CA1058074A - Feline rhinotracheitis vaccine and production thereof - Google Patents

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.

Description

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.
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; ~ 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.
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;~ 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.

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~ 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- ~
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sages in order to replicate and isolate the virus.
A particularly suitable culture medium is one derived :.":
' 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 ;
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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.
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~'` 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.
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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. -~
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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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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 ;
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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.

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~; 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
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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 ~
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~; 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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PM-l9 ~L~S1~ 74 ., 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 ,~. i ?```~: - 14-".. . .
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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 ~ .; . .
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,~ * Cat C received 2nd 1 ml in~ection 2 weeks after 1st - -,','`,! injection.
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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 -,, ` ~ ., ~, ~............................................. ' ~' "~ ., .

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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: ~
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. Table 2 .

. No. of Days ofSeverity of "
~ Clinical SignsClinical Signs ;;'` .- ~
: Control 1 9 severe `: :
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~ 2 8 severe ;
-~ - 3~ ; ``~` 8 severe ;.. ~. 4 9 severe . ``
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w`. Cat A 3 slight ~
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~, 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. ~-,;

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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.

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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
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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.

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'; ' ', 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. '~
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1058~74 PM-l9 : .

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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.
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EXAMPLE VII
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; 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.
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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.
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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. ~ ~
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", . 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 , .... : ~ .
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;' ' 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.
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1058~74 EXAMPLE VIII ~`
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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.
.~

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.
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,: ;,:, , " , ,'; , : ' ~ ` ' 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.

.
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~

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1058~74 PM~
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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 ~.

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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.

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Claims (10)

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.

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