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Patent 1058074 Summary

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(12) Patent: (11) CA 1058074
(21) Application Number: 1058074
(54) English Title: FELINE RHINOTRACHEITIS VACCINE AND PRODUCTION THEREOF
(54) French Title: VACCIN CONTRE LA RHINOTRACHEITE DES FELINS ET MODE DE PRODUCTION
Status: Term Expired - Post Grant Beyond Limit
Bibliographic Data
Abstracts

English Abstract


FELINE RHINOTRACHEITIS VACCINE
AND PRODUCTION THEREOF
ABSTRACT OF THE DISCLOSURE:
The propagation and modification of feline viral
rhinotracheitis (FVR) virus in feline tissue cultures
and the development of a vaccine useful for the
prevention of feline viral rhinotracheitis in cats,
said-vaccine comprising a modified FVR virus.


Claims

Note: Claims are shown in the official language in which they were submitted.


WHAT IS CLAIMED IS:
1. A process of attenuating virulent feline viral
rhinotracheitis virus which comprises propagating said
virus for at least 7 serial passages
through feline tissue cultures in a nutrient fluid at
an incubation temperature of about 30?2°C.
2. A process of attenuating virulent feline viral
rhinotracheitis virus according to claim 1 for the production of a vaccine
capable when injected into cats of immunizing them
against feline viral rhinotracheitis, characterized by
introducing an inoculum of live infectious feline viral
rhinotracheitis virus into a nutrient fluid culture
medium which is non-toxic to said virus and contains
viable feline tongue cells, propagating said virus by
incubating said fluid medium at a temperature of about 30?2°C
for a period of 2 to 7 days, and thereafter separating an
inoculum of said virus therefrom and serially passing the
virus through other such feline tongue cultures for a
total of at least about 7 passages.
29

3. A process of preparing a feline viral rhino-
tracheitis vaccine, characterized by attenuating live
infectious feline viral rhinotracheitis virus according
to the process of claim 1 until a virus titer of at
least about 104 tissue culture infectious doses of
virus per milliliter is obtained and harvesting the fluid
vaccine.
4. A process of preparing a feline viral rhino-
tracheitis vaccine according to claim 3, characterized
by the fact that the tissue culture comprises feline
tongue cells.
5. A process of preparing a feline viral rhino-
tracheitis vaccine in dry solid form, characterized by
attenuating live infectious feline viral rhinotracheitis
according to the process of claim 3 and drying at low
temperature the thus-obtained attenuated virus-containing
fluid.

6. A vaccine for immunizing cats against feline viral
rhinotracheitis, characterized by at least about 104 tissue
culture infectious doses of an attenuated feline viral
rhinotracheitis virus per ml, which vaccine is capable of
stimulating the production of protective feline viral
rhinotracheitis antibodies comparable to those produced by
natural infections when parenterally administered into non-immune
cats and without producing the usual pathological symptoms of
feline viral rhinotracheitis, whenever prepared by the process
of claim 3.
7. A vaccine according to claim 6, characterized by
the fact that said virus was attenuated by at least two- to
seven-day serial passages through feline tissue cultures in a
nutrient fluid at an incubation temperature of about 30?2°C.,
whenever prepared by the process of claim 2.
8. A vaccine according to claim 6, characterized by
the fact that it comprises from about 104 to about 107 tissue
culture infectious doses of the attenuated feline viral
rhinotracheitis virus per ml, said virus having been attenuated
through feline tongue cell cultures, whenever prepared by the
process of claim 4.
9. A vaccine according to claim 6, characterized by
the fact that it comprises a dry solid dosage unit form, the
fluid vaccine having been made solid by drying at low
temperature, whenever prepared by the process of claim 5.
10. A process of preparing a feline viral
rhinotracheitis vaccine which comprises propagating an attenuated
feline rhinotracheitis virus, which attenuated virus is obtained
according to the process of claim 1, by sufficient serial
passages through feline tissue cultures in a nutrient fluid at
an incubation temperature of about 32-37°C until said fluid
31

medium contains from about 104 to about 107 tissue culture
infectious doses of attenuated virus per ml and harvesting the
fluid vaccine.
32

Description

Note: Descriptions are shown in the official language in which they were submitted.


BACKGROUND OF THE INVENTION:
In~ectlous feline viral rhinotracheitis is a
specific, common and serious disease of cats caus~d
by the feline herpesvirus known as feline vlral rhino-
tracheitis (FVR) virus. Reports in the literature in-
dicate that this disease is responsible for approximately
half the clinical cases of feline respiIatory infections.
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The virus infects the epithelial cells of the nose,
pharynx, trachea and eye, cauæing epitheliolysis and
necrosis. The ocular manifestations predominantly
involve the conjunctiva; however ulcerative keratitis
~, .
can develop. rrhe virus is shed from the nose, eyes
and mouth through the course of the clinical disease. ;~
~VR virus infections are often severe and the mortality
may be signi~icant, especially in young kittens. Abor-
tion or generalized infection o~ nèwborn kittens may
occur following infection of pregnant queens with the
virus. The transmisslon of FVR virus to susceptible
.. . ..
cats iB generally by intranasal instillation, for ex-
t~ ample, by droplets expelled in sneezing or by contact
(usually nose to nose). Resistance following recovery
from natural or experlmental infectlon is of short "~
duration (1-3 months).
The causative virus of FVR was ~lrst isolated
from in~ected cats by R. A. Crandell and F. D. Maurer,
Proc. Soc. Exptl. Biol. and Med., 97, 487 (1958) and
the name "feline viral rhinotracheitis'! was first ~;
~, proposed for the disease by R. A. Crandell and E. W.
Despeaux, Proc. Soc. Exptl. Biol. and Med., 101, 494
(1959). Since then, several reports have appeared in
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the literature which confirm the isolation of FVR
virus from feline subjects in various parts of the
world, which identify the virus as a feline member of
the herpesvirus group, and which describe the trans-
,
mission, epidemiology and histologic characteristics
of the disease. For example, see J. L. Bittle et al.,
Amer. J. Vet. Res. 3 21, 547 (1960); R. A. Crandell t
et al., J.A.V.M.A., 138, 191 (1961), J. Ditch~ield
and I. GrinyerJ Virology, 26 504 (1965), R. H. John-
son and R. G. Thomas, Vet Rec., 79, 188 (1966); R.
C. Povey, Vet Rec., 82, 335 (1969), T. E. Walton and
J- H. Gillespie, Cornell Vet., 60, 232 ( 1970); Col-
loquium Report, J.A.V,M.A., 157, 2043 (1970); R. A.
Crandell, J.A.V.M.A, 158, 922 (1971); and S. I.
... .
;~ 15 Bistner et al., J A.V.M,A" 159, 1223 (1971). An
excellent up to date review is provided by R. A.
;` Crandell in the chapter entitled ~'Feline Viral Rhino-
,, tracheitis (FVR)I' in the book "Advances in Veterinary ,~
~i~' Science and Comparative Medicine~, Volume 17, Edtd.
`~!
; ~ 20 by C. A. Brandly and C. E. Cornelius, pages 201-24, Aca-
~, demic Press, Inc., New York, 1973. ;
~- Although attempts to produce a feline viral
~ .. ..
~'`r": rhinotracheitis virus vaccin~ have been reported,
-l none have proven successful. Investiga~ors in Eng-
land [Povey & Johnson, J. Small Anim. Pract., 11,
490 (1970)~ reported failure in attempts at vaccine
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prophylaxis with a live, non-attenuated FVR virus -
vaccine given intramuscularly. Although they ob-
tained a degree of success with formalinized and
beta-propiolactone inactivated FVR virus vaccines
in experimental cats, such vaccines failed to
significantly reduce the incidence of disease in
feline colonies. To datej no effective ~accine i8 ` `;;
available for protecting cats against FVR virus.
,.
. .
..
;~ DESCRIPTIO~ OF TB INVENTION:
' In accord~nce with the present invention, it
has been found that FVR virus can be propagated
`,!'1 in feline tissue cultures, preferably kidney a~d
~i tonguel and the virulence of the virus so modified
and reduced that no symptom~ of the disease are
~ observed upon parenteral inoculation.
;~!
~ 15 Accordingly, the present invention produces ;
. . .
,~` a modified or attenuated strain of live, ~eline
viral rhinotracheitis virus which when parenterally
inoculated~ preferably intramuscularly~ into catsJ `
~-! it immunizes the cats to virulent FVR disease. A
vaccine is also provided which is attenuated to an
extent that will stimulate an antibody response e~-
....
~~ fectively immunizing the cats for prolonged periods.
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The vaccine is safe in that it will not cause ~`
any disease in cats that receive it by the parenteral
route nor will the modified FVR virus pass from the
vaccinated cat to other cats in contact with lt,
thereby eliminating the possibility of increasing
the virulence of the virus by animal passage. This
constitutes a significant advance in the control of
FVR disease.
~' Live, virulent FVR virus can be obtained from
i 10 cats infected with feline viral rhinotracheitis ac-
; cording to methods of isolation and identification
. ~ !. ' ,
~,` described in the literature [e.g., see R. A. Crandell ~`
; and F. D. Maurer, Proc. Soc. Exptl. Biol. & Med., 97, ;
; 487 (1958); J. L. Bittle et al., Amer. J. Vet. Res.,
21, 547 (1960); and J. Ditchfield and I. Grin~er, Virology,
26~ ~04 (1965)]. In general, virus isolations can be -
made by swabbing the nasal and conjuntival mem~ranes
of infected cats with moist~ sterile, cotton swabs
which are then placed in a suitable feline tissue
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culture medium, followed by standard serial pas- ~
.
sages in order to replicate and isolate the virus.
A particularly suitable culture medium is one derived
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' from the cortical tissue of kidneys from 8- to 12-
~ . ,
~i 5 week old kittens which is trypsinized by a method
~' similar to that described by J. Youngner [Prod. Soc.
!$,'
'E~ptl. Bi~l. & Med'., 85,' 202 (1954)~ for monkey
, . .
,~ kidney cells.
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r'-, In preparing the vaccines of thls inven~lon, it
' 10 has been found that attenuation and modification of ~'
the virulent FVR virus can be readily accomplished by a ;
.
relatively few, at least about 7, serial passages in fe- '
~ line tissue utilizing lower incubatlon temperatures of about
!~' 30~2C, pre~erably about 32C. Purification of the viral
~'' 15 preparation may be accomplished by ~3tandard terminal di-
~,.~, .
~'', lution techniques, for example, tube or plague methods~
~' during or following the course of serial passages.
., ~ , .:
~'` The FVR virus is capable of propagation in such
feline tissue culture ~ystems, for example, lung,' ' '
; 20 testicle~ kidneyg thymusJ tongue and embryonic fetal -
tissueJ and also in established cell lines, such as~
.
for exampleJ Crandell's cat kidney cell line (CrFK)~
cat tongue cells at the third passage level (Fc3Tg) ~ '
and feline neurofibrosarcoma cell line (FNFS). Feline
ti! 25 tongue cell lines are most preferred.
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The passage time intervals should be such as to ~-
sufficiently allow the virus to replicate between pas-
sages, preferably from 2 to 7 days. The optimum pas-
. .,
sage time interval can readily be determined by stan-
dard techniques, for example, by cytopathic observa-
tions, such as by allowing the virus to grow during
a particular passage prior to the point where a gross
cytopathic effect (CPE) can be observed while con-
tinuing incubation.
': ..
The obtention of successful vaccines by the pres- -
; ent low temperature method is rather surprising in view
of the known fact that serial pasæage in feline tissue
at normal incubation temperatures, about 35-37C., does
not alter or modify the virus or it~ pathogenicity even
after 100 passages [R. A. Crandell, J.A.V.M.A., 158,
922 (1971)]. As shown hereafter (see Example III), -~
however, subsequent modification of the virus can be
accomplishe~ ~y serial pas~ages at ~ower incubation
temperatures.
In accordance with this invention, therefore, a
. . .
process is provided ror attenuating virulent feline
viral rhlnotracheitis (FVR) virus for the production
,~ of a vaccine capable when injected into cats of im- -
munizing them against FVR which comprises introducing
an inoculum of virulent FVR virus into a nutrient `
fluid feline tissue culture medium which is non-toxic
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to said virus, propagating said virus by incubating
said nutrient tissue culture medium at a temperature
of about 30+2C for a period of 2 to 7 days, and there-
a~ter separating an inoculum of said virus and serlally
passing the virus through other such feline tis~ue
cultures for a total of at least about 7 passages.
The viral preparations produced by thi~q inven-
tion may be dlluted to ad~ust their potency, and
they may have added to them stabilizers, such a~ dex-
trose and lactose, or other non-toxic substances. The
- viral preparations may also be desiccated, e.g., by
~reeze drying, ~or storage purposes or for subsequent
.: , .
` ~ormulation into liquid vaccines. Stabilizers use-
ful in the freeze drying of viruses are described
.
~, 15 in W. A. Rightsel et alO, Cryobiology, 1967, 3:~23 - ;
~ and D. Greiff et al., Advances in Freeze Dryi~g L.
;~j Rey~ Ed., pp. 103-122, Hermann, P~rlq, 1966. In ad-
dition, the vaccines may be utilized in a mixture with
other immunogenic vacCines for administration to Cats. -~
.,............... :-.
The manner in which our invention is carried out
is described in greater detail in con~unction with
;j the ~ollowing specific experiments. It is understood `
that these specific experlments are by way of illustra- ~i
~: tion, and not by limitation.
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EXAMPLE I
A sample of live virulent FVR virus cultured
and isolated according to the procedure described
by J. L. Bittle et al., Amer. J. Vet O Res. 7 21
547, (1960) is added to monolayers of a feline
diploid tongue cell line in tissue culture tubes
(16 x 125 mm) prepared as follows. The tongue
cell line used is the Fe3Tg line referred to-in
K. M. Lee et al., Cornell Veterinarian, 59, 539
~ (1969). Each cell line tube, containing 1-2 ml of
; 10 growth medium consi~ting o~ Eagles Minimum Es~ential
Medium (MEM) supplemented with 10~ fetal calf ~erum
0.1% lactalbumin hydroly~ate~ 30 units penicillin,
30 mcg streptomycln and 2.5 mcg amphotericin, is
seeded with 1 ml feline tongue cells (200,000 cells
, 15 per ml). If necessary, the pH is ad~usted with sodium
l bicarbona~e to maintain a p~ of abc)ut 7.2 - 7.8. The
.J cells in the tubes are allowed to grow at about 35+2C
.'1 : ,~ . ` '
` until a monolayer of cells is acheived. Fluids are
., .
then poured off and 1-2 ml of a maintenance medium
(same as above medium except that 1-2~ fetal calf serum
` is utilized) is added. About 4 to 6 such tubes are ;',?,:
. utilized per viral passage.
To each tissue culture tube is added the FVR virus
inoculum. The thus-seeded tube is maintained at;
30+2C until a cytopathic effect (CPE) is observed
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by microscopic examination (about 2-7 days). When
the CPE reaches about 75-90~ of the monolayer, the
..
contents of the tube are harvested and 0.2 ml inocu-
- lums are subJected to identical serial passages for
- 5 6 additional passages (7 passages total). After the 7th
, .
passage, a standard terminal dilution purification is per-
formed utilizing Eagles MEM supplemented with the ~-
aforementioned antibiotics (no serum) as the diluent
with incubation maintained at 30-2C, After 7 days,
the final tube which is positive with 75-gO~ CPE is ;
. .:
harvested and the entire procedure repeated twice
for a total of lO passages. An 11th passage is per-
formed for purposes of lncreasing volume by inoculating
.'i'''l
a 0.5 ml sample from the 10th passage into flasks con-
taining monolayers of feline diploid tongue cell cul-
; tures obtained by propagation of the tongue cells as
, ..i
previously described. At the end of the 11th passage,
the pool is harvested, identified and titrated by known
methods.
.~.~, ...
~; 20 The pool thus prepared constitutes a bulk vaccine
which may be diluted according to the titer or may have
-~ added thereto stabilizers or other nontoxic substances.
~ For use as a vaccine, it may be desiccated or it may
; be prepared in liquid ~orm.
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In propagating and attenuating the virus~ any
nontoxic nutrient fluid tissue culture medium may
be utilized. In addition to the supplemented Eagles
MEM medium described above/ it will be understood that
other nontoxic nutrient fluid tissue culture mediums
" ~ ,, . : . .
: may also be used. ~ ~
' " ' ' -.' "'-
EXA~IPLE II
','1 , , .
1 Ml of a vaccine prepared according to Example
I and titrated to a virus titer at 35~2C of 104~5
j TCID50/ml (determined by CPE) is adrninlstered intra- ~-
:, 10 muscularly to three susceptible cat~. Two other cats ~
,,,, , .: .
~; are maintained as unvaccinated controls. All 5 cats
are previously determined to be sero-negative to FVR -
virus. Evidence of the disease (FVR) is generally ob-
' served from about the 4th through the 9th day after nor- x~
,~, 15 mal contact or challenge with virulent FVR virus. On ;
,: . ,
the fourth day after vaccination~ the negative results
~; of a throat swab test on all 5 cats indicate an absence
I '
~ of shedding of the virus. The antibody titer of all 5
.. . ~ - . .
cats prior to vaccination is less that 1:2 and one month ,
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later, just prior to challenge, the antibody titer
; of the 3 inoculated cats averages 1:18 as compared to
less than 1:2 for the 2 unvaccinated controls. One
month laterg all 5 cats are challenged intranasally
with virulent FVR applled by droplets to each nostril
` (about 1OJ 000-20~ 000 TCID50/cat). The cats are observed
for 2 weeks for evidence of clinical disease. All of
the 3 vaccinated cats remain normal with no clinical
... .
;~ disease or symptoms in contrast to the 2 unvaccinated
. ,,, . ~
110 controls which become very ill with ~VR disease ex-
:, ...
~ hibiting typical symptoms such as febrile response,
: . .
running eyes and noseJ lack of appetite and general
malaise. Two months after challenge, the antibody
3`', , titer o~ all 5 animals is about the same (1:54),
;~ 15 indicating the protection a~forded the vaccinates
.. . ~ ~ . . .
~ and the successful challenge to the controls.
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EXAMPLE III
:; i
Live virulent FVR virus cultured and isolated ~ -
according to the procedure described by J. L. Bittle
et al., ibid., and denoted by said investigators
as an "F-21' isolate WQS serially passed 37 consecu-
tive times in roller tube primary feline kidney tissue
culture at 2-7 day intervals and at incubation tempera-
tures of 35-37C using the same culture medium de-
- scribed by Bittle et al. At the end of the 37th
passage, the high degree of pathogenicity retained -
by the virus made it unsuitable for immunization
:,
purposes. However, attenuation of the virus to a
suitable vaccine ~as accomplished upon subsequent
`` passage, at lower lncubation temperatures. Followlng
; the 37th passage, 44 additlonal serial passages(for
a total of 81 passages) were then made at incubation
. . ,~ .
temperatures of about 32C.
'' '
.: :
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A 0.2 ml inoculum from the 81st such passage,
having a titer of 105- TCID50/ml (50 percent end
....
point infectivity) as calculated b~ the Reed
i` Muench method, was then added to monolayers of a fe-
~- line diploid tongue cell line in tissue culture
tubes (16 x 125 mm3 and the serial passage procedure
described in Example I was repeated for 7 consecutive pas- !:
;l sages followed by 3 terminal dilutions and 1 volume-increas-
ing passage for a total of 11 serial passages. At the
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,; end of the 11th passage, the pool of bulk raw vaccine
is harvested, identified (by serum neutralization tests
with specific FVR goat antiserum) and titrated.
''. ' ;'
EXAMPLE IV
~i. ,
1 Ml o~ the vaccine prepared in Example III and
having a virus titer at 35C of 105-4 TCID50/ml (deter-
mined by CPE) was administered intramuscularly to three
susceptible cats which were previously determined to be
sero-negative to FVR virus. Two other cats were main-
tained as unvaccinated control~. On the fourth day
after inoculation, ne~atlve re~ults of a throat swab
test on all 5 cats was obtained lndicating an absence
o~ shedding o~ the virus. Two weeks a~ter the inltlal
lnoculation date, one of the three vaccinated cats was
given a second 1 ml inJection intramuscularly. One month
after the initial inoculation date, all ~ive c~ts were chal-
~; lenged intranasally with virulent FVR virus applied by drop-
lets to each nostril (10~000-20~000 TCID50/cat). During
... .
the next two week observation period, all of the three vac~
cinated cats remained normal in contrast to the two unvac-
cinated cats which evidenced symptoms of FVR disease. The
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.. results showed that one inoculation of the sub~ect
~,~ vaccine produced a minimal antibody response where- -
. ~
. as two inoculations induced a significantly higher
.:. .
response. The following antibody titers were observed:
.
`- Table 1
.... :
~ Antibody Response
, - . ,
1 Month After 2 Months After
:: Pre-Vaccination Vaccination Challenge
,:
. Control 1 0 0 1:54
:. .
i:` Control 2 0 0 1:54
;~., . ~
. Cat A 0 1:18 1:54 ~:
.:; Cat B 0 1:18 1:54
Cat C* 0 1:54 1:162
~ .; . .
, .; ......................... _ _ _ :
.,........... ~, . .
,~ * Cat C received 2nd 1 ml in~ection 2 weeks after 1st - -
,','`,! injection.
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' EXAMPr~ V
Larger pools of vaccine material were prepared
after the 11th feline tongue passage described in Ex-
ample III repeating the same method in flasks described
therein for a total of 17 consecutive serial passage in
feline tongue. 500 Ml of the virus materlal obtained
from the 17th passage was added to 500 ml of N-Z amine
lactose glutamate stabilizer and dispensed into stan-
dard vaccine vials (titer equaled 104'6 TCID50/~1) that -
were dried by conventional freeze-drying procedures.
For inoculation purposesg the vials were reconstituted
with 1 ml pyrogen-free sterile distille~ water. 1 Ml
; of the thus-prepared vaccines were admlni~tered lntra-
muscularly to two susceptible cats with one other un- -~
vaccinated cat maintained as a control. One week later,
the two vaccinated cat9 were given an identical boo ter
dose intramuscularly. Nine ~onths a~'ter the initial
: ,
,~ inoculation, all 3 ¢ats were challenged intranasally
with virulent FVR virus applied by droplets to each nostril
(lO,OOG-20,000 TCID50/cat). The cats were observed for
13 subsequent days for evldence of clinical disease.
the two vaccinated cats each showed slight symptoms on
2 out of the 13 days whereas the unvaccinated control
experienced severe clinical symptoms for 6 of the 13
days. One year after the initial inoculation, all --
_ 16 -
,, ` ~ .,
~,
~............................................. ' ~'
"~ ., .

PM-l9
.,
58Cit7~`
.,`:
3 cats were rechallenged with FVR virus as before
with no subsequent clinical disease symptoms being
observed in any of the cats. In view of the fore-
-` going, the long-term effect of the subject vaccine
on the vaccinated cats was demonstrated. The lack of
disease symptoms in the unvaccinated control cat after
the second challenge was, as expected, due to the im-
munity derived from the natural infection caused by
the first challenge.
-, ' ',
EXAMPLE VI
~; 10 Further evidence of the long-term efficacy of
$~ the subject vaccinss was demonstrated in the following
one-year challenge 6tudy. 1 Ml of the vaccine prepared ;
in Example V was administered intramuscularly to 8 sus-
ceptible cats with 4 unvaccinated cats ~aintained as
` 15 controls. All 12 cats were pre-tested and found to be
; sero-negative to FVR virus. One week later, the 8
vaccinated cats were given an identical booster dose intra-
~` muscularly. One year after the initial inoculation date,
`~ 3 cats were given a third identical booster does l.M.
;~; 20 Five days later, all 8 cats were challenged intranasally
., .~, ,
~ with virulent FVR virus applied by droplets to each nos-
`` tril (10,000-20,000 TCID50/cat). The cats were observed
i for 13 subsequent days for evidence of clinlcal disease.
` The following results were obtained: ~
.,~':,. .. .
... , . ,, .:
,, ~,'
. !, ,., 17 1~
., . ~ .. . .
.~, ................................................................... . .
~'~'` ',: '
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... .

PM-19
,
.
58~74
:~ ~
` . ` ` ` ` ` ` '` `' `' ```:
. Table 2 .
. No. of Days ofSeverity of "
~ Clinical SignsClinical Signs
;;'` .- ~
: Control 1 9 severe `: :
,, . ~:
~ 2 8 severe ;
-
~ - 3~ ; ``~` 8 severe
;.. ~. 4 9 severe . ``
.,. _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ .,. :~
,. ~ ,.
w`. Cat A 3 slight ~
... . .
~, B 3 slight
. C 4 slight
., D 1 slight
" E 6 slight ~ -
Cat F* O none
G-~ O none
H* 3 slight
: .: . . .
~ * Cats F, G and H received the 2 boosters. ~-
,;
` ` ','~;
'.
:;`'.
" .
~. - 18 - ~
:;''. :.
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.. ..
,:
:.... ~' `; `
~; .. ~, , ,

PM-19
~5~
In addition to the preparation of the instant
vaccines from live virulent FVR virus obtained from
infected cats, this invention is also concerned with
the preparation of a FVR vaccine using the FVR virus
that has been modified by the method heretofore described.
.
It would be commercially impractical for the preparation
of a vaccine to use as the starting material for each new
batch of vaccine live virulent FVR virus obtained from
infecked cats and then go through the requisite serial
. . .
passages in order to acquire the modified virus for use as
a vaccine. This invention~ therefore, embodies the method
of preparing a FVR vaccine which comprises using as the
starting virus one that has already been modified by
serial passages in feline tissue cultures as previously
; 15 described, that iSJ a "seed" viru~ from a master batch of
,, .: .
attenuated virus. Accordingly, there Ls herein provided
~` a process of preparing a ~eline viral rhinotracheitis
vaccine which comprises propagating an attenuated FVR
virus, whlch attenuated virus is produced by the process
heretofore described, by sufficient serial passages at
. ..
an incubation temperature of about 32-37C in a nutrient
~ fluid feline tissue culture medium which is non-toxic to
- - said virus until said fluid medium contains from about 104 to
.;~ 7
j~A about 10 tissue culture infectious doses of said attenuated
, 25 virus per ml and harvesting the fluid vaccine.
,
,:'.................................................................... .
;,i,''' 19 .. ': ~
.: i .,: , ~
.. ~i, .
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; ~ ' ,'
~ ' , :
:,'.' , :
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. .

PM-l9
~L~351~
. . .
By the use of the procedures described herein, a
: . ~
modified FVR virus can be readily cultivated in large
quantities and in high concentrations. Using feline -
`~ tissue culture propagated modified FVR virus in con- -
centrations of at least about 104, generally from about
104 to about 107, and preferably from about 10 5 to
about 106 ~ tissue culture infectious doses of virus
per 1.0 ml of final vaccine3 and by parenterally administer-
ing 1 ml of such vaccine to cats, there is st~mulated in such
.~, . ~- .
; 10 vaccinated cats the production of protective FVR antibodies
comparable to those produced by natural infections without
producing the usual pathological symptoms of feline viral
~ -.
rhinotracheitis. The vaccinated cats are a~so able to
~` resist challenges with the disease-producing virusO
.~,,~.,, ~`.
A marked increase in the antibody response has
been observed upon the parenteral adminlstration of a
. ~ . .
second, and even a third or more, "booster" dose of the
;,.~"
~l instant vaccines. For example, it hal been found that
`~!j; beneficial results are obtained when a second i~tra-
muscular injection is given about one week following
the initial vaccination. For best results, it is recom-
mended that the second injection be given not sooner
that about 2~3 weeks following the first in~ection.
Excellent results have even been observed with a booster
.t 25 dose given up to 12 months later.
, ~ ,,! i .
.~ `. . ' .
i,;'~.................................................................. ~, '
':' ,., ' '`
~ ;.! 20 _
~, .................................................................... .
: :: . -
~ !
': ';
','.:.i :
.,, , '~ .
, ,'................................................................... .
', '' ,
,"' .
, . . . . .
,: . , , , :

PM~l9
.
'; '
',
As a further feature of this inventionJ lt has beenfound that enhancement of antibody production can be
accomplished, in addition to the aforementioned parenteral
administration of booster doses of the instant vaccinesJ
by the exposure of catsJ which have been previously im-
. ~
m,un,ized by parenteral administration of the instant vac-
"~ cines, to FV~ virus by the intranasal routeJ which FVR
,; virus either has ~een modified according to the present
invention or is in its non-modified virulent form.
',',~ , `'
` lO Such intranasal instillation following parenteral
"' vaccination produces significantly high levels of anti-
`:;
i"~ , bodies that persist for long periods of time. Further-
~, moreJ when such treated animals are challenged with viru- -
``` lent virus, the protection afforded is much more solid, `'
as demonstratied by the lack of clinica]. disease symptoms ''
. ,, .~ . .
, after challenges with as high as 250,000 TCID50/cat of ;~
,, virulent, non-modified FVR virus~ For best resultsg it ,~;
`,';'~ is recommended that a sufficient time elapse ~or the cat
to become sensitized after the initial parenteral vac-
~;, 20 cination in order to develop at least a minimal degree
~,', of immunity as reflected by increased antibody formation
:: 1
,,~ before subjecting the animal to the subsequent intra~
; nasal contact with FVR virus. Preferablyg the intranasal
,; instillationis given within 2-4 weeks follQwing the initial
' 25 parenteral vaccination although good results have been
observed when the former is administered up to 12 months
following the parenteral vaccinatlon. '~
~- , .
, _ 21 - ~
~: ::
.' ',::
.

1058~74 PM-l9
: .
.
Intranasal instillation is readily accomplished
by inhalation of the FVR virus either by conventional
:
aerosol formulations sprayed into the nasal passages
or by droplets applied to the outer nostrils or in the
'~ 5 nasal passages. A suitable concentration of FVR virus,
whether modified as described hereinbefore or in its live
virulent unattenuated form, for intranasal instillation pur-
poses following initial vaccination by parenteral ad- -~
ministration is from about 103 to about 10 tissue cul-
ture infectious doses per ml.
"
~ It is believed that the initial vaccination by the
;;~ parenteral route followed by contact wlth FVR virus
either modified or not, by the respiratory route con-
stitutes a novel method of immuniæation against feline
viral rhinotracheitis. Such method provides the animal
with a humoral antibody response and a local immunity
:' to the respirator~ tract that ls much more protective
, . ,~ .
against the disease. Thus, a means is provided for ef- ;
' fective, long-lasting protection against feline viral
rhinotracheitis.
'.~ ,
:` }~
.. , ~ . . . .. .
; ~ , .
- 22 -
:., ':' ' .;
.:~,,.
i
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~ ... . .
:~ .. . ~ , .,
!.;,` , ..
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.. PM-l9
1~5~7~
EXAMPLE VII
.. . --. :
; This example demonstrates a method of preparing a
pool of live virulent FVR virus suitable for administra- - i
.; tion by the respiratory route after parenteral vaccination.
, . .
. , .
A. Tissues: ~
.; ,.
. Primary feline kidney tissue cultures (PFKTC) are
;;i 5 harvested and grown at about 35C by the method described
.. . . .
by J. L. Bittle et al.~ Amer. J. Vet. Res , 21, 547 (1960)
.
-: employing conventional tissue culture techniques.
: .,;
The Crandell f`eline kidney cell line (CrFK), referred
to hy Lee et al., Cornell Vet., 5~, 539 (1969) ls grown
at about 37C in stationary tubes or bottles utilizing ~
. Minimum Essential Medium (MEM) with Earle's Balanced .
'l Salt Solution (BSS) and 10% fetal calf serum, 0.1% lactalbumin
. hydrolysateJ 30 units penicillin, 30 mic~ streptomycin and :
~:. .....
.~ 2.5 mcg amphotericin. Upon completion of monolayer growth,
.~, 15 the fluid medium is removed and a maintenance medium (same
~........ as above but with 1~2% fetal calf serum) is utilized at
.. ,. .. :
~ about 37C. ~ ~
J"''~' . , ' ' '`,' ' ", .
~.~. B._ Virus_Pool: ~:
", . i . ~,
1.0 M-Llliliter portions o~ a primary feline kidney
tissue culture medium consisting of 0.5~ lactalbumin hydro- ~ :
ly~sate and 10~ horse serum in Earle's BSS with 200 units
~; penicillin and 200 mcg streptomycin per ml was allowed to
,
.... : ~ .
: - 23 - ~:
... ',: . '.:
.~ . i
. ;. '
, ~ '. .
.
.~ ;:. .

;' ' PM-19
:ilfO58~4
stand in stationary tubes at about 35C until good gro~th
of cells was observed. The fluid was then replaced by
. maintenance medium [same as before except with reduced
, (5~) horse serum] and inoculated wlth a 0.2 ml sample
., 5 of live virulent FVR virus [identifled as the "C-27"
;,~
-~ isolate by R. A. Crandell et al., Proc. Soc. Exptl.
Biol. & Med.~ 97, 487 (1958)~. The lnoculated medium
, was placed in a roller drum at about 35C and the virus
,.',` harvested when approximately 80-go~ of the cells exhibited
,~ 10 cytopathogenic effects (CPE). This procedure was re-
ci peated for a total of 10 such serial passages. At the :
1"~ , ;
~ 10th passage, a 0.2 ml sample of material was inoculated
~,.............. . . .
~.~` into stationary tubes containing the CrFK cell line and
:; :
,"~, incubated at about 35C until 80-go~ CPE. Two similar
,'. 15 passages in CrFK cell line were conducted in bottles for
harvesting of a larger pool. After the 13th passage,
,': the harvested pool, which had a tlter of approximately
~~ 7
, 10' TCID50/ml, was stored at -70C. Dilutions of this, ,.
.' material to appropriate concentrations were ~ubsequently ' .
made for administration by the respiratory route.
~,. .. . . .
, . `
,i, i~
.... . .
, .
,....................................................... : `'
j . .
:
:' ' ' ' '.''~'
.. , _ 24 -
,. .. .
'.:. ,:
.~
. ,. ~
,'".:,' ' .
, ..,;
.
. . .
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.;.

PM-19
1058~74
EXAMPLE VIII ~`
., :.
1 Ml of the vaccine prepared in Example III and ~-
having a virus titer at 35C of 105 TCID50/ml was
administered intramuscularly to three susceptible cats
.
which were previously determinated to be sero-negative
- 5 to FVR virus~ Two other cats were maintained as unvac-
cinated controls. Eleven days after the initial inocula~
tion date, one o~ the three vaccinated cats was given
a second 1 ml inJection intramuscularly. About 1 month
after the initial inoculatlon date, all three vaccinated
.. . .
cats and the two unvaccinated controls were subjected
to intranasal instillation of the live virulent FVR
virus preparation obtalned from Example VII and appropriate~
ly diluted. The head of ea~ cat ls t;ilted backwards and
'`~ droplets totaling 250,000 TCID50/cat of the virus are de-
;~` 15 posited in the nostrils using a 26 gauge needle and s~rringe.
The ~ollowing results were obtained, 'Lndicating a signi~i-
cantly hlgher antibody response and lack of clinical disease
symptoms when intranasal administration of the virus follows
` parenteral vaccination.
.~
TAB LE 3
Antibody Res~onse _ _
Titer at time Titer After Clinical Symptoms
of Intranasal Intranasal After Intranasal
Instillation Instillation** Insti lation
. ,~ .
; Cat A* 1:54 1:162 none `;
Cat B 1:~8 1:54 none -
Cat C 1:8 1:54 none ``
, .
Con-troL 1~1:2 1:54 severe
Control 2~ 1:2 1:54 severe
* Cat A received the two injections.
; ~ ** Measurements taken 1 month after intranasal instillation.
.,t,.: ,, .
~ - 25
.',:, ~, .-
. ~ .
,: ;,:, , " ,
,'; , : ' ~ ` '

lOS~74 PM-l9
~XAMPL~ IX
A 1 ml in~ection I.M. of the vaccine prepared in Example ;;
(10 TCID50/ml titer) was given to seven susceptible cats
, which were previously determined to be sero-negative to FYR
.:
virus. A second similar in~ection was administered to all
seven cats 15 days later. Three of the vaccinated cats were `.
"
-:, given an identical third inaection 5 days prior t~ intranasal
.. instillation with live virus. Two other cats were maintained
. as unvaccinated control9. Abou~ one year following the initial
.. ; inoculation date, all seven vaccinated cats and the two unvac- ,
.;~ . .
cinated controls were subjected to intranasal instillation of
the live virulent FVR virus preparation obtained ~rom Example
~II and appropriately diluted. Each cat received 250,000 tis-
sue culture infectious doses of the virus by droplets applied
~. to the nostrils. The ~ollowing results were obtained~ indicating `:~
*.1 15 the long-term effectiveness occassioned by the parenteral plus
intranasal routes of administration.
TABLE 4
.
Antibody Response~
Titer at Time Titer After Clinical Symptoms
of Intranasal Intranasal After Intranasal
Inst111~tion Instlllation* Instillation ~ -
Cat A 1:15 1:290 mild
Cat B 1:15 1:417 mild
Cat C 1:7 1:96 mild . .
Cat D 1:22 1:200 mild :.
Cat E** 1:22 1:200 none
Cat F** 1:46 1:290 none
Cat G** 1:32 1:200 very mild ~.
Control 1 ~ 1:2 1:66 severe
Control 2 ~ 1:2 1:22 severe
i;.~
_
* Measurements taken 6 weeks after intranasal instillation.
** These cats received a total of 3 I.M, in~ections~
. .,
.,
- 26 - . :
;.~`~...................................................... ...
~ , . . ... .
... . .

1058~74 PM~
. ~^
EXAMPLE X
A 1 ml injection I.M. of the vaccine prepared in .
Example V (10 ' TCID50/ml titer) was given to two ':
;~ susceptible cats which were previously determined to be ~
,- sero-negative to FVR virus. A second similar injection i~ --
, 5 w~s administered to both cats 15 days later. About 9 , ,
months following the initial inoculation date, the two
, vaccinated cats w,ere subjected to intranasal instilla-
., tion of the live virulent FVR virus preparation obtained '
`r' from Example VII and appropriately diluted. Each cat
~, 10 received 100,000 tissue culture infectious doses of the
',. virus by droplets applied to the nostrils. Three months -
~'~ later all the cats were challenged intranasally with
.~ 250,000 TCID50~cat of live FVR virus. The follwing re- ~,
-, sults were obtained: ~ ~
. . .
:;~ TABLE 5
`$:` Antibody Antibody Antibody
Titer at Time Titer at Clinical S~ymptoms Titer Six ~:
~, of Intranasal Time of 14 Days After Weeks A~te~
~ Instillation Challenge Challenge _ Challenge _
.;~ Cat A 1:7 1:45 none 1:138
`1,,'i Cat B 1 ,7 1:66 none 1:96 ~.
., ,; - ~ ,
.. , ":
:,', ' '. ~:.~, , .
. , .
.
~:,.,,.j ,, : .. '. .
~ 27 - .,.
" .
,;, .
:'." , '
,.. -
. ;, .
~ ..................................................................... .
.:

PM-l9
~158~
. ~
,`.,
,;. . ,:
The foregoing Examples VIII through X demonstrate .-
. the feature of this invention whereby cats are afforded
effective long-lasting immunization against FVR by the
., 'A .
process which comprises first adminiistering parenterally
. 5 to a cat a vaccine of at least about 104 tissue culture
, infectious doses of an attenuated feline viral rhino- :
.. tracheitis virus, which virus was attenuated by
,.,;,,
~,~ at least 7 two- to seven-day serial passages through
feline tissue cultures in a nutrient fluid at an in-
,, .
i. 10 cubation temperature of about 30+2C, followed by a
.~ subsequent administration to said cat by the respira-
,. tory route of from about 103 to about 10 tissue cul-
`.~ ture infectious doses of feline viral rhinotracheitis
virus in its live virulent unattenuated form. Similar
. 15 results are also obtainable wlth the respiratory ad-
~; ministratlon of from about 103 to about 106 tissue cul-
~ ~ .
ture infectious doses of feline viral rhinotracheitis
~`, virus which has been attenuated according to the methods ~ ~,
.~ o~ this invention.
. ,..;
,~,. . .
.....
.~,:. :
.! . :
~' .",
~'''
28 -
.
~. ~
... .
, ~ ~
, . ...

Representative Drawing

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Event History

Description Date
Inactive: IPC from MCD 2006-03-11
Inactive: Expired (old Act Patent) latest possible expiry date 1996-07-10
Grant by Issuance 1979-07-10

Abandonment History

There is no abandonment history.

Owners on Record

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Past Owners on Record
None
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Claims 1994-04-21 4 125
Abstract 1994-04-21 1 27
Drawings 1994-04-21 1 16
Descriptions 1994-04-21 28 1,034