JPS63502828A - Feline infectious peritonitis vaccine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Feline infectious peritonitis vaccine [Background of the invention] This invention is effective against coronavirus (FIPV)-induced infectious peritonitis (FIP). Concerning immunity of cats (fel1nes).

Coronaviruses isolated from cats cause two groups: PIP Divided into viruses and viruses that cause enteritis ranging from a temporary latent state to severe disease. can be done. The various isolated viruses are all morphologically and antigenically related. Yes, representative of strains of a common type of virus that infects cats, dogs, and pigs (Pederson et al., “Atopansis in Experimental ・Medicine and Biology J, Vol. 173, 365-380 (1984) )reference).

Researchers were unable to develop a vaccine for FIP for quite some time. Peda Nelson et al. (supra) showed that FIPV antibodies can indeed sensitize cats to the disease. It summarizes the level of technology that has been reached. In the literature, after immunization with another live virus, Attacking with a virulent virus increases the infection rate, shortens the incubation period, and reduces the risk of disease. It has been reported that it increases weight.

FIPV79-1146 was developed by James F. Everman (Washington Ste. It was the first virus strain isolated by the Macquarie University, and -Nan et al., “Feline: Practice”, Vol. 11, 16-20 (1981), Derson et al., supra and Pederson et al., “American Journal of Veterans Affairs” Characteristics are described in "Linary Research", Vol. 45, 2580-2585 (1984) has been done.

U.S. Pat. No. 4,195,130 describes the propagation of FIPV virus in tissue culture. This is cited as part of the explanation.

[Description of the invention]

When the feline infectious peritonitis virus WSU FIPV79-1146 is attenuated, it becomes It is possible to produce a live modified vaccine against co-infectious peritonitis (FIP”). Furthermore, this vaccine is immunocompetent and safe, i.e. in cats. was found to not induce PIF.

This invention provides a method for protecting felines from FIP and its use as a safe and effective vaccine. Method for producing non-virulent live attenuated FIPV and the produced vaccine related.

Virus attenuation is performed in cell culture, preferably non-oncogenic cell culture. growth and attenuation of the virus until a lurent immune virus is produced This is achieved by serial subculturing at temperature. Give a vaccine with an attenuated strain Cats that have been attacked by virulent strains of FIPV do not develop severe disease. There wasn't.

The cell culture used for passaging the virus may be, for example, whole fetal cells (e.g., FCWF, ellis callus whole fetus), feline kidney cells (e.g. CrFK1 Crandell cat kidney) ), feline lung cells and A-72 (canine tumor cell line) Any cell culture that can be used may be used.

Preferably the cell culture is a non-tumorigenic cell culture. Also, U.S. Patent No. 4195130 See No. 1 (supra).

The temperature at which the virus is grown is the temperature at which attenuation occurs through passage through tissue culture. good. Currently preferred temperatures are suboptimal temperatures, i.e. normal feline temperatures. Although it is lower than body temperature, it is the temperature at which the virus grows. Currently preferred temperature range The temperature range is about 3°C to about 35°C.

The number of passages necessary to obtain a safe immunoattenuated virus can be used, at least in part. vaccination and partly on the age of the feline being vaccinated. disease poison Periodic testing of cultures for sexual and immunocompetence facilitates tissue culture and temperature testing. Parameters for specific combinations can be measured.

It has been discovered that kittens are more susceptible to PIPV than adult cats.

The clear result is that it is sufficiently attenuated and safe to immunize adult cats. Even the attenuated live FIPV is still sufficiently virulent to cause noticeable disease in kittens. may cause symptoms or reactions. This is attenuated as described later in the example. This was found to be the case when the 19th passage was used. However, 40 passages Attenuated viruses are safe and effective for immunizing both adult cats and kittens. It was effective.

Number of passages required to obtain a vaccine that is generally safe and useful for all felines is at least about 35 passages and preferably at least about 40 passages. however , safe for adult felines (i.e., felines 6 months of age or older) For vaccines useful in at least about 15 passages and preferably at least It can be obtained after about 20 passages.

In a preferred embodiment of this invention, the live attenuated PIF vaccine is about 33 native Villeure at least about 40 times in CrPK at a temperature of from about 35°C to Generated by serial passage of the client virus WSU FIPV79-1146. It will be done.

The vaccines of this invention can be administered by any route that results in increased antibody titers in cats. can be done. The currently preferred routes are intranasal or oral administration. Subcutaneous administration is also available. It seems to be effective.

Example 1 The virus used in the example was developed by Dr. Everman (Washington State University). WSU FIPV79-1146 strain was first isolated by It was. It's Nails from Davis' University California ・Approximately 6th passage level to Cornell Feline Health φ Center by Pederson Supplied in The first isolate was a kitten that died of pneumonia and pleurisy shortly after birth. Manufactured from. Direct examination of viruses by electron microscopy has shown that there are two morphological clusters. It showed that a group existed. make up 95-98% of the observed virions. The other group is typical with short teardrop-shaped peplomers. It was FIPV. The other group constitutes approximately 2-5% of the virions present and is It had a longer, more bulb-shaped peplomer than the human. I think there are mixed isolates. Because of this, the virus was plaque purified three times as described below before use in these studies. was produced and cloned.

The UCDI strain of FIPV used as the attack virus was First obtained by Dr. Neils Bederson of California (Davis) . The prior history of the virus included several passages in cats with minimal illness. . A 50% liver suspension was made at the time of death of the cat at each passage, and the suspension was It was stored at -70°C before being used as a virus shot. The UCD1 virus strain is A highly virulent strain that has been used for many years by Ninel Vereen Health Sen. It has been used as an attack virus on computers. The suspension is made into a nebulizer by aerosolization. When given to cats, the cats died after developing peculiar lesions throughout.

WSU FIPV79-1146 virus strain was first introduced into one cell line. i.e. Ferris Karras whole fetal cells provided by Dr. Neils Pederson I grew it inside. The growth medium for these cells was an equal volume of Leibowitz (LI IX Gibco Grande Island, Inc., Grande Island, New York) and lO% fetal bovine blood in Dulbecco's modified minimal essential medium (DMEMX Gibco). It included Qing. Growth medium contains 0.05% lactalbumin hydrosilane (LAHX Givco), 1% MEM sodium pyruvate and 1% non-essential amino acid It was found that it was necessary to include noic acid (Gibco).

The antibiotics fungicin and gentamicin (Gibco) were also added.

Once the cells had grown into a complete monolayer, they were peeled off and transferred. FCWF cells are insufficient minutes, and in all subsequent experiments Crandell feline kidney (CRFK) cells or A- 72 cells were used. The medium used for cell growth was 20% L+s, 3% 0. It contained 1N-NaOH and 2% L-glutamine (Gibco). transfer cells To remove the growth medium, add 7c, depending on the size of the tissue culture flask. Crude pig trypsin-virgin phosphate buffered saline was added as a wash solution.

After 30 seconds, the trypsin wash was removed and 1/2 volume of trypsin was added. . The flask was incubated at 37°C for 5-10 minutes. After the above time has elapsed Once the cells are dispersed into single cells, add fresh growth medium and split into 1-3 cell splits. In contrast, the capacity was set to three times the initial capacity. Cells are then freshened as required by the protocol. The mixture was distributed into new flasks.

To grow virus stocks, the growth medium is removed from the complete monolayer and the cells are placed in MEM. Washed once with This was removed and the virus inoculum was added. Belco flask Low Profile Locker (Belco Glass, New Chassis) Top 37 It was shaken at a rate of about 2 vibrations/min for 1 hour at ℃. After 1 hour, add fresh growth medium. , flasks were incubated at 37°C. Cell change for all culture flasks Sexual activity was examined daily. When approximately 60-80% of the monolayer is infected, the flask is heated to -70°C. It was frozen. After three freeze/thaw cycles, the viral supernatant was IEC, DP at 4°C. In an R-6000 centrifuge (Damon, Needham Heights, Mass. Center) Centrifuged at 200 ORPM. The supernatant was divided into aliquots of lcc volume, −70 Frozen at ℃.

To plaque or clone the virus, transfer the growth medium to a 6-well plate. It was removed from the entire monolayer in the sheet. The monolayer was washed once with MEM, then the washing solution was removed. Serial 10-fold dilutions of virus (in MEM) 0.1-0, 5 cc was inoculated into the aliquot well. Incubate the virus for 1 hour at 37°C using a Velcro tube. It was adsorbed onto the car.

After 1 h, the inoculum was removed, the monolayer was washed once with MEM, and an overlay was added. . The overlay was made with equal volumes of 2XBME (Gibco) and 1.8% agarose (Shibuco). - ChemuME-PMC, Rockland, Maryland). . Additionally, the antibiotics fungicin and gentamicin were added. For plaque formation The plates were examined daily for If isolated plaques are observed, clone Collection using a tuberculin syringe with a 1” or 11/2” 20 gauge needle and added the clone to a vial containing lcc MEM. Inoculate the flask immediately If not, clones were frozen at -70°C for future use. Plaque collection After that, add 1 gram of crystal violet per liter of 10% buffered formalin solution. The collected plaques ( We measured which one is the purest among the two (can be more than one). The isolated cloves then collected The cells were grown and replated for further virus cloning. 3rd time After the cloning step, the virus is considered "pure" and is stored in a large flask. Grow and aliquot into vials to create stock inoculum for future experiments. It was frozen at -70°C.

This stock inoculum is considered to be a low passage virus (LP) and is at passage level 1. It was about 1.

After cloning the virus, place the LP virus in a 480ci’ roller bottle. The cells were passaged several more times (at 34°C). Large flask at passage level 1G Inoculate the virus onto the plate, allow it to adsorb with continuous rotation for 1 hour, and add maintenance medium. Ta. When the percentage of cytopathic effect reaches approximately 75%, freeze and thaw the flask three times. Frozen. The virus was then stored and designated as high passage virus (HP). It is inherited The cell level was around 17, indicating that it had only been grown at 34°C since the plaque purification process. Ta. Additionally, this HP virus is thought to be temperature sensitive and is considered to be a vaccine virus. It was used as

To perform the virus neutralization test (VN), serum samples were placed in a 56°C water bath for 35 minutes. Heat inactivated. After heat inactivation, a 2-fold dilution of the serum was added to MEM, L15 or was prepared in PBS. Then an equal volume of 100 T CI Ds. including rus was added to each serum sample. The serum virus mixture was then inked for 1 hour at 37°C. It was incubated. After incubation, 0, 2 cc of serum virus mixture was added. 48-well cosuccess freshly seeded with CRFK cells in triplicate wells. Added to plate. As a virus control, only 0.1 cc was added to each well.

As a tissue culture control, 0 and 1 cc of dilution were added to each well. then play The cells were sealed in plastic bags and incubated at 37°C for 4 days. Fourth Remove the medium and stain the plate with lθ% formalin-containing crystal violet. Soaked in color tank for IO minutes.

The plate was rinsed with water, left to dry, and the sample was titrated. Final point titration concentration complete neutralization of the virus or complete protection of the cell monolayer. The final dilution ratio was considered to be the final dilution ratio.

To evaluate the cytopathic effect brought about by WSU PIPV79-1146. For the first time, a complete monolayer in a 24-well plate with a coverslip was plated with the virus. The inoculation was carried out using a 10-fold dilution of the following. After a 1 hour adsorption period, maintenance medium was added. after that , remove infected coverslips and tissue culture control coverslips at 6 hour intervals. Ta. Coverslips were washed once in PBS and fixed in methanol for 10 minutes.

After fixation, the coverslips were stained with Maybreenwald dye (Harelco, Gebs) for 10 minutes. Town, New Chassis) and then stained with Gies at a dilution of 1/20 for 20 minutes. stained with magenta dye. The coverslips were then washed twice with distilled water and washed with acetone for 15 minutes. Wash twice for 2 seconds, dehydrate in an acetone-xylene mixture, and finally remove the pure frame for 10 minutes. Soaked in xylene. Next, remove the cover glass and mount it on a bar mount (Fisher Scientific, Fair Lawn, New Chassis) The specimen was placed on a glass microscope slide and examined.

The 11th Cornell passage FIPV79-1146 (total 17 passages) was intratracheally used as a challenge virus for virus titration (Table 1). . The virus is attenuated by rapid passage in cell culture at low temperatures (34°C). Ta. A total of 7 cold passages were performed on canine A-72 cells (passage 3) and Crandell cat cells. Sequentially performed in kidney cells (CrFKX4 passage). Attenuated toxin used in this test virus (19th passage attenuated virus) was cultured in CrFK cell culture at low temperature (34°C) seven times. This was due to culture passage and a total of 19 cell culture passages.

Filtered 14 6-month-old, specific pathogen-free cats (Liberty Labs) Low passage (12th passage) at various doses by intratracheal inoculation in isolated cages. It dawned on FIPV79-1146. Check your cat daily for signs of clinical disease and fever. I monitored it. Viral neutralizing (VN) antibodies against PIPV79-1146 weekly Serum samples were obtained for titers.

The results of the virus titration or "first vaccination" are shown in Table 1.

The lowest infectious dose was io' plaque forming units (PFU) of virus. 1 One of the two cats given 0'PFU of virus became infected 56 days after vaccination. More died. For cat Q3, serum against FIPV79-1146 virus Although the cat developed signs of PIF, including eye and chest involvement, the cat did not die until after the attack. Cat R4 developed FIP and died on day 28.

Three cats seroconverted to PIPV79-1146 (cats N4, Jl and S2 ) did not show any signs of disease.

After an observation period of 134 days, surviving cats from the previous experiment were given 10 doses by intranasal instillation. ', 10' or to'rcrDs. Attenuated WSUFIPV79-1146U virus (19th passage) 1. ：The second vaccination was given. Second vaccine No cats showed signs of illness during the post-inoculation observation period (49 days). Vaccination All inbred cats received seroconverted vaccines at a maximum titer of 1:64-1:256. The patient was seronegative at the time of vaccination. There is a correlation between virus dose and titer. I didn't. Four cats exhibiting FIP VN antibody titers at the time of revaccination Three of them showed a 2- to 4-fold increase in titer than C.

49 days after the second vaccination (day 18383 of the experiment) cats were vaccinated with low passage PIPV79 -1146 virus or UCD, using a highly virulent liver suspension of the strain. - Attacked by Rozor. The experimental design and results of this attack experiment are listed in Table 1. Ru. Low passage 79-1146 virus was used for intratracheal inoculation in the first part of this experiment. It was the same stock virus that was used. UCD, counter virus has recently been used in the IO academic year. of highly virulent viruses used in many counter-tests using PIF over a period of time. Stock liver suspension was used. Prior to this test, UCD, virus, and seropositive Most infected cats are infected within 1-2 weeks for cats and 3-4 weeks for seronegative cats. It killed 100% of the cats. 1146 challenge virus administered to one non-wax. The cutin-inoculated cat (cat Vl) became ill and died 28 days after challenge. Cat Q3 is , because she was exhibiting symptoms of chronic FIP similar to those she had been exhibiting for several weeks after the first vaccination. The animals were euthanized 60 days after challenge. Three animals administered the 79-1146 virus. One of the other antibody-positive cats and four cats administered UCD and virus were antibody-positive. Two of the cats showed temporary clinical signs (fever, anorexia and, in one case, yellow skin). However, none of these cats developed typical clinical PIF. Ne Cat H3 (against 1146) developed a fever 4-6 days after the attack, and Cat P4 (U CD I) develops fever on days 11-24 and days 43-47, and has periodic loss of appetite. Indicated. In the case of cat 52 (UCD, counter), fever occurred 8-12 days after attack, and 8-1 The patient showed anorexia on the 4th day, and jaundice serum was detected on the 14th day.

The data shows that 19th passage attenuated FIPV79-1146 was attacked by UCD virus. or sensitize cats to re-challenge with Virulent 79-1146 virus. Indicates that there was no such thing. As mentioned above, antibodies against some coronaviruses Sensitizing cats resulting in exposure to UCD or certain other feline coronaviruses This is very important, as this may lead to more acute illness. That is.

Substantial neutralizing antibody potency against FIPV79-1146 after one intranasal vaccination value was obtained. VN antibody titers are shown in Table 2.

28 0 0 64 64 0 0 128 32 0 0 Q77 0 Q 256 2048 0 0 256 512 0 0 0148 128 8 1024 2048 32 32 512 1024 16. 32 0162 128 64 512 2048 64 32 1024 512 128 32 Q169 128 128 512. 1024 128 64 512 512 128 64 0176 256 128 512 2048 25 G 64 512 1024 256 128 0200 128 512 1 024 NA NA 2048 2048 512 2048 ° 32 207 NA 256 1024 [512 NA 512 2048 NA N A NA214 512 512 2048 4096 NA NA 2048 2048 256 1024 NA221 256 256 2048 40 96 NA HA NA 2048 256 2048 NA228 512 512 NA 4096 NA NA 2048 4096 NA 2048 NAFIPV79-1146 is Kitten Mortality Complex (KMC) KMC was isolated from a newborn kitten with FIPV-induced disease. Therefore, the vaccine of this invention is also effective against KMC. This is also within the scope of this invention.

Example 2 Repeat the work (Example 1) with the 19th passage attenuated virus in adult cats. The 19th generation attenuated virus was also tested in kittens between 12 and 16 weeks of age.

Furthermore, 50 passages (38 at low temperature) were carried out for the 79-1146 virus, and the The effects of the attenuated viruses at passages 30 and 40 were measured.

Experiment 85-01 - 19th passage of 79-1146 in kittens.

Table 3 shows the experimental design for Experiment 85-01, each in kittens between 12 and 16 weeks of age. The evaluation of the attenuated virus at passage 19 is shown. Group A was used as a non-vaccinated control. Group B received 0.511Q (100OOOTCI Ds.) of vaccine virus. rus was administered intranasally, and group 0 received the same dose of vaccine subcutaneously. cat Observe on a 0-4 basis (0=normal, 1=mild, 2=moderate, 3=marked, 4=severe) ) based on clinical diseases such as functional decline, liver disease, anorexia, pneumonia, nasal discharge, and diarrhea. Clinical signs of disease were evaluated using standard assessment methods that evaluate each symptom.

Group ratings were summed each day and a group mean clinical rating was calculated for each day. Record rectal temperature daily A sample of the pharynx was collected with a cotton swab three times a week for virus isolation, and the virus was neutralized. Serum samples were collected weekly from the jugular vein for antibody titer measurements. 216 days after vaccination Attack the cat with an aerosol using Virulent F I PV-UCD in the eyes I decided to do it.

The individual and group average body temperatures of the cats in this experiment are shown in Table 4 (A, B and 0 group). Individual and group average clinical evaluations are shown in Table 5. Obtained from a pharyngeal swab sample The virus isolation results obtained are shown in Table 6, and the virus neutralization titers are shown in Table 7.

Control non-vaccinated controls (Group A) remained healthy from the beginning to the end of the experiment.

Temperature was normal, no clinical signs of disease were detected, and pharyngeal swabs showed no signs of illness until day 27. No virus was recovered from the sample, and no virus was recovered from the serum against 79-1146. Neutralizing antibody titers remained normal until day 28. Two of these controls had isolated Feed, wash, and take temperatures after kittens in groups B and 0 to monitor effectiveness. and sampled.

Six kittens (group B) vaccinated intranasally with the 19th passage vaccine. Five of the animals showed clinical signs typical of moderate to severe FIP. Vaccination Symptoms first appear 2 days after seeding, peak at 7 or 8 days, and reach a peak at 10 or 1 day after seeding. By the first day it had calmed down and I was back to normal. The symptoms reappeared on the 16th and became very severe. Between the 26th and 31st days, Nizukiko either died or was euthanized. 1 pup Ko (HF5) showed a mild exothermic reaction on days 2.3.4 and 14, and on days 6-8. I felt a little less energetic. This kitten has not developed any secondary disease and is on day 12,020. He survived in good health.

Pharyngeal swabs taken from all six kittens that received the vaccine intranasally (Table 6). In all cats, the first molar after vaccination The virus was present, and on day 6, 5 out of 6 animals were still positive for the virus. However, on day 8, the virus was present in only two of the six kittens. . All kittens tested negative by 11 days after vaccination.

Virus-neutralizing antibody titers in serum were low on day 7 and then in each weekly sample. It showed a constant increase until the 21st day. 4 out of 6 cats were 28 compared to day 21 It showed a high titer on the second day. One cat died of PIF before day 28 and no longer One cat showed a low titer.

All six cats received subcutaneously the 19th generation of attenuated 79-1146 virus. developed clinical signs of moderate to severe FIP (Tables 4 and 5). intranasal (treated) group, except that the first symptoms appeared approximately 1 day after the first symptoms appeared. , the disease progression was similar in the two groups of cats. All 6 of these kittens The animals developed secondary diseases and either died or were euthanized by FIP.

All blood samples collected by swab from kittens administered the vaccine subcutaneously throughout the experimental period. No virus was recovered from the samples (Table 6).

Virus neutralizing titers in serum were essentially the same as in the intranasal (administered) group. (Table 7).

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

Experiment 85-02 - Birth t-ta 21a generation 79-1146゜ Control sample of Experiment 85-01 Screening the virulence of the 21st passage virus (9 cold passages) using the did. Four of these cats were divided into two groups of two cats each. one side group was used as a non-vaccinated control, and the other group (Group D) received 1.0 RG live Generation 21a virus was administered subcutaneously.

Two cats in group D (HDI and HG4) received 10@TCI D! . virus of A single subcutaneous dose of the vaccine was administered.

The clinical responses of the two cats in Group D are shown in Tables 4 and 5. Responses were observed in groups B and 0. The responses were similar to those observed. Both cats developed primary disease, recovered, and then The patient developed a secondary disease typical of FIP.

One cat died and the other cat was euthanized on day 32.

2 controls were challenged with Virulent U CD + virus on day 28. both of cats developed clinical FIP. The body temperatures of these cats after challenge are shown in Table 8.

The neutralization titers of these cats after vaccination are shown in Table 9. The two control cats were 2 No neutralization titer was shown until the first day. Given a live virus from the 211i generation Two cats showed titers for the first time on day 14.

Experiment 85-03 - 30th passage 79-1146° in kittens 6 SPF pups Cats (all 14-week withdrawals were divided into three groups of two kittens each. Group E had 1.0jl12 (10'-1'TCI Da.) 30th passage F'IPV79 -1146 was administered subcutaneously. Group E received the same volume and dose intranasally. G group was used as a non-vaccinated control.

The clinical signs and thermal responses of these kittens after vaccination are shown in Tables 4 and 5. show. The response was substantially less dramatic for kittens exposed to passages 9 and 21. There was no. Primary illness was mild and short-lived in both vaccinated groups.

Both kittens given the vaccine subcutaneously (MGISME6, group E) had secondary disease. did not develop the disease. For MB2, fluid was present in one lung and cardiac sac on day 21. It piled up and was euthanized. No PIF lesions were detected in the peritoneum. Kitten MCI He was euthanized on the 31st day, but fluid accumulated in one lung, causing pneumonia and cardiomyopathy. The possibility was also recognized. Both cats developed fevers and became emaciated before being euthanized.

Two kittens given intranasal passage 30 of the virus showed mild symptoms of primary disease. However, there were almost no symptoms of secondary diseases. Kitten ME2 is 2.3. 8.18.2I and had a low grade febrile response on day 35, but on day 7 It did not show a temperature of 105.2. Kitten ME3 is 2.9.23.28.30. He developed a low grade fever on days 32 and 35. temperature daily during the secondary disease stage. Since no records were kept, it was thought that the number of days the kitten had a fever was greater than recorded.

As of the date of this report (548 days post-vaccination), both of these cats are still alive. and is reasonably healthy. They are thinner than controls and periodically develop low-grade Although he has a fever, he is healthy, alert and continues to eat.

Experiment 85-04 - FIPV79-1146 live attenuated virus of passage 40 and passage 6 Comparison of passages FIPV-Ninell-1. Tea Labo SP with 14 animals withdrawing after 14 weeks F kittens were divided into 4 groups of 2 kittens each as outlined in Table 10. divided into two groups. Group A is a non-vaccinated control, and Groups B and 0 contain attenuated live The virus was administered intranasally and subcutaneously (1:30 of 1.OxQ per kitten). 00 dilution of the 40u virus, or 10'TCr Ds. ).

Group G kittens received 6th passage FIPV Ninel-1CCU-1) Isolate 1; Oi+f2 (10'TCI Ds.) was administered subcutaneously.

As of 238 days after vaccination, intranasal vaccination with attenuated 40th passage virus Both kittens and controls remain healthy and exhibit no signs of primary disease. stomach. No fever was detected in these cats. This is a low-passage virus. This is in clear contrast to the exothermic response caused by The 40th passage vaccine was administered subcutaneously. One of the two kittens did not develop a fever, but the other kitten developed a fever after vaccination. ．． The cat developed a fever on days 7 and 8, and was euthanized on day 23 due to FIP.

Both kittens given subcutaneous CU-1 virus developed low-grade disease on day 3. He developed a fever. They were otherwise healthy until day 23.

Experiment 85-05-No. 1 in adult cats! Attenuation of FIPV79-1146 at passage 8 Re-checking efficacy, efficacy and sensitization properties. 4 mature SPF cats, (approximately 40 withdrawals) ) was vaccinated with PIPV79-1146 at passage 18 f7). this Two of these cats (E3, Z4) were treated with liver, which was the uninoculated control in the previous experiment. The cat was a tea lab cat and did not have antibodies against FIPV. the other two The cat (73,52) was a control cat used in another experiment, and most of the offspring This was also obtained from a colony that had seroconverted to feline coronavirus, These seropositive cats were sensitized and challenged with FI PV-UCD.

All four cats ranged from less than 1:100 to 1:4800 at 79-1146. The antibody titer against was expressed. In these cats, the aerosol of UCD, carried out an attack. They may exhibit a fever response or other signs of illness for a period of 56 days They were healthy with no symptoms (Table 7, Group 2). Postchallenge neutralizing antibody titer increased slightly but significantly. There were no significant changes.

Table 3 - Experimental Design, Experiment 85 - Ol-FI-P Vaccine Test Vaccine: /=FIP V-1146 passage 19 (10’・”TCI Dsolo.

1 x Q) Dilution ratio is 3 in 4.51 g PBs, 5 x Q stock virus, Each vaccination dose is 0, 5xQ or 100000T CIDS. .

Attack = Day 21 8 Virulent F I PV-UCD, virus liver suspension Aerosol attack using.

VN antibody titer = weekly.

Clinical disease assessment = daily.

Virus isolation from throat samples taken by swab = 3 times per week after vaccination times.

Temperature measurement = daily.

Table 4: Vaccination with attenuated feline infectious peritonitis virus strain 79-1146 Cat's body temperature after "seed" Table 4 “Vaccination caused by attenuated feline infectious peritonitis virus 79-1146” Cat's body temperature after "seed" Average 2+5 2.8 2.9 2.4 1.9 2. FHG2 Is 4.4 3.4 4.6・1.8 2.2 3.8 3.6 3.1 2.8 NO1, 9Nt) Table 4 "Water infection" caused by attenuated feline infectious peritonitis virus strain 79-1146 Cat body temperature after cutin vaccination Average 0 0 0 + 5. 3 2 Average 0 0 1. 5. 5 0 Average 0 0 L, 5 0 0 Average ooooo.

Table 5 “Vaccination caused by attenuated feline infectious peritonitis virus 79-1146” Average of all clinical scores for cats after species　oooooo.

Table 5 “Vaccination caused by attenuated feline infectious peritonitis virus 79-1146” Total clinical score for cats after species J Table 5 Attenuated feline infectious peritonitis virus strain 79- Average overall clinical score of cats after "7 cutin vaccination" by 11=16 NO4,6NC ) 3.5 NO Annual average of S and O 5.7 Average 3.5 5 5i f, 51° Average ND: ND, 5 Table 6 19th generation FIPV79-1146t Touched by knee t: Cat? Virus isolation from pharyngeal swab samples 0=No CPE.

+=CPE.

Isolated in A-72 cells.

★Left column = results of the first cell culture passage.

Right column 2 Results of second cell culture passage.

A = non-vaccinated control.

B = Vaccine intranasal inoculation.

C = subcutaneous vaccination.

Table 7: Cats that were inseminated with 7 mouths 7 of FIPV79-1146 of the 19u generation Virus neutralizing antibody titer tested on A-72 cells TCI Dso=48 Table 8: Vaccinated with attenuated FIPV79-1146, F IPV- Table 8 Body temperature of cats challenged with UCD-1 Attenuated FIPV79-item 4 Cats vaccinated with 6 and challenged with hlFIPV-UCD-1 Body temperature ★ = l:50 dilution of liver homogenate 0.5-1. Owl! /(cat)'s Aerosol attack.

★★=798. O0 N　D　=Not measured.

N A = Not applicable.

+=Cat died or euthanized by FIP.

Table 8 Vaccination with attenuated FIPV79-1146 7-1F IP After the attack with V-UCD-1, the temperature of the γ cat was ★; of the liver homogenate: Aerosol challenge of 0.5-1.0 iIX/(cat) of 50 dilutions.

★★=T98.0゜ N　D　=Not measured.

NA=Not applicable.

+=Cat died or euthanized by FIP.

Table 9: Kittens vaccinated with attenuated FIPV79-1146 Viral antibody titer in Table 10: Experimental design for Experiment 85-04. 40th passage F I P V 79-1 Attenuation of 14.6 and 6% of FIPV-CU-IZ>Vnorrens evaluation.

1.4. ．． 4゜Subcutaneous 29 NX3 F CCl1>129 NYI F Masa I) -1 14 week old 9 bath kitten Vaccine:/=FIPV1146-P40 titer=(10'-'TCID5oO, lI(ri, dilution rate t:3　o　o　o, in PBS. 1 mQo per 4 cats) l 4DPV revaccination group, E and F0 challenge = F IPV-UCD- Aerosol challenge with a 50% liver suspension of 1.

SN antibody titer = weekly.

Clinical disease assessment = daily.

Temperature checks – daily.

Retitration stock after experiment.

Vaccine = OL+-1-P6 Titer = lo310TCIDsO10,11e1x Q/C cat) Titration after experiment.

Starting (plaque purification 11th passage) Lye A, XWSU IIPV79-1146 ( FIPV79-1146 or 79-1146) has been deposited at A'l'CC. and was given an access number of VR2125.

The 19th passage virus was deposited with the ATCC and given accession number VR2126. It was done.

The 40th passage virus was deposited with the ATCC and given accession number VR2127. It was done.

The 50th passage virus was deposited with the ATCC and given accession number VR2128. It was done.

international search report 1memaw, la+ham+tab1,e, PcT/US 8710021 6 2ANNEX To 1LKE INTERNATIONAL 5EARC HREPORT ON

Claims (21)

[Claims] (1) Viral growth and development until non-virulent immune virus is produced. FIP in non-oncogenic cell culture where the virus grows at temperatures that result in attenuation. Modified live feline coronavirus produced by serial passage of V79-1146 Feline Infectious Peritonitis Virus (FIPV) ) how to protect felines from infection. 2. The method of claim 1, wherein the continuous passage comprises at least about 15 passages. 3. The method of claim 2, wherein the continuous passage comprises at least about 40 passages. (4) The passage temperature is lower than the normal body temperature of felines, as set forth in claim 1. How to put it on. (5) The method according to claim 4, wherein the passage temperature is about 34°C. (6) the non-tumorigenic cell culture is selected from the group consisting of FCWF and CrFK; The method described in item 1 of the scope of the request. (7) The claim that the FIPV79-1146 strain is ATCC VR2125 The method described in box 1. (8) Viral growth and development until non-virulent immune virus is produced. FIP in non-oncogenic cell culture where the virus grows at temperatures that result in attenuation. Feline infectious peritonitis virus produced by serial passage of V79-1146 A modified live vaccine to protect felines against russ (FIPV)-induced infections. (9) The virus according to claim 8, wherein the continuous passage consists of at least about 15 passages. Luz. (10) The utensil according to claim 9, wherein the continuous passage consists of at least about 40 passages. ilus. (11) Claim 8, wherein the passage temperature is lower than the normal body temperature of a feline. The virus described. (12) The virus according to claim 11, wherein the passage temperature is about 34°C. (13) the non-tumorigenic cell culture is selected from the group consisting of FCWP and CrFK; The virus according to claim 8. (14) The claimed FIPV79-1146 strain is ATCC VR2125. Virus according to scope item 8. (15) Protecting cats against feline infectious peritonitis virus (FIPV)-induced infections A method for producing a modified live vaccine for protection, comprising: (a) virus growth and FIPV79-1146 in non-tumorigenic cell culture at temperatures that result in attenuation. (b) Passage the virus until a non-virulent immune virus is obtained. periodically test A method consisting of things. (16) The continuous passage of claim 15 comprises at least about 15 passages. Method. (17) The continuous passage of claim 16 comprises at least about 40 passages. Method. (18) Claim 15, wherein the passage temperature is lower than the normal body temperature of felines. The method described in section. (19) The method according to claim 18, wherein the passage temperature is about 34°C. (20) the non-tumorigenic cell culture is selected from the group consisting of FCUF and CrFK; The method according to claim 15. (21) The claimed FIPV79-1146 strain is ATCC VR2125. The method according to scope item 15.