Note: Descriptions are shown in the official language in which they were submitted.
9~z
1 This invention relates to the art of vaccines.
In particular, the invention relates to a modified live
~eline viral rhinotracheitis vaccine, to a combination
feline viral rhinotracheitis-calicivirus vaccine prepared
therefrom and to processes for preparing and using such
vaccines.
Feline viral rhinotracheitis (FVR) disease was
first described by Crandell and Maurer, Proc. Soc. Exptl.
Biol. & Med., 97:487-490 (1958). The virus is a herpesvirus
and is responsible for approximately forty percent of the
upper respiratory infections of cats.
A vaccine against FVR is described by Bittle and
Rubic, Am. J. Vet. Res., 36:89-91 (1975) and Scott,
Feline Practice, Jan.-Feb. 197;, pgs. 17-22. That vaccine
was obtained by serial passage of a virulent FVR virus 81
times in primary feline kidney tissue culture and then 17
times in a feline diploid tongue cell line. German
Offenlegungsschrift 25,12,903 also discloses a FVR vaccine
prepared from virulent FVR virus by serial passage in
?0 _eline tissue cultures.
The prior art vaccines are administered
intramuscularly at the normal body temperature of the cat
(39C), at which temperature the virus is unable to grow and
behaves essentially as a killed vaccine. During injection
or the vaccine, the respiratory tract of the animal must be ;
protected from infection by the virus. Other disadvantages
of the prior art vaccines are that multiple doses are
reco~nended to effect immunity and that vaccination o,
pregnant female animals is not recommended.
The present invention consists of a safe and
effective vaccine for prophylaxis or aninals belonging to
the genus felidae against virulent FVR infection, which
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1 vaccine can be administered intranasally such as by
dropping or s?raying a suitable vaccine solution into the
nasal passages, intraocularly by absorption of vaccine
solution dropped into the eyes into the susceptible tissues
with nasal-lachrYmal duct drainage into the nose and throat
areas or by injection of a suit:able solution of the vaccine.
The intranasa} and a combination intranasal-intraocular ~ -
(one drop in each eye with the remainder dropped into the
nose) routes or administration are preferred. Preferably,
from about lO TCID50 to about lO8-0 TCID50 per dose is
administered. The vaccine of this invention may also be
acministered to pregnant ,emale animals with no abortion or
teratogenic effects. Processes for preparing and usins sùch
vaccines are also considered as objects of this invention.
The vaccine of this invention is prepared by
exposing to ultraviolet light a FVR virus which has itself
been subjected to chemical treatment with mutagens. In its ;
preferred form, the process of preparation consists of
exposing to ultraviolet light a FVR virus which has been
~0 subiected to chemical mutation with 5-fluorouracil,
5--luorodeoxyuridine and bromodeoxyuridine so that the
virus grows well at 30C + 2C, but not as well at 37C.
Any virulent FVR virus may be used in the
treatment with the chemical mutagens. The virus may be
isolated in a suitable host cell culture and propagated and
maintained in media known to the art. Suitable host cell
cultures include any cells cultured from the genus felidae
such as kidney, tongue, tracheae, turbinate, tonsil and lung.
Feline kidney cells are preferred. The virus is then
~0 subjected to chemical mutagens (DNA inhibitors) after a
p_etreatment of the substrate cells with 5-fluorouracil,
5-fluorodeoxyuridine, bromodeoxyuridine or any other known
~ .
3LOgZ~7Z
DNA inhibitor. A mixture of 5-fluorouracil, 5-fluoro-
deoxyuridine and bromodeoxyuridine is preferred to mutate
the virus. The virus cultures are then cloned at 30C - 2C,
preferably at 31C, to purify the virus and those that grow
well at that temperature are used for repeated treatment.
The mutation treatment is repeated for from 12 to 36 hours,
about 24 hours being preferred.
Virus obtained from the mutation treatment, ATTC
No. VR 814, is then subjected to ultraviolet light until from
10 about 90% to about 99%, preferably from about 95% to about
99% and advantageously about 99%, of the virus particles are
killed. Before being administered to cats, the remainder of
the ultraviolet treated virus is cloned at 31C. Advantageously,
the cloned virus is serially passaged at 30C - 2C from one
to about 12 times, 10 to 12 times being preferred. Any clone
of the virus may also be treated repeatedly with ultraviolet
light, preferably one additional treatment, and cloned before
final passage and/or use.
Although virus growth (both chemically mutated virus
and ultraviolet treated virus) is optimum at 31C, the virus
may also be grown at temperatures up to and including about
37C. Growth of the virus is naturally restrictive at 39C.
Preferably, growth of the virus is carried out at 30C - 2C.
Thus, in accordance with the present teachings, a
combination modified live feline viral rhinotracheitis~
calicivirus vaccine capable of inducing immunity in animals
of the genus felidae without serious side effects is provided
which comprises a modified live feline viral rhinotracheitis
virus which has been chemically modified so that the virus
30 grows well at 30C - 2C but not at 39C with ultraviolet light
until from about 90% to about 99% of the virus particles are
killed, a vaccinal feline calicivirus and a carrier therefor.
~.A.' ~
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72
Detailed Description of the Vaccine
A FVR virus used to make the modified virus was
cultured from a three-month old cat. The virus was isolated
during a routine study of upper respiratory infections and
came from the throat of a cat with no clinical symptoms of
upper respiratory disease. To determine if the virus indeed :
was infectious for susceptible animals, it was passed once
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1 in feline kidney cells (NLKF-1) and then administered by
intranasal inoculation into two SPF (specific pathogen
free) cats. Anorexia, pyrexia, difficulty in breathing and
sluggishness were o~served rrom three to eight days after
inoculation. Thus, it was apparent that this virus was
infectious for susceptible cats and not a naturally occurring
non-pathogenic variant.
The host~cell used ~or isolation and all further
studies was a stable serially plantable feline kidney cell
line designated as NLFK-l. Hanks' Balanced Salt Solution
plus lactal~umin (HAL medium) was used for cell propagation
and as maintenance medium for viral growth. The medium was
supplemented with 10% foetal calf serum for cell growth and
2% for maintenance.
Exposure of the FVR to chemical mutagens was
- carried out according to procedures outlined by Pringle
et al., V rology 55:495-505 (1973). A monolayer of NLFX-l
cells was exposed to 5-1uorouracil (10 mcg/ml) incorporated
into serum-free HAL medium for 18 hours prior to virus
infection. After the 18 hour exposure, the monolayer was
washed with serum-free HAL medium and 102 virus particles
adsorbed for one hour at 37C. The cells were then fed
with HAL serum-free medium containing 5-fluorouracil
(10 mcg/ml), 5-fluorodeoxyuridine (10 mcg/ml) and bromo-
deoxyuridine (5 mcg/ml). Incubation was carried out at
37QC for 24 hours. The cell monolayer was disrupted by
rapid alternate freezing and thawing and the entire
process repeal_ed. After each exposure to the DNA inhibitors,
the viral cultures were cloned at 31C in 24 well Linbro
plastic culture plates and only clones which grew well at
31C were used for repeat treatment. Virus was harvested
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l only when one clone was observable a~er seven days
incubation. All isolated clones were cultivated at 31 C
in ~LFK-l cells grown in milk dilution bottles. Titrations
were made at 31C and 37C to detect any temperature sensi-
tive (ts) properties. After ~he first mutagen exposure,no ts properties were detected and the mutagenic process
was repeated with a clone. A clone which showed very small
ts differences was chosen from each of four subsequent
cloning procedures and subjected to further treatment.
Thirty clones were obtained rom the first treatment, twenty-
five from the second, fifty from the third and fiteen fromthe fourth.
Cloned virus from the rourth mutagenic exposure
W2S tested in susceptible SPF cats. Each cat received
approximatelY 0.25 ml (10 -lO TCID50/dose) of virus
in each nostril and one drop of virus suspension in each
eve. Throat swabs taken 3 days post-inoculation were
~ositive for FVR. This virus was deposited in the American
Type Culture Collection and given the accession number
~R 814.
The virus obtained from the fifteenth clone of the
rourth DNA-inhibitor treatment was then exposed to ultra-
violet light (sterilamp). The virus was exposed in a plate
having a fluid depth of 5 mm at 15 cm from the light source.
Agitation during ultraviolet treatment was by magnetic
bar at approximately 50 rpm. Samples were removed at three
minute intervals. Ultraviolet light was particularly lethal
to the FVR virus with a reduction in virus titer of
ap2roximately 99% in eight minutes. Clones of the
ultraviolet treated virus which grew at 31C after 99~ of
the virus particles were killed were prepared. Although any
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297Z
1 of these clones is suitable for further use, a clone
having a virus titer of 107'5 TCID50 at 31C and
106-63 TCID50 at 37C was again exposed to ultraviolet light
and clones were picXed from it at 31C. A clone having a
virus titer of 108- TCID50 at 31C and 106-68 TCID50 at
37 (other clones may also be used) was selected for
further study and was serially passed at 31C before
administration to animals.
The viral material isolated as described above is
used directly to vaccinate cats or is preferably formulated
as lyophilized material, advantageously in combination with
a suita~le stabili~er known to the art. The lyophilized
product is rehydrated by mixing with sterile water or other
acce~table diluent prior to administration. All such forms
o~ viral material suitable for vaccinating cats are
considered to be within the-scope of this invention.
The FVR virus altered as described above (FVRm)
was cultivated at 31C in all studies. Roux and Povitsky
bottles were used for stationary cultures and 10 liter
~0 roller bottles used for viral growth. An NLFK-l cell mono-
layer was allowed to form in all culture vessels prior to
inoculation of the FVRm virus. Although io% foetal calf
` serum (pretested for BVD contamination) has been used for
cell growth, the ~LFK-l cell line will grow very well with
~5 reduced serum content and in up to about 10% of other
homologous or heterologous sera. The FVRm virus multiplies
well in the N~FK-l cells in the presence of 2% foetal calf
serum. Virus harvest was made when cytopathogenic changes
indicated maximum growth. The virus obtained from this
harvest was deposited in the America~ Type Culture
Collection and given accession number VR 815.
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~09~:~72
1 This virus was used to vaccinate cats as described below.
Studies for confirmation of the identity of the
virus consisted of growth in feline tissue culture with
typical cytopathologic changes accompanied by type A
inclusion bodies, failure to grow in cells of other species
(host specificity), fluorescent antibody staining, organic
solvent inacti~ation, growth inhibition by DNA synthesis
inhibiting chemicals and neutralization by specific antiserum.
The host specificity of the FVR virus has been
well established, Andrews et al., Viruses of Vertebrates,
pp. 348-349 (1972). The FVRm virus likewise has a host cell
specificity for feline established and primary cell
types when compared with various rabbit, canine, bovine,
equine, mouse, monkey and human cell types. The mutation
which has occurred in the ~VR virus to form the FVRm virus
has not altered the host specificity, but has altered the
virulence of the virus when introduced into the animal by the
normal route of infection. Fully virulent FVR virus will
not cause any type of disease in the susceptible cat when
introduced by the intramuscular or intravenous routes.
The respiratory tract, however, must be adequately protected
during the inoculation procedure. Intravenous inoculation
of as much as 10 5 TCID50 of virulent FVR virus, although
not producing disease~ will stimulate high titer of humoral
2S antibody with one injection. Intramuscular injection
with the same virus engenders only a slight humoral response
with one injection.
Fluorescent antibody, obtained from the National
Animal Disease Laboratory, has been used to identify the
FVRm virus. Specific staining has been observed in cells
infected with FVR but not with cells infected with caliciviru5.
A 20% concentration of ethyl e~her has been shown
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1 to totally inactivate the FVRm virus in 18 hours.
Virulent FVR virus was used as a controLA A 50~ chloroform
concentration will inactivate totally the FVRm virus in
approximately 30 minutes. These experiments indicate that
the ~ virus has an essential. lipid which is extractable
with organic solvents identical to the unmodified FVR virus.
Desoxycholate, a chemical known to inhibit DNA
synthesis, specifically inhibit.s the FVRm and FVR viruses
to the same degree.
,a~ntiserum prepared in rabbits against a well
estabIished strain of FVR virus specifically inhibits the
FVRm virus. This antiserum does not inhibit a calicivirus
strain whicn is a well established pathogen of the cat~
Accelerated stability studies were perormed on
lyophilized samples of F~Rm by combining the FVRm virus with
_ a stabilizing fluid and incubating at 37C for one~ two and
three w~eks.. The stabilizer had the following composition:
Solution A
PancrPatic digestive casein (type M) 40 g
Gelatin (Knox 250 A) 40 g
Distilled Water (q.s.) 1000 ml `~
- Solution B
Sucrose 150 g
Distilled Water (q.s.) 1000 ml
Solutions A and B were combined at 1:1 concentration
and added to a solution of the virus to 33-1/3~ total
volume.
Results of the stability studies are shown in
Table 1.
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~092~
1 TABLE I
Virus ~iter (TCID50/ml)
Berore
Test Number Incubation 1 Week 2 Weeks 3 Weeks
1o6.32 106 2~ 1o5-6g 105-76 ,'
2 1o6.32 105 69 105 3 105-4
From this data, it is apparent that the FVRm
virus in combination with a stabilizing solution remains
potent (as indicated by virus titers) for at least three
weeks under accelerated stability studies5which is equivalent ~
to about two yea~s at re~rigeration temperatures. Other `
combination ratios of FVRm virus and stabilizer may also
be used provided the virus titer remains in the range of
about 105' TCID50 to about 108' TCID50.
Use of the .~odified Feline Viral Rhinotracheitis Vaccine
- The FV ~ virus was tested by administering it to
SPF minimal disease and conventional colony-reared cats and
then challenging the cats with virulent virus. All cats
were vaccinated by administering approximately 0.25 ml
or virus containing fluid (105'-108- TCID50/dose) into
each nostril. The vaccine was dropped into the nose while
tne head or the animal was held upright with the nose in as
perpendicular a position as possible consistent with comfort
o the animal. Alternatively, one drop o. the vaccine
may be dropped into each eye with the remainder being
dropped into the nostrils. ;~
Antibody titers were determined by microtiter
techniques. Serum dilutions were made in two-fold steps,
mixed with 50 TCID50 of virulent virus, allowed to incubate
for one hour and mixed with sufficient NLFK-l cells to
plant 8 wells in a 96 well microtiter plate. The cell-
antibody-virus titrations were incubated at 37C for six
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~9Z97z
1 days and observed daily on a Leitz inverted microscope.
Other well established methods in virology for incubation
and determination of antibody ~iters may also be used.
Three weeks following vaccination, the animals
were exposed to an aerosol of virulent virus. The aerosol
was accomplished by use of a DeVilbiss atomizer utili7ing
compressed air as a vehicle. The aerosol was directed
into the animal's face and consisted of 107-3 TCID50 f
virulent virus. Control unvaccinated animals were treated
10 - in an identical manner. Daily examinations were made in an
attempt to discern clinical disease. Viral isolations were
attempted from throat swabs taken three days after challenge.
FVRm virus obtained from the second treatment with
` ultraviolet light described above was inoculated intranasa.Lly
into two SPF cats prior to serial passage. Although these
animals ~xperienced a pyrexia for one to four days, no other
symtoms of FVR were discernable.
Serial passage 10 was inoculated into four
susceptible cats. The vaccination was made by the intranasal
route. Serial passage 10 was inoculated into fo-lr kittens
that were three weeks of age. No clinical disease was
observed over a nine week observation period in any of these
animals. Although the four kittens had substantial antibody
titers, probably maternally derived, these titers were
2S boosted 2-4 fold after intranasal inoculation.
Minimal disease cats were obtained fro~ Fauna
Laboratories, Hudson, Mass. (now Liberty Laboratories,
Liberty Corner, New Jersey) to test their reactions to the
intranasal inoculation of passage 10. These animals were
12 weeks of age when received and had serum antibody titers
~1/2. The dosage of the FVRm virus was 0.25 ml into each
nostril and one drop into each eye. Serum antibody
:~09Z~7~
responses ranged from 1/5 to 1/25 after vaccination. No
signs of clinical disease were observed post-vaccination or t
post-challenge. The minimal disease animal is highly sus-
ceptible to FVR infection and is adequately protected when
vaccinated intranasally with FVR virus. Results of this
study are given in Table 2.
TABLE 2
Reaction of Minimal Disease Cats to
Intranasal FVRm Vaccination
Serum Antibody Response Clinical Signs
Pre- Post- Post- Post-
CatVaccination Vaccination Vaccination Challenge
1 <1/2 1/8 Negative Negative
2 <1/2 1/8 Negative Negative
3 <1/2 1/12 Negative Negative
4 <1/2 1/5 Negative Negative
<1/2 --- --- F, ND, S
6 <1/2 --- --- F, ND, S
7 <1/2 --- --- F, ND, S
8 <1/2 --- --- F, ND, C, S
F = Fever
ND = Nasal Discharge
S = Sneezing
C = Conjunctivitis
To test the boostering effect of a second intranasal
vaccination, five susceptible animals were given one dose and
one month later another dose was administered. A definite
boostering effect was noted following a second intranasal
vaccination (Table 3).
- 12 -
lV~Z~7Z
1 TABLE 3
Reaction of Cats to Two Intranasal FVRm Vaccinations
.
Serum Antibody Response
Pre- Post- Post-
S Cat Vaccination Vaccination l Vaccination 2 Clinical Signs
l l/3 l/12 l/56 Negative
2 ~l/2 1~6 l/32 Negative
3 ~l/2 l/5 l/28 Negative
4 ~l/2 l/5 l/48 Negative '
~; lO Fourteen minimal disease kittens, approximately
12 wee.~s of age were obtained from Liberty Laboratories and `;~
vaccinated intranasally with FVRm - passage ll. All animals
had serum antibody response of <1/2. FVRm was adjusted to
105' TCID50/animal. In all animals, serum antibody re-
sponses ranged from l/8 to l/24 post-vaccination. None oî
the animals exhibited cli~ical signs of disease.
Three six week old kittens and the mother queen ~ `
were also vaccinated intranasally with FVRm - passage 11.
The kittens and the queen were susceptible to FVR (serum
~0 antibody responses ~1/2). Three weeks following vaccination
all animals had serum antibody titers of l/20 or greater.
Although this queen was nQt selected as a seronegative ;~ ;
animal, the evidence certainly demonstrates that FVRm virus
will immunize totally susceptible kittens of six weeks of age
2~ with no evidence of deleterious side effects.
Five ~ack-passages of FVRm - passage ll in totally
susceptible minimal disease animals has not shown in any
case a reversion to virulence. The animals were administered
ap~roximately 106-5 TCID50/animal of each back passage.
Clinical disease was not seen and no pyrexia observed.
The virus was administered intranasally and into the eyes
of each of two animals at each passage.
109~7;~
1 A further aspect of this in~ention is a
combination feline viral rhinotracheitis-feline calicivirus
vaccine prepared from the FVRm virus described above and a
naturally-occurring modified calicivirus capable of creatins
immunity.
The calicivirus used in these studies was cultured
from the nasopharyngeal regio~ of a six month old cat and was
shown not to produce any manirestation of disease in
susceptible animals. Studies for confirmation of identity
of the virus consisted of rapid growth in feline tissue
culture with typical calicivirus cytopathic changes and
absence of inclusion bodies in stained preparations, no
inactivation by organic solvents and neutralization by
specific antiserum.
Preparation and use of the feline viral rhinotracheitis-
calicivirus combination vaccine-
The FVRm virus and the calicivirus were combined
with stabilizer in the following proportions: ;,
O.25 ml FVRm virus
0.25 ml Calicivirus
0.50 ml Stabilizer
Each 0.25 ml of virus had 106-5 TCID50 or greater.
The stabilizer had the following composition:
Solution A
Pancreatic digestive casein (type A) 4.0 g
Gelatin (Knox 250 A) 4.0 g
Distilled water ~q.s.) 100.0 ml
Solution B
Sucrose 15.0 g
Distilled water (q.s.) 100.0 ml
Final stabilizer solution was prepared by adding
- 14 -
. . ,
~09;~:~7Z
1 equal portions of Solution A and Solution B. The pH was
adjusted to 7Ø
Accelerated stability studies were performed on
lyophilized samples of the combination product by incubating
samples at 37C for one week and two weeks. After the
incubation period, the samples were rehydrated and titrated
in NLFK-l cells. Other combination ratios of FVR virus,
calicivirus and stabilizer may also be used provided
tne virus titer remains in the range of about 105- TCID50
to about 10 ~CID50-
The lyophilized vaccine was rehydrated to 0.5 ml
with sterile diluent and all cats were vaccinated by
administering approximately 0.25 ml of virus containing
fluid into each nostril. One drop of virus containing
fluid was often placed into each eye to determine safety
of the vaccine and as a test for irritation to mucous
membranes. The virus was dropped into the nose while the
head of the animal was held upright with the nose in as
perpendicular a position as possible consistent with
~ comfort of the animal.
To determine the effects of a feline viral
rhinotracheitis-calicivirus combination vaccine when
a~ministered intranasally, cats were administered 0.5 ml
of a l:l-combination of FVRm and calicivirus vlruses.
?5 These viruses had titers o 107-5 TCID50 for the FVRm virus
and i08 6 TCID50 for the calicivirus. All of the animals
were pre-bled and the serum from each pen pooled. Each pen con-
sisted of at lleast five an~mals. The serum neutralization
studies can be found in Table 4.
~ - 15 -
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o ~ ~ ~ ~
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-- 16 -- `
` :. ` . . :
1~92~7;:
l A combination of the two vaccine viruses profoundly
influenced the serum neutralization titers without any
evidence of interference when administered intranasally.
All of the animals were carefully observed for indications
of upper respiratory tract infection after the vaccination
and none were seen. An identical control group of cats
which were located in pens across a walk-way demonstrated
no upper respiratory tract infection during this period.
Thirty-one colony reared kittens (varying from
two weeks to five months of age) were vaccinated with a
combination feline viral rhinotracheitis-calicivirus vaccine.
The vaccine contained lO6-0 TCID50 of each virus per dose.
In all instances but one, a significant increase in serum
neutralizing titer was seen.
The abortigenic effect of the feline viral
r~inotracheitis-calicivirus combination vaccine~was tested
i~ four SPF pregnant queens. The queens were approximately
30 days into gestation when a full dose of vaccine
(l~6-5 TCID50 of each virus) was administered intranasally.
The queens were devoid of antibody to either virus prior to
vaccination. All queens responded to the combination
- vaccine by developing significant antibody titers prior to ~ .
parturition. Normal kittens were born to each queen and
remained-normal throughout the experimental study. It is
apparent that the feline viral rhinotracheitis-calicivirus
combination can safely be administered to pregnant queens
and will not cause abortions or disease in the neonatal
animals.
~our kittens born to SP~ unvaccinated queens were
given the feline viral rhinotracheitis-calicivirus
combination vaccine at two weeks of age. The kittens had
no prevaccination titer to either virus but developed
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~9~7Z
protective titers to both viruses at three weeks post-
vaccination. Thus, the feline viral rhinotracheitis-
calicivirus combination vaccine may be safely administered
intranasally to very youn~ kittens.
The feline viral rhinotracheitis-calicivirus
combination vaccine can be formulated and administered in
the same manner as described above for the modified feline
viral rhinotracheitis vaccine. The intranasal or combination
intranasal-intraocular routes of administration of
1~ rehydrated lyophili~ed vaccine are preferred.
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