WO1993014190A1 - Vaccins contre le virus de la leucemie des felins - Google Patents

Vaccins contre le virus de la leucemie des felins Download PDF

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Publication number
WO1993014190A1
WO1993014190A1 PCT/US1993/000221 US9300221W WO9314190A1 WO 1993014190 A1 WO1993014190 A1 WO 1993014190A1 US 9300221 W US9300221 W US 9300221W WO 9314190 A1 WO9314190 A1 WO 9314190A1
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felv
cell
subtype
feline
vaccine
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PCT/US1993/000221
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English (en)
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Keith Haffer
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Smithkline Beecham Corporation
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Priority to EP93903066A priority Critical patent/EP0621893A4/fr
Priority to JP5512609A priority patent/JPH07502897A/ja
Publication of WO1993014190A1 publication Critical patent/WO1993014190A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This application relates generally to the field of veterinary vaccines, and more specifically to compositions and methods for preventing persistent infection in felines caused by feline leukemia virus.
  • Feline leukemia is debilitating viral disease of felines, particularly domestic cats, which can often result in death of the animal.
  • the pathogenesis of feline leukemia can be divided into the following stages: primary disease, recovery or remission, and terminal illness.
  • primary disease Generally, only cats which become persistently viremic will show significant clinical or hematological signs of illness in the primary stage of infection.
  • the clinical symptoms include varying degrees of fever, malaise, weight loss, generalized lymphadenopathy and hematological abnormalities.
  • Cats infected with feline leukemia virus may appear asymptomatic or may manifest the symptoms of feline leukemia disease.
  • FeLV feline leukemia virus
  • death occurs in the primary stage of infection, it is usually a direct consequence of severe bone marrow suppression and/or secondary infection.
  • a carrier cat may remain asymptomatic throughout its lifetime, most of these animals will develop some FeLV-related illness.
  • Diseases which are related directly to the effect of the virus upon FeLV carriers include neoplastic disease, bone marrow suppressive disorders, immunological disorders and reproductive problems. Indirectly related illnesses include viral and bacterial secondary infections and protozoal diseases.
  • FeLV is usually transmitted by physical contact between infected and naive animals.
  • Three subtypes of FeLV are associated with disease in felines.
  • Subtype A is present in 100% of all FeLV infections.
  • virus is isolated from a cat infected with FeLV, 50% of the time FeLVA is found alone, 49% of the time FeLVA is found together with FeLVB, and only 1% of the time is an infection found to be caused by FeLV subtypes A, B and C together.
  • the virus is highly unstable in the environment, rarely surviving more than a few hours at room temperature, and can be inactivated with most disinfectants.
  • the present invention provides a stable feline T cell lymphoma cell line transfected with a FeLV subtype which produces antigens useful in protecting against disease caused by FeLV.
  • a cell line is provided comprising FeLV subtype A.
  • Other embodiments employ one or more of subtypes A, B and C.
  • the transfected cells are characterized by the presence of the CD4 and CD8 T cell antigens on the cell membrance and are capable, in culture, of producing the selected FeLV subtype virus proteins.
  • the present invention provides a selected FeLV subtype protein produced by culturing a transfected cell, described above, and isolating from the cell lysate or medium, the viral protein.
  • a FeLV vaccine composition which comprises the selected FeLV antigens produced by a cell line of the invention.
  • the vaccine is capable of protecting the vaccinated animal against infection by more than one subtype of FeLV in the substantial absence of neutralizing antibodies to the FeLV virion gp70 neoantigen or feline oncorna virus cell associated membrane antigen (FOCMA).
  • a vaccine composition which comprises antigens produced by the cell line of the invention in combination with other feline leukemia viral antigens, the vaccine capable of protecting against infections with different strains of feline leukemia virus.
  • Still a further aspect of the present invention entails a method for preparing a vaccine against FeLV infection which comprises combining with a suitable carrier inactivated antigenic fluids derived from a cell line of the invention and an optional adjuvant.
  • this invention provides a method of preventing the clinical symptoms of feline leukemia by inoculating a naive feline with a suitable vaccine composition of the invention.
  • the invention provides a method of enhancing the immune response to a selected feline vaccine by concurrently or sequentially administering an FeLV vaccine of the invention with the selected vaccine.
  • Suitable vaccines for administration in connection with an FeLV vaccine according to the invention include, e.g. feline panleukopenia, calici, rhinotracheitis and chlamydia vaccines or other antigens against which protection is sought in the vaccinee.
  • Fig. 1 is a graph illustrating the results of a study of the Clone E
  • FeLVA vaccine (U, vaccine group) upon viremia through 12 weeks post challenge with Rickard FeLVA.B ( ⁇ , control group).
  • Fig. 2 is a graph illustrating the FeLV infection rates upon Clone
  • a selected stable feline T cell lymphoma cell line is infected by conventional techniques with at least one FeLV subtype.
  • the cell line employed in the exemplified embodiment of this invention was the T cell lymphosarcoma cell line 3201, passage 81, obtained from Dr. Richard Olsen of Ohio State University.
  • feline T cell lymphoma cell lines sharing the characteristics of cell line 3201, i.e., a stable karyotype through multiple passages, and the retention of the T cell CD4 and CD8 antigens on the cell membranes, and the absence of Feline Oncorriavirus Cell Associated Membrane Antigen (FOCMA), may be infected in a similar manner to produce vaccinal compositions of this invention.
  • Other feline T ce lines may be obtained from private sources or public depositories. " Therefore, this invention need not be limited to the use of the specifically exemplified cell line.
  • the cell line may be transformed or infected with one or more of FeLV subtypes A, B, and C.
  • FeLV subtypes may be obtained from the American Type Culture Collection, Rockville, MD; the National Veterinary Service Laboratory, Ames, Iowa; the National Institutes of Health, or from private research sources.
  • the cell line is infected with FeLV subtype A, because of its association with all FeLV infections.
  • the infected cell line is then cultured in serum-free medium. Cell culture supernatants are collected to check for the FeLV gp70 envelope antigen and cells are collected and cloned.
  • a clone having optimal cell viability and activity in a p27 (FeLV core protein) ELISA, described below, is selected and multiply passaged.
  • the number of cell passages may be selected by one of skill in the art to enable selection of a stable cell line that is capable of producing FeLV protein or polypeptide antigens that are capable of producing immunity against at least one strain of FeLV and also retains on the cell membrane the CD4 and CD8 T cell antigens.
  • the number of passages ranged between 15 to 35. Similar numbers of passages may be employed with other feline cell lines.
  • the cells are cultured and passaged in serum-free medium, of which many recipes are available in the art.
  • the infected cells secrete the whole gp70 viral envelope protein into serum-free medium. It is not desirable to culture the infected cells in serum- containing medium, because when infected cells are cultured in serum- containing medium, infectious whole virus particles are secreted into the medium.
  • the cell line of this invention may be used to prepare subunit vaccines or whole cell vaccines as detailed below. subunit vaccines
  • the cell culture from a selected passage is then harvested and suspended.
  • the culture may be stored in a serum-free medium for approximately 3-10 days. Cultures are harvested when 50% viability is observed.
  • the cultures and antigen-containing culture fluids are clarified by an appropriate conventional method which can be determined by one of skill in the art. As one example, the culture fluids may be clarified by filtration to remove unwanted cell debris or medium components.
  • any residual nucleic acid in the clarified culture fluids is inactivated by conventional means.
  • antigens isolated after filtration may be inactivated with bromoethyleneimine, betapropiolactone, or some other appropriate inactivating agent.
  • the resulting inactivated antigenic culture fluids are then admixed with an optional, selected adjuvant and may be formulated for use as a vaccine.
  • the culture fluids may be further purified to isolate the antigenic proteins by resort to conventional protein purification methods known to the art.
  • a T cell lymphoma cell line 3201 Clone E is infected with FeLV subtype A and passaged several times to select high producing cells with FeLVA.
  • Cell culture supernatants are collected to check for FeLV antigen and cells are collected and cloned.
  • a clone having optimal cell viability and p27 ELISA activity is selected and passaged either 15 times in order to produce a Master Cell Stock (MCS) or 35 times in order to produce (MCS+20).
  • MCS or MCS +20 is then harvested suspended and stored in a serum-free medium for 3-10 days.
  • FeLV antigens are secreted by the FeLV infected feline T cells of this invention into surrounding serum-free media and the culture fluids, clarified by filtration, are inactivated.
  • the resulting inactivated FeLVA gp70 antigenic proteins in the culture fluids are capable of providing specific active immunity against FeLV subtype A.
  • Cell line 3201 Clone E is being maintained by the inventors and the assignee, SmithKline Beecham Laboratory, Inc., King of Prussia, Pennsylvania.
  • the clarified, inactivated antigenic culture fluids, or more purified antigenic proteins are aclministered to naive felines to protect them against infection by FeLV.
  • the antigenic viral proteins secreted into the medium, either isolated or present in culture fluids, according to this invention are capable of generating in inoculated animals circulating antibodies which bind epitopes or portions of epitopes of the FeLV viral envelope protein gp70, rather than the whole viral protein itself. These antibodies have not been shown to be cross-reactive to other epitopes than that of the gp70 of the particular FeLV which was used to infect the cell line.
  • the antibodies generated by exposure to the antigenic proteins or culture fluids of this invention bind to FOCMA.
  • the antigens produced by Clone E induce the formation of antibodies in inoculated animals which antibodies target the gp70 epitope, or a portion thereof, of FeLVA but do not bind to subtype B and subtype C gp70 or FOCMA.
  • the virus proteins produced by the infected T cell line exhibit epitopic rather than whole protein binding, the induced antibody response to, e.g., FeLVA is not cross reactive with FeLVB or FeLVC. It is similarly expected that where the selected FeLV subtype used to infect the T cells is subtype B or subtype C, a similar lack of cross-reactivity will be evidenced.
  • these inactivated, adjuvanted antigens produced by Clone E or other infected T cells according to this invention can be used to produce a vaccine which is capable of protecting an immunized animal against persistent infection with FeLVA, or one or more of the other FeLV subtypes.
  • these antigens do not generate enhancing antibodies to other FeLV antigens, than the gp70 epitope as described above. whole cell vaccines
  • the infected cell line of this invention may be administered to the feline. It is naturally preferred that non-infective material should be used as a vaccine providing it is sufficiently immunogenic. Whole cells can be rendered inactive through a variety of techniques known to those in the art.
  • All the vaccine formulations of the present invention are preferably presented for administration to felines in unit dose forms, such as sterile parenteral solutions or suspensions, sterile non- parenteral solutions or suspensions and the like, containing suitable quantities of the inactivated viral antigens (or culture fluids) or whole cells of the invention as an active ingredient.
  • unit dose form refers to a dose for a cat, each unit containing that amount of the viral antigens or whole cells of the invention which is required in order to produce the desired therapeutic effect (i.e., an effective amount) in association with an adjuvant if needed and, optionally a pharmaceutically acceptable diluent, carrier or vehicle.
  • fluid unit dose forms are prepared utilizing the active ingredient, either suspended or dissolved in the vehicle, which should be sterile.
  • Each unit dose of a vaccine compositions of the present invention preferably contains between about 1000 ng/mL to about 10,000 ng/mL total viral gp70 protein, and more preferably between about 1500 ng/mL to about 8,0000 ng/mL total viral protein.
  • a method of preventing the clinical symptoms of feline leukemia is described.
  • a previously uninfected feline may be vaccinated with a vaccine of the invention as described herein.
  • the dosage administered will be dependent upon the identity of the viral infection, the age, health and weight of the animal, kind of concurrent treatment, frequency of treatment and therapeutic ratio.
  • the amount of inactivated FeLV antigens produced according to the invention required to obtain protection against FeLV disease is expected to be the same or less than if the inactivated FeLV antigens of the invention were administered in the absence of other antigens.
  • Suitable pharmaceutically acceptable vehicles are well known to those of skill in the art.
  • Some exemplary vehicles include water, liquid alcohols, liquid glycols, liquid polyalkylene glycols, liquid esters, liquid amides, liquid protein hydrolysates and liquid lanolins.
  • the vaccine of the invention is formulated with the antigen described above and one or more conventional adjuvants.
  • Quil A is added to the final product in a final concentration of between 10 and 50 aeg mL, most preferably about 25 aeg/mL
  • aluminum hydroxide is added in a final concentration of between about 1 to about 10 percent by volume, most preferably about 5% by volume.
  • other suitable adjuvants may be substituted in the place of either the Quil A or the aluminum hydroxide.
  • Such other suitable adjuvants include saponins, oil based adjuvants, polymers, liposomes, bacterial antigens and other cell wall derivatives, colloids, vegetable oils, conjugated mitogens, plant extracts and toxoids, hydrocarbons, cholesterol and its derivatives, and plastic polymers.
  • the vaccine formulation of the invention may also include other conventional vaccine composition components.
  • Chemical stabilizers such as citric acid, sodium hydroxide may be used in suitable conventional amounts.
  • Other components and buffering agents may be used to adjust pH, such as Hepes buffer.
  • the vaccine formulation may include a local anesthetic.
  • the vaccine formulation may also include preservatives such as merthiolate, gentamicin, and parabens.
  • merthiolate is added in a dilution from between about 1:10,000 to 1:50,000, most preferably about 1:20,000.
  • a surfactant or wetting agent may be included in the composition to facilitate uniform distribution of the active ingredient.
  • the inactivated viral antigens or whole cells of the invention may be used in conjunction with other feline leukemia viral antigens, to produce a vaccine composition capable of protecting against infections with different strains feline leukemia.
  • Such other viral antigens may be produced in a manner similar to that described for the antigens of the invention by infecting the cell line with another desired FeLV type or strain, e.g., FeLVB or FeLVC.
  • other antigens for use in a vaccine of the present invention may be prepared by methods known to those of skill in the art, e.g. in the manner described in Olsen, U. S. Patent No. 4,332,793, incorporated herein by reference.
  • Clone E may also be desirable to co-infect a desired cell line, e.g., Clone E, with an additional virus.
  • Clone E may be co-infected with another FeLV subtype, e.g., FeLVB and/or FeLVC to obtain gp70 viral proteins of more than one FeLV subtypes.
  • an FeLV-infected cell line of this invention may be co-infected with another feline virus, such as those mentioned herein.
  • the invention also provides a method of enhancing the immune response to a selected non-FeLV feline vaccine by concurrently or sequentially administering a vaccine of the invention with the selected non-FeLV vaccine.
  • a suitable amount i.e. a unit dose form
  • the FeLV vaccine of the invention acts not only to confer protection against disease caused by infection with FeLV, but also acts as a general immune stimulant capable of enhancing the animal's immune response to the selected non-FeLV vaccine.
  • antigens derived from cell membrane i.e. CD4+ and CD8+ antigens, may be incorporated into the vaccine formulation of the invention.
  • Suitable vaccines for co-administration (concurrent or sequential) in connection with a FeLV vaccine according to the invention include vaccines to other feline infectious agents, e.g. feline panleukopenia, calici, rhinotracheitis, rabies, feline immunodeficiency virus, and chlamydia vaccines or other antigens against which protection is sought in the vaccinee.
  • feline infectious agents e.g. feline panleukopenia, calici, rhinotracheitis, rabies, feline immunodeficiency virus, and chlamydia vaccines or other antigens against which protection is sought in the vaccinee.
  • the FeLV vaccine of the invention may be used as a diluent for the selected on-FeLV vaccine.
  • the FeLV vaccine will not be administered in a suitable amount, i.e. a unit dose form, to confer protection against disease caused by FeLV. Rather, when used as a diluent, the FeLV vaccine of the invention will act
  • a T cell lymphosarcoma cell line (3201) at passage 81 [Dr. Richard Olsen, Ohio State University] is centrifuged at 1000 x g for 10 minutes at 5°C. After the supernatant is discarded, the cells are resuspended in growth medium, which is made up of 15% fetal bovine serum, 2% L- Glutamine (200 mM), 1% sodium pyruvate (100 ml), 1% penicillin/streptomycin, ' the remainder being 50% RPMI 1640/50% Leibowitz-15.
  • the transfer ratio is 1 part cells:8 parts growth medium and transfer occurs every 4 to 5 days. Forty milliliters of the cell suspension is then aliquotted into each of eight 75 cm flasks. The culture flasks are then loosely capped and placed into a C02 incubator at 37°C.
  • the cells were again centrifuged (1500 rpm, 10 minutes) and the 3201-DEAE medium was aspirated off. The cell pellet was then resuspended in an amount (approximately 4 mL) of the 3201 growth medium needed to give 3 x 106 cells/mL. These cells were then incubated at 37°C in 5% C02 until ready to infect.
  • FeLV p27 the group specific antigen or core protein of FeLV
  • virus titer by ELISA. Detection of p27 is the primary diagnostic indicator of FeLV infection.
  • Virus isolation in CL 81 cells [ATCC] was performed as described in P. Fishinger et al, J. Virol., 14(1): 177-179 (1974). The cells were consistently FeLV negative. Cells were subsequently examined for Mn2+ and Mg2+-reverse transcriptase (RT) activity according to known techniques, and for FeLV (Mn2 +- dependent) and Feline Immunodeficiency Virus (Mg2 ---dependent) detection.
  • RT reverse transcriptase
  • the 3201 cells were infected with FeLV subtype A (FeLVA) by resuspending them in 24 mL (2 aeg/mL polybrene) of 0.45 aem filtered supernatant obtained from the centrifuged (100 x g for 10 minutes) CT600 cells [Dr. Niels Pederson, University of California at Davis]. These CT600 cells secrete FeLVA into the culture supernatant. After five passages of the FeLV-infected 3201 cells, p27 ELISA activity and virus liters of 3 x 104 FFU/mL (Focus Forming Units) supernatant was obtained. After two additional passages, cells were cloned in 96-well plates by limiting dilutions with RPMI 1640 + 10% calf serum.
  • FeLVA FeLV subtype A
  • the clone (clone E) containing optimal cell viability and the greatest p27 ELISA activity was selected and passaged 15 times to produce a Master Cell Stock (MCS). Passage 15 was harvested, suspended in a serum-supplemented media and placed in a serum-free environment.
  • An appropriate serum-supplemented media may be, e.g. RPMI 1640, 10% adult bovine serum (ABS) and 10% dimethyl sulfoxide (DMSO).
  • the concentration of the MCS in the media should be between about 1 x 108 cells/mL to about 5 x 108 cells/mL.
  • the diploid and modal chromosome number of the domestic cat is 38.
  • seventy-five metaphases of MCS preparations were analyzed.
  • Eleven metaphases (14.6%) had chromosome counts of 37 and eight metaphases (10.6%) had 36 chromosomes.
  • Twenty-two percent of the cells had counts ranging from 18 to 35.
  • One hypermodal metaphase was observed. Marker chromosomes were not detected.
  • Clone E cells (MCS at 1 x 106 cells/mL, prepared as described above in Example 1) from frozen storage are incubated in roller bottles and scaled up to 50 L fermentors. Each cell culture fluid- harvest is washed by centrifugation with maintenance medium, pH 6.95-7.3, to remove bovine serum from cells. Cells are suspended in maintenance medium to a cell density of approximately 1 to 5 x 106 cells/mL. Culture fluid is harvested after 2 to 12 days incubation at 36° to 37°C in serum- free maintenance medium, at a time when cell viability is reduced to less than 50 percent. Cell lysis and disruption are observable by light microscopy. For example, the trypan blue exclusion test is one technique useful for judging cell death.
  • the harvested culture fluids are frozen at -40°C or below for clarification at a later date or clarified to separate the remaining intact cells from the soluble fraction.
  • the minimum acceptable yield of harvested material is 100 ng/mL of gp70 antigen.
  • Harvested material having less than this required minimum yield are either concentrated, by the methods described below, to achieve the minimum yield or pooled with other harvested material so that the pool meets the minimum yield.
  • the Clone E fluids are inactivated as follows. After determining the volume of the culture fluids from Clone E cells, Binary ethylimine (BEI), an inactivating agent, is added to the culture filtrate at 35° + 2°C to a final BEI concentration of 1 mM (1.0 percent).
  • BEI solution is prepared from sterilized solutions of 200 mM 2-bromoethylamine in distilled water and 0.4 M NaOH in distilled water. Equal volumes of each are mixed and incubated in a 37°C water bath for 1 hour. The final solution may be used within 2 hours of preparation if stored at 4°C.
  • the culture filtrate is maintained at approximately 37°C with constant stirring during the inactivation for a period of at least 2 days.
  • NL-FK-1 feline kidney cells [SmithKline Beecham Animal Health] are treated with DEAE- dextran, inoculated with 1.0 mL per lot of inactivated virus fluid, and maintained at 36°C + 1°C for 21 days, including at least five subculturings.
  • Crandel Feline Kidney Cells [American Type Culture Collection] may be used in place of the NL-FK-1 cells.
  • the cell monolayers are examined for the presence of FeLV p27 antigen.
  • The can be done using a fluorescein isothiocyanate (FITC) conjugated immunoglobulin or a commercially available monoclonal-based licensed diagnostic test kit [e.g. Cambridge Bio Science Co.].
  • FITC fluorescein isothiocyanate
  • a commercially available monoclonal-based licensed diagnostic test kit [e.g. Cambridge Bio Science Co.].
  • the absence of detectable p27 antigen constitutes a satisfactory inactivation test.
  • BEI-inactivated fluids are neutralized with a sterile 1 M sodium thiosulfate solution in distilled water added to a final concentration of approximately 0.25% (w/v).
  • the inactivated culture fluids may be concentrated by ultrafiltration equipment. This is desirable when a high antigen yield has been obtained or when the inactivated culture fluids are to be used in a combination product in which space is at a premium. If concentrated, the fluids should have a concentration of at least 1.5 times the original inactivated culture filtrate volume. Inactivated fluids, whether concentrated or not, are stored at approximately 4°C until adjuvanted.
  • diluents may be added to standardize higher titered fluids prior to the addition of adjuvants.
  • Quil A (Superfos, Sweden] is added as an adjuvant to the product in a final concentration of approximately 25 asg/mL.
  • Aluminum hydroxide gel (Alhydrogel, 2% A1203) is added as an adjuvant to the product in a final concentration of 5 percent by volume.
  • merthiolate is added to a final concentration of up to 1:10,000. After the addition of the above components, the concentration of gp70 antigen in ng/mL is determined by a monoclonal antibody capture test.
  • the monoclonal capture assay (MAb-ELISA) for gp70 is performed in 96-well bottom plates (Immunolon 2) which is coated with a 16,000 dilution (100 aeL) of monoclonal antibody IC-2 [SmithKline Beecham].
  • IC-2 is diluted in 0.01 M borate buffer, pH 9.0. Coated plates are incubated at 4°C overnight. After incubation, the coating solution is discarded and plates are post-coated with 2% BSA in borate buffer, 100 seL/well for 30 minutes at 37°C. The plates are washed 3 times with PBS-Tween.
  • the gp70 standard is diluted to approximately 1640 ng/mL in PBS-0.25% BSA-0.25% Triton X-100 [Sigma]. Four serial 2-fold dilutions of the standard are then made in the same diluent (82-103 ng/mL). Samples are directly diluted 1:8 or as otherwise required in PBS-0.286% BSA-0.286% Triton. All 5 standards and sample dilutions are loaded onto washed plates, 100 aeL per well in triplicate. The plates are incubated for 1 hour at 37°C, and then washed 5 times with PBS- Tween.
  • Goat anti-gp70 (calf serum absorbed) diluted 1:3200 in 3% BSA in PBS-Tween is added to each well (100 aeL well). Plates are again incubated for 1 hour at 37°C. Plates are washed 3 times with PBS- Tween and 100 selJ well of rabbit anti-goat IgG in 0.5% BSA in PBS- Tween is added.
  • the conjugate dilution used provides an optical density for the lowest reference dilution replicates of 1.0 to 1.3 at 405 nm/490 nm in approximately 20 minutes. Plates are incubated at 37°C for 1 hour and washed 3 times with PBS-Tween. ABTS substrate (chromagen) is added, 100 aeL/well, and plates are allowed to incubate at room temperature until the first standard (1640 ng/mL) reaches an OD value of 1.3 at 405 nm/490 nm reference.
  • OD values for the standards and samples are corrected for background absorbance.
  • a standard curve is then generated from the log concentration of the reference standard and the value of each test sample dilution is determined from this curve.
  • the gp70 content of each test sample is calculated as the mean of the values obtained for the dilutions of that sample which fall within the limits of the standard curve.
  • the tested value by MAb-ELISA for gp70 was approximately 295 ng/mL, using the above described technique.
  • each dose of vaccine contains at least 1768 ng/mL total gp70 at release.
  • Example 4 Clinical Effect of Inoculation with Vaccine Formulation of the Invention The following clinical study was performed to determine the effectiveness of a FeLVA vaccine in preventing transient and persistent FeLV viremia through 12 weeks post challenge.
  • the FeLVA inactivated fluids were prepared as described in Example 3 above. To prepare the subunit vaccine used in this study, 0.445 mL of these fluids were mixed with 0.05 mL AlOH and 0.5 mL RPMI 1640 medium.
  • FeLV-negative, SPF cats [Liberty Labs, Liberty, N ] were used at 10 - 14 weeks of age. Ten cats were assigned to the vaccine group and a control group. Vaccination was by the subcutaneous route on day 0 and 21. The temperature of each cat was taken on day -1,0, and +1 post each vaccination. Complete blood analysis was performed on days, 0, 21, 35 and biweekly through 12 weeks post challenge. Antibody titer to gp70 were conducted by ELISA, FOCMA titers by IFA and VN response by an indirect ELISA as described in K. Haffer et al, Vaccine, 8:12-16 (1990) (5 weeks post vaccination and 12 weeks post challenge only).
  • Transient viremia (cumulative infection incidence) was significantly reduced in vaccinates (60%), as compared to 100% in controls (p ⁇ 0.01, Fisher's Exact Test) [See Fig.l]. Similarly, persistent viremia was also significantly reduced to 30% in the vaccinates, while 80% of the controls were persistent viremic (p ⁇ 0.01) [See Fig.2].

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Abstract

On décrit un vaccin contre le virus de la leucémie des félins (FeLV) contenant des fluides antigènes inactivés obtenus à partir d'une lignée de cellules T infectée, stable, ou de cellules T infectées, entières, inactivées et qui peut protéger contre plus d'une souche de leucémie féline en l'absence d'anticorps neutralisant détectables dirigés contre contre la gp70 et l'antigène FOCMA.
PCT/US1993/000221 1992-01-06 1993-01-06 Vaccins contre le virus de la leucemie des felins WO1993014190A1 (fr)

Priority Applications (2)

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EP93903066A EP0621893A4 (fr) 1992-01-06 1993-01-06 Vaccins contre le virus de la leucemie des felins.
JP5512609A JPH07502897A (ja) 1992-01-06 1993-01-06 ネコ白血病ウイルスワクチン

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US81745092A 1992-01-06 1992-01-06
US07/817,450 1992-01-06

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3056214A1 (fr) * 2008-06-27 2016-08-17 Zoetis Services LLC Nouvelles compositions d'adjuvants

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US4034081A (en) * 1973-09-18 1977-07-05 University Of Glasgow Vaccines against feline leukemia
US4117112A (en) * 1973-09-18 1978-09-26 University Of Glasgow Vaccine for prevention of feline leukemia
US4332793A (en) * 1979-12-18 1982-06-01 The Ohio State University Research Foundation Method of recovering cell antigen and preparation of feline leukemia vaccine therefrom
US4406885A (en) * 1978-12-28 1983-09-27 Research Corporation Preparation of native oncornavirus envelope subunits and vaccines therefrom
US4434157A (en) * 1979-12-18 1984-02-28 The Ohio State University Research Foundation Method of recovering cell antigen and preparation of feline leukemia vaccine therefrom
US4657761A (en) * 1985-06-05 1987-04-14 Pinto Cesar M Polyvalent non-specific immuno-stimulating vaccine and method
US4699785A (en) * 1984-09-18 1987-10-13 The Regents Of The University Of California Cell line producing feline leukemia virus
US4711778A (en) * 1980-07-30 1987-12-08 Norden Laboratories, Inc. Inactivated rabies vaccine for veterinary use
US4789702A (en) * 1984-05-18 1988-12-06 Cetus Corporation Feline leukemia virus vaccine

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Publication number Priority date Publication date Assignee Title
US4034081A (en) * 1973-09-18 1977-07-05 University Of Glasgow Vaccines against feline leukemia
US4117112A (en) * 1973-09-18 1978-09-26 University Of Glasgow Vaccine for prevention of feline leukemia
US4406885A (en) * 1978-12-28 1983-09-27 Research Corporation Preparation of native oncornavirus envelope subunits and vaccines therefrom
US4332793A (en) * 1979-12-18 1982-06-01 The Ohio State University Research Foundation Method of recovering cell antigen and preparation of feline leukemia vaccine therefrom
US4434157A (en) * 1979-12-18 1984-02-28 The Ohio State University Research Foundation Method of recovering cell antigen and preparation of feline leukemia vaccine therefrom
US4434157B1 (fr) * 1979-12-18 1987-12-29
US4711778A (en) * 1980-07-30 1987-12-08 Norden Laboratories, Inc. Inactivated rabies vaccine for veterinary use
US4789702A (en) * 1984-05-18 1988-12-06 Cetus Corporation Feline leukemia virus vaccine
US4699785A (en) * 1984-09-18 1987-10-13 The Regents Of The University Of California Cell line producing feline leukemia virus
US4657761A (en) * 1985-06-05 1987-04-14 Pinto Cesar M Polyvalent non-specific immuno-stimulating vaccine and method

Non-Patent Citations (2)

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Title
See also references of EP0621893A4 *
Virology, Volume 179, issued 1990, T.S. TOCHIKURA et al., "In Vitro Replication and Cytopathogenicity of the Feline Immunodeficiency Virus for Feline T4 Thymic Lymphoma 3201 Cells", pp. 492-497, see entire document. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3056214A1 (fr) * 2008-06-27 2016-08-17 Zoetis Services LLC Nouvelles compositions d'adjuvants
US9662385B2 (en) 2008-06-27 2017-05-30 Zoetis Services Llc Adjuvant compositions

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AU3441693A (en) 1993-08-03
CA2127466A1 (fr) 1993-07-22
JPH07502897A (ja) 1995-03-30
EP0621893A1 (fr) 1994-11-02
EP0621893A4 (fr) 1995-07-12

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