Note: Descriptions are shown in the official language in which they were submitted.
6~3
INJECTABLE RABIES VACCINE COMPOSITION AND METHOD FOR PRE-
PARING SAME
The present invention relates to an improved rabies vaccine
composition and a method for preparing same using viral-
laden suckling mice or rat brain tissue.
Rabies vaccines are well known in the prior art and such
vaccines are widely used in the treatment of both humans
and animals. As is also well known, these vaccines, in
order to be completely acceptable and fully effective,
must produce or increase immunity to rabies with minimal
side effects and the immunity imparted thereby must endure
for a reasonably long period of time. Moreover, for these
vaccines to be completely acceptable and fully effective,
it is necessary that the same be a standardized preparation
having a potency which remains relatively constant over
reasonably long periods of time. Rabies virus can be grown
in a number of tissues and tissue cultures.
In recent years, brains of suckling mice have been used
as a suitable source for propagating rabies virus in the
preparation of commercial animal vaccines, and the brains
of suckling rats have been proposed (see, e.g., Lavender:
Purified Rabies Vaccine (suckling rat brain origin) Appl.
^~5~73
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Microbiol. 19, (1970), pp. 923-927). ~enerally, in pre-
paring rabies vaccines, a suspension of the viral-laden
tissues in a buffered aqueous solution is prepared and
then is inactivated. The pH-value of the buffered sus-
pension and the inactivated vaccine composition is ad-
justed to a slightly basic value. A phosphate saline
buffer solution is conventionally used for this purpose.
However, several serious dificulties are encountered in
conventional rabies vaccines containing a phosphate buffer:
a) in order to maintain the desired slightly alkaline
reaction and the buffering capacity of the phosphate
buffPr during the inactivation period, the pH-value of
the suspension has to be repeated:Ly adjusted by adding
potassium hydroxide solution;
b) the potency of the inactiva~ed vaccine composition
relative to the amount of viral-laden tissue is adversely
affected by the presence of the phosphate buffer; and
c) the pH-value of the vaccine composition is stabi-
lized only for a limited period of time after which the
pH-value slowly decreases. This change towards an acid
reaction causes serious problems in that acidity is
detrimental to potency and is a false indication of
bacterial contamination of the vaccine. Since contamination
-- 3 --
of a vaccine with bacteria usually leads to acidification
of the vaccine composition, determining the reaction of
a vaccine composition with the p~ indicator agent phenol
red is used as a simple method for detecting bacterial
contamination of vaccine compositions. After a storage
period of 9 to 1~ months conventional phosphate buffer
containing rabies vaccine compositions often react posi-
tive to the phenol red test, even though they are not
contaminated at all. Due to this false indication of
bacterial contamination large amounts of vaccines are
unnecessarily discarded.
The present invention provides a rabies vaccine compo-
sition, wherein the drawbacks of the prior art rabies
vaccines are avoided or substantially reduced.
In particular, the present invention provides such a
vaccine composition wherein the potency relative to the
amount of viral laden tissue therein is increased, which
is storage-stable and wherein the p~-value is stabilized
within a slightly basic range for a prolonged period of
time. Also, the vaccine is a standardized preparation
and will remain potent over a relatively long period of
time. The vaccine will produce or increase immunity to
a rabies with minimal side effects, and, when used, will
61J~
provide immunity over a relatively long period of time.
This invention also provides a method for preparing such
vaccines, in particular a method wherein no repetitive
addition of potassium hydroxide solution is needed for
maintaining the pH-value of the composition during the
inactivation period.
The rabies vaccine composition of the present invention
comprises a sterilized suspension of a minor concentration
by weight of proteinous suckling mice or rat brain par-
ticles of injectable particle size laden with inactivatedrabies virus, in an aqueous buffer solution having a
slightly basic pH-value and an amount, dissolved thereln,
of a buffer composition comprising a mixture of an organic
base of the formula
CH20H
R~
N C CH OH
R / 1 2
2 CH2H
wherein Rl and R2 each are hydrogen or CH2CH20H and an
acid addition salt thereof with an acid the anion of
which is-compatible with virus replication.
~:15~
According to the present invention there is further pro-
vided a process for preparing the above defined rabies
vaccine composition which comprises the steps of:
(a) suspending asufficient amount of vïral laden suckling
mice or rat brain tissue material in a sterilized aqueous
buffer solution having a slightly basic pH value and an
amount, dissolved therein, of a buffer composition suffi-
cient to stabilize the pH at said value, said buffer com-
position comprising a mixture of an organic base of the
formula
CH2OH
N 1 CH2H
2 CH2H
where~n Rl and R2 each are hydrogen or CH2CH20H, and an
acid addition salt thereof with an acid the anion of which
is compatible with virus replication, to obtain a concen-
15 ~ trated suspension having suspended therein an amount of
` at least about 20 percent by weight of proteineous viral
laden suckling mice or rat brain tissue material;
(b~ comminuting the suspended viral laden suckling mice
or rat brain tissue material within the concentrated sus-
_
:.
.
- ~c~ .t3~3
~; _
pension into particles of injectable particle size;
(c) diluting the concentrated suspension with a suffi-
cient amout of saidsterilized aqueous buffer solution
to obtain a dilute suspension having a concentration of
the proteineous viral laden brain particles of no greater
than about 10 percent by weight;
(d3 inactivating the dilute suspension; and
(e) adjusting the concentration of viral laden brain
particles in the inactivated suspension to obtain the
vaccine composition.
As used herein MLD50 refers to the dilution at which
death loss of 50% of the mice occurs.
The rabies vaccine composition according to the present
invention is prepared and used in form of a sterilized
suspension of proteineous suckling mice or rat brain
particles of injectable particle size laden with the
inactivated rabies virus in the aqueous buffer solution
having a slightly basic pH-value. In this regard, it
should be noted that the proteineous viral laden material
will generally be suspended in the aqueous buffer solution
such that the same will provide the virus in the final .
- :
,
~ 673
.~
-- 7 --
produc~ in a concentration sufficient to provide the
desired antigenic response.
The aqueous buffer solution which is used within the
vaccine composition according to the present invention
contains a buffer composition comprising a mixture of an
organic base of the formula:
CH20
Rl~
~ f CH OH
? CH2H
wherein Rl and R2 each are hydrogen or CH2CH20H and an
acid addition salt thereof with an acid the anion of
1~ which is compatible with virus replication.
~mong the above bases tris(hydroxymethyl)aminomethane is
preferred. ~owever, N-(2-hydroxyethyl)amino-tris(hydroxy-
methyl)methane and N,N-bis(2-hydroxyethyl)amino-tris(hydroxy-
methyl)methane can also be used.
Any organic or inorganic acids which are not detrimental
to virus replication are suitable within the acid addition
salts. Hydrochlorides are preferred. Other suitable acid
salts include salts of inorganic-acids such as carbonates,
6~
nitrates, and salts of organic acids such as acetates,
benzoates, maleates, oxalates, and succinates.
Most preferred is a mixture of Tris(hydroxymethyl)amino-
methane (~hich in the following will be abbreviated as
Tris) and its hydrochloride (which in the following will
be abbreviated as Tris-HCl). The ratio between the free
base and its salts within the above buffer composition
of course will vary depending an the desired pH-value
in the vaccine composition and the particular buffer sub-
stances which are used.For example, in order to achieve apH-value of between 7.5 and 8.4 at a temperature of about
25C, a by weight ratio of Tris/Tris~HCl of between about
1.18/6.35 and about 4.03/2.6~ is suitable. Both Tris and
Tris-HCl are commercially available.
Preferably the vaccine composition is buffered to a pH-
valuè of between about 7.8 to 8.0, and most preferably
to a pH-value of about 7.8 at a temperature of about
25C. Accordingly, the buffer solution most preferable
is an about 0.05 M Tris/Tris HCl buffer solution containing
about 5.32 g/l of Tris-HCl and about 1.97 g/l of Tris and
having a pH-value of about 7.8 at a temperature of 25C.
The buffer solution can be prepared by dissolving appro-
'
'
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priate amounts of the base and its salt; e.g., of Tris
and of Tris-~C1, in deionized water, or by dissolving an
appropriate amount of the base, e.g., Tris, in deionized
water and forming the salts in situ, by adding such an
amount of the respective acid, e.g., a hydrochloric acid
solution, to the solution that the pH of the initially
basic solution is adjusted to the desired value. Equally
the pH of an initially acidic solution can be adjusted to
the desired pH-value by addition of a solution of the
base.
An indicator dye such as phenol red may be added, when
desired, to facilitate the adjustment of the pH and to
monitor the pH during storage. Generally, when phenol
red is employed the same will be added in a concentra-
tion ranginy between about 1 and about 2~ and the same
will, generally, be used as a 1% solution thereof.
Following the pH adjustment, the solution will be steri-
lized in accordance with methods known in prior art. For
example, the same may be sterilized by heating to a
temperature between 120 and 121C for a period of tima
between about 30 and 60 minutes.
When desired, preservatives, e.g., antibiotics such as
penicillin, streptomycin and amphotericin B, may be added
~ 6~;~3
-- 10 --
to the buffer solution or to the suspension of proteineous
viral laden particles when the same is prepared.
The viral laden brain tissue from suckling mice or rats
is mixed with a sufficient amount o~ the buffer solution
to form a concentrated suspension, preferabl~ containing
between about 30 and about 60 wt% of the vixus laden brain
tissue. Suitably, the buffer solution will contain the
buffer composition and all antibiotics required to ma~e
a final vaccine concentration between about 10 and 30
units of penicillin, 10 and 30 mcg of streptomycin sul-
fate and between about 5 and 10 mcg of amphotericin B
per milliliter of final vaccine product. The suspension
will then be subjected to high shear agitation so as to
reduce the size of the brain particles to a size suit-
able for injection. Suitably, the high shear agitationwill be continued until the particle size of all particles
is within the range of about 1 to about 10 microns.
The resulting suspension may be stored at a temperature
between about -40 and about -60C. All tests required
to insure the viru~ containing suspension of satis-
factory quality will be completed prior to use of the
stored virus containing suspension. Generally, this sus-
pension comprising living, fully virulent virus will be
.
tested for purity, safety, and potency. When the sus-
pension is to be used in the preparation of a vaccine
product, it is essential that the suspension exhibit
satisfactory purity and safety and that the same have
a virus titer of at least 105MLD50 per 0.01 ml at a con-
centration of 3 wt% (for a one-year vaccine), and at
least 10 MLD50 per 0.01 ml at a COnCentratiQn of 6 wt%
~for a three-year vaccine~.
As has been noted, supra, the brain tissue material which
is laden with the living, fully virulent viruses to be
used in the vaccines of this invention will, generally,
be stored in a frozen state and it will be necessary
to thaw the same prior to preparation of the vaccine.
When the virus laden brain tissue material to be used
in the vaccine is frozen, the same will yenerally be
rapidly thawed and then diluted with the buffer solution
to a concentration ranging between about 2 and about
10 wt%.
After the suspension of viral laden brain tissue particles
has been diluted, the same may then be filtered to remove
particles having a size greater than 10 microns or the
same may be first treated so as to inactivate the virus
contained therein and subsequently be filtered.
~ 6~J3
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After the viral laden brain particles have been suitably
suspended in the buffer solution, and the pH thereof ad-
justed, the same may be inactivated, directly, or the
same may be first formulated into a vaccine and the
viruses inactivated at this point. In either case, any of
the techniques known in the prior art to be useful for
this purpose may be employed. For example, chemical in-
activation may be accomplis~ed with compounds such as ~-
propiolactone, or formalin or other suitable aldehydes,
or the virus may be inactivated with ultraviolet light.
Beta propiolactone inactivation is, however, preferred
since this method results in minimal antigenic distortion.
When ~-propiolactone is used to inactivate the virus,
an aqueous solution containing between about 5 and about
15 volume percent of unhydrolyzed ~-propiolactone will
be prepared by dissolving a pharmaceutical grade o~ un-
hydrolyzed ~-propiolactone in either distilled or
deionized water. Generally, ~he solution containing the
unhydrolyzed ~-propiolactone will be prepared at a tem-
perature between about 4 and 5C and the same will beadded to the diluted suspension at or near this tempe-
rature such that the composite product contains ~-pro-
piolactone in a dilution within the range of about
1:1,000 and 1 10,000. The composite product will then be
~ 3
allowed to warm to room temperature and be su~jected to
periodic agitation for a period of time of between about
24 to 30 hours. During this time, the ~-propiolactone
will be hydrolyzed and upon complete hydrolysis, the virus
will be inactivated~ Either before or after inactivation
of the virus a conventional suspension stabilizing agent
ma~ be added and if necessary the pH-value may be read-
justed to a range of between about 7.8 and 8Ø The vaccine
thus prepared will have a storage life of at least 24
months at a temperature between about 4 and 5C. It will,
of course, be appreciated that the vaccine should be
stored under sterile conditions. It will also be appre-
ciated that the vaccine may be stored in single or
multiple dose containers and that t:he same will be used in
accordance with the techniques wel] known in the art.
Generally, a single dose will range between about 1 and
5 ml.
At this point, it should be noted that the suckling
mouse or rat brain tissue cannot effectively be separated
from the living, full~ virulent rabies virus propagated
therein without reducing the virus titer of the resulting
viral suspension to an unsatisfactory level. For this
reason, the brain tissue should be retained in the vaccine
of the present invention. In this regard, it should be
:
~ 3
- 14 -
noted that a series of tests completed on test animals
with the vaccine of this invention indicate that there
is no reaction whatever to the presence of this tissue
in the vaccine. It should also be noted that the rabies
vaccine compositions prepared in accordance with the
present invention exhibit a potency, as determined in
mice, of at least ten times the NI~ minimum standard.
It is believed that this increased potency is the result
of the relatively high virus titers obtained when the
virus is supplied with a high antigenic mass source
such as the viral laden suckling mice or rat brain tissue
and the vaccine composition is buffered by means of
the buffer composition heretofore described. In this
regard, it should be noted that these relatively high
potencies will be consistently obtained when care is
e~ercised to insure the high titers heretofore described
and to insure that the final vaccine contains at least
about 2 wt% of the viral laden brain tissue.
In the following a method of obtaining the viral laden
suckling mice or rat brain tissue which is used as a
starting material for the presently claimed rabies vaccine
composition will be described:
It has been found that a high antigenic mass rabies virus
.
6~3
- 15 -
can be propagated in suckling mouse brain or rat brain
tissue and that standardized vaccine preparations can be
prepared with rabies virus propagated in this manner.
Generally, vaccine production quantities of a high anti-
genic mass rabies virus can be propagated in suckling
mouse or rat brain tissue starting with any of thP strains
of rabies virus generally availa~le for this purpose. In
this regard, the CVS strain of rabies virus has been
found particularly effective for use in the propagation
of such a high antigenic mass virus. Such viruses are
available from the National Institute of Health, Bet:hesda,
Maryland, U.S.A. as well as from other Culture Depositories
and the same may be obtained as a lyophilized suspension.
The high antigenic mass rabies vir~us can be propagated
starting from a lyophilized suspension of a suitable
rabies virus obtained from one of the Culture Depositories.
Generally, the lyophilized suspension, when reconstitut~d,
will have a virus titer between about 10 and 107 MLD50
per 0.03 ml. This suspension~will then be intracerebrally
inoculated into several suckling ~ice or rats. The actual
size of the dose is not, of course, critical so long as
each of the mice or rats receive a dose sufficient to
induce rabies in the inoculated species. Suitably, suckling
~15~6 d 3
- 16 ~
mice will be injected with a dose having a virus titer
between about 100 and 150 Ml,D50.
As will be readily apparent, the intracerebral inoculation
of the live rabies virus will produce typical rabies
symptoms in the mice or rats and permit the rabies virus
to propagate through the brain cell tissues. Generally,
the virus will be permitted to incubate for a period
between about 4 and 5 days and the virus may be harvested
after this period. As will be pointed out more fully,
hereinafter, uninoculated control litters from the same
source of mice or rats will be observed during and after
the incubation period to insure that the animals used
for propagation did not suffer from abnormal symptoms.
Af~er the necessary tests have been completed to insure
that the inoculated animals did not suffer from other
diseases, the propagated rabies virus will be harvested
by removing the brain of the inoculated suckling animals.
This can, of course, be accomplished by any suitab~e
technique known in the art. For example, inoculated
sucXling mlce, after having become moribund will be held
at a temperature of between about -40 and -60C after the
virus incubation period and prior to harvesting. The frozen
mice will, then, be thawed just prior to harvest and the
~ 6~3
rabies virus laden brain tissue removed, generally, at
or near room temperature. Following the harvest, the
rabies virus laden brain will be suspended in a suitable
medium and stored at a temperature between about -40 and
~60C. A portion of the brain tissue buspension will,
generally, of course, be withdrawn and tested befoxe the
remaining portion thereof is used for any purpose~
Generally, the first batch of harvested viral laden
brain tissue will be used as a master seed for all future
production of liviny, fully virulent viruses for use in
the vaccine of this invention. When this is done, the
first batch will be extensively tested to insure that
the same is completely satisfactory for its intended
purpose. As will be readily appare!nt, when the first
batch of harvèsted brain is used exclusively to inoculate
suckling animals for the purpose of propagating the virus
for subsequent vaccine production, a relatively large
source of seed virus will be provided and each batch of
vaccine produced therewith will be more uniform since each
will be started with the virus from the same source.
This results in a more uniform vaccine product and this
method of subsequent propagation lS preferred for this
reason. Notwithstanding this, however, a continuing supply
of living, fully virulent virus could be provided by
- 18 -
using a portion of each batch of viral laden brain tissue
to inoculate additional suckling mice or rats with the
remaining portion used to produce a vaccine in accordance
with this invention. As will be readily apparent, however,
propagation in this manner would be unfeasible for indu-
strial vaccine production due to the extensive tests that
must be completed on each batch of seed virus to insure
the high quality product of this invention.
When the first batch of harvested viral laden brain tissue
is used as a master seed, the same will, generally, be
suspended in a suitable media at a concentration of about
20% fetal bovine serum and the suspension will contain
about 1000 units penicillin, 1000 mcg streptomycin sul-
fate and 10 mc~ amphotericin B per milliliter thereof.
The particular concentration of fetal bovine in suspension
is not, of course, critical and the particular concentration
employed can be varied. Moreover~ the concentration of
antibiotics in the suspension is also not critical~ It is,
however; essential that the master seed have a virus titer
~0 of at least 10 MLD50 per 0.01 ml at a 20% concentration
to insure the high potency of the vaccine of the present
invention.
.
~c~6'7~
-- 19 --
Generally, when the first batch of harvested viral laden
brain tissue is to be used as a master seed, the same
will be subjected to high shear agitation so as to reduce
the particle size of the suspended brain tissue to between
about 1 and 10 microns. This can, of course, be most
easily accomplished by subjecting the suspension to high
shear agitation at a relatively high concentration of at
least about 20 wt~ and thereafter diluting the same to
the desired concentration for storage. Moreover the master
seed will, generally, be stored, thawed and used, as
required, for subsequent testing and/or the production
of working seed.
After a first batch of viral laden brain tissue has been
prepared, all subsequent batches of brain tissue con-
taining the living, fully virulent rabies virus will beproduced by inoculating suckling mice or rats with viral
laden brain tissue. Generally, this will be accomplished,
exclusively, with the first batch of viral laden brain
tissue which will be preserved as a master seed. As has
been noted, supra, however, this can be accomplished by
using a portion of the first or any subsequent batch of
viral laden brain tissue therefor. In either case, the
viral laden brain tissue will be suspended in a suitable
~5'~ 3
- 20 -
diluent and diluted such that each inoculated suckling
animal receives a dose which is sufficient ~or inducing
rabies. Suitably a suckling mouse receives a dose having
a strength of between about 100 and 500 MLD50. Generally,
this will be achieved with a dose of about 0.01 ml,
although other size doses could be used.
Broadly, any number of mice or rats could be inoculated
and the viral laden brains thereof subsequently pooled
to produce a vaccine in accordance with this invention.
Generally, however, the number of inoculated animals will
range between about 1,000 and 10,000 and about 50,000
and 500,000 doses of a vaccine product will be produced
from each such batch. After the inoculation of each batch
of suckling animals, the animals will be observed for
typical rabies symptoms and the viral laden brain tissuè
harvested after the animals become moribund. In this
regard, it should again be noted that the moribund ani-
mals can be stored at a temperature between about -40
and about -60C for a period of time of about two weeks.
The moribund animal will, generally, then be stored and
alI ~ests necessary to insure that healthy animals were
inoculated will be completed before the viral laden brain
tissue is harvested. When these tests are completed, the
brains will be harvested in the same manner as indicated
~ 6
- 21 -
previously with respect to the first batch prepared and
then suspended in a suitable medium. Following this sus-
pension, the viral laden suspension will also be tested
so as to insure that the same is suitable for the pre-
paration of a vaccine within the scope of this invention.
When the rabies virus is to be harvested directly after
the animals have become moribund, this can be accomplished
directly by extraction of the brain. When the virus is
to be harvested from stored animals, however, it will be
`~ 10 necessary to thaw the frozen, moribund animals prior to
removing the brain. This will, ~enerally, be accomplished
by immersing the frozen animal in a water bath at a
temperature between 5 and 1~C. The viral laden brains
will then be removed at or near room temperature. Though
lS this is not necessarily re~uired, the skin surface of the
animals may be treated with tincture of iodine prior to
extraction of the brain, and the extraction may ke accomp-
lished with a suitably sized hypodermic needle inserted
tangentially in the forward aspect of the cranial cavity.
Generally, the hypodermic needle will be attached to a
safety-trappe~ vacuum system. Generally, the extracted
brains will be cooled to a temperature between about
-50 and +5C immediately after removal thereof.
. . .
~5~67~
A~ter the brains have been removed from the inoculated
suckling mice or rats the obtained virus laden brain
tissue material can be frozen or can directly be used
within the process accordiny to the present invention.
For example, it may be directly diluted with the buffer
solution to form the above mentioned concentrated sus-
pension which again can be frozen and for a period of
time be stored in frozen form for example until all tests
which are required have been performed as samples of sus-
pension.
At this point, it should be noted that the various testsperformed on both the primary seed and subsequent batches
of viral laden brain tissue form no part of the present
invention and all may be completed in accordance with
techniques well known in the prior art. Nonetheless, it
should be noted that the batch used as the primary master
seed would normally be tested for purity, safety, potency,
sterility, and identity while subsequent batches of viral
laden brain tissue would be tested only for purity, safety,
and potency. Safety, on the other hand, may be determined
by intracerebrally inoculating any of several animal
species with the inactivated virus and observing the in-
oculated species for a period of about 21 days. Identity,
on the other hand, may be determined by inoculating guinea
~ 6~3
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pigs and or mice intracerebrally and observing the develop-
ment of typical rabies symptoms; in cell cultures by fluores-
cent antibody microscopy, using specific fluorescein labeled
rabies antiserum; and by the virus' ability, when inacti-
vated, to protect guinea pigs and/or mice against lethalchallenge of rabies virus. Finally, potency may be deter-
mined by inoculating any of several animal species, in
accordance with known procedures, with a vaccine containing
the inactivated virus and determining the minimum concen-
tration or dose required to protect the species thus inocu-
lated.
In ~eneral, any strain o~ mice or rats may be used to pro-
pagate the rabies virus which is most useful in the present
invention. Care should be exercised, however, to remove
any animals evidencing disease symptoms from the colony.
Any such animals thus removed should then be autopsied
and examined grossly and microscopically for specific
disease lesions so as to insure that the animals actually
used for the propagation of the rabies vaccine are suit- `
able therefor. In addition, representative litters of
suckling animals should be routinely examined for LCM virus.
Moreover, the pooled brain su~pensionsshould be routinely
tested for murine leukemia particles and serologic end-
point titrations should be run on individual samples to
,. ..
~ 3'
- 24 -
determine murine virus antibodies for the following: -:
reovirus type 3 (Hl), pneumonia virus of mice (PVM) (HI),
K virus (HI), Theiler's encephalomyelitis (GDVII) ~HI),
polyoma ~HI~, Sendai (HI), minute virus of mice (MVM)
(HI), mouse advenovirus (CF), mouse hepatitis virus (CF),
lymphocytic choriomeningitis virus (LCM) (CF) and ectromelia
(vaccine virus HI test). In addition, routine cultures
should be obtained from the vital organs of several pro-
duction females for the purpose of determining the absence
of bacterial pathogena. Generally, all of these tests will
be completed in accordance with procedures well known
in the prior art and all will be completed on the suckling
mice or rats used to prepare the master. seed and the same
will be routinely accomplished thereafter.
Notwithstanding the fact that esse.ntially any strain of mice
or rats could be used to propagate the rabies virus use~
ful in the vaccinesof this invention, it is preferred that
a strain known to be useful for medical purposes be usad
and most preferred that a germ-free strain be employed for
propagation of the virus. In this regard, it should be
noted that a germ-free colony of suitable mice has been
develo~edand the same is designated as Strain ICR-MCR ~y
the supplier, Mld-Continental Research Animals, IncO of
Shawnee, Kansas, U.S.A.
~ ,a6~3
In the following the preparation of a rabies vaccine com-
position comprising a sterilized suspension of viral laden
proteineous suckling mice brain particles according to
a preferred embodiment of the invention will be described:
a~ Preparation of starting viral laden suckling mice brain
tissue material.
The rabies virus for vaccine production is propagated by
injecting suckling mice intracerebrally at an age between
two and five days with a working seed containing living,
fully virulent rabies virus, which working seed is pre-
pared by diluting a master seed to a virus titer between
about 100 to S00 MLD50 per dose. The master seed, in turn,
is prepared by inoculating 2 - 5 day old suckling mice
from the ~ame source as that used for future propagation
with a CVS strain rabies virus and then harvesting the
viral laden brain tissue. The master see~ has a virus
titer of at least 10 MLD50 per 0.01 ml at a concen-
tration of 20 wt%~ (In the definition of the virus titer
107-2 indicates the number of virus particles at the
specifled quantity and concentration of the mice brain
suspension.) The production virus is then harvested from
- the inoculated mice after the development of typical rabies
symptoms, when the mice have become moribund (4-5 days)
.
- 26 -
and when 2-5~ of the inoculated mice have died. The thus
propagated virus will be harvested and used only if there
is no evidence of atypical rabies virus propagation and
only if the results of all tests noted, heretofore, are
satisfactory.
Until it is determined that the virus are satisfactory
for harvest, the virus laden mice are stored in plastic
containers at a temperature between about -45 and -55C.
After satisfactory results have been obtained and the
mice are ready for harvest, the mice are thawed by immer-
sing the same in water (in the plastic containers) at a
temperature between about 5 and 15C. Once the mice have
been thawed, the skin surface thereof is treated with
tincture of iodine and the brains withdrawn from the
cranial cavity using a 15 gauge hypodermic needle inserted
tangentially in the forward aspect of the cr~nial caviky
in combination with a vacuum aspirator. After the brains
have been harvested, the same are pooled together.
b) Preparation of rabies vaccine.
The pooled viral laden brain material is subsequently sus-
pended in a 0.05M solution of Tris-HCl/Tris buffer con-
taining a sufficient amount of antibiotics to provide 10
6~3
27 -
to 30 units of penicillin, 10 to 30 mcg streptomycin sul-
fate and 5 to 10 mcg amphotericin B per milliliter of
final vaccine product. In the preferred embodiment, the
viral laden mouse brains will be suspended, initially,
at a concentration of 30 to ~0 wt% and then subjected to
high shear agitation so as to reduce the particle size
of the suspended mouse brains to between about 1 and 10
microns.
Between about 2 and 10 ml of-the suspension thus pre-
pared are withdrawn and diluted for purposes of further
tests. The remaining portion is stored at a temperature
between about -45 and -55C and subsequently used ~after
satisfactory test results have been obtained) in the
preparation of a rabies vaccine.
In the preparation of the rabies vaccine, the concen-
trated (30 - 60 wt%) frozen suspension is thawed at a
temperature of between 4 and 5~C and diluted to a con-
centration of about 2.5 to about 6 wt%. Dilution is
effected with an aqueous 0.05M solution of Tris/Tris-HCl
buffer and may further contain any additional antibiotics
which might be required to provide the desired antibiotics
concentration in the final product. The diluted suspension
then is filtered under sterile conditions to remo~e par-
~iLl~C.-a.673
- 28 -
ticles greater than 10 microns. The pH-value of the re-
sulting suspension is between about 7.5 and 8.4.
During the dilution, filtration and adjustment of the pH,
the suspension is maintained at a temperature of b~tween
0 and 5C. Following the dilution, filtration and pH ad-
justment, the rabies virus then ls inactivated with un-
hydrolized B-propiolactone. As has been noted, supra,
this can be accomplished with a solution containing be-
tween aboutl5~and !15 wt~ of unhydrolized ~-propiolactone.
10 The B-propiolactone solution is suitably added to the 1
diluted suspension with both at a temperature of between ¦
about 4 and-5C. Thereafter the combined mixture will be
allowed to become fully hydrolized. Generally, this will
be accomplished at room temperature within about 24 to
about 30 hours. The suspension should be agitated perio-
dically throughout the hydrolyzation period.
To insure standardization of the product, the concentration
of suspended tissue in the product must be between àbout
2.5 and 6 wt~. The resultiny product may be packaged or
stored under sterile conditions.
6'~3
- 29 -
The following Examples will further illustrate the present
invention and demonstrate the effectiveness thereof.
Example 1
A) Preparation of viral laden suckling mice brain tissue
starting material for a master seed suspensionO
For preparing a concentrated suspension of living, fully
virulent rabies virus three 1.0 ml ampules of a 10 wt%
lyophilized mouse brain suspension were obtained from
the Division of Biologic Standards, National Institute of
Health, Bethesda, Maryland, containing the CVS rabies virus
strain. The ampules were identified by Serial No. CVS-31.
The three ampules were then pooled, reconstituted and
diluted with an aqueous 0.05M Tris/Tris HCl buffer solution
and the diluted suspension was used to intracerebrally
inoculate 132, three day old, suckling mice. The inocu-
lation was accomplished with a calibrated, automatic syxinge
inserted approximately midway into-the cranial cavity at
an adequate depth into one of the cerebral hemispheresand
each of the inoculated mice received 0.01 ml of the viral
suspension. The inoculated mice developed typical rabies
:` ` :
;' -~ , .
.' ..
' '.
~:l.'t`~6
- 30 -
symptoms after three days and became moribund in about
four days. The moribund, inoculated mice were then stored
at a temperature of -50C for about two days. Following
this period of storage, the virus laden brain tissue was
separated from the inoculated mice by first warming the
frozen mice to a temperature of 5C, treating the skin
surface of the inoculated mice with a 2% tincture of
iodine and then withdrawing the brain by inserting a 15
gauge hypodermic needle tangentially in the forward aspect
of the cranial cavity. The hypodermic needle was attached
to a safety trapped vacuum system which was closed be-
tween the harvest of each individual mouse brain. Twenty-
five grams of brain tissue were withdrawn from 120 of the
132 inoculated mice.
The stock breeders used to produce the mice which were
inoculated in this and subsequent Examples were from the
germ-free strain designated ICR-MCR and available from
Mid-Continent Research Animals, Inc., Shawnee, Kansas,
U.S.A. These breeders were housed in sterile facilities
and used solely for the purpose of producing a gnoto-
biotic colony of mice to be used to propagate the rabies
virus useful in the vaccines of this invention. In this
regaxd, it should be noted that this was accomplished by
retaining a portion of the progeny of the first and sub-
- 31 -
sequent generations for future breeding of suckling mouse
breeders and using the remaining portion of the first and
subsequent genera~ions directly for breeding of suckling
mice tw be used in the propagation of the rabies virus.
B) Preparation of a sterilized buffer solution-
A buffer solution was prepared by mixing 5.32 g/l Tris
HCl and 1~97 g/l Tris base in 1000 ml deionized water and
2 ml of a 1~ solution of phenol red. The pH of the solution
is then at a value of 7.8. The solution was then steri-
lized by autoclaving at 121C for 30 minutes and tested
for sterility. The suspension medium was found satis-
factory for use in the preparation of vaccines and was
stored under sterile conditions.
~.
C) Preparation of a concentrated primary master seed
suspension of viral laden suckliny mice brain tissue:
The tissue, which was obtained as described above in
step ~ was suspended in 100 ml of a sterilized, aqueous
0.05M solution of Tris-HCl/Tris buffer which contained
a sufficient amount of antibiotics to provide 1,000 units
of penicillin per liter, 1,000 mcg per liter of strepto-
mycin sulfate and 10 mcg amphotericin B per milliliter
~ 6~'3
- 32 -
of suspensionO The resulting suspension contained 20 wt%
of the virus laden brain tissue and the same was subjected
to high shear agitation for a period of six minutes so
as to reduce the size of the brain particles to a si~e be-
tween about 1 and 10 microns. Twenty-five milliliters of
the 20 wt% suspension was then removed for testing and
the remainder of the 20 wt% solution was then dispensed
in 0~5 ml amounts in 200 ampules and stored at -50C.
The concentrated viral suspension prepared by the method
of Step C of this Example was tested for purity, safety,
potency, sterility and identity. The virus titer of the
suspension was found to be 10 ~SLD50 per 0.01 ml there-
of and the suspension was found to be satisfactory for use
as a primary master seed.
D) Preparation of a diluted secondary master seed sus-
pension of viral laden suckling mice brain tissue:
.
A secondary mas~er seed was prepared by diluting 0.~ ml
of the primary master seed suspension prepared in Step C
with 399.6 ml of a sterilized 0.05 M aqueous solution of
Tris-HCl/Tris buffer. A portion of the secondary master
seed was tested for sterility and the remainder dispensed
in 3 ml ampules and stored at -50C. The secondary master
~L5~ 3
-- 33 --
seed of this Example was found to be satisfactory for use
in the preparation of additional rabies virus.
E) Preparation viral laden suc~ling mice brain tissue
starting material for a vaccine composition:
3 ml of the secondary master seed obtained in Step D were
thawed and diluted with a sterilized 0.05M aqueous solu-
tion of Tris-HCl/Tris buffer prepared in the mannex des-
cribed in Step B so as to provide a suspension of living,
fully virulent rabies virus having a virus titer between
100 and 500 MLD50 per 0.01 ml thereof. 0.01 ml of this
suspension was then injected intracerebrally into each
of 92, two to four day old, suckling mice produced with
the breeders described in Step A. Following these inocu-
lations, the suckling mice were re!turned to their cages
to incubate the virus. ~fter about three days, the inocu-
lated mice developed typical rabies symptoms and all of
the inoculated mice became moribund after 4-5 days. The
moribund mice were then stored at -50C until tests on
uninoculated lltters of mice from the same source were
~0 completed. ~
'~'..
:.
~fter these tests were completed and satisfactory results
obtained, 75 of the stored, moribund mice were then thawed
6~3
~ 34 -
and the living, ~ully virulent rabies viruses harvested
by xemoval of the brain tissue from the mice in the
same manner as that set forth in Step A. After separation,
the brains from these 75 inoculated mice were pooled to
y7 eld 15 grams of brain tissue.
F) Preparation of concentrated suspension of viral
laden suckling mice brain tissue for producin~ vaccine
compositions:
The viral laden brain tissue obtained in Step E was
then suspended in a sterilized, 0.05M solution of Tris~
HCl/Tris buffer further containing, for example, 600
units of penicillin, 600 mcg streptomycin sulfate and
50 mcg amphotericin B per ml thereof. Two different sus~
pensions were prepared containing respectively 30 and
60% by weight of the viral laden mice brain tissue in
the buffer solution. The 30% suspension is suitable for
preparing a vaccine composition providing a one-year
immunization period; and the 60% suspension is suitable
for preparing a vaccine composition providing a three-
year immunization period.
.
The 30 and 60 wt~ suspensionswere subjected to high shear
agitation in a homogenizer at a temperature of about
~.~5~
5C so as to reduce the size of the brain tissue to avalue of between about 1 and 10 microns. Two milliliters
of each suspension was then removed for testing and the
remainder stored at -50C. The suspensions were tested
for potency, mycoplasma and sterility. As a result of
these tests, the 60 wt% suspension was found to have a
virus titer of 107-38MLD50 per 0.01 ml at a concentration
of 6 wt% and to be satisfactory for use in the preparation
of a three-year rabies vaccine. The 30 wt~ suspension was
10found to have a virus titer of 105-37MLD50 per 0.01 ml
at a concentration of 3 wt%, satisfactory for a one-year
vaccine.
.-
;
G) Preparation of inactivated rabies vaccine compo-
sitions:
After satisfactory results were obtained in the quality
control tests, the concentrated 30 and 60 wt% viral laden
mouse brain suspensions obtained in Step F were thawed .
by warm.ing to a temperature of 4-5C and then diluted to
form 3 and 6 wt% suspensions respectively by adding 9 ml
of the buffer solution prepared-in Step B to one ml of
the concentrated suspension so as to produce 375 ml each.
. The diluted suspensions which are maintained at about
4-5'C throughout were then filtered through a sterile.
, _
~.
.. . : .
- : :
~ 6
- 36 -
Millipore clarifying screen to remove particles having a
particle size greater than 10 microns. Subsequently 25 ml
of each of the suspensions were withdrawn for subsequent
testing. The living, fully virulent viruses contained in
the remaining portion of each dilute suspension were in-
activated with B-propiolactone. The deactivation was
accomplished with a solution containing 10 wt~ of ~-propio-
lactone. The ~-propiolactone solution was prepared by
adding a medicinal grade of unhydrolyzed ~-propiolactone at
a temperature of -20C to deionized water at a temperature
of 5~C. After the ~-propiolactone was added to the water,
the mixture was agitated briefly to insure the formation
of a solution and the same was then used directly in the
inactivation of the rabies virus in the diluted suspensions.
In this regard, it should be noted that best results are
obtained when the ~-propiolactone solution is added to
the dilu~ed suspension within five minutes after prepa-
ration thereof. The ~-propiolactone solution was trans-
ferred to the diluted suspension with sterile filtered
air pressure at a rate of 4.0 ml per liter of diluted sus-
pension in an amount providing a 1:2500 dilution of ~-
propiolactone in the combined product. The suspension was
then allowed to warm to room temperature and the same wa~
agitated periodically for a period of 24 hours.
~ ~5
- 37 -
In order to insure a standardized product, it is essential
that the final vaccine product contain at least 2.5 wt%
of the viral laden mouse brain tissue extracted from the
suckling mice and used in the preparation of the vaccine
for a one-year immunization period and 5 wt% for a three-
year immunization period in~dogs and cats.
Each of the one and three year vaccines prepared in this
Example yielded 310 ml. The vaccine compositions were
packaged in a plurality of single and 10 dose vials. The
packaging was, of course, accomplished in sterile con-
ditions and the containers employed were of the con-
ventional borosilicate type. Each of the single dose
bottles contained 1.3 ml of the vaccine and the 10 dose
bottles contained 11.4 ml each.
Example 2
The vaccine compositions obtained in Example 1 were
tested for inactivation by inoculating 0.03 ml intra-
cerebrally into mice, 21 days old. In one series of tests,
10 mice were injected with the vaccine composition as
20 ` prepared. In the second series, 10 mice were injected
with a l:10 dilution of the vaccine composi~ion and in
a third series of tests, 10 mice were injected with the
composition dilution of 1:100. The mice in all three
t~
- 38 -
series survived during the entire 21 day observation
period thus indicating that the rabies virus in the
vaccine had been inactivated.
Example 3
The relative potency or antigenic factor of the vaccine
prepared in Step G of Example 1 was determined in accor-
dance with the procedure set forth for the Modified
National Institue of Health potency test, which pro-
cedure is summarized in the World Health Organization
"Laboratory Techniques in Rabies" 2nd Edition, 1966. The
materials and methods used, and results obtained are as
follows~
~ . .
Six 100 ml test samples of a concentrated suspension con-
taining 60% of viral laden mice brain particles each
were diluted tenfold to obtain a 6% suspension.
Three of the test samples were diluted with a buffer
solution according to the present invention which had
been prepared by mixing 5.32 g/l of the Tris HCl with
1.97 g/l of the Tris base and 7.2 g/l NaCl. Phenol red
was added to the solutions at a concentration of 3 mls/l
of a 1% solution.
: . ~
,,- . ' ~ .
- 39 -
The remaining three test samples were diluted with a con-
ventional phosphate buffered saline solution comprising
0.01M dibasic potassium phosphate and NaCl t7 g/l).
The six resulting dilute test suspensions were inactivated
with ~-propiolactone at a concentration of 1:2500 for
a 24-hour period carried out at a room temperature (22-23C).
The pH of the six dilute 6~ mouse brain test suspensions
was maintained at a pH-value of 7.8 during the inacti-
vation period, by using lN KOH if necessary. Table No. 1
below gives the amount of lN KOH which was needed to
maintain the pH of the test suspensions at 7.8.
At the end of the 24-hour inactivation period, samples
from each of the six test suspensions were aseptically
drawn with a volumetric pipet ànd tested for the pre-
sence of live virus using the mouse safety test. Dilutionsof 10 , 10 , and 10 were inoculated into 10 mice for
eàch dilution of the six test suspensions and observed
for 21 days. No rabies deaths attributable to live virus
were observed.
An amount of 20% v/v stabilizer was added to each of the
~% mouse brain test suspensions to obtain six test-vaccine
.
~c~ 73
- 40 -
compositions. The stabilizer employed was pharmaceutical
grade Carbopol supplied by B.F. Goodrich, and was added
at a weight/volume concentration of 3 g/l to provide a
final stabilizer content of 0.5 g/l in the vaccine com-
positions.
Vaccine compositions 1 - 3 contained the Tris/Tris HCl
buffer solution and ~accine compositions 4 - 6 contained
the phosphate bu~fer solution.
The completed test vaccine compositions were thoroughly
mixed by agitation for 2 hours after the addition of the
stabilizer.
Samples of the eompleted test vaccine eompositions were
taken for safety and potency testing.
VaccinesNo. 1 through 6 were tested for potency using the
NIH potency test after eompletion of the final produet
to determine the antigenic value of the six experimental
lots.
Vaceinesl and 4 were incubated at 37C for 230 hours and
the potency of each lot tested.
~ 73
- 4~. -
Vaccines 2 and 5 were incubated 54 days at room tempe-
rature (22 - 23C) and the potency of each lot tested.
Vaccines 3 and 6 were held in the cooler at 5C for 59 days
and the potency of each lot tested~
~.
The antigenic values obtained from the NIH potency test
after incubation for all samples are listed in Table 2
below.
Table No. l
Addition of l N KOH during the 24 hour Inactivation Period
l0 Tris buffered suspensions Phosphate buffered suspensions
Suspension No. l N KOH Added Suspension No. l N KOH Added
(ml) (ml~
l 0 4 16
2 0 5 14
3 0 6 20
6;~'3
~2 -
Table No. 2
Antigenic Values of Rabies Vaccine Formulated Tris/Tris-HCl
Buffered Saline and Phosphate Buffered Saline.
The potencies of the six vaccines were tested by the NIH
Potency test after incubation for 230 hours at 37C, 54 days
at room temperature and 59 days at 5C.
Antigenic Values of the VaccinesBefore and
After Incubation at 37C for 230 hours.
Antigenic Value Before Incuba-...... Antigenic Value After Incuba-
tion tion
Vaccine No. 1 Vaccine No. 1
*EPD50 Test Vaccine 156.3 EPD50 Test Vaccine 130.0
EPD50 Ref~ Vaccine 23.07 EPD50 Ref. Vaccine 22.03
Antigenic Value 6.78 Antiqenic Value 5.90
].5 Vaccine No. 4 Vaccine No. 4
EPD50 Test Vaccine 208.4 EPD50 Test Vaccine 112.2
EPD50 Ref. Vaccine 23.17 EPD50 Ref. Vaccine 17.42
Antiqenic Value . 4.99 Antigenic Value 2.99
_ , ,, ,,, , . . _ . _
.
'
.
-- 43 --
Antigenic Values of Vaccines Incubated
At Room TempO(22-23C) for 54 ~ays
AV Before Storage AV Afer Storage
Vaccine No. 2 Vaccine No. 2
-
EPD50 Test Vaccine 146 EPD50 Test Vaccine 176
EPD50 Ref. Vaccine 31 EPD50 Ref. Vaccine 27.6
Antigenic Value 7.26 Antigenic Value 6.4
Vaccine No. 5 Vaccine No. 5
EPD50 Test Vaccine 167 EPD50 Test Vaccine 218
EPD50 Ref. Vaccine 23.3EPD50 Ref. Vaccine 37.1
Antigenic Value 4.7 Antigenic Value 2.7
Antigenic Valuesof Vaccine refri~erated at
5C for 59 Days
Vàccine No. 3 Vaccine No. 3
EPD50 Test Vaccine 184.2EPD50 Test Vaccine 228
EPD50 Ref. Vaccine 28.5EPD50 Ref. Vaccine 32-.5
~ntigenic Value 6.46 Antigenic Value 5.07
Vaccine No. 6 Vaccine No. 6
EPD50 Test Vaccine 133 EPD50 Test Vaccine 163
EPD50 Ref. Vaccine 28.5EPD5~ Ref. Vaccine 32.6
Antigenic Value 4.67 Antigenic Value 3.67
*EPD50 is that dose where 50% of the mice are protected when
challenged with live rabies virus.
-
- ~4 -
The results from the above test suspensions confirmed
that the pH of ~he Tris/Tris-HCl buffered suspensions
did not fluctuate after the addition of ~-propiolactone
as did the mouse brain virus suspensions buffered with
the phosphate buffer. (See Table 1.)
i,
The pH of the Tris/Tris~HCl buffered suspensions was
buffered at a pH of 7.~ throughout the inactivation pro-
cedures and there was lowering of pH (6.9 - 7.2) of the
phosphate buffered lots during the same inactivation
period and lt was necessary to add 1 N KOH to raise the
pH of the phosphate buffered experimental lots. I
Results of the safety test in mice revealed no rabies
deaths attributable to live virus in all groups tested~
The results of the NIH potency show that there were dif-
ferences in the antigenic values of the vaccines prepared
using Tris/Tris-HCl buffer and the vaccines prepared
using phosphate buffer (See Table 2.)
As will be readily apparent, the foregoing Examples clearly
indicate that the vaccines of this invention are completely
acceptable and fully effective for the purpose intended.
~ ,
.. .
~ 7
- 45 -
As is also readily apparent, the rabies vaccine prepared
in accordance with this invention does produce immunity
in all tested animal species to this disease.
