CA1092972A - Feline viral rhinotracheitis vaccine and combination feline viral rhinotracheitis-calicivirus vaccine prepared therefrom - Google Patents

Abstract

CANADA

ABSTRACT OF THE DISCLOSURE

A modified live feline viral rhinotracheitis vaccine is prepared by treating with ultraviolet light a feline viral rhinotracheitis virus which has been subjected to chemical treatment with mutagens. A combination feline viral rhinotracheitis-calicivirus vaccine is prepared by combining a modified live feline viral rhinotracheitis virus and naturally-occurring calicivirus.

Description

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 ~ .

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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.

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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 :, -4a-:3 092~7Z

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 .. .: ,:

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 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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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 " ~

~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.

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).

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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.

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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 . . , ~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.

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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 ~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.

Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A combination modified live feline viral rhinotracheitis-calicivirus vaccine capable of inducing immunity in animals of the genus felidae without serious side effects comprising a modified live feline viral rhinotracheitis virus which has been chemically modified so that said virus grows well at 30°C ? 2°C but not at 39°C with ultraviolet light until from about 90% to about 99% of the virus particles are killed, a vaccinal feline calicivirus and a carrier therefor.

2. The combination of claim 1 wherein the modified live feline viral rhinotracheitis virus was passaged ten to twelve times in stable feline kidney cells containing from about 2% to about 10% of foetal calf serum at 31°C.

3. The combination of claim 2 wherein the modified feline viral rhinotracheitis virus is ATCC No. VR 814.

4. The combination feline viral rhinotracheitis-calicivirus vaccine of claim 1 where the modified live feline viral rhinotracheitis virus and the calicivirus are present in a 1:1 ratio.