FELINE INFECTIOUS PERITONITIS VACCINE AND PROCESS FOR ITS PREPARATION

Abstract: Feline infectious peritonitis is a disease of members of the genus Felix which is almost invariably fatal, and for which there is currently no vaccine available. Indeed many attempts to isolate and grow the virus have failed. By passaging a virulent isolate of felix infectious peritonitis virus in feline embryonic cell cultures until the infected cell cultrues exhibit a cytopathic effect, then inactivating the virus and presenting it in association with a pharmaceutically acceptable carrier, an inactivated vaccine suitable for immunisation purposes can be produced.

Description: FELINE INFECTIOUS PERITONITIS VACCINE This invention relates to a method of preparing an inactivated feline infectious peritonitis vaccine, hereinafter referred to as FIP vaccine, and in particular to a vaccine made by the method, which is useful in the prevention of FIP. In a further aspect the invention relates to a method for developing immunity to FIP in animals of the genus Felix. Feline infectious peritonitis is an important disease of both domestic and wild felidae because it is almost invariably fatal. The disease was first recognised in about 1950 and reported under the name chronic fibrinous peritonitis (J. Amer. Vet. Med. Assoc., 144, 1409-1420, (1963)). Since then FIP has been diagnosed in North America, Europe, South Africa Australasia and Japan. ore recently the causative organism has been described as a corona-like virus (Nature, 266, 682, (1977)). Animals which contract the disease become lethargic and inappetant. Fluid collects in the peritoneal cavity with the result that the abdomen becomes enlarged and the cat eventually dies in a jaundiced state. whilst therapy and supportive treatment may prolong the course of the disease, all animals showing clinical signs eventually die, (J. Amer. Vet. hecl. Assoc., 154, 26-35, (1969)). It has long been recognised that a vaccine is required to prevent FIP outbreaks (J. After. Vet. Med. Assoc., 155, 1728-1733, (1969)), however many attempts to isolate and grow the virus have failed. It has recently been reported that FIP virus has been grown in feline small intestine organ culture, (The Cornell Veterinarian, 68, 411-417, (1978)), but this is not a practical method for vaccine production. It has now been found that an inactivated FIP vaccine can be prepared b passaging an isolate of virulent FTP in feline embryonic cell cultures until the infected cultures exhibit a characteristic cytopathic effect (CPE), as hereinafter described, inactivating the virus so produced, and presenting the virus in associ,tion with a pharmaceutically acceptable carrier. Accor-ding to the present invention in one aspect there is provided a method eor preparing an inactivated feline infectious peritonitis vaccine comprising serially passaging an isolate of virulent FIP in feline embryonic cell cultures until the infected cultures exhibit a CPE characteristic of PIP, inactivating the virus so produced, and presenting the inactivated virus in association with a pharmaceutically acceptable carrier. Virulent F1P virus may be isolated from the peritoneal fluid, blood, urine and various organs of a cat suffering from the disease. In the case of the peritoneal fluid, the urine, the infected fluid may be inoculated directly on to the selected cell cultures. For isolating virus from the blood the infected animal may be bled from the jugular or any other suitable vein. The sample is subsequently allowed to clot, is then removed and triturated, for example in a pestle and mortar, and resuspended in a suitable medium, such as phosphate buffered saline. The suspension is then centifuged to remove gross tissue debris the supernatant fluid is removed and used to inoculate cell cultures. Virus may be obtained from such organs as the liver, spleen or omentum; the procedure for preparing the virus for inoculation into cultures is esselltiallb similar to that for the clotted blood, in that the organ is triturated, resuspended, followed by centrifugation and removal of the supernaWant fluid for inoculation purposes. reline embryonic cell cultures suitable for serially passaging tSe virulent virus may be derived from such organs as lung, kidney, heart, liver or gut, or a mixture thereof, or alternatively from the skin, muscle, amnion (placenta) or tongue. Cell cultures may be prepared from the various parts of a feline embryo, or even the whole embryo, by the method described in British Patents Nos. 1 177 635 and 1 286 250 and also in J.Hygiene, 67, (1969), 115-124. The former patent describes a method for preparing diploid cell strains, whilst the latter describes the preparation of heteroploid cell lines. The methods comprise taking the embryo, or the required part of it, and disaggregating it mechanically or enzymatically. The resulting cell suspension is transferred into suitable containers holding a nutrient medium suitable for culturing cells, and then incubating at 320 to 390C. To facilitate subculturing it is customary to treat the confluent sheets of cells with trypsin and an innocuous chelating agent, before each transfer into fresh medium. Alternatively primary feline cell cultures may be used for growing FIP virus, and these are prepared by techniques well known in the art. The isolated virulent FIP virus may be propagated by inoculating a confluent monolayer or sheet of the selected feline embryonic cells with the isolate from the infected animal, followed by incubation at 350C to 39 C, preferably 37 0C, for from 3 to 10 days, preferably 5 to 8 days, in the presence of a suitable medium. Desirably the medium is chemically defined with no biological additives, for example the medium is preferably serum-free. Such medina as Eagle 's Basal Medium (Eagle H. , Science, (1959) , 130, 432) or 199 Maintenance Medium, (see Morgan, J.P. et al, Proc. Soc. Exp. Biol. (N.Y.), 73, 1, (1950)) are suitable. Antibacterial agents such as penicillin and/or streptomycin may be added to the medium. After incubation the virus can be harvested by mechanical means such as freezing and thawing, ad thereafter used for serially passaging in monolayer cultures of feline embryonic cells by standard tissue culture techniques (see "A manual of Basic Virological Techniques" by G.C. Rovozzo and C.N. Burke, published by Prentice-Hall Inc., Eagiewood Cliffs, N.J. U.S.A., 1973). During the fist few passages of the virus usually up to 5 subcultures, no obvious cytopath.c effect (CPE) is visible. Thereafter FIP virus infection of cell monolayres characteristically causes the infected cells to produce irregular shapes, when examined microscopically as wet preparations, and ultimately round up and/or become detached fro the solid surface on which they are growling Thus ultimately the virus completely destroys the monolayer cultnre. Under the electron microscope FIP virus has an appearance similar to other members of the corona- viridae, which are essentially spherical in shape with a diameter of 80 to 160 nm, together with outstanding projections about 25 nm longr which give the viruses the appearance of prossessing a halo or corona. The titre of virus produced from esch passage will vary from between about 10 4.0 TCID50 and 10 10.0 TCID50 per ml. (TCID50 means the tissue culture infective dose which produces a cytophatic effect in 50% of the culture cells exposed to virus). Once the FIP infected cultures exhibit a CPE, the cell culture is harvested and the virus is inactivated by, for example, chemical means such as formaldehyde, acetylethyleneimine, ss-propiolactone, ss-ethyleneimine or other known virus inactivating agents. When form aldehyde is the selected inactivating agent, the virus suspension, obtained after harvesting of the cultures, is incubated in the presence of for example, 0.08% formaldellyde, until complete inactivation of the virus is achieved; this usually requires more than 72 hours incubation Following this the formaldehyde is neutralised with for example sodiu-n metabisulphate, and the suspension is dialysed against phosphate buffered saline to remove excess sodium metabisulphate, which otherwise would interfere with subsequent safety tests Alternatively the virus may be inactivated by phisical means such as UV radiation. Before formulating the inactivated virus into a vaccine, the preparation must first be tested for freedom from live virus, for sterility and potency. To ensure freedom from live virus a sample of the preparation is incubation in feline embryonic cell cultures. Lack of CPE in the cell cultures indicates the absence of live virus. To test for sterility, nutrient and meat broths are inoculated with the suspension Potency is judged by the antibody titres produce in cats as a result of injecting them with known dilutions of the inactivated virus suspension The pharmaceutically acceptable carrier for the vaccine can be a liquid, such as an aqueous solution containing nutrients and stabilizers, e.g. Hank's balanned salt solution or other similar media The carrier may, in some instances, include stcrile sealed container, , sucli as an ampoule or vial. According to a second aspect of the invention there is provided a vaccine for developping immunity in animals of the genus Felix against discase caused by FIP, which comprises FIP virus propagated, inactivated and formulated, as hereinbefore described, in an effective dosage, or multiples thereef. The effective dosage for vaccination may be from about 104.0 to about 108.0 TCID50/ml preferably 10 to 10 TCID50/ml. The TCID50/ml of the virus preparation is determined prIor to inactivation. Preferably the inactivated FIP virus is combined with an adjuvant for stimulating the immune response of the animal to be vaccinated, and therefore increasing the effectiveness of the vaccine. Suitable adjuvants include aluminium or calcium salts such as the phosphate or silicate; aluminium hydroxide gel is preferably used. An alternative type of adjuvant is that of the oil-water- lanolin type such as that described by Freund or mineral oil mixtures such as Bayol F (Esso Ltd.) and Falba (Pfaltz and Bauer Inc. New York). Vaccines may be prepared by homogenising the selected oily adjuvant with an equal volume of the inactivated virus preparation. Prior to formulatg the vaccine the virus may be concentrated by any of several means including for exampe diafiltration with a protein precipitant such as organic solvents. Good results can be achieved with water miscible lower aliphatic alcohols and ketones, particularly methanol and acetone. After formulation the vaccine is conveniently in- corporated into a sterile container which is then sealed and stored at 2 tu 8 he until the vaccine is required for use. Alterna atively the virus suspension may be frozen in ampoules or vialsr and stored in liquid nitrogen, A further alternative is to add stabilizers, such as sorbitol and/or protein hydrolystate, e.g. Sol-u-pro, to the virus suspension to enable the pleparation to be freeze dried. The vaccine of the present invention is administered to animals of the genus Felix dosirably by intraperitoneal, subcutaneous or intramuscular injection or via the oral, nasal or intraocular route. The dose may be administered as a single unit, or as a multiplicity of sub-doses ever a period of time. The preferred schedule is to administer two doses of 2 ml containing the effective dosage of PIP virus, which are administered at not less than 21 days apart. In addition to containing the attenuated FIP virus, the vaccine of the present invention may also contain other vaccines comprising antigenic material derived from other micro-organisms that cause disease in meitbers of tiie genus Felix, sch as parvoviruses for example feline panleucopenia (feline infectious enteritis), feline calicivirus, feline rabies, feline retrovirus, e.g. felinc leukaemia virus, and feline herpes viruses. A particularly preferred combination, presented as a vaccine pack, comprises a wet preparation of inacti- vated PIP virus in a sterile vessel together with a vessel containing a freeze dried preparation of one or more feline vaccines suitably adapted to be combined before usage. Alternatively the inactivated FIP virus may be freeze-dried in combination with the other vaccine viruses and packaged with a vessel containing sterile water for use in reconstituiting the freezedried vaccine prior to administration. The vessels are packaged in a box or container together with instructions for use, which include the instruction to reconstitute the freeze-dried vaccine with the wet preparation or sterile water prior to administration. In p. further aspect of the present invention there is provided a method for developing immunity in animals against FIP which comprises the administration to the aniiliai of an effective dosage of a vaccine as hereinbefore defined. The following Examples illustrate the invention but do not limit it in any may. EXAMPLE 1 Preparation of inactivated virus a) Propagation A 6 month old cat suffering from FIP, as demon strated by pyrexia and loss of condition, was bled from the jugular vein. The blood was left for 20 minutes to allow it to clot. The serum/plasma was removed from the clot, the clot was then triturated in a tissue disintegrater, i.e. a Stomacher (COLWORTH) in phosphate buffered saline for 60 seconds. The suspen- sion was then centrifuged at 1108 g after which the supernatant was decanted and used to inoculate confluent monolayers of diploid feline embryonic lung cells in serum-free 199 Medium and incubated at 370C for approximately 7 days. After incubation the cells were harvested by freezing and thawing. An aliquot of the medium and disrupted infected cells was then inoculated on to fresh monolayer cultures of diploid feline embryonic lung cells in 199 Medium, and incubated at 37 C. After serially passing the virus in a similar manner six times, small areas of characteristic CPE started to appear, that is the infected cells produced irregular snapes and eventually rounded up when viewed as wet preparations under the microscope. From the fifth passage onwards the CPE was very evident and rapidly destroyed the cell sheet, that is within 48 to 72 hours. b) Inactivation After the sixth passage the infected cells where harvested by freezing and thewing and gave a virus titre of 109.0 TCID50/ml. An alignot (100 ml) of the harvest was mixed with 40% formaldehyde (0.4 ml) and stood en a magnetic stirrer at OC for 90 hours, after which time the virus was totally inactivated. Sodium metabisulphate (2 ml) at 1:16 of standard 32% was added to the virus preparation to neutralize the formaldehyde. In order to remove the sodium metabisulphate for the purposes of carrying: out the safety test, the virus preparation was dialysed cvernight in Sorensens phosphate buffered saline. c) Control Standard (i) Sterility Test Aligouts (5 ml) of the inactivated virus preparation were added to 300 ml of nutrient broth and meat broth respectively and incubated at 32 C for 7 days and then at 18 to 20 C for a further 7 days. No growth was observed in either medium showing that the preparation was free from contamination. (ii) Safety Test Diploid Feline embryonic lung cells were inoculated with the inactivated virus preparation and incubated at 37 C. After 7 days the cells were versenized and re-seeded and incubated at 37 C for a further 7 days. No CPE was observable at 72 hours or 96 hours indicating that there was no virus replication. EXAMPLE 2 Peritolleal fluid was removed aseptically from a 3 to 4 month old cat which had died from FIP r by pipette and inoculated on to confluent monolayers of diploid feline whole embryo cells in serum free 199 Medium and incubated at 37 0C for 7 days. The virus was then harvested, passaged and inactivated as described in Example 1. EXAMPLE 3 The liver was removed from an 11 month old cat, which had died from FIP. The tissue was triturated in a tissue disintegrater, Stomacher 80 (COLWROTH) with phosphate buffered saliile for 60 seconds. The suspension was then centrifuged at 1108 g alter which the supernatant was decanted and inoculated onto confluent monolayers of heteroploid felline embryonic lung cells in serum free 199 Medium and incubated at 37 0C for 7 days. (Heteroploid cells are cells whicli have been passaged more than 70 times from primary culture and have, in consequence, become an established cell line.) The virus was then harvested passaged and inactivated as described in Example 1. EXAMPLE 4 Preparation of FIs Vaccine Inactivated viral material having a titre of 10 9.0 TCID50/ml prior to inactivation was prepared and TCI tested as described in Example 1. Equal volumes of the virus preparation, alhydrogel and phosphate buffered saline were mixed together and agitated on a magnetic stirrer for 1 hou at top speed. EXAMPLE 5 A vaccine, as prepared in Example 4 was administered to 12 week old cats, which had previously been shown to be free of FIP antibody, by intraperitoneal injection as a dose of 2 ml. After 21 days a further 2 ml dose of vaccine was administered intraperitoneally; at this time the cats were also bled and the sera exa- mined for the presence of neutralising FIP antibody. The cats were similarly bled 21 days after the second injection. Significant levels of neutralising antibody were found to be present as was demonstrated below: - Pre-vaccination antibody titre = less than 10 Antibody titre 21 days after 1st injection = 40 Antibody titre 21 days after 2nd injection - 80 EXAMPLE 6 A vaccine pack for lirmunising members of the genus felix against FIP and feline infectious enteritis (FIE) is composed of a vial containing a freeze dried preparation of live attenuated FIE vaccine and a second vial containing an inactivated FIP virus suspension as prepared in Example 4, together with instructions to reconstitute the freeze-dried vaccine with the wet preparation prior to administration. EXAMPLE 7 A vaccine pack for irrnising members of the genus felix against YIP, feline infectious enteritis (FIE) and feline viral rhinotracheitis is composed of a vial containing a freeze-dried preparation of live attenuated FIE vaccine and live modified feline viral rhinotraceitis and a second viral containing an inactivated FIP virus suspension as prepared in Example 4, together with instructions to reconstitute the freeze-dried vaccine with the wet prepa ration prior to administration.

Claims: Claims 1. A method for preparinq an inactivated feline infectious peritonitis vaccine characterised in that an isolate of virulent feline infectious peritonitis virus is serially passaged in feline embryonic cell cultures until the infected cultures exhibit a cytopathic effect, that is the cells produce irregular shapes when examined microscopically as wet preparations, the virus so produced is inactivated and presented in association with a Pharmaceutically acceptable carrier. 2. A method as claimed in claim 1 further characterised in that the embryonic cell cultures comprise diploid feline embryonic lung cells. A A method as claimed in claim 1 further characterised in that the feline embryonic cell cultures comprise heteroploid feline embryonic lung cells. 4. A method as claimed in any one of claims 1 to 3 further characterised in that the virus is inactivated by chemical means. 5. A me as c3 aimed in any one of claims 1 to 3 further characterised in that the virus is inactivated by physical means. 6. A vaccine for developing immunity in animals to feline infectious peritollitist prepared according to method of claim 1, in an effective dosage, or multiples thereof 7. A vaccine as claimed in claim 6 further characterised in that the effective dosage is from 104.0 to TCID50 per ml.

Inventors: FISHMAN, BERNARD, HITCHCOCK, LOUISE MARY, O'REILLY, KEVIN JOSEPH
Applicants: THE WELLCOME FOUNDATION LIMITED
Application year: 1979
Main classification: A61K39
IPC codes: A61K39