheal, intranasal, or by aerosol spray and the vaccines are contemplated for beneficial use in a variety of animals, including humanJ equine, porcine, and avian groups.
The viral preparations produced by this invention may be diluted with water to adjust their potency, and they may have added to them stabilizers such as sucrose, dextrose, lactose, or other non-toxic substances. The viral preparations may be desiccated by freeze drying for storage purposes or for subsequent formulation into attenuated vaccines or thcy may be chemically inactivated for the preparation of killed virus vaccines.
It should be understood that the above e~amples are merely illus-trative and that the scope of this disclosure should be limited only by the following claims.

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of preparing an influenza virus vaccine which comprises the steps of:
(a) infecting a portion of the cells of a liquid cell culture with an influenza virus;
(b) incubating the infected culture in the presence of a proteo-lytic enzyme under conditions sufficient to assure further infection and replication of the virus in cells other than the originally infected cells;
(c) harvesting the virus from the culture, and (d) preparing a vaccine from the harvested virus.

2. The method of claim 1 wherein the cell culture of step (a) comprises a confluent monolayer of cells.

3. The method of claim 1 wherein the cell culture of step (a) comprises a liquid suspension of cells.

4. The method of claim 1 wherein the vaccine preparation of step (d) includes the step of inactivating the harvested virus.

5. The method of claim 1 wherein the vaccine preparation of step (d) includes the step of attenuating the harvested virus.

6. The method of claim 1 wherein the proteolytic enzyme of step (b) is selected from trypsin, chymotrypsin, pepsin, pancreatin, papain, pronase and carboxypeptidase.

7. The method of claim 1 wherein the virus is an equine influenza virus.

8. The method of claim 7 wherein the virus is a strain selected from equine influenza Al and equine influenza A2 virus strains.

9. The method of claim 1 wherein the virus is a human influenza virus.

10. The method of claim 9 wherein the virus is selected from B/Hong Kong, A/Texas, and A/USSR virus strains.

11. The method of claim 1 wherein the cells of the cell culture are dog kidney cells.

12. The method of claim 1 wherein the cell culture of step (a) is a dog kidney cell culture, the virus of step (a) is an equine influenza virus, and the enzyme of step (b) is trypsin.

13. The method of claim 1 wherein the cell culture of step (a) is a dog kidney cell culture, the virus of step (a) is a human influenza virus, and the enzyme of step 9(b) is trypsin.

14. The method of claim 1 wherein the enzyme of step (b) is present in an amount sufficient to assure infectivity and replication of the virus in cul-ture cells other than the cells comprising that portion infected in step (a).

15. The method of claim 14 wherein the amount of trypsin ranges from about 4 to about 25 micrograms per ml of culture media.

16. An influenza vaccine preparation comprising an effective amount of influenza virus particles whenever prepared by a process according to claim 1 and a pharmaceutically acceptable carrier, the preparation being substantially free of egg proteins.

17. The preparation of claim 16 wherein the virus particles are attenuated.

18. The preparation of claim 16 wherein the virus particles are killed.

19. The preparation of claim 16 wherein the virus is selected from the group consisting of human, equine, avian and porcine strains.

20. The vaccine preparation of claim 16 wherein the virus is an equine influenza virus selected from equine influenza A1 and equine influenza A2 strains.

21. The vaccine preparation of claim 16 wherein the virus is a human virus selected from B/Hong Kong, A/Texas and A/USSR viruses.

22. The vaccine preparation of claim 16 wherein the carrier is liquid.

23. The vaccine preparation of claim 22 wherein the liquid carrier in-cludes an adjuvant selected from aluminum hydroxide, alum, aluminum phosphate, Freund's, and an acrylic acid polymer cross-linked with a polyallylsaccharide.

24. A method of propagating influenza viruses in a liquid cell culture medium which comprises infecting a liquid cell culture with an influenza virus, incubating the infected culture in the presence of a proteolytic enzyme under conditions sufficient to assure virus propagation, and terminating the in-cubation.

25. The method of claim 24 wherein the influenza virus is an equine influenza virus.

26. The method of claim 25 wherein the virus is selected from equine influenza A1 and equine influenza A2 viruses.

27. The method of claim 24 wherein the virus is a human influenza virus.

28. The method of claim 27 wherein the virus is a strain selected from B/Hong Kong, A/Texas, and A/USSR viruses.

29. The method of claim 24 wherein the proteolytic enzyme is selected from trypsin, chymotrypsin, pepsin, pancreatin, papain, pronase and carboxy-peptidase.

30. The method of claim 24 wherein the enzyme is trypsin in an amount ranging from about 4 to about 25 micrograms per ml of liquid cell culture.

31. The method of claim 24 wherein the tissue culture comprises dog kidney cells, the influenza virus is selected from equine and human virus strains, and the proteolytic enzyme is trypsin.

32. An influenza virus-inoculated liquid cell culture comprising influenza viruses, a cell culture capable of hosting propagation of the viruses, and a proteolytic enzyme, the amount of the enzyme being sufficient to assure infec-tivity and replication of the viruses in substantially all the cells of the liquid cell culture.

33. The cell culture of claim 32 wherein the virus is an equine influenza virus.

34. The cell culture of claim 33 wherein the virus is a strain selected from equine influenza A1 and equine influenza A2 viruses.

35. The cell culture of claim 32 wherein the virus is a human influenza virus.

36. The cell culture of claim 35 wherein the virus is a strain selected from B/Hong Kong, A/Texas, and A/USSR viruses.

37. The cell culture of claim 32, wherein the proteolytic enzyme is selected from trypsin, chymotrypsin, pepsin, pancreatin, papain, pronase and carboxypeptidase.

38. The culture medium of claim 32 wherein the cell line tissue culture comprises dog kidney cells, the influenza virus is selected from equine and human influenza virus strains, and the proteolytic enzyme is trypsin.

39. The culture medium of claim 38 wherein the amount of trypsin ranges from about 4 to about 25 micrograms per ml of liquid cell culture.