EP0261168A1

EP0261168A1 - Feline infectious peritonitis vaccine

Info

Publication number: EP0261168A1
Application number: EP87901809A
Authority: EP
Inventors: Charles A. Baldwin; Fredric W. Scott
Original assignee: Cornell Research Foundation Inc
Current assignee: Cornell Research Foundation Inc
Priority date: 1986-02-06
Filing date: 1987-02-05
Publication date: 1988-03-30
Also published as: JPS63502828A; AU604682B2; AU7037287A; CA1303500C; WO1987004624A1

Abstract

On a découvert que le virus de la péritonite infectieuse des félins, WSU FIPV 79-1146, peut être affaibli pour former un vaccin vivant modifié contre la péritonite infectieuse des félins (PIF), qui transmet l'immunité, est sûr et ne provoque pas la PIF, chez le félin.It has been discovered that infectious feline peritonitis virus, WSU FIPV 79-1146, can be weakened to form a modified live vaccine against infectious feline peritonitis (FIP), which transmits immunity, is safe and does not cause PIF, in the feline.

Description

-I-

FELINE INFECTIOUS PERITONITIS VACCINE

BACKGROUND OF THE INVENTION

This invention relates to the immunization of felines against σoronavirus (FIPV) induced infectious peritonitis (FIP).

Coronaviruses which have been isolated from cats can be divided into two groups: viruses which cause FIP and viruses which cause a transient subclinical to severe enteritis. The various isolates are all morphologically and antigenically related and probably represent stains of a common species of virus that infects cats, dogs and swine; (see Pedersen et al, Adv. Exp. Med. Biol., 173:365-380 (1984)).

The development of a vaccine against FIP has eluded investigators for some time. Pedersen et al, supra, summarize the state of the art which indicates that FIPV antibodies actually can sensitize felines to the disease. The literature has reported that following immunization with live alternated virus, challenge with virulent virus caused increased infection rates, reduced latency periods and enhanced disease severity.

FIPV 79-1146 is a virus strain originally isolated by James F. Evermann, Washington State University and has been characterized inter alia by McKeirnan et al. Feline Practice, 11.16-20 (1981);

SUBSTITUTE SHEET Pedersen et al supra and Pedersen et al. Am. J. Vet. Res., _45_:2580-2585 (1984).

U.S. Patent 4,195,130 is drawn to the propagation of FIPV virus in tissue culture and is hereby incorporated by reference.

DESCRIPTION OF THE INVENTION

It has now been discovered that feline infectious peritonitis virus, WSU FIPV 79-1146 can be attenuated to form a live modified vaccine against feline infectious peritonitis (FIP) which vaccine both imparts immunity and is safe, that is does not cause FIP in the feline.

The present invention relates to a method of protecting felines against FIP, a method of producing a non-virulent attenuated living FIPV for use as a safe, efficacious vaccine, and the resultant vaccine.

The attenuation of the virus is accomplished by serial passaging in cell culture, preferably non- oncogenic cell culture, in which virus growth occurs, at a temperature which causes virus growth and attenuation until a non-virulent immunizing virus is produced. Felines vaccinated with the attenuated strain did not suffer significant illness when challenged by a virulent strain of FIPV.

The cell culture employed in passaging the virus can be any cell culture in which the virus repli¬ cates, for example, whole fetus cells (e.g. FCWF, felis catus whole fetus), feline kidney cells (e.g. CrFK, Crandell Feline Kidney) , feline lung cells and A-72 (a canine tumor cell line). Preferably the cell culture is a non-oncogenic cell culture. See also U.S. Patent 4,195,130, previously incorporated.

The temperature at which the virus is grown can be any temperature at which with tissue culture passage

SUBSTITUTE SHEET attenuation occurs. The presently preferred temperature is a sub-optimal temperature, that is a temperature lower than the normal body temperature for a feline but at which virus growth occurs. The presently preferred temperature range is between about 33°C'and about 35°C.

The number of passages required to obtain safe, immunizing attenuated virus is dependent at least in part on the conditions employed and in part on the age of the felines being vaccinated. Periodic testing of the culture for virulence and immunizing ability can readily determine the parameters for a particular combination of tissue culture and temperature.

It has been discovered that kittens appear more susceptible to FIPV than do adult felines. As an apparent consequence, an attenuated living FIPV which is sufficiently attenuated to be safe for and will immunize adult felines, may still be sufficiently virulent to cause significant disease symptoms or reactions in kittens. This was found to be the case with the 19 passage attenuated as set forth in the Examples. However, a 40 passage attenuated virus was both safe and effective in imparting immunity in both adult felines and kittens.

Typically the required number of passages in obtaining a safe useful vaccine for all felines is at least about 35 passages and preferably at least about 40 passages. Although a safe useful vaccine for adult felines (i.e. felines six months old or older) can be obtained after at least about 15 passages and preferably at least about 20 passages.

In the preferred embodiment of the invention, an attenuated live FIP vaccine is produced by serially passaging the native virulent virus WSU FIPV 79-1146 at least about forty times in CrFK at a temperature between about 33°C and about 35°C.

SUBSTITUTE SHEET The vaccine of this invention can be administered by any route which causes an increase in antibody titer in the feline. The presently preferred route is intranasal or oral administration. Subcutaneous administration is also contemplated as effective.

EXAMPLE 1

The virus used in the Examples was the strain WSU FIPV 79-1146, originally isolated by Dr. J.F. Evermann at Washington State University. It was supplied, at approximately the sixth passage level, to the Cornell Feline Health Center by Dr. Neils Pedersen from the University of California at Davis. The original isolate was made from a kitten that had died soon after birth of pneumonia and pleuritis. Direct examination of the virus by electron microscopy demonstrated two popula¬ tions present morphologically. One population, constituting 95-98% of the virions seen, was the typical FIPV with short, more tear-dropped-shaped peplomers. The other population was approximately 2-5% of the virions present and had longer, more bulbous-shaped peplomers present. Since it appeared that a mixed isolate was present, the virus was plaque purified and cloned, as described below, three times prior to use in these experiments.

The UCD1 strain of FIPV employed as a challenge virus was originally obtained from Dr. Neils Pedersen at , the University of California, Davis. The prior history of the virus included several passages through minimal disease cats. A 50% liver suspension was made upon the death of the cats at each passage and the suspension was stored at -70°C prior to its being used as a challenge virus. The UCDl virus strain is a highly virulent one and has been used as a challenge virus at the Cornell Feline Health Center for many years. It consistently

S produced specific lesions followed by death when the suspension was given by aerosolization to cats.

All of the WSU FIPV 79-1146 viral strains were first grown in one cell line, Felis catus whole fetus cells (FCWF's), which were supplied by Dr. Neils Pedersen. Growth media for these cells included equal volumes of Leibowitz's (L15) (Gibco Grande Island Co., Grande Island, New York) and Dulbecco's modified minimal essential media (DMEM) (GIBCO) supplemented with 10% fetal bovine serum. It was found to be necessary to include in the growth media 0.05% Lactalbumin hydrosylate (LAH) (GIBCO), 1% MEM sodium pyruvate and 1% nonessential amino acids (GIBCO) . The antibiotics Fungizone and Gentamicin (GIBCO) , were also added. When the cells had grown to a complete monolayer, they were split and transferred. The FCWF cells did poorly and in all later experiments Crandell feline kidney ^«(CRFK) cells or A-72 cells were used. Media used to propagate the cells consisted of 20% L₁₅, 3% 0.1 N NaOH, 2% L-glutamine (GIBCO). To transfer the cells, the growth media was removed and, depending on the size of the tissue culture flask, up to 7 cc of crude porcine trypsin-versine in phosphate buffered saline were added as a wash. After 30 seconds, this trypsin wash was removed and a second volume of the trypsin was added. The flask was incubated for 5-10 minutes at 37°. After the time had elapsed and when the cells had been dispersed into single cells, new growth media was added to a volume three times the original volume, for a one to three cell split. The cells were then dispensed into the new flasks as the protocol demanded.

To grow the virus strains, the growth medium was removed from complete monolayers and the cells were washed once with MEM. This was removed and the viral inoculum was added. The flask was rocked on a Belco Low-

SUBSTITUTE SHEET Profile Rocker (Belco Glass, Inc., New Jersey) for one hour at 37°C at a speed of approximately 2 oscillations per minute. After one hour, new growth medium was added and the flasks were incubated at 37°C. All the culture flasks were examined daily for cytopathic effect. When approximately 60-80% of the monolayer was infected, the flasks were frozen at -70°C. After three freeze/thaw cycles, the virus supernatant was centrifuged at 2000 RPM at 4°C in a IEC, DPR-6000 centrifuge (Damon, Needham Heights, Mass.). The supernatant was aliquoted into lcc volumes and frozen at -70°C.

To plaque-pick or clone a virus, growth medium was removed from confluent monolayers in 6-well plates. The monolayer was washed once with MEM and the wash was then removed. Serial ten-fold dilutions, in MEM, of the virus, and 0.1 to 0.5 cc was inoculated into duplicate wells. The virus was allowed to adsorb for one hour at 37°C on a Belco Rocker. After one hour, the inoculum was removed, the monolayer was washed once with MEM, and the overlay added. The overlay consisted of equal volumes of 2X BME (Gibco) and 1.8% Agarose (Seakem-ME-FMC Corp., Rockland, ME) . In addition, the antibiotics Fungizone and Gentamicin were added. The plates were examined daily for plaque -production. When isolated plaques were seen, the clones were picked with the use of tuberculin syringes with 1" or 1 1/2" 20 gauge needles, and the clone was added to a vial containing 1 cc of MEM. If not inoculated immediately into flasks, the clones were frozen at -70°C for future use. After the plaques were picked, the plates were stained with a solution consisting of one gram crystal-violet per liter of 10% buffered formalin solution and examined to determine which was the most isolated of the plaque(s) picked. The isolated clones that had been picked were then grown up and the virus was replaqued for more clones. After the

SUBSTITUTE SHEET third cloning procedure, the virus was considered "pure", was grown up in large flasks, aliquoted into vials, and frozen at -70°C as the stock inocula for future experiments. This stock inocula was considered to be low passage virus (LP) and was approximately at passage level 11.

After the virus had been cloned, the LP virus was passaged several more times in 480 cmr roller bottles but at 34°C. At passage level 16 a large flask was inoculated with virus, adsorbed for one hour with continuous rolling and maintenance media added. When the amount of cytopathic effect had reached approximately 75%, the flask was frozen and thawed for three times. The virus was then stored and was designated high passage virus (HP) . It was approximately passage level 17 and had only been propagated at 34°C since the plaque purifi¬ cation procedure. This HP virus was also considered to be a temperature-sensitive and was used as vaccine virus.

To perform virus neutralization tests (VN) , serum samples were heat inactivated for 35 minutes in a 56°C water bath. After heat inactivation, two fold dilutions of serum were made in either MEM, L15 or PBS. An equal volume of virus containing 100 TCID₅₀ was then added to each serum sample. The serum-virus mixture was then incubated for one hour at 37°C. Following incubation, 0.2 cc of the serum-virus mixture was added in triplicate wells to a 48-well Costar plate that had been just seeded with CRFK cells. For the virus control, only 0.1 cc was added to each well. The tissue culture control had 0.1 cc of the diluent added to each well. The plates were then sealed in plastic bags and incubated at 37°C for 4 days. On the fourth day, the medium was removed and the plate immersed in a crystal violet staining tank containing 10% formalin for 10 minutes. The plates were rinsed with water, allowed to dry and the

SUBSTITUT sample was titered. The end point titer was considered to be the last dilution in which there was complete neutralization of the virus or in which there was complete protection of the cell monolayer.

To assess the cytopathic effect produced by WSU FIPV 79-1146, confluent monolayers in 24-well plates with coverslips were inoculated with ten-fold dilutions of the virus. After a one-hour adsorption period, maintenance medium was added. Thereafter, at 6-hour intervals, an infected coverslip and a tissue culture control coverslip were removed. The coverslips were washed once in PBS and then fixed for ten minutes in methanol. After fixation, the coverslips were stained for ten minutes in May- Breenwald stain (Harelco, Gebbstown, NJ) , followed by a 20 minute staining period in a 1/20 dilution of Gies a stain. The coverslips were then washed twice in distilled water, washed twice for 15 seconds in acetone, dehydrated in acetone-xylene mixtures, and finally immersed in pure xylene for ten minutes. The coverslips were then removed, mounted on glass microscope slides for a permanent record using Permount (Fisher Scientific, Fairlawn, NJ) and examined.

^• The 11th Cornell passage of FIPV 79-1146 (total 17th passage) was used as challenge virus for the intra- tracheal "1st vaccination" or virus titration (Table 1). Attenuation of virus was done by rapid passage in cell cultures at low temperature (34°C). A total of 7 low temperature passages were done in a sequence of canine A-72 cells (3 passages) , and Crandell feline kidney cells (CrFK) (4 passages). The attenuated virus (19 passage attenuated virus) used in this study had 7 low temperature (34°C) CrFK cell culture passages and a total of 19 cell culture passages.

Fourteen (14) 6-month old specific pathogen free (SPF) cats (Liberty Lab) were placed into filter

SUBSTIT isolation cages and exposed to varying doses of low passage (12th passage) FIPV 79-1146 via intratracheal inoculation. Cats were monitored daily for signs of clinical disease and fever. Weekly serum samples were obtained for virus neutralizing (VN) antibody titer against FIPV 79-1146.

The results of the viral titration or "1st vaccination" are listed in Table 1. The minimal infectious dose was 10³ plaque forming units (PFU) of virus. One of 2 cats receiving 10³ PFU of virus became and infected and died on day 56 after inoculation. Cat Q3 seroconverted to FIPV 79-1146 virus and developed signs of FIP including ocular and chest involvement, but this cat did not succumb until after aerosol challenge. Cat R4 developed FIP and died on day 28. Three cats that seroconverted to FIPV 79-1146 (cats N4, Jl and S2) did not show any signs of illness.

After an observation period of 134 days, the cats remaining from the previous experiment were given a "2nd vaccination" with 10⁴, 10⁵, or 10⁶ TCID₅₀ of attenuated WSU FIPV 79-1146 virus (19th passage) via intranasal drops. None of the cats showed any signs of illness during the observation period (49 days) after the 2nd vaccination. All vaccinated cats were seronegative at the time of vaccination seroconverted with maximum titers of 1:64 to 1:256. There did not seem to be any correlation between the dose of virus and the resulting titer. Three of the 4 cats with FIP VN antibody titers at the time of revaccination had a 2 to 4 fold rise in titer.

Forty-nine days after the second vaccination (day 183 of the experiment), cats were challenged via aerosol with either low passage FIPV 79-1146 virus or the highly virulent liver suspension of strain UCD-_j_. The experimental design and results of this challenge experi- ent are listed in Table 1. The low passage 79-1146 virus was the same stock virus used in the intratracheal inoculation in the first part of this experiment. The UCD^ challenge virus was the stock liver suspension of highly virulent virus used in numerous challenge trials with FIP over the last ten years. Prior to this study, the UCD-_j^ virus has been fatal for nearly 100% of cats infected within 1 to 2 weeks in seropositive cats and within 3 to 4 weeks in seronegative cats. The one unvaccinated cat (cat VI) that received 1146 challenge virus developed illness and died on day 28 after challenge. Cat Q3 was euthanized on day 60 after challenge because it was showing chronic FIP similar to what it had shown for several weeks following the initial inoculation. One of 3 other antibody positive cats receiving 79-1146 virus and 2 of 4 antibody positive cats receiving UCD-_j_ virus showed transient clinical signs (fever, anorexia, and in one case, icterus), but none of these developed typical clinical FIP. Cat H3 (1146 challenge) had fever on days 4-6 after challenge, cat P4 (UCD^ challenge) had a fever on days 11-24 and 43-47 as well as periodic anorexia. Fever was detected in cat S2 (UCD- challenge) on days 8-12, anorexia on days 8-14, and icteric serum on day 14 after challenge.

The data demonstrates that the 19 passage attenuated FIPV 79-1146 did not sensitize felines to either challenge with the UCD-_|_ virus or rechallenge with virulent 79-1146 virus. This is of considerable significance since, as set forth above, antibodies against several coronaviruses appear to sensitize felines so that a more acute disease is produced with exposure to UCDi or certain other feline coronaviruses.

Substantial neutralizing antibody titers against FIPV 79-1146 were produced following a single intranasal vaccination. The VN antibody titers were listed in Table 2.

SUBSTITUTE SHEET Table 1 - Response of cats to virulent and attenuated WSU FIPV 79-1146

1st Vaccination 2nd Vaccination* Challenge**

Pre Vac. (Low passage 1146 Intratracheal) (High passage of 1146 Intranasal) (Aerosol)

VN Viral VN Viral VN VN

Titer Dose Titer FIP Death Dose Titer FIP FIP Titer FIP Death Cat 1146 (TCI ₀) 1146 Signs (days) (TCIDJQ) 1146 Signs Death Virus 1146 Signs (days)

VI 0 0 0 - - Control 0 1146 32 28

B3 0 0 0 - — Control 0 Control 0

M2 0 10} 0 _ _ lo 256 1146 512

H3 0 10¹ 0 10⁶ 64 1146 128 + D ) P4 0 1()2 0 10;? 256 UCD_χ 2048 +

03 0 10² 0 10⁵ 128 UCD_X 256

A3 0 10³ 0 10⁴ 256 TC Control 256 π D2 0 10³ 1024 + 56 t t t t t

03 0 ¹⁰a 2048 + _ ¹⁰1 2048 1146 4096 60

N4 0 10⁴ 256 - - 10⁴ 1024 1146 2048

Jl 0 10⁵ 512 _ _ 10⁴ 1024 UCD. 4096

R4 0 10⁵ 32 + 28 t t t

Virus strains. Titer = recipricol of serum dilutions that

1146 = FIPV 79-1146 protected 100% of cell cultures

UCD-i = Liver suspension of virulent virus infected with 100 TCID5_Q of virus

- = Negative; + = ϊbsitive; t = Cat died; * = day 134 of experiment; ** = day 183 of experiment

Table 2 - Virus Neutralizing Antibody Titers of Senm against FIPV 79-1146 in Cats "Vaccinated" with Low or High Passage Virus.

FIPV-1146 Virus neutralizing Antibody Titer (Cat No. )

Cays A3 P2 N4 Q3 M2 H3 S2 Jl Q3 P4 VI

0 0 0 0 0 0 0 0 0 0 0 0

14 0 0 32 32 0 0 8 32 0 0 0

28 0 0 64 64 0 0 128 32 0 0 0

42 0 0 64 512 0 0 256 64 0 0 0

49 0 0 128 1024 0 0 512 128 0 0 0

56 0 0 128 1024 0 0 512 256 0 0 0

63 0 0 256 2048 0 0 1024 256 0 0 0

70 0 0 256 1024 0 0 1024 256 0 0 0

77 0 0 256 2048 0 0 256 512 0 0 0

129 0 0 256 2048 0 0 512 512 0 0 0

141 0 0 128 2048 0 0 128 512 0 0 0

148 128 8 1024 2048 32 32 512 1024 16 32 0

162 128 64 512 2048 64 32 1024 512 128 32 0

169 128 128 512 1024 128 64 512 512 128 64 0

176 256 128 512 2048 256 64 512 1024 256 128 0

183 256 64 1024 2048 256 128 1024 1024 128 256 0

193 256 128 1024 2048 128 64 512 1024 128 ^'512 0

200 128 512 1024 NA NA NA 2048 2048 512 2048 32

207 NA 256 1024 NA 512 NA 512 2048 NA NA NA

214 512 512 2048 4096 NA NA 2048 2048 256 1024 NA

221 256 256 2048 4096 NA NA NA 2048 256 2048 NA

228 512 512 NA 4096 NA NA 2048 4096 NA 2048 NA

As FIPV 79-1146 was isolated from a neonatal kitten with kitten mortality complex ( KMC) , it would appear that KMC is a FIPV caused disease and that thus the vaccine of the invention is also effect ive agains t KMC , which is therefore included within the scope of this invention.

SUBSTITUTE SHEET EXAMPLE 2

The work with 19th passage attenuated virus in adult cats (Example 1) was repeated and the 19th passage attenuated virus was also tested in 12 to 16 week old kittens.

Further the 79-1146 virus was carried through 50 passages (38 at low temperature), and the effect of the 30th and 40th passages of attenuated virus in kittens was determined.

Experiment 85-01 - 19th passage of 79-1146 in kittens: Table 3 lists the experimental design for experiment 85-01, the evaluation of passage 19 attenuated virus in 12- to 16-week old kittens each. Group A served as unvaccinated controls, Group B received 0.5 ml (100,000 TCID₅Q) of vaccine virus intranasally, and Group C received the same dose of vaccine subcutaneousl . Cats were observed and scored for clinical signs of illness as per a standard scoring procedure wherein each sign of clinical disease such as depression, liver, anorexia, pneumonia, nasal discharge, diarrahea were scored on a 0 to 4 basis 0 = normal, 1 = slight, 2 = moderate, 3 = marked, 4 - severe) . Daily group scores were totaled and a group mean clinical score for each day calculated. Rectal temperatures were recorded daily, pharyngeal swabs were taken 3 times per week for viral isolation, and weekly serum samples were obtained from the jugular vein for virus neutralizing antibody titer determination. Cats were to be challenged by aerosol with virulent FIPV- UCD-L on day 21 after vaccination.

The individual and group mean temperatures of cats in this experiment are listed in Table 4 (Groups A, B, and C) . The individual and group mean clinical scores are listed in Table 5. The viral isolation results from pharyngeal swabs are listed in Table 6, and the virus neutralizing titers are given in Table 7.

SUBSTITUTE SHEET The control, nonvaccinated kittens (Group A) remained healthy throughout the experiment. Temperatures were normal, clinical signs of illness were not detected, no virus was recovered from pharyngeal swabs through day

27, and virus neutralizing antibody titers in serum against 79-1146 remained normal through day 28. Two of these controls were fed, cleaned, temperatured and sampled after the kittens in Groups B and C in order to monitor the ef ectiveness of the isolation.

Five of the 6 kittens vaccinated intranasally (Group B) with the passage 19 vaccine showed moderate to severe clinical signs typical of FIP. Signs first appeared on day 2 after vaccination, reached a peak on day 7 or 8, and subsided to normal by day 10 or 11. Signs redeveloped around day 16 and became very severe until the kitten either died or was euthanized between day 26 and day 31. One kitten (HF5) had a mild febrile response on days 2, 3, 4, and 14, and was slightly depressed on days 6 to 8. This kitten did not develop the secondary disease and has remained healthy through day 120.

Virus was isolated from pharyngeal swabs from all 6 kittens receiving intranasal vaccine (Table 6). Virus was present by 1 day after vaccination in all cats, 5 of 6 were still positive for virus on day 6, but only 2 of 6 kittens had virus on day 8. All kittens were negative by day 11 after vaccination.

Virus neutralizing antibody titers in sera were low at day 7, then increase in each weekly sample through day 21. Four of 6 cats had increased titers on day 28 compared to day 21. One cat had died of FIP prior to day

28, and a second cat had a decreased titer.

All 6 cats that received the 19th passage of attenuated 79-1146 virus subcutaneously developed moderate to severe clinical signs of FIP (Tables 4 and 5) . Other than signs first appearing about one day after signs first appeared in the intranasal group, the progression of the disease was similar in the 2 groups of cats. All 6 of these kittens developed secondary disease and either died or were euthanized with FIP.

No virus was recovered from any of the pharyngeal swabs from kittens vaccinated subcutaneously throughout the duration of the experiment (Table 6). Virus neutralizing titers in sera were essentially identical to those in the intranasal group (Table 7).

Since most of the vaccinated kittens in this experiment eventually developed clinical FIP, there was no need to challenge the control kittens with virulent virus.

Experiment 85-02 - Living 21st passage 79- 1146: The control cats (Group A) from experiment 85-01 were utilized to screen the virulence of the 21st passage of virus (9 low temperature passages). Four of these cats were divided into 2 groups of 2 cats each. One group served as unvaccinated controls, and the second group (Group D) received 1.0 ml of live 21st passage virus subcutaneously.

The 2 cats in group D (HDl and HG4) received a single subcutaneous dose of a vaccine containing 10⁶TCID₅₀ of virus.

The clinical response of the 2 cats in Group D are listed in Tables 4 and 5. The response was similar to that observed in Groups B and C. Both cats developed primary disease, recovered, then developed secondary disease typical of FIP. One cat died and the other cat was euthanized on day 32.

The 2 controls were challenged on day 28 with virulent UCD^ virus. Both cats developed clinical FIP. The temperatures of these cats after challenge are listed in Table 8.

SUBSTITUTE SHEET The neutralizing titers of these cats after vaccination are listed in Table 9. The 2 control cats did not have neutralizing titers through day 21. The 2 cats receiving the 21st passage of live virus first had titers on day 14.

Experiment 85-03 - 30th passage of 79-1146 in kittens: Six SPF kittens 14 weeks of age were divided into 3 groups of 2 kittens each. Group E received 1.0 ml (10^7,5TCID₅₀) of the 30th passage of FIPV 79-1146 subcutaneously. Group F received the same volume and dose intranasally. Group G served as the nonvaccinated controls.

The clinical signs and temperature responses of these kittens after vaccination are listed in Tables 4 and 5. The response was substantially less dramatic than that for kittens exposed to passages 19 and 21. The primary disease was milder and of shorter duration in both of the vaccinated groups. Both of the kittens vaccinated subcutaneously (MGl, ME6, Group E) did develop secondary disease. ME6 was euthanized on day 21 with fluid in one lung and in the heart sac. Lesions of FIP were not detected in the peritoneum. Kitten MGl was euthanized on day 31, and had fluid in one lung, pneumonia, and possible cardiomyopathy. Both cats were running fevers and were thin prior to euthanasia.

The 2 kittens that received the 30th passage of virus intranasally showed only mild primary disease, and little in the way of secondary disease. Kitten ME2 had a low grade febrile response on days 2, 3, 8, 18, 21, and 35, but did have a temperature of 105.2 on day 7. Kitten ME3 had low grade fever on days 2, 9, 23, 28, 30, 32, and 35. Since temperatures were not recorded on every day during the secondary disease phase, kittens in all like¬ lihood had fever on more days than were recorded. As of the date of this report (day 54 after vaccination) , both of these cats are still alive and reasonably healthy. They are thinner than the controls and are periodically showing low grade fevers, but they are bright, alert, and continue to eat.

Experiment 85-04 - Comparison of 40th passage of FIPV 79-1146 attenuated live virus with the 6th passage of FIPV-Cornell-1: Fourteen 14-week-old Liberty Lab SPF kittens were divided into 4 groups of 2 kittens each as outlined in Table 10. Group A was the nonvaccinated control, Groups B and C received attenuated live virus intranasally and subcutaneously (1.0 ml/kitten of a 1:3,000 dilution of 40th passage of virus, or 10⁴TCID₅₀/kitten) .

Group G kittens received 1.0 ml (10 TCIDς_Q) subcutaneously of the 6th passage of the Cornell-1 (CU-1) isolate of FIPV.

As of day 23 after vaccination both of the kittens vaccinated intranasally with the attenuated 40th passage of virus, plus the controls, have remained healthy and have not exhibited signs of primary disease. Fever has not been detected in these cats. This is in distinct contrast to the febrile response with the lower passages of virus. One of the 2 kittens vaccinated with 40th passage subcutaneously has not developed a fever, but the other kitten has had a fever on days 3, 7, and 8 after vaccination and was euthanized on day 23 with lesions of FIP.

The 2 kittens receiving the CU-1 virus subcutaneously both exhibited low grade fever on day 3. Otherwise they have been healthy through day 23.

Experiment 85-05 - Recheck of the attenuation, efficacy, and sensitizing properties of the 18th passage of FIPV 79-1146 in adult cats: Four adult SPF cats, approximately 40 weeks of age, were vaccinated with the 18th passage of FIPV 79-1146. Two of these cats (W3, Z4)

SUBSTITUTE SHEET were Liberty Lab cats that had been uninoculated controls in a previous experiment and did not have antibodies to FIPV. The other 2 cats (73, 52) were control cats from another experiment, had come from a colony where most every kitten seroconverted to feline coronaviruses, and these seropositive cats were sensitized to challenge with FIPV-UCD^

All 4 cats developed antibody titers to 79-1146 ranging from slightly under 1:100 to 1:4,800. These cats were challenged via aerosol with UCD-ι_. They have remained healthy with no febrile response or other signs of illness for a period of 56 days (Table 7, Group Z). Neutralizing antibody titers rose slightly but not dramatically after challenge.

SUBSTITUTE SHEET Tctble 3 - Experimental Cfesign, Experiment 85-01 FIP Vaccine Study

Route of Cage Cat FIPV

Group Vaccination # # Sex Challenge

A None 36 Jl F UCD-, F4 F

40 G4 M Dl M

23 D2 M A6 M

B Intranasal 21 G2 M UCD-,

F5 M

27 F6 M A5 M

35 Cl F Bl F

C Subcutaneous 24 A7 M UCD-, E4 M

25 G3 M C2 M.

26 B2 F

E3 F

Vaccine = FIPV-1146 Passage 19 ( Dilute 3 .5 ml stock vi Each vaccinate received 0.5 ml or 100,000 TCID^Q

Challenge = aerosol challenge on day 21 with liver suspension of virulent FIPV-UCD-, virus.

VN antibody titers = weekly.

Clinical disease evaluation = daily.

Viral isolation from pharyngeal swabs = three times per week after vaccination.

Temperature = E&ily .

SUBSTITUTE SHEET Table 4 - Temperature of Cats after "Vaccination" with attenuated Beline Infectious Peritonitis Virus Strain 79-1146

Temperature (°F. +100)

Vaccine Cats (Cays After "Vaccination")

Passage Dose Gat Age

Group level Itoute (ml) # (wks) 0 1 2 3 4 5

HJ1 15 1.6 1.6 1.4 1.4 1.0 1.8

HF4 15 2.4 2.2 2.4 3.2 1.6 3.2

A. Control HD1 15 1.2 2.4 1.4 1.6 1.8 2.2

HG4 15 1.2 2.2 2.0 1.8 0.6 1.8

HD2 15 1.8 2.4 2.2 2.0 2.0 2.4

HA6 15 1.8 2.8 2.2 2.2 2.4 2.8

Mean 1.7 2.? 1.9 2.0 1.6 2.4

HA7 15 2.0 2.0 2.6 3.6 3.6 3.0

HE4 15 0.8 2.6 2.8 2.8 2.2 2.4

B P19 SC 0.5 HG3 15 2.2 2.0 2.2 3.6 3.0 3.8

HC2 15 2.6 2.4 2.0 4.0 3.8 2.8

HB2 15 2.0 1.4 2.2 3.6 4.2 2.8

HE3 15 2.0 2.2 1.8 3.8 3.4 2.6

Mean 1.9 2.1 2.3 3.6 3.4 2.9

HG2 15 2.0 2.0 3.8- 3.6 3.0 2.6

HF5 15 2.0 2.6 3.2 3.0 3.0 1.6

P19 IN 0.5 HE6 15 2.2 2.8 2.8 2.8 2.4 1.8

HA5 15 2.6 2.8 3.2 4.2 3.2 2.8

HO. 15 1.2 2.4 3.2 5.0 4.0 3.0

HB1 15 2.2 3-0 4.0 4.0 2.2 3.0

Mean 2.0 2.6 3.4 3.8 3.0 2.5

P21 SC 1.0 HD1 21 1.4 2.8 2.8 3.6 4.8 3.6

HG4 21 1.6 1.2 2.0 2.2 4.2 3.6

Mean 1.5 2.0 2.4 2.9 4.5 3.6

P30 SC 1.0 M31 14 1.8 2.6 3.6 2.4 3.2 1.0

ME6 14 2.8 2.8 3.4 3.0 2.8 2.2

Mean 2.3 2.7 3.5 2.7 3.0 1.6

P30 IN 1.0 ME2 14 2.4 3.8 3.2 2.8 1.8 2.2

ME3 14 2.4 2.6 3.6 2.6 2.8 2.2

Mean 2.4 3.2 3.4 2.7 2.3 2.2

Control MF5 14 1.2 2.0 2.2 2.6 2.0 1.8

MI5 14 1.8 2.4 2.2 2.4 2.4 2.2

Mean 2.0 2.2 2.2 2.5 2.2 2.0

* i-iimal Died

ND Not Determined

SUBSTITUTE SHEET Table 4 - Ttemperature of Cats after "Vaccination" with attenuated Feline Infectious Peritonitis Virus Strain 79-1146

Temperature (°F. +100)

Vaccine Cats (Cays After "Vaccination ")

E-issage Dose Cat Age

Group Level Route (ml) # (wks) 6 7 8 9 10 11

HJ1 15 1.0 1.8 1.4 1.6 2.2 2.0

HF4 15 1.8 1.8 5.8 1.8 2.0 4.6

A Control - HD1 15 1.6 1.8 1.2 1.4 2.0 1.8

HG4 15 1.4 2.2 1.2 1.6 1.2 1.2

HD2 15 2.4 2.0 2.0 1.8 2.0 1.6

HA6 15 2.2 2.0 2.2 1.4 1.0 2.2

Mean 1.7 1.9 2.3 1.6 1.7 2.2

HA7 15 4.0 3.6 3.2 4.4 3.0 4.0

HE4 15 1.4 2.8 4.8 3.6 1.8 2.0

B P19 SC 0.5 HG3 15 4.8 3.6 4.0 2.4 2.4 4.2

HC2 15 2.4 4.0 2.0 1.0 1.4 2.2

HB2 15 2.0 2.2 2.0 1.6 1.4 1.8

HE3 15 0.6 0.6 1.2 1.4 1.6 1.8

Mean 2.5 2.8 2.9 2.4 1.9 2.7

HG2 15 4.4 3.4 4.6- 1.8 2.2 3.8

HF5 15 1.4 2.2 1.8 ^•2.0 1.8 1.8

C P19 IN 0.5 HF6 15 1.0 2.2 1.4 1.8 1.8 2.2

HA5 15 5.2 3.8 3.2 1.8 2.4 4.4

HCL 15 3.6 2.2 1.8 1.2 1.6 2.2

HBl 15 3.6 3.8 2.8 1.6 1.8 1.0

Mean 3.2 2.9 2.6 1.7 1.9 2.6

D P21 SC 1.0 HDl 21 3.2 2.8 4.0 ND 2.0 ND

HG4 21 4.0 3.4 1.6 ND 1.8 ND

Mean 3.6 3.1 2.8 ND 1.9 ND

E P30 SC 1.0 MGl 14 0.8 1.6 1.8 1.8 1.4 2.0

ME6 14 1.6 3.4 1.4 2.2 1.0 1.4

Mean 1.2 2.5 1.6 2.0 1.2 1.7

F P30 IN 1.0 ME2 14 5.2 3.8 1.8 2.2 2.2 2.2

ME3 14 2.4 2.2 2.0 3.0 1.0 2.2

Mean 3.8 3.0 1.9 2.6 1.6 2.2

G Control — ME5 14 2.2 1.0 2.0 2.4 1.8 1.8

MI5 14 2.0 1.4 0.8 1.8 1.4 1.8

Mean 2.1 1.2 1.4 2.1 1.6 1.8

* = __ιimal Died

ND = Not Determined

SUBSTITUTE SHEET Table 4 - Temperature of Cats after "Vaccination" with attenuated Feline Infectious Peritonitis Virus Strain 79-1146

Temperature (°F. +100)

Vaccine Cats ⁽cays After "Vaccination ")

Passage Dose Cat Age

Group level Route (ml) # (wks) 12 13 14 15 16 17

HJ1 15 1.6 1.0 1.0 1.4 1.0 0.8

HF4 15 1.6 2.4 2.0 3.0 2.0 2.2

A Control - - HD1 15 2.0 1.4 0.6 2.1 2.0 1.2

HG4 15 1.2 1.2 1.4 1.7 1.5 0.8

HD2 15 2.0 2.2 1.8 2.6 2.9 2.2

HA- 15 2.2 2.2 1.8 2.6 2.7 2.0

Mean 1.8 1.7 1.4 2.2 2.0 1.5

H ? 15 2.0 5.4 5.8 4.4 6.0 5.0

HE4 15 3.6 2.4 2.6 3.7 4.6 6.0

B P19 SC 0.5 HG3 15 5.4 5.2 6.0 3.5 4.4 4.6

HC2 15 2.2 3.2 5.0 2.2 4.2 3.6

HB2 15 ^' 2.2 1.6 1.2 2.0 1.1 2.2

HE3 15 2.0 1.8 1.8 1.8 2.6 3.6

Mean 2.9 3.3 3.7 2.9 3.8 4.2

HG2 15 4.2 4.8 5.2- 4.0 5.0 5.2

HE5 15 2.0 1.8 3.2 2.3 2.2 2.6

C P19 IN 0.5 HP6 15 2.0 2.2 2.0 2.6 1.8 1.6

HA5 15 4.4 5.2 5.0 2.0 5.0 5.0

HO. 15 1.8 3.2 2.4 1.4 0.8 1.8

HB1 15 1.8 2.2 3.4 4.3 5.1 5.2

Mean 2.7 3.2 3.5 2.8 3.3 3.7

D P21 SC 1.0 HD1 21 3.6 3.6 3.6 ND ND ND

HG4 21 3.4 4.4 3.8 ND ND ND n Mean 3.5 4.0 3.7 ND ND ND

E P30 SC 1.0 MGl 14 ND 3.2 ND 3.4 3.4 ND

ME6 14 ND 2.2 ND 2.8 3.0 ND

Mean ND 2.7 ND 3.1 3.2 ND

F P30 IN 1.0 ME2 14 ND 2.6 ND 2.2 3.0 ND

ME3 14 ND 2.8 ND 2.6 2.0 ND

Mean ND 2.7 ND 2.4 2.5 ND

G Control — — MF5 14 ND 2.0 ND 1.8 2.2 ND

MIS 14 ND 2.4 ND^" 1.2 2.4 ND

Mean ND 2.2 ND 1.5 2.3 ND

* = Animal Died ND = Not Determined

ET Table 4 - Temperature of Cats after "Vaccination" with attenuated Eeline Infectious Peritonitis Virus Strain 79-1146

Temperature (°F. +100)

Vaccine Cats (Cavs After "Vaccination ")

Passage Dose Cat Age

Group level Route (ml) # (wks) 18 19 20 21 22 23

HJ1 15 1.4 1.0 1.0 0.6 1.2 ND

HF4 15 2.4 2.4 2.6 1.8 2.0 ND

A Cbntrol - - HD1 15 2.2 1.8 1.8 1.2 1.4 ND

HG4 15 2.0 1.4 1.4 1.0 1.6 ND

HD2 15 1.8 2.4 2.2 2.2 2.4 ND

HA6 15 2.0 2.0 2.2 2.0 1.8 ND

Mean 2.0 1.8 1.9 1.6 1.7 ND

HA7 15 6.0 6.4 6.4 6.0 5.4 ND

HE4 15 5.6 5.4 5.2 5.4 5.0 ND

B P19 SC 0.5 HG3 15 4.4 4.4 4.0 4.2 4.0 ND

HC2 15 4.4 3.4 3.4 2.8 2.2 ND

HB2 15 1.8 2.0 1.8 2.2 3.0 ND

HE3 15 4.2 4.6 4.8 4.2 4.0 ND

Mean 4.4 4.4 4.3 4.1 3.9 ND

HG2 15 4.6 5.0 4.8- 5.2 5.0 ND

HE5 15 3.0 3.2 2.6 2.2 2.4 ND

C P19 IN 0.5 HP6 15 2.4 3.0 3.8 2.8 3.8 ND

HA5 15 5.8 5.4 5.2 5.2 5.0 ND

HO. 15 2.0 3.8 2.2 2.4 2.0 ND

HB1 15 6.0 5.4 5.0 4.6 4.6 ND

Mean 4.0 4.3 3.9 3.7 3.8 ND

D P21 SC 1.0 HD1 21 5.4 ND ND 2.4 ND 1.8

HG4 21 5.2 ND ND 2.4 ND 2.0

Mean 5.3 ND ND 2.4 ND 1.9

E P30 SC 1.0 M31 14 3.0 ND ND 3.8 ND 4.2

ME6 14 4.2 ND ND 3.2 *

Mean 3.6 ND ND 3.5 ND 4.2

F P30 IN 1.0 ME2 14 4.2 ND ND 3.8 ND 2.6

ME3 14 2.4 ND ND 2.8 ND 3.0

Mean 3.3 ND ND 3.3 ND 2.8

G Cbntrol — — MF5 14 2.0 ND ND ND 4.6 ND

MI5 14 1.8 ND ND^' ND 4.8 ND

Mean 1.9 ND ND ND 4.7 ND

* = Animal Died ND = Not Determined

SUBSTITUTE SHEET Table 4 - Temperature of Cats after "Vaccination" with attenuated Eeline Infectious Peritonitis Virus Strain 79-1146

Temperature ( °F. +100)

Vaccine Cats (Cays After "Vaccination" )

Passage Dose Cat Age

Group level Route (ml) # (wks) 24 25 26 27 28

HJ1 15 ND 2.0 ND 1.0 ND

HF4 15 ND 1.8 ND 2.4 ND

Control HD1 15 ND 2.2 ND 1.8 ND

HG4 15 ND 2.0 ND 1.2 ND

HD2 15 ND 2.0 ND 1.6 ND

HAβ 15 ND 1.6 ND 2.4 ND

Mean ND 1.9 ND 1.7 ND

HA7 15 ND *

HE4 15 ND 5.6 ND 4.8 ND

B P19 SC 0.5 HG3 15 ND 4.6 ND 5.2 ND

HC2 15 ND 3.2 ND 3.4 ND

HB2 15 ND 2.4 ND 3.4 ND

HE3 15 ND 0.2 *

Mean ND 3.2 ND 4.2 ND

HG2 15 ND 3.4 ND 0.4 *

HF5 15 ND 2.6 ND 2.8 ND

P19 IN 0.5 HF5 15 ND 5.0 ND 5.0 ND

HA5 15 ND 3.8 ND 3.6 ND

HO. 15 ND 1.8 ND 1.6 ND

HB1 15 ND 4.0 ND 3.0 ND

Mean ND 3.4 ND 2.7 ND

P21 SC 1.0 HD1 21 ND ND ND ND 2.0

HG4 21 ND ND ND ND 2.0

Mean ND ND ND ND 2.0

P30 SC 1.0 MGl 14 ND ND ND ND 4.8

ME6 14

Mean ND ND ND ND 4.8

P30 IN 1.0 ME2 14 ND ND ND ND 2.0

ME3 14 ND ND ND ND 3.8

Mean ND ND ND ND 2.9

Control MF5 14 ND ND ND ND 3.4

MI5 14 ND ND ND ND 2.6

Mean ND ND ND ND 3.0

* Animal Died

ND Not -fetermined

SUBSTITUTE SHEET Table 5 - Total Clinical Scores of cats after "Vaccination" with

Attenuated Feline Infectious Peritonitis Virus Strain 79-1146

Clinical Scores

Vaccine Cats (Days After "Vaccination" )

Passage Dose Cat Age

Group level Route (ml) # (wks) 0 1 2 3 4 5

HJ1 15 0 0 0 0 0 0

HF4 15 0 0 0 1 0 1

A Cbntrol - - HD4 15 0 0 0 0 0 0

HG1 15 0 0 0 0 0 0

HD2 15 0 0 0 0 0 0

HA6 15 0 0 0 0 0 0

Mean 0 0 0 .2 0 .2

HA7 15 0 0 0 1 2 4

HE4 15 0 0 0 0 1 3

B P19 SC 0.5 HG3 15 0 0 0 1 1 3

HC2 15 0 0 0 2 1 2

HB2 15 0 0 0 1 2 2

HE3 15 0 0 0 1 1 2

Mean 0 0 0 1.0 1.3 2 1.7

HG2 15 0 0 1 1 1 0

-HF5 15 0 0 1 1 1 0

C P19 IN 0.5 HP6 15 0 0 0 0 0 0

HAS 15 0 0 1 2 1 0

HO. 15 0 0 1 3 2 1

HB1 15 0 1 2 2 0 1

Mean 0 .2 1.0 1.5 .8 .3

D P21 SC 1.0 HD1 21 0 0 0 1 3 2

HG4 21 0 0 0 0 3 2

Mean 0 0 0 .5 .3 2

E P30 SC 1.0 MGl 14 0 0 1 0 1 0

ME6 14 0 0 1 1 0 0

Mean 0 0 1 .5 .5 0

F P30 IN 0.5 ME2 14 0 0 1 1 0 0

ME3 14 0 0 1 0 0 0

Mean 0 0 1 .5 0 0

G Control — _ MF5 14 0 0 0. 0 0 0

MI5 14 0 0 0 0 0 0

Mean 0 0 0 0 0 0

* = Animal Died ND = Not Determined

SUBSTITUTE SHEET Table 5 - Ibtal Clinical Scores of cats after "Vaccination" with

Attenuated Feline Ihfectious Peritonitis Virus Strain 79-1146

Clinical Scores

Vaccine Cats ( Cays After °' Vaccination " )

Ε-issage Dose Cat Age

Group level Route (ml) t (wks) 6 7 8 9 10 11

HJ1 15 0 0 0 0 0 0

HF4 15 0 0 3 0 0 2

A Control - HC4 15 0 0 0 0 0 0

HG1 15 0 0 0 0 0 0

HD2 15 0 0 0 0 0 0

HA6 15 0 0 0 0 0 0

Mean 0 0 0 .5 0 .3

HA7 15 6 6 8 5 2 2

HE4 15 4 5 8 4 1 0

B P19 SC 0.5 HG3 15 7 9 10 4 1 2

HC2 15 4 11 10 4 1 0

HB2 15 4 8 8 3 1 0

HE3 15 4 4 4 3 1 0

Mean 4 .8 7.8 8 .6 3.8 1 .2 .7

HG2 15 5 10 9 3 1 1

HF5 15 1 2 2 0 0 0

C P19 IN 0.5 HP6 15 1 2 2 0 0 0

HA5 15 4 7 6 1 0 2

HC1 15 4 4 3 2 0 0

HB1 15 4 ^■ 5 3 2 0 0

Mean 3.2 5.0 4.2 1.3 .2 .5

D P21 SC 1.0 HD1 21 2 1 5 ND 0 ND

HG4 21 3 1 1 ND 0 ND

Mean 2.5 1 3 ND 0 ND

E P30^' SC 1.0 MGl 14 0 0 0 0 - 0 0

ME6 14 0 4 1 0 0 0

Mean 0 2 .5 0 0 0

F P30 IN 0 .5 ME2 14 0 6 3 0 0 0

ME3 14 0 0 0 1 0 0

Mean 0 3 1.5 .5 0 0

G Control — MF5 14 0 0 0 0 o^' 0

MI5 14 0 0 0 0 0 0

Mean 0 0 0 0 0 0

* = Animal Died

ND = Not Determined

Attenuated Feline Ihfectious Eeritonitis Virus Strain 79-1146

Clinical Scores

Vaccine Cats ( Cays After "Vaccinatioi n" )

Passage Dose Gat Age

Group level Route (ml) # (wks) 12 13 14 15 16 17

HJ1 15 0 0 0 0 0 0

HF4 15 0 0 0 1 0 0

A Cbntrol - - HD4 15 0 0 0 0 0 0

HG1 15 0 0 0 0 0 0

HD2 15 0 0 0 0 0 0

HA6 15 0 0 0 0 0 0

Mean 0 0 0 .2 0 0

HA7 15 0 3 4 5 9 13

HE4 15 1 0 0 1 2 7

B P19 SC 0 .5 HG3 15 3 3 5 1 2 3

HC2 15 0 0 1 4 0 2

HB2 15 0 0 0 0 0 0

HE3 15 0 0 1 0 0 4

Mean .7 1 .2 2.3 1.2 2.5 4 .8

HG2 15 2 2 4 3 4 5

HF5 15 0 0 1 0 0 0

C P19 IN 0. 5 HF6 15 0 0 0 0 0 0

HA5 15 2 3 4 0 3 5

HO. 15 0 1 0 0 0 0

HB1 15 0 0 1 2 3 ^' 5

Mean .7 1.0 1.7 .8 1.7 2.5

D P21 SC 1.0 HD1 21 1 1 1 ND ND ND

HG4 21 1 2 1 ND ND ND

Mean 1 1.5 1 ND ND ND

E P30 SC 1.0 MGl 14 ND 1 ND 1 1 ND

ME6 14 ND 0 ND 0 1 ND

Mean ^' ND .5 ND .5 1 ND

F P30 IN 0 .5 ME2 14 ND 0 ND 0 0 ND

ME3 14 ND 0 ND 0 0 ND

Mean ND 0 ND 0 0 ND

G Oontrol _ _ MF5 14 ND 0 ND 0 0 ND

MI5 14 ND 0 ND 0 0 ND

Mean ND 0 ND 0 ^■ 0 ND

* = Animal Died ND = Not Determined

Attenuated Feline Ihfectious Peritonitis Virus Strain 79-1146

Clinical Scores

Vaccine cats (Davs After ^πι Vaccinatioin")

E&ssage Dose Cat Age

Group Level Route (ml) # (wks) 18 19 20 21 22 23

HJ1 15 0 0 0 0 0 ND

HF4 15 0 0 0 0 0 ND

A Control - HD4 15 0 0 0 0 0 ND

HG1 15 0 0 0 0 0 ND

HD2 15 0 0 0 0 0 ND

HA6 15 0 0 0 0 0 ND

Mean 0 0 0 0 0 ND

HA7 15 14 15 12 13 13 ND

HE4 15 5 7 7 8 10 ND

B P19 SC 0.5 HG3 15 3 4 4 6 5 ND

HC2 15 2 3 3 3 3 ND

HB2 15 0 0 1 0 0 ND

HE3 15 6 7 7 9 9 ND

Mean 5.2 6.0 5.7 6.5 6.7 ND

HG2 15 4 9 6 10 9 ND

HF5 15 1 1 0 0- 0 ND

C P19 IN 0.5 HP6 15 0 1 1 0 1 ND

HAS 15 5 12 12 12 12 ND

HQ 15 0 1 0 0 0 ND

HB1 15 6 6 6 6 8 ND

Mean 2.7 5.0 4.2 5.0 5.0 ND

D P21 SC 1.0 HD1 21 4 ND ND 1 4 1

HG4 21 5 ND ND 2 5 4

Mean 4.5 ND ND 1.5 4.5 2.5

E P30 SC 1.0 M31 14 1 ND ND 1 ND 2

ME6 14 2 ND ND *

Mean 1.5 ND ND ND 2

F P30 IN 0.5 ME2 14 1 ND ND 6 ND 0

ME3 14 0 ND ND 0 ND 1

Mean .5 ND ND 3 ND .5

G Control — MF5 14 0 ND ND. 0 ND 0

MI5 14 0 ND ND 0 ND 0

Mean 0 ND ND 0 ND 0

* = Animal Died

ND = Not Cetermined

SUBST Table 5 - Total Cl inical Scores of cats after "Vaccination" with

Attenuated Feline Ihfectious Peritonitis Virus Strain 79-1146

Clinical Scores

Vaccine Cats ( Cays After "Vaccination" )

__ιssage Dose Cat Age

Group level Route (ml) # (wks) 24 25 26 27 28

HJ1 15 ND 0 ND 0 ND

HF4 15 ND 0 ND 0 ND

Control HD4 15 ND 0 ND 0 ND

HG1 15 ND 0 ND 0 ND

HD2 15 ND 0 ND 0 ND

HA6 15 ND 0 ND 0 ND

Mean ND 0 ND 0 ND

HA7 15 ND *

HE4 15 ND 7 ND 4 ND

B P19 SC 0.5 HG3 15 ND 4 ND 5 ND

HC2 15 ND 3 ND 4 ND

HB2 15 ND 0 ND 1 ND

HE3 15 ND 9 *

Mean ND 4.6 ND 3.5 ND

HG2 15 ND 10 ND ^' 10 *

HF5 15 ND 1 ND 1 ND

P19 IN 0.5 HP6 15 ND 6 ND 5 ND

HA5 15 ND 4 ND 8 ND

HCL 15 ND 0 ND 0 ND

HB1 15 ND 10 ND 10 ND

Mean ND 5.0 ND 5.7 ND

D P21 SC 1.0 HD1 21 1 3 4 5 9

HG4 21 6 7 8 10 11

Mean 3.5 5 6 7.5 10

P30 SC 1.0 MGl 14 ND ND ND ND 2

ME6 14 ND ND

Mean ND ND ND ND 2

P30 IN 0 .5 ME2 14 ND ND ND ND 0

ME3 14 ND ND ND ND 1

Mean ND NF ND ND .5

Control MF5 14 ND ND ND. ND 0

MI5 14 ND ND ND ND 0

Mean ND ND ND ND 0

* = Animal Died ND = Not Determined

UBSTITUTE SHEET Table 6 - Virus Isolation from Pharyngeal Swab Samples from Cats Vaccinated with the 19th Passage of FIPV 79-1146

Cat Virus Isolation (Days Post Vaccination)

Group No. 0 1 4 6 8 11 13 21 27

A Jl 0 0* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

F4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

G4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Dl 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

D2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

A6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

B G2 0 0 + + + 0 0 0 0 0 0 0 0 +? 0

F5 0 0 + + + + 0 0 0 0 0 0 0 0

F6 0 0 + + + + 0 0 0 0 0 0 0 0

A5 0 0 + + 0 0 0 0 0 0 0 0 0 0 0 0

Cl 0 0 + + + 0 0 0 0 0 0 0 0 0 0

Bl 0 0 + + + 0 0 0 0 0 0 0 0 0 0

C A7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 died

E4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

G3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

C2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

B2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

E3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 died

0 = no CPE

+ = CPE

Isolations done in A-72 cells

* left column = lesults of 1st cell culture passage

Right col-mn = Results of 2nd cell culture passage A = nonvaccinated controls B = vaccinated intravasally C = vaccinated subcutaneously

SUBSTITUTE Table 7 - Virus Neutralization Antibody Titers in Cats Vaccinated with the 19th Passage of FIPV 79-1146.

Cat Neutralizing liter (Days Post Vaccination)

Group No. 0 7 14 21 28

A Jl 0 0 0 0 0

F4 0 0 0 0 0

G4 0 0 0 0 0

Dl 0 .0 0 0 0

D2 0 0 0 0 0

A6 0 0 0 0 0

B G2 0 6 96 384 died

F5 0 3 32 768 8192

F6 0 3 24 384 4096

A5 0 8 32 512 6144

Cl 0 3 12 384 1534

Bl 0 4 48 768 512

C A7 0 <2 24 384 died

E4 0 4 16 192 2048

G3 0 4 32 768 8192

C2 0 2 48 512 2048

B2 0 2 32 256 4096

E3 0 3 24 96 died

Test done in A-72 cells

SUBSTITUTE SHEET Table 8 - Temperature of Cats Vaccinated with Attenuated FIPV 79-1146 and Challenged* with FIPV-UCD-1

Tteitperature ( °F + 100)

Vaccine Cats ( Cays After Challenge)

Passage Dose Cat Age

Group level Route (ml) # (wks) 0 1 3 4 6 7

P18 SC 1.0 W3 48 1.0 ND 1.6 1.0 1.6 1.4

Z4 48 1 .6 ND 0.8 0 .6 2.8 2.8

73 48 2.0 ND 1.0 1.2 1.6 1.2

52 48 1.8 Too : mean to temp .

Mean 1.6 ND 1.1 0.9 2.0 1.8

Control HA6 25 1.6 1.6 ND 3.6 3 .0 1 .8

HD2 25 1.8 2.0 ND 2.4 1.8 1.4

Mean 1.7 1.8 ND 3 .0 2.4 1.6

T-ible 8 - Temperature of Cats Vaccinated with Attenuated FIPV 79-1146 and Challenged* with FIPV-UCD-1

Temperature ( °F + 100)

\&ccine Cats (Cays After Challenge)

Passage Dose Cat Age

Group level Route (ml) # (wks) 8 9 11 12 13 14

P18 SC 1.0 W3 48 ND ND 1.6 ND 1.8 ND

Z4 48 ND ND 2.0 ND 2.0 ND

73 48 ND ND 1.0 ND 2. 4 ND

52 48

Mean ND ND 1.5 ND 2.1 ND

Cbntrol HAfi 25 2.0 1 .6 ND 3 .0 3 .6 3 .6

HD2 25 3.0 3.2 ND 3.8 4.4 4.0

Mean 2.5 2.4 ND 3.4 4 .0 3 .8

* = Aerosol challenge of 0.5 to 1.0 ml/cat of a 1:50 dilution of liver homogenate.

** = 198.0 ND = Not Determined NA = Not Applicable + = cat died or was euthanized with FIP Table 8 - Temperature of Cats Vaccinated with Attenuated FIPV 79-1146 and Challenged* with FIPV-UCD-1

Temperature ( °F + 100) Vaccine cats ( Cays After Challenge)

E-issage Dose Cat Age Group level Route (ml) # (wks) 15 20 25 28

Z P18 SC 1.0 W3 48 ND 1.8 ND 2.0

Z4 48 ND 2.0 ND 2.4

73 48 ND 2.8 ND 2.0 52 48

Mean ND 2.2 ND 2.1

I Control - - HA6 25 4.0 4.6 **+ NA

HD2 25 4.4 3.4+ NA NA

Mean 4.2 4.0 NA NA

* __ Aerosol challenge of 0.5 to 1.0 ml/cat of a 1:50 dilution of liver homogenate. ** = 198.0 ND = Not Determined NA = Not Applicable

+ __ cat died or was euthanized with FIP

Tctble 9 - Virus Antibody Titers in Kittens after Vaccination with Attenuated FIPV 79-1146.

Cat Neutralizing Titer (Days Post Vaccination)

Group No. 0 7 14 21

Cbntrol

I HD2 <2 <2 <2 <2

HD6 <2 <2 <2 <2

1146-P21

III HD1 <2 <2 6 48

HG4 <2 <2 4 96

^!3ST_TUTE SHEET Table 10 - Experimental Design of Experiment 85-04. Evaluation of attenuation of the 40th Passage of FIPV 79-1146, and the Virulence of the 6th Passage of FIPV-CU-1.

Route of Cage Cat FIPV

Group Vaccination # # Sex Challenge

A None 31 NTI M UCD-1 Control 31 NR2 M UCD-1

B Intranasal 30 NW6 F UCD-1 1146-P40 30 NP4 F UCD-1

C Subcutaneous 29 NX3 F UCD-1 1146-P40 29 NY1 F UCD-1

G Subcutaneous 21 NRL F UCD-1 CU1-P6 21 NV4 F UCD-1

14-week-old Liberty Kittens

Vaccine = FIPV 1146-P40 Titer = (10^{7 •}5TCID₅₀/0.1 ml⁾

Dilute 1:3000 in FBS

1 ml per cat Re vaccinate Groups D, E, and F at 14 DPV

Challenge = Aerosol challenge with 50% liver suspension of FIPV-UCD-1 SN Antibody Titers = weekly Clinical Disease evaluation = Daily. Temperature = Daily Retitrate stock after exp .

Vaccine = Cu-1-P6 Titer = 10³/° TCIDςø/O .l ml

1 ml/cat Retitrate after Exp.

The s tarting ( plaque purif ied 11th passage) virus WSU FIPV 79-1146 (also referred to as FIPV 79-1146 or 79-1146) has been deposited with the ATCC and has been ass igned access No . VR 2125.

SUBSTITUTE SHEET The 19th passage virus has been deposited with the ATCC and has been assigned accession No. VR 2126.

The 40th passage virus has been deposited with the ATCC and has been assigned accession No. VR 2127.

The 50th passage virus has been deposited with the ATCC and has been assigned accession No. VR 2128.

SUBSTITUTE SHEET

Claims

WE CLAIM

1. A method of protecting felines from infection caused by feline infectious peritonitis virus (FIPV) comprising inoculating an animal with modified living feline coronavirus vaccine prepared by serially passaging FIPV 79-1146 in non-oncogenic cell culture, in which virus growth occurs, at a temperature which causes virus growth and attenuation until a non-virulent, immunizing virus is produced.

2. The method of claim 1 wherein the serial passaging comprises at least about 15 passages.

3. The method of claim 2 wherein the serial passaging comprises at least about 40 passages.

4. The method as in claim 1 wherein the passage temperature is at a temperature lower than the normal body temperature for a feline.

5. The method as in claim 4 wherein the passage temperature is approximately 34°C.

6. The method as in claim 1 wherein the non- oncogenic cell culture is selected from the group consisting of FCWF and CrFK.

7. The method as in claim 1 wherein the FIPV 79-1146 strain is ATCC VR 2125.

8. A modified live vaccine for protecting felines against feline infectious peritonitis virus (FIPV) caused infection prepared by serially passaging FIPV 79-1146 in non-oncogenic cell culture in which virus growth occurs at a temperature which causes virus growth and attenuation until a non-virulent, immunizing virus is produced.

9. The virus of claim 8 wherein the serial passaging comprises at least about 15 passages.

10. The virus of claim 9 wherein the serial passaging comprises at least about 40 passages.

11. The virus as in claim 8 wherein the passage temperature is at a temperature lower than the normal body temperature for a feline.

12. The virus as in claim 11 wherein the passage temperature is approximately 34°C.

13. The virus as in claim 8 wherein the non- oncogenic cell culture is selected from the group consisting of FCWF and CrFK.

14. The virus as in claim 8 wherein the FIPV 79-1146 strain is ATCC VR 2125.

15. The method of producing a modified live vaccine for protecting felines against feline infectious peritonitis virus (FIPV) caused infection which comprises:

(a) serially passaging FIPV 79-1146 in non- oncogenic cell culture at a temperature which causes virus growth and attenuation;

(b) periodically testing the passaged virus until a non-virulent immunizing virus is obtained.

T.TUTE '

16. The method of claim 15 wherein the serial passaging comprises at least about 15 passages.

17. The method of claim 16 where the serial passaging comprises at least about 40 passages.

18. The method as in claim 15 wherein the passage temperature is at a temperature lower than the normal body temperature for a feline.

19. The metho as in claim 18 wherein the passage temperature is approximately 34°C.

20. The method as in claim 15 wherein the non- oncogenic cell culture is selected from the group consisting of FCUF and CrFK.

21. The method as in claim 15 wherein the FIPV 79-1146 strain is ATCC VR 2125.

SUBSTITUTE SHEET