CA1198368A - Rabies vaccine - Google Patents

Abstract

RABIES VACCINE

ABSTRACT OF THE DISCLOSURE

The preparation of a vaccine for immunization of canine and feline animals from inactivated HCP-SAD strain 25 of rabies virus is disclosed.

Description

R~BIES- VACCINE

~hîs invention relates to veterinary rabies vaccines. More particularly, the invention relates to the propagation of rabies virus in swine testicle cell cul-ture~ to mo~o- and polyvalent vaccines containing the inact~vated rabies virus so propagated and to the use o 20 such vaccine~ to vaccinate canine an~ feline animals. The inactivate~ rabies vaccine of this invention is particularly useful for vaccinating dogs and cats and produces advantageously high antibody responses in cats.
For many years, research has been directed toward 25 the preparation of sa~e and effective veterinary rabies vaccines [Crick et al., Vet. Rec. 99(9):162 (1976);
Plotkin et al., Ann. Rev. Med. 29:583 (1978)1. A number of rabies waccines are currently marketed for use in dogs, cats and other animals. These vaccines are classified as 30 nervous tissue vaccines~ avian embryo vaccines and tissue culture vaccines, depending on the ~edium in which the virus was propagated~ The inactivated rabies vaccines currently marketed are of mur ine (nervous tissue) and hamster cell line origin (tissue culture~ ~"Compendium of 1 Animal Rabies Vaccinesl', 1980, J. Amer. Vet. Med. Asssc.
176(5):~99 (1980)] and are Icnown to be o~ limited safety, particularly in use with cats. In fact, high cell pas5age SAD rabies strain vacoines are no longer approved by the United States Department of Agriculture for vaccination of cats.
Many known veterinary rabies vaccines comprise virus which has been propagated in tissue culture. For example, the Flury HEP ~train was grown in canine kidney 10 cells [Brown et al., Amer. J. Vet. Res. 28(124)~751 (1967)], the ERA strain was propagated and attenuated in porcine kidney cells (U. S. Patent NoO 3,423,505), the PRI
strain was produced by repeated passage of the ERA strain in porcine kidney cells (U. S Pa~ent No. 4,040,904) and 15 the ERA strain has been attenuated in bovine kidney cells (U. S. Patent No. 3,585,266; German Patent No.

2,163,013). Other cells used for the production of attenuated or inactivated rabies vaccines include hamster fibroblasts [C. R. Hebd. Seances Acad. Scio Ser. D. Sci.
20 Natur. 265(25):2143 (1967)J, baby hamster kidney cells [Crick et al., Res. Vet. Sci. 12(2) o].56 (1971); U. S.
Patent No~ 3,769,415], chick embryo fibroblasts (U. S.
Patent No. 4,115,195; Belgian Patent No. 863,368), fetal calf kidney cells (French Patents Nos. 2,261,779 and 25 2,290,220), fetal canine lung diploid cells (Belgian Patent No. 859,178), human diploid cells (U. S. Patent No.

3,397r267), a diploid porcine embryonic cell strain (U. S.
Patent No. 4,070,453), human and murine neuroblastoma cells [Clark, Science, 199(4333):1072 (1978) and Infect.
30 Immun~ 27(3):1012 (1980)], African green monkey kidney cells [Nawathe et al., Bull. Anim. Health Prod. Afr.
` 26(1):1 (1978)] and quail embryo primary cells [Bektemirova et al., Arch. Virol. 61~1-2):61 (1979)].

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1 Until the present work, rabie~ virus has not been adapted for growth in swine testic]e cell cultures~ The present invention consists of the growth of rabies virus in swine testicle cell cultures, particularly in a diploid swine testicle cell line designated the NL-ST-l cell line, and the preparation of safe and highly effective mono- and polyvalent vaccines from the inactivated virus for immunization of canine and feline animals against rabies.
Swine testicle cell cultures used to propagate the rabies 10 virus are described by McClurkin et al., Can. J. Comp.
Med. Vet. Sci., 30:190 (1966). Use of the NL-ST-l cell line for virus production was approved by the United States Department of Agriculture, Animal and Plant Health Inspection Service in November, 1976; a pseudorabies 15 vaccine containing virus propagation on this cell line was licensed and marketed in the United States in 1977.
The monovalent vaccine of this invention is administered parenterally, preferably by lntramuscular injection, in one or more doses. Preferably, a single 1.0 20 ml to 1 2 ml dose of vaccine containing 0.93 ml of inactivated virus containing fluids having from about 104- to about 109- TCID 0/ml, preferably from about 106- to about 108-~ TCID50/ml, combined with a suitable carrier, adjuvant and/or stabilizer is 25 administered. Animals younger than three months of age when initially vaccinated should be revaccinated after reaching the age of three months. Annual revaccination is recommended.
The rabies virus used to prepare the inactivated 30 virus vaccine of this invention is the high cell culture passage of the Street Alabama Dufferin (HCP-SAD) rabies virus strain. This virus was initially isolated from a rabid dog at CDC-Dufferin Laboratories, Montgomery, Alabama in 1935. The isolate was passaged 54 times in 35mice, followed by 25 passages in hamster kidney cell 1 culture, 10 passages in embryonated chicken egys and 40 serial passages in porcine kidney cell culture. The virus is then adapted for growth in the swine testicle cell cultures by passaging at least once. The virus may be passaged up to about 25 times in swine testicle cell cultures, with from about 6 to about 12 passages being preferable. sefore passaging in swine testicle cellsl the virus may be passaged in other mamalian cell cultures such as bovine kidney cell cultures.
After growth in swine testicle cells at from about 34C. to about 38C., preferably about 36C, the virus is inactivated with an inactivating agent which does not destroy the virus particles or antigenicity according to standard methods known to the art. Examples of such 15 inactivating agents are beta-propiolactone or ethyleneimine derivatives, preferably beta-propiolactone.
To prepare the vaccine of this invention, the inactivated rabies virus is combined with an adjuvant, a suitable carrier and/or a stabi]izer according to - 20 standard, known to the art methods. Any known adjuvant which enhances the antigenicity of the vaccine, for example aluminum hydroxide gel, may be used.
It has been found that a single 1.0 ml intramuscular vaccination with the inactivated rabies 25 vaccine of this invention having a titer of about 106-; to about 10 TCID50/ml elicited si~nificant serological responses in 100~ of vaccinated dogsO At one year following vaccination, 96% of these dogs remained protected against challenge with virulent virus which 30 killed 100% of the unvaccinated controls. In cats, vaccination with a single 1.0 ml intramuscular vaccination of the inactivated rabies vaccine of this invention having a titer of about 106- to about 108- TCID50/ml produced a surprising and highly significant serological 35 response in 100% of the vaccinated animals. At one year ..

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5_ 1 following vaccina-tion, 100% of these cats rernaine~ protected against challenge wi~h virulent v:irus whi.ch killed 90~ of the unvaccinated controls. Thus, the inactiva-ted rabies vaccine of -the present invention is a safe and effective veteri.nary vaccine, being particularly safe, effect:ive and superior -to prior art vaccines for protecting felines agains-t rabies.
The inactivated rabies vaccine of this invention has been licensed for use in dogs and cats by the United States Department of Agriculture, Animal and Plant Heal-th Inspection Services on June 16, 1980. It is believed to be the only vaccine containing the SAD strain of rabies virus approved for use in cats.
Thus, in accordance with the present teachings, a com-bination vaccine is provided capable of inducing immunity to feline animals without serious side effects comprising vaccinal amounts of inactivated HCP-SAD strain of rabies virus which has been adapted to grow, before inactivation, in swine testicle cell cultures and one or more vaccinal feline viruses selected from modiEied fel.ine viral rhinotracheitis virus, calvicivirus and panleukopenia virus.
DETAILED DESCRIPTION OF THE INVENTION
Preparation oE the Inactivated Rabies Vaccine The high cell passage of Street Alabama D~fferin (HCP-SAD) strain of rabies virus was used to prepare the inactivated ~5 r~ibies vaccine of this inven-tion~ This virus was obtained from a commercial rabies vaccine manufactured by Jensen-Salsbury Laboratories, Kansas City, Missouri a-t the 129th passage level and was further transferred for 15 additional serial passages in a bovine kidney cell culture and 6 serial passages in the swine testicle cell line, NI.-ST-l. The 150th passage was designated as the master seed virus. The virus was identified by specific immunofluorescence of infected cell cultures stained with fluorescein conjugated rabies specific antiserum.
Viral antigen was demonstrated in the cytoplasm of infected cells. The virus was also identi~ied by neutralization with specific rabies antiserum. ~7irulence was demonstrated by intracerebral inoculation of the master seed virus into young adult mice.

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1 For propagation of the master aeed virus, frozen ampoules of NL-ST-l cells were thawecl and reconstituted to sufficient volume for seeding in production flasks. The production flasks were allowed to incubate at 36C until a confluent cell monolayer formed (3 to 5 days). Passage of these cells was accomplished by use of a combination of versene-trypsin and the cells were expanded in volume to plant sufficient numbers in production flasks for inoculation. While the cells were suspended in the growth 10 medium, master seed virus fluids were added to the suspension at 0.1~ of total medium volume. Production flasks were planted with the virus-inoculated growth medium and the cells allowed to multiply until 90-100%
confluency of cell monolayer was achieved (3 to 5 days).
15 These virus-infected cells were removed from the production flasks with versene~trypsin and suspended in maintenance medium. Production flasks were planted with - maintenance medium containing the virus-infected cell suspension and maintained at 36C until harvest.
20 Replication of the rabies virus is complete at the time of appearance of cytopathogenic effects, characterizetl by rounded cells which subsequently become detached from the cell monolayers. Contamination was detected by gross and microscopic examination. Titer of the master seed virus 25 inoculum was at least 106- TCID50/ml.
Harvest of virus-laden fluids following production flask planting, which is dependent upon the cytopathogenic changes occurring in the infected monolayer, occured after 3 to 6 days. The virus-laden fluid was transferred and 30 pooled into sterile storage con~ainers under sterile environmental conditions. Samples of harvested fluids were tested for sterility and viru~ titer. Only fluids found to be pure were used for vaccine preparation. Viru titer of the harvested fluids must be from about 104-35 to about 10 TcID5o/ml-1 To inactivate the harvested vlrus, beta-propiolactone was added to the pooled fluids at pH
6.8 to 7.4 to a final concentration of 1:6000 (0.017~).
The virus containing fluids were maintained at 4C with constant stirring for 48 hours. Following inactivation, merthiolate (thimersol, N.F.~ was added as a preservative to give a final concentration of 1:10,000. After the addition cf merthiolate, the product was stored at 4C
until addition of ad~uvant.
The inactivated virus fluids were pooled, the pH
was adjusted to 6.0 with 4N HCl and aluminum hydroxide gel (2~ A12O3) was added as adjuvant until a final concentration o~ 5~ by volume. The inactivated virus-aluminum hydroxide gel mixture was stirred for 2 15 hours at 4C. The vaccine thus prepared was tested ~or safety and potency and stored at 4C.
An accelerated stability study was conducted to determine stability of the vaccine. After incubation of samples of a vaccine prepared as described above at 37C
20 for one week, there was a loss of 0.04 relative potency as compared with a sample stored at 4C.
The relative potency of the vaccine was determined by the National Institutes of Health (NIH) mouse potency test described in Chapter 33 of "Laboratory Techniques in 25 Rabies", 3rd edition, WHO, Geneva (1973) by comparing test results of five samples of the vaccine to test results of five samples of NIH standard reference vaccine. The geometric mean relative potency value was 0.15.

30 Use o~ the Inactivated Rabies Vaccine The inactivated rabies virus vaccine was used to vaccinate susceptible dogs and cats. Rabies-susceptible dogs were obtained from commercial sources and ranged in age from 4.5 to 7.5 months when vaccinated. Rabies-sus-35 ceptible cats were obtained from commercial sources and 3~;~
~3-1 ranged in age from 7 to 13 months when vaccinated. Each animal was vaccinated intramuscularly at one site in the thigh with a single 1 ml dose of vaccine having a titer of 106- TCID50/ml, either undiluted (full dose) or diluted 1:2 (half dose). The half dose vaccine was prepared by ~ixing equal amounts of the undiluted vaccine and diluent containing 5% aluminum hydroxide gel. All vaccinated animals were bled prior to vaccination and at 30, 60, 90, 180, 270 and 365 days following vaccination 10 Serum was collected and evaluated for the presence of serum neutralizing rabies antibody in accordance with the Quantitative Assay and Potency Test of Antirabies Serum and Immunoglobulin in Chapter 40 of "Laboratory Techniques in Rabies", 3rd edition, WHO, ~eneva (1973). Serum-neutra-15 lization (SNj antibody titer was determined by the methodof Reed and Muench, J. Amer. J. Hy~iene, 27:493 (1938).
At 365 days following vaccination, the immunity of the test animals was challenged with virulent NYC strain of street rabies virus. The challenge virus was a 20 dilution of a 20~ fox brain suspension~
Vaccination and Challenye of Dogs Twenty-six dogs determined to be serologically negative to rabies virus were vaccinated with a full dose of the inactivated rabies vaccine. At 30 days following 25 vaccination, the sera of the vaccinates contained a geometric mean antibody titer of l:109, with titers ranging from 1:32 to 1:431. During a one year serological evaluation, titers gradually declined to a geometric mean antibody titer of 1:10 at 365 days following vaccination.
30 Twenty susceptible control doss were inoculated with virulent NYC strain of street rabies virus, ten animals receiving virus diluted 1:150 and ten receiving virus diluted 1:300. One year (365 days) following vaccination, the vaccinated dogs were challenged with a 10150 dilution 350f the virulent rabies virus (NYC strain of street rabies 1 virus). I'wenty-five out of twenty-six (96%) of the vaccinates remained normal throughout the 90 day period following challenge. The results from this test appear in Table l; results frorn the control animals appear in Table 3.
Twenty-five dogs determined to be serologically negative to rabies virus were vaccinated with a half dose (1:2) of the inactivated rabies vaccine. At 30 days following vaccination, the sera of the vaccinates 10 contained a geometric mean antibody titer of 1:42, with titers ranging from 1~8 to 1:204. Titers gradually declined to a geometric mean antibody titer of 1:7 at 365 days following vaccination. The immunity of the animals was challenged with virulent NYC strain of street rabies 15 virus 365 days following vaccination. Twenty-three owt of twenty-five (92%) of the vaccinated dogs survived challenge. The results from this test appear in Table 2;
results from the control animals appear in Table 3.
Vaccination and Challen~e of Cats Twenty-five cats determined to be serologically negative to rabies virus were vaccinated with a full dose of ~he inactivated rabies vaccine~ At 30 days following vaccination, the sera of the vaccinates contained a geometric mean antibody titer of 1:13~8, with titers 25 ranging from 1:214 to 1:8192. At 365 days following vaccination, the geometric mean antibody titer was 1:88 with titers ranging from 1:26 to 1:1024. As controls/
twenty susceptible cats were inoculated with virulent NYC
strain of street rabies virus, ten animals receiving virus 30 diluted 1O30 and ten receiving virus diluted 1:60. The immunity of the vaccinated cats was challenged at 365 days following vaccination with virulent rabies virus (NYC
strain of street rabies virus). All (100%~ of the vaccinated cats remained normal throughout the 90 day 35 period following challenge. The results from this test 1 appear in Table 4; resul~s from the control animals appear in Table 5.

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l TABLE l Protection A~uL~e~ DocJs b~ V~ ;n~;~n With Inactivated Rabies ~accine, S~ine Testicle Cell Line Orig m (Full Dose) Dog Serum N~utrA~ ;~n Titer* Following Vac. (days) Status No. 0 30 60 90 180 270 365 Post ~h~ n~
Neg.81 16 8 4 13 64 Normal 11 Neg.38 5 4 3 Neg.Nes. Normal 10 16 N~g.38 41 13 5 ~ 2 ~ormal Neg.206 l9 8 3 5 10 Normal 22 Neg.128 10 4 3 4 13 Died (Rabies, 19) ~3 Neg.~2~ 54 64 l9 19 2 Nbrmal 15 27 Neg.64 76 81 54 64 51 Normal 29 Ne~.203 41 19 25 32 38 ~brmal 31 Neg.~03 41 65 54 ~5 32 Normal Neg.431 12 ll. 1.3 5 5 Normal 6~ Neg.128 5 4 6 2 4 ~bxmal 20 79 Neg.98 5 8 13 25 16 Normal 82 Neg.87 214 54 13 11 lO No~nal 83 Neg.431 13 64 76 64 128 Norn~il 94 Neg.182 l9 l9 l9 l9 54 Normal 97 Ne~.203 25 21 ~9 13 6 Normal 25 38 ~eg.38 16 64 16 6 3 Normal 99 Neg.203 54 16 16 10 6 N~rmal 100 Neg.32 5 54 25 13 13 Nbrmal 101 Neg.256 19 54 l9 16 10 N~rmal 107 ~eg.81 3 3 4 4 lO Normal 30 112 Neg.38 10 54 6 4 2 Nvrmal 144 Neg.81 128 46 13 lO 19 Normal . 145 Ne~.107 20 16 6 16 10 N~rmal 154 ~eg.81 41 6 2 ~eg. 2 NDrmal 155 Neg.85 32 19 2 3 2 Normal 35 Geo. - ~
Mean Neg. lO921 l9 lO lO lO
*Titer expressed ~s reciprocal of serum-neutralization end point.

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Protection Aff~rded Dogs by Vaccination with Inactivated ~abies Vaccine, Swine Testicle Cell Line Oriqin (HAI f Dose) D~g Ser~m NeutrAl;~A~;~n Titer* F~l ~c~ng VaC. (days) Status 5 No. 0 30 60 90 180 270 365 post-rh~ nge 2 Neg. 8 3 5 5 3 2 Normal 3 ~eg.20 4 2 4 5 3 Normal

4 Neg.204 54 25 13 6 16 Normal 19 N~g.20 5 5 4 5 3 Normal 1021 Neg.81 5 10 4 4 3 Normal 42 Neg.51 41 10 3 2 Neg. Normal 43 Neg.151 46 25 5 4 3 Normal 64 Neg.32 5 14 8 13 4 Normal 67 Neg.27 Neg. 2 5 3 Neg. Normal 1574 Neg.32 6 3 Neg. 6 3 Normal 78 Neg.16 2 2 2 2 4 Normal 81 ~g. 32 3 3 13 6 10 Normal 103 Neg.13 4 3 4 3 4 Normal 104 Neg.13 22 10 Neg. 3 2 Died (Rabies, day 14) 108 Neg.51 13 6 13 10 32 Normal 109 Neg.51 11 8 4 4 5 Normal 110 Neg.32 5 5 3 3 4 Normal 111 Neg.28 4 2 3 Neg. Neg. ~ormal 25 115 Neg.38 10 3 8 6 6 Died (Rabies, day 14) 118 Neg.166 32 41 13 20 21 Normal 123 N~g.81 41 25 25 11 54 Normal 125 Neg.81 13 3 N~g. 4 Neg. ~ormal 30 126 Neg.~7 13 16 13 ~0 13 N~rmal 143 Neg.12B 19 19 16 20 19 Normal 147 ~eg.151 16 ~1 41 102 54 Normal Gec)~ , ~b~n Neg.42 10 7 7 6 7 35 *Titer e~pr s5ed as reciprocal of serum n~LLdlization ~nd pomt.

-TP.e;LE 3 Dog Controls - 20% F~x Bram 5~ o:E N~C StrAin of Street Rabies Virus ~0.5 ml ~;1A~r~1 rL~e~e, r~lscles) ~h~ n~e Dog. Cbservations Dilution No.
Rabies - Da~ 11 71 R~bies - Day 10 87 Rabie~ - Day 10 1:150 106 Rabies - Da~ 10 116 Rabies Day 10 130 Rabies - Day 13 132 Rabies ~ Day 12 135 Rabie6 ~ D~y 13 136 Rabi~s - Day 10 137 Rabies - ~ay 14 1 Rabies - Dk~y 13 Rabies - Day 13 9 Rabies - Day 12 1-300 4g Rabîes - Day 13 86 Rabie~ - Day 11 91 Rabie~ - Day 10 105 Su~7iv~d 124 Rab.ies - Day 13 134 Rabies Day 11 150 Rabies - D~y 10 1 T~BLE 4 Protection Afforded Cats by Vaccination w~.th :Lnac~ivated Rabies Vaccine, Swine Testicle Cell Line Ori.gi~ (~Llf Dose)

5 Cat Serum Neu~r~ tion Titer* Foll~ ~ng Vac. (days) Status Nb. 030 60 90 180 270 365 post-rh~ nge N~6 NegO6166 64 102 64 100 64 ~ormal NF3 Ney. 407 646 407 151 263 64 Normal 1o WXl Neg.8192 102 151 128 407 101 Normal W~3 Neg.2028 64 32 32 64 64 Normal S~2 ~eg. 302 64 23 32 42 40 NoLmal WV4 Ney. 304 64 41 20 51 64 Normal NCl Neg.5248 256 128 324 214 64 N~rmal Neg.1024 64 32 64 64 50 N~rmal N~l Neg~ 512 1024 1230 603 1622 256 Normal XB2 ~e~.1622 64 64 51 51 64 Nbrmal SB3 Neg.1024 64 91 128 214 64 Normal WD2 Neg.1445 302 813 603 817 215 Normal 20 NF4 Neg.1660 76 G4 128 54 54 Normal ~3 Neg.8192 302 813 151 76 64 Normal NC4 Neg. 407 64 54 128 162 54 ~brmal B~l Neg.6457 302 128 151 407 215 ~ormal LD3 Neg. 256 56 32 46 32 16 Normal 25 SBl Neg.8192 1622 1122 1738 1445 406 Normal SC3 Neg.1318 102 162 151 427 101 Mormal S~l Neg. 512 64 16 64 107 64 Normal LC3 ~eg. 407 64 81 107 128 40 Normal SG5 ~eg.4096 646 512 1230 1024 1024 ~ormal 3o BCl Neg. 21~ 102 151 427 646 304 Normal OFl Neg. 407 54 38 64 256 64 Normal BB2 Neg.6456 214 43 50 51 ~4 Nbrma~
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Mean Ne~.1328 138 112 130 178- - 88 *Titer expressed as reciprocal of serum neutralization end point.

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Cat c~ntrDl~ - 20% F~x Bram ~l~rPn.~i~n of N~C Strain Street Rabies Virus (0.25 ml Bilateral Neck Mhtscles) I'h~ .n~e Cat C~5~ vd~ s Dilut~^on No.
22 Rabies - Day 15 23 Rabies - Day 20 24 Rabies - Day 14 1-30 WQ2 Rabies - Day 13 S~vived Rabies - Day 20 26 Rabies - Day 29 28 Rabies - Day 35 aF6 ~abies - Day 57 SC2 Rabi.es - Day 29 NDl Survived ND2 Rabies ~ ~ay 12 NC3 Rabies - Da~ 31 1:~0 N~4 Survived NF2 Rabies - Day 17 BF2 Rabies - Day 11 EA3 Rabies - Day 34 ~53 ~abies - Day 36 OG2 Rabies - Day 13 OF3 R~bies - Day 36 1 A further aspect of this invention is the preparation and use of combination polyvalent vaccines comprising vaccinal amounts of the adjuvanted inactivated rabies virus described herein and one or more canine or feline viruses. For example, feline vaccines comprising vaccinal amounts of modified feline rhinotracheitis virus, calicivirus, and/or panleukopenia virus combined with the inactivated rabies virus can be prepared. Such polyvalent vaccine will, preferably, contain from about 30% to about 10 70% total volume of the inactivated rabies virus, depending on the number of viruses in combination. An example of such a polyvalent feline vaccine contemplated by this invention comprises about 40% of the inactivated rabies virus, about 20% of feline rhinotracheitis virus, 15 about 20% of calicivirus and about 20% of panleukopenia virus (all percentages based on total volume).
Likewise, canine vaccines comprising vaccinal amounts of distemper virus, canine adenovirus type 2 and para-influenza virus combined with the inactivated rabies 20 virus can be prepared. I.eptospira bacterin may also be added to such polyvalent vaccine. The polyvalent vacclne will, preferably, contain from about 30~ to about 40~
lotal volume of the inactivated rabies virus. An example of a polyvalent canine vaccine contemplated by this 25 invention comprises about 40% of the inactivated rabies virus, about 40% of distemper virus and about 20% total of all other micro-organisms in the combination (all percentages based on total volume).
The polyvalent vaccines of this invention are 30 administered parenterally, preferably by intramuscular injection.

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Claims (3)

CLAIMS:

1. A combination vaccine capable of inducing immunity in feline animals without serious side effects comprising vaccinal amounts of inactivated HCP-SAD strain of rabies virus which has been adapted to grow, before inactivation, in swine testicle cell cultures and one or more vaccinal feline viruses selected from modified feline viral rhinotracheitis virus, calvicivirus and panleukopenia virus.

2. The combination vaccine of claim 1 which contains from about 30% to about 70% total volume of the inactivated rabies virus.

3. The combination vaccine of claim 2 which contains about 40% total volume of the inactivated rabies virus, about 20% total volume of modified feline rhinotracheitis virus, about 20% total volume of calicivirus and about 20% total volume of panleukopenia virus.