U.S. patent number RE33,164 [Application Number 07/015,827] was granted by the patent office on 1990-02-13 for influenza vaccine production in liquid cell culture.
This patent grant is currently assigned to Mobay Corporation. Invention is credited to Karen K. Brown, Richard C. Stewart.
United States Patent |
RE33,164 |
Brown , et al. |
February 13, 1990 |
**Please see images for:
( Certificate of Correction ) ** |
Influenza vaccine production in liquid cell culture
Abstract
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 production from liquid cell
cultures instead of from more costly embryonated chicken eggs.
Resulting vaccine is thus substantially free of egg proteins.
Inventors: |
Brown; Karen K. (Kansas City,
MO), Stewart; Richard C. (Merriam, KS) |
Assignee: |
Mobay Corporation (Pittsburgh,
PA)
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Family
ID: |
26687845 |
Appl.
No.: |
07/015,827 |
Filed: |
February 18, 1987 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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39236 |
May 15, 1979 |
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Reissue of: |
428495 |
Sep 29, 1982 |
4500513 |
Feb 19, 1985 |
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Current U.S.
Class: |
424/209.1;
435/235.1; 435/236; 435/237; 435/238; 435/239 |
Current CPC
Class: |
A61K
39/145 (20130101); A61K 39/12 (20130101); A61K
2039/5252 (20130101); A61K 2039/5254 (20130101); A61K
2039/543 (20130101); A61K 2039/55555 (20130101); C12N
2760/16134 (20130101); C12N 2760/16234 (20130101) |
Current International
Class: |
A61K
39/145 (20060101); A61K 039/12 (); C12N 007/00 ();
C12N 007/02 (); C12N 007/04 () |
Field of
Search: |
;424/89
;435/235-239 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Klenk et al., Virology 68, 426-439 (1975). .
Lazarowitz et al., (I), Virology, 68, 440-454 (1975). .
Lazarowitz et al., II, Virology 52, 199-212 (1973). .
Effect of Trypsin on Reproduction of Type 4 Parainfluenza Virus in
Vero Cell Cultures Under Fluid Overlay--Morimoto et al.-pp. 1-11,
Japan. J. Med. Sci. Biol., 23, 1970. .
Effect of Trypsin on Viral Susceptibility of Vero Cell
Cultures-Cercopithecus Kidney Line-Itoh et al.-pp. 227-235, 1970,
Japan J. Med. Sci. Biol., 23, 1970. .
Genetic Recombination for Antigenic Markers of Antigenically
Different Strains of Influenza B Virus-Tobita et al.--pp. 347-352,
Journal of Virology-Feb. 1974. .
The Use of a Continuous Cell Line for the Isolation of Influenza
Viruses-Davies et al.-pp. 991-993, Bulletin of the World Health
Organization 56(6) (1978). .
Identification of the Defective Genes in Three Mutant Groups of
Influenza Virus-Ritchey et al.-pp. 1196-1204, Journal of Virology,
21(3) (1977). .
Activation of Influenza A Viruses by Trypsin Treatment-Klenk et
al., pp. 426-430-Virology 68, (1975)..
|
Primary Examiner: Nucker; Christine M.
Attorney, Agent or Firm: Gil; Joseph C. Whalen; Lyndanne
M.
Parent Case Text
This application is a continuation-in-part application of Ser. No.
039,236, filed May 15, 1979, abandoned.
Claims
We claim:
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
substantially free of proteolytic enzyme with an influenza
virus;
(b) incubating the infected culture in the presence of a
proteolytic enzyme under conditions sufficient to assure further
infection and replication of the virus in cells other than the
originally infected cells and an EID.sub.50 /ml titer of at least
about 10.sup.7 ;
(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 A1 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 (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 culture 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.
.Iadd.
16. 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
substantially free of proteolytic enzyme with an influenza
virus;
(b) incubating the already infected culture in the presence of a
proteolytic enzyme present in an amount sufficient to assure
further infection and replication of the virus in cells other than
the originally infected cells until the EID.sub.50 /ml titer has
increased by a factor of at least about 10.sup.2 ;
(c) harvesting the virus from the culture; and
(d) preparing a vaccine from the harvested virus. .Iaddend.
.Iadd.
17. The method of claim 16 wherein the proteolytic enzyme is
present at a concentration of between about 4 and 25 micrograms per
ml of culture medium. .Iaddend. .Iadd.18. The method of claim 16
wherein the cells are incubated with the infecting inoculum for at
least about one hour before the proteolytic enzyme is added.
.Iaddend. .Iadd.19. The method of claim 16 wherein the proteolytic
enzyme is selected from the group consisting of trypsin,
chymotrypsin, pepsin, pancreatin, pronase and carboxypeptidase.
.Iaddend. .Iadd.20. The method of claim 19 wherein the proteolytic
enzyme is trypsin. .Iaddend. .Iadd.21. The method of claim 20
wherein the trypsin is present at a concentration of between about
4 and 25 micrograms per ml of culture medium. .Iaddend. .Iadd.22.
The method of claim 21 wherein the cells are incubated with the
infecting inoculum for at least about one hour before the trypsin
is added. .Iaddend. .Iadd.23. The method of claim
22 wherein the virus is an equine influenza virus. .Iaddend.
.Iadd.24. The method of claim 23 wherein the virus is a strain
selected from the group consisting of the A1 and A2 equine
influenza strains. .Iaddend. .Iadd.25. The method of claim 22
wherein the virus is a human influenza virus. .Iaddend. .Iadd.26.
The method of claim 25 wherein the virus is selected from the group
consisting of the B/Hong Kong, A/Texas, and A/USSR human influenza
strains. .Iaddend. .Iadd.27. The method of claim 23 or claim 24 or
claim 25 or claim 26 wherein the cells of the cell culture are dog
kidney cells. .Iaddend. .Iadd.28. The method of claim 16 wherein
the cell culture which is infected is a liquid cell suspension
culture. .Iaddend. .Iadd.29. The method of claim 28 wherein the
cell culture is maintained as a liquid cell suspension culture
until the virus is harvested. .Iaddend. .Iadd.30. The method of
claim 16 wherein the cell culture is incubated with the proteolytic
enzyme as a confluent monolayer of cells. .Iaddend. .Iadd.31. The
method of claim 30 wherein the cell culture is incubated with the
proteolytic enzyme in a roller bottle, Roux bottle or Povitsky
flask. .Iaddend. .Iadd.32. The method of claim 31 wherein the cell
culture is incubated with the proteolytic enzyme in a
roller bottle. .Iaddend. .Iadd.33. A method of preparing an equine
influenza vaccine which is essentially free of egg proteins which
comprises the steps of
(a) incubating a liquid cell culture with an amount of an inoculum
of the influenza virus sufficient to infect a portion but not all
of the cells in the absence of any substantial amount of
proteolytic enzyme for between about 1 to 72 hours;
(b) combining the infected cell culture with between about 4 and 25
micrograms per ml of culture of trypsin and incubating until
maximum cytopathic effect is observed and the EID.sub.50 /ml titer
has increased by a factor of at least about 10.sup.2 ;
(c) harvesting the virus from the culture; and
(d) preparing a vaccine from the culture. .Iaddend. .Iadd.34. The
method of claim 33 wherein the cells of the culture are dog kidney
cells. .Iaddend.
Description
BACKGROUND OF THE INVENTION
1. Field
This disclosure is concerned generally with a novel influenza virus
propagation medium and specifically with the use of that medium for
influenza vaccine production.
2. Prior Art
Influenza vaccines have been in use since the early 1940's for
human vaccination and since the late 1960's for equine vaccination.
All influenza vaccines presently 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 influenza 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 described, for example,
by Davis et al., MICROBIOLOGY, Harper and Row Publishers, Chapter
44, pp. 1138-39 (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 preparation of virus vaccines. Thus, liquid cell
cultures have not been 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 preparation of
vaccines. Unfortunately, chick embryo-grown viruses usually require
concentration, and, in the case of human vaccines, also require
some form of purification to reduce toxic reactions due to the
undesirable egg proteins. The use of the eggs for vaccine
production is time consuming, labor 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. Thus, the
manufacture of millions of doses requires innoculating and
harvesting millions of embryonated eggs.
Recently, it has been noted that a wide variety of influenza 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 by K.
Tobita et al., "Plaque Assay and Primary Isolation of Influenza A
Viruses in an Established Line of Canine Kidney Cells (MDCK) in the
present 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 above reports the effects of
trypsin on influenza virus propagation were observed in semi-solid
cultures in plaque formation assays and isolation techniques,
neither of which are concerned with liquid cell cultures or the
large scale or commercial propagation of influenza viruses for
vaccine production. The term liquid cell culture used herein
describes the in vitro growth of cells and propagation of virus 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 infection of
successive numbers of cells in the same cell culture. By thus
overcoming the limitations of the "one-step growth cycle" of past
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 techniques 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.
SUMMARY OF THE INVENTION
Our influenza virus propagation medium comprises a cell culture
capable of being infected with an influenza 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, incubating the inoculate in the presence of a
protein-hydrolyzing enzyme under conditions sufficient to assure
maximum 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 attenuating by further cell culture passage for vaccine use.
Especially preferred embodiments involve the use of the protease
trypsin in conjunction with a dog kidney cell line to propagate any
of several types of influenza viruses.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1-5 are bar graphs illustrating the increases in titer
achieved when the indicated virus strains were incubated in a
liquid cell culture in the presence of various amounts of a typical
protease, trypsin. Results are reported as geometric mean
titers.
SPECIFIC EMBODIMENTS
The influenza virus and vaccine production techniques of this
disclosure contemplate the use of influenza vaccine viruses. As
used herein, the term influenza virus includes any viruses capable
of causing a febrile diease state in animals (including man) marked
by respiratory symptoms, inflamation 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.
The vaccine preparations contemplates by this disclosure include
any preparation of killed, living attenuated, or living fully
virulent influenza viruses that can be administered to produce or
artificially increase 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 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 the
replication of, one or more given influenza virus strains. Although
a number of such cells are known and thought to be useful for the
techniques disclosed herein, we 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 U.S. Department of Agriculture for use in producing veterinary
vaccines and is similar to the madin Darby Dog Kidney Cell Line
(ATCC No. CCL 34) and to the dog kidney cell line described in U.S.
Pat. No. 3,616,203 to A. Brown. A brief history and description of
the specific master cell stock used for the cell line of the
Examples follows although it should be understood that the
techniques disclosed herein are thought to be useful with any
influenza virus susceptible cell culture.
CUTTER LABORATORIES DOG KIDNEY (CLDK) CELL LINE HISTORY
The parent line of CLDK was initiated and established at Cutter
Laboratories, Inc., Berkeley, Calif. 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 Earle'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 cm.sup.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 manner and medium to prepare the
frozen Master cell stock at passage 148.
An ampoule of the Master cell stock was thawed, planted, and
serially subcultured 20 times to obtain bottle cultures of the
168th passage. These cells were then frozen.
DESCRIPTION OF MASTER CELL STOCK (MSC)
Number of Serial Subcultures from Tissue of Origin: 148
Freeze Medium: Minimum essential medium (Eagle) in Earle's BSS with
reduced bicarbonate (1.65 gm/L) 80%; fetal bovine serum 10%;
dimethyl sulfoxide 10%.
Viability: Approximately 75% (dye exclusion).
Culture Medium: Minimum essential medium (Eagle) in Earle's BSS
with reduced bicarbonate (1.65 gm/L) 90%; fetal bovine serum 2-10%;
antibiotic penicillin and streptomycin 100 U. or .gamma./ml.
Growth Characteristics of Thawed Cells: An inoculum of
3.times.10.sup.6 viable cells/ml cultured in the above culture
medium at 37.degree. C. in a closed system, multiplies
approximately 6-8 fold in 5 days.
Morphology: Epithelial-like.
Karyology: Chromosome Frequency Distribution 100 Cells: 2N=72.
______________________________________ Cells 1 1 2 4 6 9 69 5 3
Chromosomes 60 65 68 69 70 71 72 73 74
______________________________________
No marker chromosomes.
Sterility Tests: Free of Mycoplasma, bacteria and fungi.
Species: Confirmed as canine by immunofluorescence test.
Virus Susceptibility: Susceptible to influenza viruses, rabies
virus, infectious bovine rhinotrachetis, infectious canine
hepatitis, canine distemper virus, and possibly other viruses.
The protein hydrolyzing enzymes (proteolytic enzyme or protease)
contemplated as being useful for purposes of this disclosure
include well known proteases such as trypsin, chymotrypsin, pepsin,
pancreatin, papain, pronase, carboxypeptidase, and the like, with
trypsin being an especially preferred enzyme. The exact
mechanism(s) by which a protease such as trypsin enhances the
influenza virus infectivity is not fully known. One possible
mechanism has been suggested in the above cited article by Klenk 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 growth cycle but, in the
case of confluent monolayer cultures, not so much as to cause a
sloughing of confluent cells from the surfaces of the tissue
cultivation vessel (i.e. the 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 about 4 to
25 micrograms per ml (.mu.g/ml) of liquid tissue culture medium,
preferably about 10 .mu.g/ml.
VIRUS PROPAGATION METHODS
The influenza virus propagation method of this disclosure comprises
the three general steps of infecting a portion 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 assure maximum cytopathic (CP) effect, and harvesting the
viruses from the culture. Vaccine preparation comprises the
subsequent step of modifying the harvested virus by known
techniques to result in a live virulent, attenuated, or killed
(inactive) vaccine preparation. The vaccine may be available in dry
form, to be mixed with a diluent, or in liquid, ready to use form.
Suitable adjuvants may be included, as described below, to enhance
immunogenicity.
The protease may be added to an aqueous suspension cell culture or
before or after formation of a confluent monolayer culture.
Examples of some of the various propagation methods are described
below.
METHOD A-Infection of Pre-formed Monolayers
1. Cells are grown to confluency in culture containers such as
roller bottles, Povitsky flasks, or Roux bottles utilizing cell
culture growth media known 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 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 the diluent containing virus is added to the cell
monolayer in quantities ranging from 10% to 100% of the final
harvest volume.
5. The infected monolayers are incubated at 34.degree.-37.degree.
C. for 1 to 72 hours.
6. Protease, alone or in combination with the virus propagation
media, is added at a concentration which will stimulate multiple
cycle growth without producing cell slough. For trypsin, this
optimum concentration is between about 8 and 15 .mu.g/ml.
7. The virus growth containers are once again incubated at
34.degree.-37.degree. C. until maximum cytopathic effects are
observed. At this point the virus fluids are harvested.
8. Harvest involves shaking the containers vigorously to remove
cells and transferring fluids and cells to a sterile container for
further processing.
METHOD B-Infection of Liquid Cell Suspension Prior to Monolayer
Formation
1. Cells are removed from growth containers using conventional
procedures.
2. Cells are 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.degree. C.-37.degree.
C.) in a sterile, closed container (such as a screwcap Ehrlenmeyer
flask) while being mixed on a magnetic type stirrer or rotary
shaker for 10 minutes to 4 hours.
5. Aliquots of the cell virus suspension are placed into growth
containers (roller bottles, Roux bottles, Povitsky 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.degree.-37.degree. C.
until confluent monolayers are formed (approximately 2-4 days).
7. After monolayers have formed, protease is added at a
concentration which will stimulate multiple cycle growth without
producing cell slough. For trypsin, this optimum concentration is
between 10 and 25 .mu.g/ml.
8. Growth containers are incubated at 34.degree.-37.degree. C.
until maximum cytopathic effects are observed. Virus is then
harvested.
9. Harvest involves shaking containers vigorously to remove cells
and transferring cells and fluids to a sterile container for
further 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.degree.-37.degree. C. for 10 minutes to several hours.
4. Fetal calf serum may be added and the culture suspension is
further incubated at 34.degree.-37.degree. 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
.mu.g/ml.
6. Incubation at 34.degree.-37.degree. C. is continued until
maximum cytopathic 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 described here
in detail.
EXAMPLE 1
The 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 chick embryo. The seventh
passage was made at and obtained from Lederle Laboratories. The
strain underwent further chick embryo passage 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 planted in roller bottles, using Hank's
Minimum Essential Medium (MEMH) containing the following
ingredients:
Fetal Bovine Serum, 5-10%
Non-Essential Amino Acids, 10 ml/l (Gibco)
L-Glutamine, 10 ml/l (Gibco)
Neomycin Sulfate, 30,000 mcg/l
Polymyxin B, 30,000 units/l
Mycostatin, 25,000 units/l
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 Infective Dose.sub.50
(EID.sub.50) titer of approximately 10.sup.3.0 /ml in MEMH
supplemented with the following ingredients:
50% Dextrose, 2.6 ml/l
MEM Vitamins, 30 ml/l (Gibco)
Non-Essential Amino Acids, 10 ml/l (Gibco)
L-Glutamine, 10 ml/l (Gibco)
Neomycin Sulfate, 30,000 mcg./l
Polymyxin B, 30,000 units/l
Mycostatin, 25,000 units/l
Inoculating medium equivalent to 14% of the final volume is added
to each roller bottle and the containers are incubated at
34.degree.-35.degree. C. for 72 hours. At this time, the remaining
medium (86%) containing 12 .mu.g/ml of sterile trypsin (Sigma,
1:250) solution (0.1 g/100 ml), is added to each roller bottle.
Containers are again incubated at 34.degree.-35.degree. C. until
maximum cytopathic effect is observed (48-72hours) at which time
the fluids are harvested. 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 EID.sub.50 titers of A2 Influenza Virus grown using this
technique are shown in Table 1. Also, see FIG. 1. A comparison is
made with A2 virus grown by the same method but without adding
trypsin.
TABLE 1 ______________________________________ EID.sub.50 OF MIAMI
STRAIN EQUINE INFLUENZA VIRUS GROWN IN CLDK CELLS WITH AND WITHOUT
TRYPSIN Amt. Trypsin Titer (EID.sub.50 /ml) Fold Increase
(.mu.g/ml) Input Harvest With Trypsin
______________________________________ None 10.sup.2.6 10.sup.6.2
-- None 10.sup.3.2 10.sup.6.2 -- 5 10.sup.2.9 10.sup.8.3 126 5
10.sup.2.9 10.sup.8.1 79 5 10.sup.2.6 >10.sup.9.2 >1.000 10
10.sup.2.9 10.sup.9.2 1.000 10 10.sup.2.9 10.sup.8.5 200 10
10.sup.3.2 10.sup.8.2 100 10 10.sup.3.2 .sup. 10.sup.10.0 6.310 10
10.sup.3.1 .sup. 10.sup.14.5 199,526.232 15 10.sup.3.1 10.sup.8.9
501 ______________________________________
Incorporation of trypsin into the growth medium produced a
geometric mean increase of 3.2 logs or 1711 times as many virus
particles/ml during production of the Miami strain.
EXAMPLE 2
A sample of virulent type A1 Equine Influenza virus was obtained
from the University of Pennsylvania Medical School. The strain,
designated Pennsylvania (A1), 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 culture
production at passages 12-17.
The preferred method of tissue culture propagation of the A1 strain
involves infection of a suspension of CLDK cells prior to monolayer
formation. CLDK cells at a concentration of approximately
10.sup.5.5 /ml, Pennsylvania strain of virus at an EID.sub.50 titer
of 10.sup.3.0 /ml to 10.sup.5.0 /ml, and Hank's Minimum Essential
Medium (MEMH) supplemented with the ingredients listed below are
incubated at 25.degree. 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.
Supplemented MEMH
50% Dextrose, 2.6 ml/l
MEM Vitamins, 30 ml/l (Gibco)
Non-Essential Amino Acids, 10 ml/l (Gibco)
L-Glutamine, 10 ml/l (Gibco)
Neomycin Sulfate, 30,000 meg./l
Polymyxin B, 30,000 units/l
Mycostatin, 25,000 units/l
After this suspension incubation, 10 ml aliquots of the cell virus
suspension are added to roller bottles containing 1 liter of MEMH
supplemented as listed above and containing 5% Fetal Calf Serum.
The roller bottles are incubated at 34.degree.-35.degree. C. until
the monolayer is confluent (approximately 48-72 hours) after which
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.degree.-35.degree. C. until the maximum cytopathic effect is
observed (3-5 days). The virus fluids are harvested by vigorously
shaking each roller bottle to remove cells which remain attached
and transferring cells and fluids to a sterile container for
further processing.
Harvest EID.sub.50 titers of the A1 Influenza virus grown using
this technique are shown in Table 2. Also, see FIG. 2. A comparison
is made with A1 virus grown by the same method excluding
trypsin
TABLE 2 ______________________________________ EID.sub.50 TITERS OF
PENNSYLVANIA STRAIN EQUINE INFLUENZA VIRUS GROWN IN CLDK CELLS WITH
AND WITHOUT TRYPSIN Amt. Trypsin Titer (EID.sub.50 /ml) Fold
Increase (.mu.g/ml) Input Harvest With Trypsin
______________________________________ None 10.sup.5.3 10.sup.5.9
-- None 10.sup.4.9 10.sup.5.4 -- 10 10.sup.5.3 10.sup.7.4 50 10
10.sup.4.9 10.sup.6.8 13 10 10.sup.5.1 10.sup.8.0 200 20 10.sup.5.3
10.sup.8.7 1.000 20 10.sup.4.9 10.sup.8.5 631 20 10.sup.5.0
10.sup.7.1 25 20 10.sup.4.9 10.sup.8.3 398 20 10.sup.3.2 10.sup.9.2
3.162 20 10.sup.3.2 10.sup.7.5 63 20 10.sup.3.2 10.sup.8.2 316
______________________________________
Incorporation of trypsin into the growth medium produced a
geometric mean increase of 2.3 logs or 187 times as many virus
particles/ml during production of the Pennsylvania strain.
EXAMPLE 3
A strain of Human Influenza virus designated B/Hong Kong/5/72
(BX-1) was received from The Center for Disease Control in Atlanta,
Ga. This was passaged once in embryonated chicken eggs and frozen
at -70.degree. C. as working seed virus.
The preferred method of tissue culture propagation of the B/Hong
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 infected with virus diluted in the
inoculating medium listed in Example 1 to an EID.sub.50 titer of
approximately 10.sup.3.0-5.0 /ml. The inoculum consists of a volume
equivalent to 33.3% of the final harvest volume. Containers are
incubated at at 34.degree.-35.degree. C. for 40-48 hours after
which the remaining medium (66.7%) containing 12 .mu.g/ml of
trypsin solution (0.1 g/100 ml) is added to each container.
Incubation at 34.degree.-35.degree. C. is continued until the
maximum cytopathic effect is observed (48-72 hours) at which time
the virus fluids are harvested. Harvest involves vigorously shaking
each container to remove cells and transferring cells and fluids to
a sterile container for further processing.
Harvest EID.sub.50 titers of B/Hong Kong/5/72 Influenza virus grown
using this technique are shown in Table 3. Also, see FIG. 3. A
comparison is made with this strain grown by the same method but
without adding trypsin.
TABLE 3 ______________________________________ EID.sub.50 TITERS OF
B/HONG KONG/5/72 STRAIN HUMAN INFLUENZA VIRUS GROWN IN CLDK CELLS
WITH AND WITHOUT TRYPSIN Amt. Trypsin Titer (EID.sub.50 /ml) Fold
Increase (.mu.g/ml) Input Harvest With Trypsin
______________________________________ None 10.sup.3.2 10.sup.6.0
-- None 10.sup.5.7 10.sup.6.4 -- 8 10.sup.3.0 10.sup.8.7 316 8
10.sup.2.0 10.sup.9.0 631 8 10.sup.4.5 10.sup.9.5 1.995 8
10.sup.3.5 .sup. >10.sup.10.2 >10.000
______________________________________
Incorporation of trypsin into the growth medium produces a
geometric mean increase of 3.1 logs or 1412 times as many virus
particles during 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, Ga. This
was passaged once in embryonated chicken eggs and frozen at
-70.degree. 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 Example 3.
Harvest EID.sub.50 titers of A/Texas/1/77 Human Influenza virus
grown using this technique are shown in Table 4. Also, see FIG. 4.
A comparison is made with this strain grown by the same method but
without adding trypsin.
TABLE 4 ______________________________________ EID.sub.50 TITERS OF
A/TEXAS/1/77 STRAIN HUMAN INFLUENZA VIRUS GROWN IN CLDK CELLS WITH
AND WITHOUT TRYPSIN Amt. Trypsin Titer (EID.sub.50 /ml) Fold
Increase (.mu.g/ml) Input Harvest With Trypsin
______________________________________ None -- 10.sup.5.2 -- 8
10.sup.5.1 10.sup.8.9 5.012 8 10.sup.4.1 .sup. >10.sup.10.2
>100.000 10 10.sup.3.0 10.sup.8.3 1.259 10 10.sup.2.0 10.sup.9.8
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.
EXAMPLE 5
A strain of Human Influenza virus designated A/USSR/90/77 was
received from the Center for Disease Control in Atlanta, Ga. This
was passaged once in embryonated chicken eggs and frozen at
-70.degree. 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 EID.sub.50 titers of A/USSR/90/77 Human Influenza virus
grown using this technique are shown in Table 5. Also, see FIG. 5.
A comparison is made with this strain grown by the sane method but
without adding trypsin.
TABLE 5 ______________________________________ EID.sub.50 TITERS OF
A/USSR/90/77 STRAIN HUMAN INFLUENZA VIRUS GROWN IN CLDK CELLS WITH
AND WITHOUT TRYPSIN Amt. Trypsin Titer (EID.sub.50 /ml) Fold
Increase (.mu.g/ml) Input Harvest With Trypsin
______________________________________ None 10.sup.4.0 10.sup.6.3
-- 10 10.sup.4.0 10.sup.8.7 251 10 10.sup.4.0 10.sup.9.0 501 10
10.sup.4.5 10.sup.8.4 126
______________________________________
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 production of the A/USSR strain. VACCINE
PREPARATION
EXAMPLE 6-VIRUS ATTENUATION
Attenuation of the virus from harvested fluids of Example 1 is
accomplished chemically or by standard serial passages including
terminal dilution passage techniques wherein a sufficient number of
passages in a susceptible cell culture is employed 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
sysmptoms 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.
EXAMPLE 7-VIRUS INACTIVATION
The technique is similar to that described in Examples 1 and 2 but
the harvest viral ledan 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.degree. C. for 10
to 14 days. Testing of the final inactivated viral preparation
showed it to be free from live virus. Adjuvants known to the art,
such as aluminum hydroxide, alum, aluminum phosphate, Freund's, or
those described in U.S. Pat. Nos. 3,790,665 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-INACTIVATED VIRUS VACCINE PREPARATION AND USE
A 1.0 ml equine dose consists of 0.45 ml of the Pennsylvania (A1)
strain, 0.45 ml of the Miami (A2) strain and 0.10 ml of the
Carbopol adjuvant. Equal parts of the inactivated vaccine strains
obtained from Example 7 were mixed and 1 ml aliquots administered
to 19 horses by the intramuscular route. No clinical disease or
symptoms of influenza were noted in any of the horses after
vaccination. Antibody titers against both Equine Influenza A1 and
Equine Influenza A2 were obtained on blood sera of all animals at
2, 4, and 8 weeks following inoculation. These are compared to the
pre-inoculation levels (Table 6) using the standard
haemagglutination inhibition test (DIAGNOSTIC PROCEDURES for Viral
and Rickettsial Infections, Fourth Edition; Lenette and Schmidt,
pp. 665-66 (1969). American Public Health Association, New York,
N.Y. 10019).
TABLE 6 ______________________________________ ANTIBODY RESPONSES
(HAEMAGGLUTINATION INHIBITION) Equine Influenza A1 Equine Influenza
A2 2 4 8 2 4 8 Horse No. Pre. wk. wk.* wk. Pre. wk. wk.* wk.
______________________________________ 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 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 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 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
______________________________________ *Day of booster
As can be seen from Table 6, antibody developed to both viral
antigens in horses receiving the inoculations. These vaccinates
would be immune to Equine Influenza since antibody titers in excess
of 1:20 to A2 and 1:60 for A1 are accepted as being protective by
the National Veterinary Services Laboratories of the U.S.
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
intramuscular inoculation.
EXAMPLE 9-ATTENUATED VIRUS VACCINE USE
Three horses were given 5 ml of the live, attenuated Equine
Influenza 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).
TABLE 7 ______________________________________ ANTIBODY RESPONSE
(HAI) EQUINE INFLUENZA A2 Horse No. Pre 1 week 2 weeks 4 weeks
______________________________________ 19 <8 128 256 128 23 8 64
128 128 61 <8 128 256 128
______________________________________
Once again, the antibody titers obtained are greater than required
for 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 influenza vaccine. The liquid cell culture system used in
this invention entains 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 production of viral fluids of sufficiently high titers for the
preparation of vaccines.
The influenza vaccine preparation itself comprises effective
amounts of one or more strains of given influenza virus particles
and a pharmaceutically acceptable carrier, the total preparation,
preferably in aqueous 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 the vaccine preparations is the seed virus
which is diluted >1:100,000.
The vaccines are administered to animals by various routes,
including intramuscular, intravenous, subcutaneous, intratracheal,
intranasal, or by aerosol spray and the vaccines are contemplated
for beneficial use in a variety of animals, including human,
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 they may be chemically inactivated for the
preparation of killed virus vaccines.
It can be appreciated that all of the virus strains of the above
Examples fall into the Genus Influenza virus within the
Orthomyxoviridae Classification. See, for example, Principles of
Animal Virology, W. K. Joklik, Appleton-Century-Crofts/ 1980, pages
52 and 53. Accordingly, it is intended that this disclosure should
enable one skilled in the art to use the above-described methods to
prepare high-titered (e.g. EID.sub.50 /ml of at least about
10.sup.7) vaccines for any virus of the Orthomyxoviridae class.
Regarding the tissue culture cell use, it is only necessary that
the cell be susceptible to the specific virus strain of the Genus
Influenza from which a vaccine is to be prepared. It is thought to
be well within the skill of a component microbiologist or
virologist to select such a susceptible cell vis-a-vis a given
virus strain of the Orthomyxoviridae class. In a typical situation
a virologist would select the best growing cells from known cell
stocks, infect them with the virus strain in the presence of
protease, and observe for production of cytopathic effect. Fluids
from all infected cells producing cytopathic effects would be
titrated for virus concentration. Experiments in our laboratory
indicated that many cell types including Bovine Kidney. Vero, and
Canine Kidney support growth of Influenza virus when using the
procedure described herein.
As described above, an essential feature of the present invention
is that the incubation (virus replication) step be in the presence
of a protease under conditions sufficient to overcome the one-step
growth cycle of the virus. As can be seen from the data, and
especially as shown by the Figures, when a protease (such as
trypsin) is included (in the range of about 4-25 micrograms/ml
culture medium) in the culture, an EID.sub.50 /ml titer of at least
about 10.sup.7 is obtained. Obtaining such a high titer (in many
cases .gtoreq.10.sup.9 EID.sub.50 /ml) now provides a means for
economically making Influenza Virus vaccines without the use of
costly embryonated eggs. It should be pointed out that the
surprisingly high titer results shown in the Tables and Figures are
due solely to the presence of the indicated amounts of the protease
(trypsin) in the infected tissue culture. In cases where
trypsinization techniques are used (e.g. in removing cell layers
from a surface, using significantly larger amounts of the enzyme,
0.5 to 1.0 mg/ml) it is common practice, prior to virus infection,
to remove or inactivate the enzyme to avoid digestion of the cells.
Removal of the trypsin is accomplished by simply centrifuging the
trypsin/cell mixture and pouring off the supernatant trypsin
solution or by simply diluting the active trypsin out of the
system. Alternatively, any remaining active trypsin may be
neutralized (inactivated) by assuring the presence of known trypsin
activity neutralizers (i.e. the presence of serum, used for
nutrative purposes in the cell culture, will assure the
inactivation of trypsin and preclude the carry over of active
trypsin to the virus-infected cell culture). In either case
(trypsin removal or inactivation), it is important to note that the
pre-infection liquid cell culture of this disclosure is
substantially free of proteolytic activity. As used herein, the
expression substantially free of proteolytic activity means that
whatever activity may remain it is not sufficient to overcome
single step growth cycle. Additional fresh trypsin may be added to
accomplish the virus replication required by the invention
disclosed herein. Even if there were some residual trypsin which
somehow escaped removal or inactivation techniques such residual
amounts would correspond to the "zero" amounts in the Figures (the
controls) since those cells had been transferred using conventional
trypsinizing technique and, hence, not be present in quantities
sufficient to lead to the high titer results. Because of the use of
the protease during the replication step (in contrast to any prior
trypsination steps which would eliminate any such protease
activity), the final vaccine product of this invention will contain
measurable amounts of protease activity. In the case of trypsin
use, the final Influenza virus vaccine will typically contain at
least 1 to 10 units of trypsin activity per milliliter of Vaccine
preparation (e.g. vaccine in an aqueous carried), a feature not
associated with known Influenza virus vaccines. One unit is defined
as the .DELTA.A.sub.253 of 0.001/min. with N alpha-benyl-L-arginine
ethylester (BAEE). This feature helps distinguish the vaccine of
this invention from those of the prior art. Thus, the typical
vaccine of this invention contains virus grown to at least 10.sup.7
EID.sub.50 /ml, a trypsin activity of at least about 1 to 10 units
per mil, and includes a pharmaceutically acceptable liquid carrier
which is relatively clear, non-cloudy, and essentially free of egg
protein. As used herein, the expression pharmaceutically acceptable
liquid carrier includes a tissue culture fluid which consists of a
basal salts solution, amino acids, vitamins, other growth
nutrients, antibiotics and a fungistat.
It should be understood that the above examples are merely
illustrative and that the scope of this disclosure should be
limited only by the following claims.
* * * * *