A BIOMASS FOR PRODUCING VIRUS ANTIGEN AND METHOD OF
PRODUCTION OF VIRUS ANTIGEN USING SUCH BIOMASS
BACKGROUND OF THE INVENTION
Conventional methods for producing a virus antigen include in ovo production
i.e., production within an infected embryonated egg; or cell culture production i.e.,
primary cells or a cell line which have been infected. A typical in ovo method for
producing antigen involves many steps which are difficult to automate and are labor
intensive, time consuming and subject to contamination. In general, cell culture
production entails the use of individual cells and cell aggregates which are as small
as possible and which require the tissue to be disintegrated or disassociated to the
utmost extent mechanically or enzymatically. This treatment may lead to the decay
of many cells which may result in a high degree of contamination of cell proteins
which are difficult to separate from the desired product. Methods for producing tick-
born encephalitis virus antigen and influenza virus vaccine are described in US
5,391,491 and US 5,698,433, respectively. These methods use avian embryo cell
aggregates having a diameter of >100 urn to <1,000 urn and require an enzymatic
step.
Therefore, it is an object of this invention to provide a biomass useful for the
production of virus antigens which eliminates the disadvantages of in ovo antigen
production and antigen production using individual cells, cell lines or cell aggregates
of micron dimensions.
It is another object of this invention to provide a method for the production of
virus antigens which leads to high production output of virus antigen and which may
be used on a commercial scale.
It is an advantage of this invention that the biomass is easy to handle and the
antigen production method offers an economy of steps and significantly reduced
opportunity for contamination.
It is a feature of this invention that vaccines effective against viral infection
and disease may be more readily and economically produced.
Further objects and features of the invention will become more apparent from
the detailed description set forth hereinbelow.
SUMMARY OF THE INVENTION
The present invention provides a biomass for producing virus antigen which
comprises avian embryo particles having a particle size of about 0.5 mm to 10.0 mm
wherein said particles are infected with a virus.
The present invention also provides a method for the production of a virus
antigen which comprises:
a) infecting avian embryo particles having a particle size of about 0.5 mm to 10.0
mm with a virus in a culture medium to form a biomass;
b) oxygenating said biomass at an elevated temperature to form an oxygenated
mixture; and
c) filtering said mixture to give a filtrate containing the desired virus antigen
product.
The present invention further provides a method for the preparation of a vaccine
which comprises:
a) infecting avian embryo particles having a particle size of about 0.5 mm to 10.0
mm with a virus in a culture medium to form a biomass;
b) oxygenating said biomass at an elevated temperature to form an oxygenated
mixture;
c) filtering said mixture to give a filtrate containing the desired virus antigen product;
d) mixing said filtrate with a pharmacologically acceptable liquid carrier; and
e) optionally adding an immunogenically stimulating adjuvant.
DETAILED DESCRIPTION OF THE INVENTION
Conventional methods for producing a virus antigen include production in an
infected embryonated egg such as a hen's egg, production in a primary cell culture
which has been infected or on an infected cell line or production using avian embryo
cell aggregates of micron dimensions. All of these methods involve multiple steps
and/or enzymatic cleavage and separation techniques such as centrifugation,
sedimentation, or the like.
Surprisingly, it has now been found that a biomass comprising avian,
preferably chicken, embryo particles having a particle size of about 0.5 mm to 10.0
mm, preferably about 1.0 mm to 3.0 mm, wherein said particles are infected with a
virus may be used to effectively and efficiently produce a virus antigen. The avian
embryo particles of the biomass according to the invention may be obtained by
conventional mechanical size reduction methods such as high shear blending, rapid
multi-baffled stirring, homogenizing or the like, preferably homogenizing. For
example, chicken embryos which have been harvested from 9-12, preferably 11, day
old incubated hen eggs may be washed with a sterile buffer solution, diluted with a
culture medium such as tryptose phosphate broth to a concentration of about 50 to
250, preferably about 80-120, more preferably about 100, embryos per liter and
mechanically reduced in size to give a suspension of avian embryo particles having a
particle size of about 0.5 mm to 10.0 mm, preferably 1.0 mm to 3.0 mm. This
suspension may then be infected with a virus or virus master seed to give a biomass
in accordance with the invention. Suitable viruses include any virus or other antigen
capable of reproducing in avian embryonic cells, such as Reovirus, Infectious Bursal
Disease Virus (IBDV), Marek's Disease Virus (MDV), Newcastle Disease Virus
(NDV), Infectious Bronchitis Virus (IBV), Poxvirus, Chicken Anemia Virus (CAV), Egg
Drop Syndrome (EDS), Turkey Rhinotracheitis (TRT) Virus, Pneumovirus, Infectious
Laryngotracheitis (ILT) Virus, Encephalomyelitis Virus, Influenza Virus, Rabies Virus,
Distemper Virus, Hemorrhagic Enteritis Virus, Hepatitis Virus, Chlamydia Psittaci,
Haemophilus Paragallinarum, or the like, preferably avian viruses, more preferably
Infectious Bursal Disease Virus.
Accordingly, the present invention provides a method for the production of
virus antigen which comprises infecting avian embryo particles having a particle size
of about 0.5 mm to 10.0 mm, preferably about 1.0 mm to 3.0 mm, with a virus,
preferably an avian virus, more preferably infectious bursal disease virus, in a culture
medium to form a biomass; oxygenating said biomass at an elevated temperature to
form an oxygenated mixture; and filtering said mixture to give a filtrate containing the
desired virus antigen product.
Culture media suitable for use in the method of invention include tryptose
phosphate broth, EMEM, DMEM, (manufactured by Anhui Chemicals) or any
conventional media suitable for biological cultures, preferably tryptose phosphate
broth.
Elevated temperatures suitable for use in the method of invention are
temperatures of about 90°F to 110°F, preferably about 95°F to 105°F, more
preferably about 98°F to 102°F.
Oxygenated mixtures obtained in the method of the invention may contain
about 40% to 60%, preferably 45% to 55%, more preferably 50%, dissolved oxygen.
In actual practice a suspension of avian embryo particles having a particle
size of about 0.5 mm to 10.0 mm, preferably about 1.0 mm to 3.0 mm, in a culture
medium such as tryptose phosphate broth having a concentration of about 50 to 250,
preferably about 80-120, more preferably about 100 embryos per liter of culture
medium is infected with a virus, preferably an avian virus, more preferably infectious
bursal disease virus, to give a biomass; said biomass is oxygenated at an elevated
temperature of about 90°F to 110°F, preferably about 95°F to 105°F, more
preferably about 98°F to 102°F, to give an oxygenated mixture having about 40% to
60%, preferably about 45% to 55%, more preferably about 50% dissolved oxygen;
this oxygenated mixture is filtered through a screen of about 50 micron to 100
micron, preferably about 70 micron to 80 micron, more preferably about 75 micron, to
obtain a filtrate containing the desired antigen product. The antigen stock thus-
obtained may be treated with conventional excipients such as stabilizers,
antioxidants, antifoam agents, or the like and stored via freezing or lyophilazation for
use in future vaccine preparation or may be used as is in a vaccine preparation.
Accordingly, the present invention also provides a method for the preparation
of a vaccine which comprises mixing the antigen stock produced by the method
described hereinabove with a pharmacologically acceptable carrier and optionally
adding an immunogenically stimulating adjuvant.
Pharmacologically acceptable carriers suitable for use in the vaccine
preparation method of the invention include any conventional liquid carrier suitable
for veterinary pharmaceutical preparations, preferably a balanced salt solution
suitable for use in tissue culture media.
Immunogenically stimulating adjuvants suitable for use in the vaccine
preparation method of the invention include any compound which is capable of
potentiating or stimulating an immune response in an animal when administered in
combination with an antigen such as surfactants, i.e. as hexadecylamine,
octadecylamine, lysolecithin, dimethyl dioctadecyl ammonium bromide, N,N-
dioctadecyl-N'-N-bis(2-hydroxyethyl-propane diamine), methoxyhexadecylglycerol,
Pluronic® polyols, saponin, Quil® A, or the like; polyanions such as pyran, dextran
sulfate, polynucleotide complex of polyinosinicpolycytidylic acid, polyacrylic acid,
carbopol, aluminum hydroxide, aluminum phosphate, or the like; peptides such as
muramyl dipeptide, dimethyl glycine, tuftsin or the like; oil emulsions;
immunomodulators such as interleukin-1, interleukin-2, interleukin-12, GM-CSF or
the like; or a combination thereof.
In actual practice, virus antigen stock preferably avian virus antigen, more
preferably infectious bursal disease virus antigen, obtained according to the antigen
production method of the invention as described hereinabove is mixed with a
pharmacologically acceptable carrier such as phosphate buffered saline solution and
optionally an immunogenically stimulating adjuvant to give a vaccine product having
an antigen concentration of about 1.2 log above the minimum protective dose.
The vaccine thus prepared may be further treated with conventional
excipients commonly used in veterinary vaccines such as stabilizers, antioxidants,
antifoam agents or the like.
In one embodiment of the invention the virus antigen stock may be inactivated
prior to the vaccine preparation. The virus antigen stock prepared according to the
antigen production method of the invention may be inactivated by conventional
inactivating means, for example chemical inactivation using chemical inactivating
agents such as binary ethyleneimine, beta-propiolactone, formalin, merthiolate,
glutaraldehyde, sodium dodecyl sulfate, or the like, or a mixture thereof, preferably
formalin. Said antigen may also be inactivated by heat or psoralen in the presence of
ultraviolet light.
For a more clear understanding of the invention, the following examples are
set forth below. These examples are merely illustrative and are not understood to
limit the scope or underlying principles of the invention in any way. Indeed, various
modifications of the invention, in addition to those shown and described herein, will
become apparent to those skilled in the art from the following examples and the
foregoing description. Such modifications are also intended to fall with the scope of
the appended claims.
Unless otherwise noted, all parts are parts by weight.
EXAMPLE 1
Preparation of Infectious Bursal Disease Virus (IBDV) Antigen
Embryos from hen eggs which have been incubated at 99°F for 11 days are
harvested, washed three times with a solution of gentamicin in phosphate buffer
solution (30 mg/mL) at room temperature. The washed embryos are diluted to
approximately 100 embryos per liter in tryptose phosphate broth. The diluted
embryos are homogenized to a particle size of 1.0 to 3.0 urn using a W250V (Gifford-
Wood) model homogenizer, manufactured by Chemineer (Greerco) with a preset gap
setting of 3.0 mm. The resultant suspension is mechanically mixed with 0.2 mL per
embryo of X+4 Lukert strain working seed (USDA-APHIS approved IBDV)(5.5
TCIDEo/mL) for 20-30 minutes. The resultant mixture is oxygenated at pH 7.1, 3.0
psi, 100.4°F and a concentration of 20 embryos/Liter to a final dissolved oxygen (DO)
content of 50% DO. After 48h, the oxygenated mixture is filtered through a 75 micron
screen, mixed 1:1 with Stabilizer H1 for 1h at room temperature and stored at