WO2005080416A1 - Feline calicivirus vaccines - Google Patents

Feline calicivirus vaccines Download PDF

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Publication number
WO2005080416A1
WO2005080416A1 PCT/IB2005/000069 IB2005000069W WO2005080416A1 WO 2005080416 A1 WO2005080416 A1 WO 2005080416A1 IB 2005000069 W IB2005000069 W IB 2005000069W WO 2005080416 A1 WO2005080416 A1 WO 2005080416A1
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fcv
vaccine
diva
strain
seq
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PCT/IB2005/000069
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French (fr)
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Sing Rong
David Ewell Slade
Robert Gerard Ankenbauer
David Walter Wheeler
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Pharmacia & Upjohn Company Llc
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/16011—Caliciviridae
    • C12N2770/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/16011—Caliciviridae
    • C12N2770/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2770/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/16011—Caliciviridae
    • C12N2770/16061—Methods of inactivation or attenuation

Definitions

  • the present invention relates to a vaccine for immunizing a cat against feline calicivirus.
  • the present invention also relates to a nucleic acid clone that encodes the capsid protein of the isolated feline calicivirus.
  • the present invention further relates to a live or killed vaccine comprising the isolated feline calicivirus, a subunit vaccine comprising the capsid protein of the isolated feline calicivirus, a nucleic acid vaccine comprising a nucleic acid clone of the isolated feline calicivirus, and a recombinant virus vector vaccine comprising nucleic acid encoding the capsid protein of the isolated feline calicivirus.
  • the present invention also relates to a method for identifying a feline calicivirus useful for producing a vaccine composition and for assays for diagnosing cats infected with feline calicivirus.
  • the caliciviruses are a family of small viruses that possess a plus-strand, non- segmented, polyadenylated RNA genome.
  • the buoyant densities of these non- enveloped viruses range from 1.36 to 1.39 g/ml (Oglesby, A.S., et al., Biochemical and biophysical properties of vesicular exanthema of swine virus, Virology 44, pp. 329-341 (1971); Burroughs, J.N and Brown, F., Physico-chemical evidence for re- classification of the caliciviruses, J. Gen. Virol, 22, pp. 281-285 (1974); Soergel,
  • FCV feline calicivirus
  • SMSV San Miguel sea lion virus
  • VMV vesicular exanthema virus of swine
  • human calicivirus Schond-Conrat, H. and Wagner, R., eds
  • strains of low virulence produce no or only moderate pyrexia but do produce ulcers of the tongue, hard palate and nose.
  • Strains of high virulence produce pyrexia, anorexia, depression, dyspnea or polypnea and frequently death, especially in neonatal animals. Ulcers of the tongue, hard palate and nose may, but do not always, occur in addition to pneumonic signs produced by the strains of high virulence.
  • a FCV strain has also been implicated to some extent in causation of the urethritis-cystitis complex. FCV has been shown to be shed from the throat and in the feces of carrier cats for at least a year.
  • FCV infection tends to be high, and mortality in young kittens with severe signs of pneumonic disease may approach 100%.
  • the mild form of the disease characterized by lingual ulcers, is seldom fatal.
  • the disease caused by highly virulent or pneumonic FCV is first manifested as fever (104-105° F) and anorexia.
  • the fever tends to fluctuate after the initial elevation, but the temperature tends to remain elevated through the course of the disease.
  • Polypnea or dyspnea appears shortly after the initial fever rise, and moist rales may be ausculated.
  • the invention includes a vaccine for immunizing cats against feline calicivirus comprising an FCV-Diva strain, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier.
  • the invention also provides a vaccine wherein the Diva strain comprises a nucleic acid encoding SEQ ID NO:2 which is disclosed below.
  • the invention also provides a vaccine wherein the Diva strain comprises a nucleic acid comprising SEQ ID NO:l which is also disclosed below.
  • the vaccine may contain an adjuvant.
  • the FCV-Diva strain of the vaccine may be inactivated or it may be live or live attenuated.
  • the vaccine may contain at least one other feline calicivirus strain selected from the group consisting of FCV-F9, FCV-M8, FCV-255, and FCV-2280.
  • the vaccine may contain at least one other feline pathogen selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica.
  • the invention also comprises a vaccine to immunize cats against feline calicivirus which comprises: a nucleic acid encoding an FCV-Diva capsid protein or a specific immunogenic fragment thereof, wherein the nucleic acid is operably linked to a heterologous promoter sequence, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier.
  • the nucleic acid may optionally be inserted in a plasmid or in a recombinant virus vector.
  • the recombinant viruses used may include but are not limited to feline herpesvirus, raccoon poxvirus, canary poxvirus, adenovirus, Semlicki Forest virus, Sindbis virus, and vaccinia virus.
  • the nucleic acid encodes the capsid protein comprising the amino acid of SEQ ID NO: 2 or a specific immunogenic fragment thereof.
  • the nucleic acid encoding the capsid protein is SEQ ID NO: 1 or a fragment thereof which encodes a specific immunogenic fragment.of SEQ ID NO:2.
  • SEQ ID NO: 1 and the encoded amino acid sequence SEQ ID NO:2 are reproduced below.
  • the FCV-Diva capsid protein comprises the amino acid sequence of SEQ ID NO:2 or a specific immunogenic fragment thereof.
  • the invention includes a method of immunizing a cat against feline calicivirus comprising administering to the cat an effective dose of any of the compositions described herein.
  • the invention includes any of the substances or compositions described herein for use as vaccine.
  • the invention also includes any of the compositions described herein for the manufacture of a vaccine for treating or preventing a disease or disorder in an animal caused by infection with feline calicivirus..
  • the invention also includes a method for immunizing a cat against feline calicivirus comprising administering to the cat an effective dose of a vaccine selected from the group consisting of a live FCV-Diva strain, a live attenuated FCV-Diva strain, a killed FCV-Diva strain, an isolated FCV-Diva capsid protein, and a nucleic acid comprising a a sequence encoding an FCV-Diva capsid protein or a specific immunogenic fragment thereof, in a pharmaceutically acceptable carrier.
  • a vaccine selected from the group consisting of a live FCV-Diva strain, a live attenuated FCV-Diva strain, a killed FCV-Diva strain, an isolated FCV-Diva capsid protein, and a nucleic acid comprising a a sequence encoding an FCV-Diva capsid protein or a specific immunogenic fragment thereof, in a pharmaceutically acceptable carrier.
  • the method may include administration of the vaccine in combination with an effective dose of a vaccine derived from other feline pathogens selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica.
  • a vaccine derived from other feline pathogens selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica.
  • the invention further provides a method which comprises the administration of an isolated FCV-Diva capsid protein comprising the amino acid sequence of SEQ ID NO:2.
  • the invention provides a method which comprises the administration of a nucleic acid encoding an FCV-Diva capsid protein wherein the nucleic acid comprises SEQ ID NO: 1 or a fragment thereof which encodes a specific immunogenic fragment.of SEQ ID NO:2.
  • the invention also provides a method which comprises the administration of a nucleic acid encoding an FCV-Diva capsid protein wherein the nucleic acid is operably linked to heterologous promoter.
  • the nucleic acid may be contained within a plasmid or a recombinant virus vector.
  • the invention also comprises an isolated FCV-Diva capsid protein comprising SEQ ID NO: 2 or a specific immunogenic fragment thereof..
  • the invention further comprises a nucleic acid encoding a FCV-Diva capsid protein comprising the nucleic acid sequence of SEQ ID NO: 1.
  • the invention comprises an antibody specific for an FCV-Diva strain or capsid protein.
  • the antibodies may be polyclonal or monoclonal.
  • the invention also provides a self contained kit containing the antibodies of the invention.
  • a specifically disclosed monoclonal antibody which is specific for Diva strains is identified herein as "1-4 mAb" and a hybridoma producing that antibody has been deposited with the American Type Culture Collection (ATCC) as described in Example 3.
  • ATCC American Type Culture Collection
  • a composition comprising the antibody specific for an FCV Diva strain or capsid protein optionally comprises an acceptable carrier or diluent.
  • the invention also comprises a method of determining whether a cat has been vaccinated or infected with a Diva strain comprising determining whether the cat has produced antibodies specific for an FCV-Diva capsid protein.
  • Such a method may include an antigen capture assay, an antibody capture assay or an ELISA assay.
  • the invention comprises a method of determining whether a particular FCV strain is a Diva strain which comprises determining what residues occupy positions 455 and 458 of the capsid protein wherein a the presence of a methionine at residue 455 and a serine at position 458 of the capsid protein indicates the strain is a Diva strain.
  • the invention includes a method of determining whether a particular FCV strain is a Diva strain which comprises determining whether the antibodies selected from the group consisting of FCV 8-1 A and 1-4 mAb bind specifically to said FCV strain wherein specific binding indicates the strain is a Diva strain Detailed Description of the Invention
  • the present invention includes feline calicivirus vaccines derived from FCV-Diva strains.
  • the vaccines comprise a modified live vaccine and/or a killed vaccine and a pharmaceutically acceptable carrier, the vaccine may also comprise an adjuvant.
  • Nucleic acid vaccines are also contemplated.
  • Subunit vaccines are additionally contemplated.
  • FCV-Diva or FCV-Diva strains
  • FCV-Diva strains includes but is not limited to the two Diva feline calicivirus strains reported by Schorr-Evans at al J. Feline Medicine and Surg. 5:217-226 (2003).
  • FCV-Diva strains described by Schorr-Evans at al. contain unique amino acid residues at position 455 and 458 of the FCV capsid protein sequence.
  • FCV-Diva capsid protein refers to a capsid protein encoded by a FCV-Diva strain.
  • the capsid protein encoded by SEQ ID NO:l refers to a capsid protein encoded by SEQ ID NO:l.
  • a specific immunogenic fragment thereof when used in conjunction with the term FCV-Diva capsid protein, means a fragment of an FCV- Diva capsid protein which comprises the unique residues at position 455 and 458 and which stimulates production of antibody or sensitized cells during an immune response.
  • antibody refers to an immunoglobulin molecule with the capacity to bind with a specific antigen as the result of a specific immune response. Immunoglobulins are serum proteins made up of light and heavy polypeptide chains and divisible into classes, which contain within them antibody activities toward a wide range of antigens.
  • polyclonal antibody refers to a mixed population of antibodies made against a particular pathogen or antigen.
  • the population contains a variety of antibody groups, each group directed towards a particular epitope of the pathogen or antigen.
  • the whole pathogen or an isolated antigen is introduced by inoculation or infection into a host that induced the host to make antibodies against the pathogen or antigen.
  • the term "monoclonal antibody” refers to antibodies produced by a single line of hybridoma cells all directed towards one epitope on a particular antigen.
  • the antigen used to make the monoclonal antibody can be provided as an isolated protein of the pathogen or the whole pathogen.
  • a hybridoma is a clonal cell line that consists of hybrid cells formed by the fusion of a myeloma cell and a specific antibody- forming cell.
  • monoclonal antibodies are of mouse origin; however, monoclonal antibody also refers to a clonal population of an antibody made against a particular epitope of an antigen produced by phage display technology or method that is equivalent to phage display or hybrid cells of non-mouse origin.
  • the term "specific for,” when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind FCV-Diva capsid proteins or Diva strains exclusively (i.e., are able to distinguish FCV-Diva capsid protein from other known proteins or FCV strains from other FCV strains by virtue of measurable differences in binding affinity. It will be understood that specific antibodies may also interact non-specifically with other proteins and strains (for example, S.
  • aureus protein A or other antibodies in ELIS A techniques through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule.
  • Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), Antibodies A Laboratory Manual: Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6.
  • Antibodies that recognize and bind fragments of the FCV-Diva capsid proteins of the invention are also contemplated, provided that the antibodies are specific to FCV-Diva capsid proteins.
  • Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
  • active immunity includes both humoral immunity and/or cell mediated immunity against feline calicivirus induced by vaccinating a cat with the vaccine of the present invention.
  • passive immunity refers to the protection against feline calicivirus provided to a cat as a result of vaccinating the cat with a vaccine comprising antibodies against the FCV- Diva or an immunogenic component or fragment of a component thereof.
  • an "immunologically protective amount” or “effective amount to produce an immune response" of an antigen is an amount effective to induce an immunogenic response in the recipient that is adequate to prevent or ameliorate signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with feline calicivirus. Either humoral immunity or cell-mediated immunity or both may be induced.
  • the immunogenic response of an animal to a vaccine composition may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain.
  • the protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number and overall physical condition and overall health and performance of the subject.
  • the immune response may comprise, without limitation, induction of cellular and/or humoral immunity.
  • the amount of a vaccine that is therapeutically effective may vary depending on the particular virus used, the condition of the cat and can be determined by a veterinary physician.
  • "Isolated” as used herein and as understood in the art, whether referring to an "isolated” nucleic acid or polypeptide, is taken to mean separated from the original cellular environment in which the polypeptide or nucleic acid is normally found. As used herein therefore, by way of example only, a recombinant cell line constructed with a nucleic acid of the invention makes use of the "isolated" nucleic acid.
  • the immunogenic and vaccine compositions of the present invention can include one or more pharmaceutically-acceptable carriers.
  • a pharmaceutically-acceptable carrier includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the components of the invention and not deleterious to the subject to be immunized. Typically, the carriers will be will be sterile and pyrogen free.
  • the vaccine compositions optionally may include vaccine-compatible pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media.
  • Diluents can include water, saline, dextrose, ethanol, glycerol, and the like.
  • Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others.
  • Stabilizers include albumin, among others.
  • any adjuvant known in the art may be used in the vaccine composition, including oil-based adjuvants such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, mycolate-based adjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (i.e., mureins, mucopeptides, or glycoproteins such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), BiostimTM (e.g., 01K2), the "Iscoms" of EP 109942, EP 180564 and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pl
  • Adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), alum, aluminum hydroxide gel, cholesterol, oil-in water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), AMPHIGEN® adjuvant, saponin, Quil A, QS-21 (Cambridge Biotech Inc.,
  • the immunogenic compositions can further include one or more other immunomodulatory agents such as, e.g., interleukins, interferons, or other cytokines.
  • the immunogenic compositions can also include gentamicin and Merthiolate.
  • the present invention contemplates compositions comprising from about 50 ⁇ g to about 2000 ⁇ g of adjuvant and preferably about 500 ⁇ g/2 ml dose of the vaccine composition.
  • the present invention contemplates vaccine compositions comprising from about 1 ⁇ g/ml to about 60 ⁇ g/ml of antibiotic, and more preferably less than about 30 ⁇ g/ml of antibiotic.
  • the immunogenic compositions of the present invention can be made in various forms depending upon the route of administration.
  • the immunogenic compositions can be made in the form of sterile aqueous solutions or dispersions suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized immunogenic compositions are typically maintained at about
  • a stabilizing solution e.g., saline or/and HEPES, with or without adjuvant.
  • the virus was purified once by limiting dilution on 96 a well plate and the purified viruses were amplified and frozen stocks were made.
  • the cells used were NLFK (Norden Laboratories Feline Kidney) cells.
  • the growth medium is OptiMEM (Life Technologies; Gaithersburg, MD), supplemented with antibiotics and
  • RNA sample Approximately 1 ug of viral RNA was used for RT- PCR (Ready-To-GoTM RT-PCR beads, Amersham Biosciences; Piscataway, NJ). The conditions used were: 30 min at 42°C; 7 min at 95°C; followed by 40 cycles of 1 min at 94°C, 1 min at 52°C and 1 min at 72°C; followed by a final incubation at 72°C for 7 min, and storage at 4°C. Three sets of gene-specific primers were used for each RNA sample.
  • FCV-N1 (5'-TTCGGCCTTTTGTGTTCC-3' SEQ ID NO:3) with FCV-Cl (5'- TTGAGAATTGAACACATC-3' SEQ ID NO:4) generates a DNA fragment of 670 bp.
  • FCV-N1 with FCV-C2 (5'-TCCTCGCCAATCCCAGTGTA-3' SEQ ID NO:5) generates a DNA fragment of 580 bp.
  • FCV- N2 (5'--TTCGGCCTTTTGTGTTCC-3' SEQ ID NO:3
  • CATTTCGACTTTAACCAAGA-3' SEQ ID NO:6 with FCV-C2 generates a DNA fragment of 478 bp.
  • different methods were used to purify the product. If there was a single band of the correct size, a QIAquick PCR Purification Kit (Qiagen) was used. If there were multiple bands, a Qiaquick Gel Extraction Kit (Qiagen) was used to purify the band of correct size. About 1-5 ng/ul of purified DNA was used for sequencing.
  • FCV-33585 is very closed related to FCV-Diva 5 and FCV-Diva 24 as reported by Schorr-Evans et al (J. Feline Medicine and Surg. 5:217-226 (2003)).
  • a ClustalW alignment comparing the amino acid sequence of region E from 83 known FCV capsid protein sequences contained within Genbank to Diva strains FCV-Diva 15, FCV-Diva 24 and FCV-Diva 33585, is shown in Figure 1.
  • the sequences used for the comparison and their Genbank accession numbers from which they were derived are shown in Table 1. Table 1
  • FCV strain 103-1991 Genbank Accession #: AP186244. :SEQ ID NO: 7
  • FCV 182cvs5A (AF031875) : SEQ ID NO:8
  • FCV strain 184cvsllA (AF184952) : SEQ ID N ⁇ :9
  • FCV strain 2024 SEQ ID NO: 10
  • FCV 213-95 (AF283778) : SEQ ID N ⁇ :ll
  • FCV strain 2280 (X99445) : SEQ ID N ⁇ :12
  • FCV strain 254cvs465 (AF043227) : SEQ ID N ⁇ :13
  • FCV strain 255 (U07130) : SEQ ID N ⁇ :14
  • TIPEKLVPAGHYAIANGTGNDITTA DYDSATVIQNNTNFKGMYICGSLQRAWGDKKISNTA
  • FCV strain 255 PI persistent infection clone FCU83970: SEQ ID N ⁇ :15
  • FCV strain 255 P5 persistant infection clone (FCU83974) : SEQ ID N ⁇ :16
  • FCV strain 255 P7 persistant infection clone (FCU83976) : SEQ ID NO:18
  • FCV strain 255 P8 persistant infection clone (FCU83977) : SEQ ID N ⁇ :19
  • FCV strain 255 P9 persistant infection clone (FCU83978) : SEQ ID N ⁇ :20
  • FCV strain 259cvs48A (AF184953) : SEQ ID NO:22
  • FCV strain 431 capsid (AX078768) : SEQ ID NO:24
  • FCV strain 63-1991 (AX186246) : SEQ ID NO:25
  • FCV strain 82857-91 (U06648) : SEQ ID NO:26
  • FCV strain 89243-91 (U06651) : SEQ ID NO:27
  • FCV strain 91-1 (AB029581) : SEQ ID NO:28
  • FCV strain 91-10 (AB029588) : SEQ ID NO:29
  • FCV strain 91-11 (AB029589) : SEQ ID N ⁇ :30
  • FCV strain 91-12 (AB029590) : SEQ ID NO:31 IPETLTPAGDYSITTADGNDITTATQYDAASVIKNTTNFRGMYICGSLQRAWGDKKISATA
  • FCV strain 91-13 (AB029591) : SEQ ID NO:32
  • FCV strain 91-14 (AB029592) : SEQ ID NO:33
  • FCV strain 91-16 (AB029593) : SEQ ID NO:34
  • FCV strain 91-17 (AB029594) : SEQ ID NO:35
  • FCV strain 91-19 (AB029596) SEQ ID NO:37
  • FCV strain 91-2 (AB029582) : SEQ ID NO:38
  • FCV strain 91-20 (AB029597) : SEQ ID NO:39
  • FCV strain 91-22 (AB029598) : SEQ ID N ⁇ :40
  • FCV strain 91-25 (AB029599) : SEQ ID N ⁇ :41
  • FCV strain 91-26 (AB029600) : SEQ ID NO:42
  • FCV strain 91-27 (AB029601) : SEQ ID NO:43
  • FCV strain 91-3 (AB029583) : SEQ ID NO:44
  • FCV strain 91-31 (AB029602) : SEQ ID NO:45
  • FCV strain 91-33 (AB029603) : SEQ ID NO:46
  • FCV strain 91-34 (AB029604) : SEQ ID NO:47
  • FCV strain 91-37 (AB029607) : SEQ ID N ⁇ :50
  • FCV strain 91-38 (AB029608) : SEQ ID NO:51
  • FCV strain 91-40 (AB029609) : SEQ ID NO:52
  • FCV strain 91-41 (AB029610.1) : SEQ ID NO:53
  • FCV strain 91-5 (AB029584) : SEQ ID NO:54
  • FCV strain 91-6 (AB029585) : SEQ ID NO:55
  • FCV strain 91-7 (AB029586) : SEQ ID NO:56
  • FCV strain 91-9 (AB029587) : SEQ ID NO:57
  • FCV strain A4 (AF109468) : SEQ ID NO:58
  • FCV strain C01 (AF260320) : SEQ ID NO:59
  • FCV strain C28 (Af260319) : SEQ ID N ⁇ :60
  • FCV strain C46 (AF259956) : SEQ ID N ⁇ :61
  • FCV strain C58 (AF259394) : SEQ ID NO:62
  • FCV strain CFI-68 (FCU13992) : SEQ ID NO:63
  • FCV strain Fl-76-77 (AF184951) : SEQ ID NO:64
  • FCV strain F4 (P27405) : SEQ ID NO:65
  • FCV strain F65 (AF109465) : SEQ ID NO:66
  • FCV strain F9 (Z11536) : SEQ ID NO:67
  • FCV strain fcv255 (U06646) : SEQ ID NO:68
  • FCV strain fcvLLK (U06649) : SEQ ID NO:69
  • FCV strain fpl-Bolin (U06652) : SEQ ID N ⁇ :70
  • FCV strain fpl-n ⁇ i (U06653) : SEQ ID N ⁇ :71
  • FCV strain fri-nci (U06647) : SEQ ID NO:72
  • FCV strain FS (U06650) : SEQ ID NO:73
  • FCV strain FT (E12464) : SEQ ID NO:74
  • FCV strain Gl (AX078766) : SEQ ID NO:75
  • FCV strain JOK63 (AF109466) : SEQ ID NO:76
  • FCV strain KCD (L09719) : SEQ ID NO:77
  • FCV strain KS109 (X99446) : SEQ ID NO:78
  • FCV strain KS20 (X99447) : SEQ ID NO:79
  • FCV strain KS8 (X99449) : SEQ ID N ⁇ :81
  • FCV strain LS012 (AF109467) : SEQ ID N ⁇ :82
  • FCV strain LS015 (AF109464) : SEQ ID N ⁇ :83
  • FCV strain NADC (L09718) : SEQ ID N ⁇ :84
  • FCV strain ⁇ l (AF357012) : SEQ ID NO:85
  • FCV strain U2 (AY053460) : SEQ ID NO:86
  • FCV unkno n-1 (AR198747) : SEQ ID NO:87
  • FCV unknown-2 capsid (AF486286) : SEQ ID NO:88
  • FCV unknown-2 (AR198746) : SEQ ID NO:89
  • FCV unkno n-3 (AR198745) : SEQ ID N ⁇ :90
  • FCV strain ⁇ rbana (L40021) : SEQ ID N ⁇ :91
  • FCV strain V16 (AF031869) : SEQ ID NO:92
  • FCV strain V179 (AF031870) : SEQ ID NO:93
  • FCV strain V20 (AF031872) : SEQ ID NO:94
  • FCV strain V25 (AF038382) : SEQ ID NO:95
  • FCV strain V274 (AF031877) : SEQ ID NO:96
  • FCV strain V276 (AF032106) : SEQ ID NO:97
  • FCV strain V280891 (AF186247) : SEQ ID NO:98
  • FCV strain V290892 (AF184594) : SEQ ID NO:99
  • FCV strain V3 (AF038381) : SEQ ID N ⁇ :100
  • FCV strain V310792 (AF186245) : SEQ ID N ⁇ :101
  • FCV strain V61 (AF031871) : SEQ ID N ⁇ :102
  • FCV strain V66-97 (FCA9721) : SEQ ID N ⁇ :103
  • FCV strain V77 (AF038126) : SEQ ID N ⁇ :104
  • FCV strain V83 (AF031876) : SEQ ID N ⁇ :105
  • FCV-Diva strains (FCV-Diva 15, FCV-Diva 24 and FCV-Diva 33585) are shown in Figure 1 to have unique amino acid residues at positions 32 and 35 of the truncated sequence used for the comparison. These residues correspond to positions 455 and 458 of the full-length FCV capsid protein sequence.
  • FCV-Diva 33585 we have determined the complete sequence of the nucleic acid molecule encoding the capsid protein of FCV- Diva 33585, and report that nucleic acid sequence as SEQ ID NO:l and the encoded polypeptide sequence as SEQ ID NO:2. Identifying an FCV-Diva strain Identification of an FCV-Diva strain can be accomplished by determining what residues occupy positions 455 and 458 of the capsid protein.
  • RNA virus calicivirus RNA can be analyzed directly, but that it is more convenient to create a DNA copy of the RNA by reverse transcription. DNA is more stable than RNA, and the methods for determining DNA sequence information are technically simpler. Hybridization methods are also easier to perform with DNA targets, primers and probes.
  • a preferred method of preparing nucleic acid to determine whether a particular calicivirus isolate is a Diva strain is by reverse transcriptase- polymerase chain reaction (RT-PCR).
  • feline calicivirus RNA is extracted from a biological sample using methods well known in the art, such as the Qiagen QIAmp Viral RNA kit (Qiagen, Valencia, CA).
  • the RT-PCR reaction is performed by any one of the methods well known in the art such as the BRL One-Step RT-PCR Kit (Life Technologies, Bethesda, Md.).
  • a RT-PCR primer pair flanks a target sequence from within the region of the viral genome encoding the capsid protein.
  • US Patent 6,355,246 and 6,355,246 describe primer sequences suitable for capsid gene amplification. The primers described in Example 2 are preferred.
  • nucleotide Sequence Analysis of the nucleotide sequence encoding region E of the FCV capsid protein as described in the Example above can be accomplished using either the dideoxy chain termination method or the Maxam Gilbert method (see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., CSHP, New York (1989); Zyskind et al., Recombinant DNA Laboratory Manual; Acad. Press, (1988)). Although the sequence determination performed above was done manually, it should be recognized that the field of DNA sequencing has advanced considerably in the past several years, and that the invention contemplates such advances. Most notably, within the past decade, there has been increasing reliance on automated DNA sequence analysis.
  • Allele-specific probes can be designed that hybridize to a segment of target nucleic acids from FCV-Diva strains, but which do not hybridize to the corresponding segment from other known FCV strains, due to the presence of the unique sequence found in the FCV-Diva strains. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between FCV- Diva strains and other known strains.
  • Some probes are designed to hybridize to a segment of target nucleic acid such that the site of interest aligns with a central position (e.g., in a 15 mer at the 7 position; in a 16 mer, at either the 8 or 9 position) of the probe.
  • This design of probe achieves good discrimination in hybridization between different allelic forms.
  • These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising the E region of FCV-Diva capsid to hybridize thereto.
  • the G+C content in the probes of the invention usually ranges between 10 and 75 %, preferably between 35 and 60 %, and more preferably between 40 and 55 %.
  • the length of these probes can range from 10, 15, 20, or 30 to at least 100 nucleotides, preferably from 10 to 50, more preferably from 18 to 35 nucleotides.
  • a particularly preferred probe is 25 nucleotides in length.
  • the Diva alleles are within 4 nucleotides of the center of the probe.
  • the Diva marker is at the center of the probe. . Shorter probes may lack specificity for a target nucleic acid sequence, and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes are expensive to produce and can sometimes self-hybridize to form hairpin structures.
  • Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example, peptide nucleic acids which are disclosed in International Patent Application WO 92/20702 and Morpholino analogs which are described in U.S. Patents Numbered 5,185,444; 5,034,506 and 5,142,047.
  • the probe may have to be rendered "non-extendable" in that additional dNTPs cannot be added to the probe.
  • nucleic acid probes can be rendered non-extendable by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation.
  • the 3' end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group.
  • the 3' hydroxyl group simply can be cleaved, replaced or modified.
  • the probes of the present invention are useful for a number of purposes. They can be used in Southern hybridization to DNA or Northern hybridization to RNA. The probes can also be used to detect PCR amplification products.
  • hybridization assays By assaying the hybridization to an allele-specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample.
  • High-throughput parallel hybridizations in array format are specifically encompassed within "hybridization assays" and are described below.
  • Diva-specific probes are often used in pairs, one member of a pair showing a perfect match to a Diva strain and the other member showing a perfect match to another FCV strain. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple variants of the target sequence. 3.
  • FCV-Diva Specific Primers An FCV-Diva specific primer hybridizes to a site on a target nucleic acid overlapping a Diva specific allele and only primes amplification of an allelic form to which the primer exhibits perfect complementarily (See Gibbs, Nucleic Acid Res. 17, 2427-2448,1989). This primer is used in conjunction with a second primer that hybridizes at a distal site. Amplification proceeds from the two primers leading to a detectable product signifying the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the Diva specific site and the other of which exhibits perfect complementarily to a distal site.
  • the single-base mismatch prevents amplification and no detectable product is formed.
  • the method works best when the mismatch is included in the 3 '-most position of the oligonucleotide aligned with the site in question, because this position is most destabilizing to elongation from the primer (See, e.g., WO 93/22456).
  • the invention contemplates such primers with distal mismatches as well as primers that, because of chosen conditions, form unstable base pairing and thus prime inefficiently.
  • Denaturing Gradient Gel Electrophoresis Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. See Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co, New York, 1992), Chapter 7.
  • Single-Strand Conformation Analysis Alleles of target sequences can be differentiated using single-strand conformation analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989).
  • Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single- stranded amplification products.
  • Single-stranded nucleic acids may refold or form secondary structures that are partially dependent on the base sequence.
  • the different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences.
  • FCV-Diva specific hybridization on filters including allele-specific PCR, PCR plus restriction enzyme digest (RFLP-PCR), denaturing capillary electrophoresis, primer extension and time-of- flight mass spectrometry, and the 5' nuclease (TAQMAN TM) assay.
  • RFLP-PCR PCR plus restriction enzyme digest
  • TAQMAN TM 5' nuclease
  • TAQMAN PCR-based TAQMAN technology
  • TAQMAN is a registered trademark of Roche Molecular Systems, Inc. (Alameda, CA).
  • Methods and apparatus for performing TAQMAN-based reactions and detecting the reaction products are available from Applied Biosystems Division, Perkin-Elmer (Foster City, CA).
  • the TAQMAN methodology is based upon RT-PCR being carried out using an oligonucleotide probe labeled at the 5' end with an energy donor fluor, and at the 3' end with an energy quencher.
  • the probe is complementary to a sequence within the target sequence which is generally 100 nucleotides or less from either the upstream primer or the downstream primer.
  • Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., Nature Biotechnology, 16:49-531 1998).
  • Preferred techniques for FCV genotyping should allow for large scale, automated analysis. Examples of the later are DASH (Dynamic Allele-Specific hybridization) that is amenable to formatting in microtiter plates (Hybaid) and "single- stringency" DNA-chip hybridization (Affymetrix). It should be recognized of course, that this list is not inclusive.
  • Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants. Efficient access to FCV sequence information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (the chip) at selected positions.
  • the chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime. The chip technology has already been applied with success in numerous cases.
  • Chips of various formats for use in detecting particular sequences can be produced on a customized basis by Affymetrix (GeneChipTM), Hyseq (HyChip and HyGnostics), and Protogene Laboratories. In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments.
  • EP785280 describes a tiling strategy for the detection of various sequences. Briefly, arrays may generally be "tiled" for a large number of specific sequences.
  • tileing is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as pre-selected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e. nucleotides. Tiling strategies are further described in PCT application No. WO 95/11995.
  • arrays are tiled for a number of specific, identified marker sequences.
  • the array is tiled to include a number of detection blocks, each detection block being specific for a specific sequence.
  • a detection block may be tiled to include a number of probes, which span the sequence segment that includes an area of interest.
  • the array is scanned to determine the position on the array to which the target sequence hybridizes.
  • the hybridization data from the scanned array is then analyzed to identify which allele or alleles are present in the sample.
  • Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 and US patent No. 5,424,186.
  • the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length.
  • the chip may comprise an array including at least one of the sequences comprising between 6-800 contiguous nucleotides of SEQ ID NO. 1 and the sequences complementary thereto, or a fragment thereof at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides, including at least one Diva-specific site.
  • the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these nucleic acids of the invention.
  • Other methods to determine whether an FCV strain is a Diva strain can be envisaged by one skilled in the art and include methods that include antibody-based detection methods.
  • irnmunofluorescense assay a stock of FCV- 33585 was used to infect a 24-well plate with NLFK cells of about 90% confluency. Approximately 20 hours post-infection, the plate was rinsed twice with lx PBS, and fixed with 80% acetone. Monoclonal antibodies were diluted to approximately 2 ug/ul and added to the plate (0.2 ml well). Following a 1 hour incubation (rocking) at RT, each well was washed twice with lx PBS. Secondary antibody (anti-mouse FITC, 10 ug/ml). After 30 min. rocking at RT, the plate covered with foil, each well was washed twice with lx PBS, and air dried. Each well was then observed under a microscope for FITC staining intensity.
  • IFA irnmunofluorescense assay
  • a 96-well ELISA plate was coated with 100 ul of purified FCV- 33585 or F9 virus at a concentration of 5 ug/ml in PBS buffer, pH 7.4. The plate was incubated (uncovered) at 37°C overnight in a non-humidified incubator. The following day, virus on the plates was fixed by applying 0.1 ml of methanol, followed by incubation at RT for 5 minutes. The plate was then washed six times with distilled water. Blocking followed by adding 200 ul of 10% of horse serum in lx PBS, and incubating overnight at 4°C. The plate was again washed six times with distilled water.
  • Monoclonal antibodies were added to each well (100 ul/well) at a concentration of 1 ug/ml (Table 3). or diluted 1:100, 1:400, and 1:2000 in PBST (lx PBS with 0.5% Tween-20) (Table 4). Each antibody was tested in triplicate. Following incubation at 37°C for 1 hour, wells were washed 6 times with PBST. 100 ul of a 1:2000 dilution of peroxidase-conjugated AffiniPure goat anti-Mouse IgG (H+L; Jackson ImmunoResearch; cat. No.715-035- 150) was added, and the plate was incubated for 1 hour at 37°C.
  • FCV Diva isolates are useful for developing efficacious vaccines for immunizing cats against disease caused by heterologous feline calicivirus strains. Therefore, the present invention provides vaccines that are based upon live or killed FCV-Diva strains, nucleic acid vaccines encoding for an FCV-Diva capsid protein or a specific immunogenic fragments thereof, and isolated capsid protein or a specific immunogenic fragment thereof.
  • the vaccine of the present invention is generally intended to be a prophylactic treatment which immunizes cats against disease caused by virulent strains of feline calicivirus. However, the vaccine is also intended for the therapeutic treatment of cats already infected with a virulent strain of feline calicivirus.
  • a vaccine comprising antibodies produced by immunizing a heterologous host with FCV-Diva, or immunogenic component thereof, is used for the therapeutic treatment of a feline calicivirus-infected cat.
  • vaccines that provide active immunity i.e., vaccines comprising FCV-Diva or mutants thereof, or a specific immunogenic fragments thereof, would be expected to be effective when given as a therapeutic treatment against various diseases.
  • the immunity that is provided by the present invention can be either active immunity or passive immunity and the intended use of the vaccine can be either prophylactic or therapeutic.
  • the route of administration for any one of the embodiments of the vaccine of the present invention includes, but is not limited to, oronasal, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral as well as transdermal or by inhalation or suppository.
  • the preferred routes of administration include oronasal, intramuscular, intraperitoneal, intradermal, and subcutaneous injection.
  • the vaccine can be administered by any means that includes, but is not limited to, syringes, nebulizers, misters, needleless injection devices, or microprojectile bombardment gene guns (Biolistic bombardment).
  • the vaccine for any one of the embodiments of the present invention is formulated in a pharmaceutically accepted carrier according to the mode of administration to be used.
  • a vaccine that comprises a live or killed FCV-Diva, an FCV-Diva capsid protein or an a specific immunogenic fragment thereof, a recombinant virus vector encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof, or a DNA molecule encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof.
  • an isotonic formulation is preferred.
  • additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose.
  • isotonic solutions such as phosphate buffered saline are preferred.
  • the formulations can further provide stabilizers such as gelatin and albumin.
  • a vaso-constrictive agent is added to the formulation.
  • the pharmaceutical preparations according to the present invention are provided sterile and pyrogen free.
  • the preferred formulations for the pharmaceutically accepted carrier which comprise the vaccines of the present invention are those pharmaceutical carriers approved in the regulations promulgated by the United States Department of Agriculture, or equivalent government agency in a foreign country such as Canada or Mexico or any one of the European countries, for live feline calicivirus vaccines, killed feline calicivirus vaccines, polypeptide (antigen) subunit vaccines, recombinant virus vector vaccines, antibody vaccines, and DNA vaccines. Therefore, the pharmaceutically accepted carrier for commercial production of the vaccine of the present invention is a carrier that is already approved or will be approved by the appropriate government agency in the United States of America or foreign country.
  • the vaccine can further be mixed with an adjuvant that is pharmaceutically acceptable.
  • the vaccine is combined with other feline vaccines to produce a polyvalent vaccine product that can protect cats against a wide variety of diseases caused by other feline pathogens.
  • feline pathogens Currently, commercial manufacturers of feline vaccines, as well as end users prefer polyvalent vaccine products. Therefore, in a preferred embodiment, the present invention provides a polyvalent vaccine which immunizes cats against feline calicivirus and at least one other feline pathogen, preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus. Inoculation of a cat is preferably by a single vaccination that produces a full, broad immunogenic response.
  • the cat is subjected to a series of vaccinations to produce a full, broad immune response.
  • the vaccinations can be provided between about one day to four weeks or longer apart.
  • the cat is vaccinated at different sites simultaneously.
  • Live Vaccines In one embodiment of the vaccine of the present invention, the vaccine comprises a live vaccine comprising FCV-Diva. It is well known in the art that a calicivirus strain which is virulent when administered to a cat by the oronasal route, the natural route of infection, is generally apathogenic when administered by another route such as the intraperitoneal, intramuscular, intradermal, or subcutaneous routes.
  • the UV light-treated virus-containing supernatant was directly plaqued on 24 hr old Fc3Tg cell monolayers, as were 1 ml dilutions of the supernatant. All were then allowed to incubate at 30°C for 90-120 minutes. Agarose overlays were applied to the monolayers and the plates incubated at 30°C for 3-4 days to allow for plaque development for the purpose of enumeration. The end result was a reduction in virus titers of approximately 3 logs. Selection of Temperature Sensitive Variants
  • FCV33585 TS25 was characterized further. This variant of FCV-33585 shows reduced viral replication at 39°C compared to 30°C as demonstrated by Table 6 Table 6
  • FCV-Diva Strain 33585 Serial dilutions of FCV-Diva 33585 (10 "2 to 10 "7 ) were prepared and 0.1 ml of each dilution was added to monolayers of Fc3Tg and NLFK cells in 6-well plates. After 3-4 days incubation at 30°C, wells were examined under a microscope for CPE (cytopathic effect).
  • FCV-Diva attenuated variants of FCV-Diva are made by serial passage of the virus on a cell line such as Crandall-Reese Feline Kidney (CRFK) cells for between about 10 and 100 passages.
  • CRFK Crandall-Reese Feline Kidney
  • the virus loses its ability to cause disease in the cat, e.g., becomes avirulent or non-pathogenic, while maintaining its ability to replicate in the cat and produce a protective immune response.
  • feline calicivirus isolate FCV-Diva or an attenuated or mutated variant thereof, is grown in cell culture on a suitable feline cell line, i.e., CRFK cells, to titers sufficient for producing a vaccine.
  • the calicivirus is harvested according to methods well known in the art. The calicivirus is then concentrated, frozen, and stored at -70°C, or freeze-dried and stored at 4°C.
  • the calicivirus Prior to vaccination the calicivirus is mixed to an appropriate dosage, which is from about 10 3 to 10 8 tissue culture infectious doses per ml (TC-D 5 o/ml)) with a pharmaceutically acceptable carrier, such as a saline solution and/or an optionally adjuvant, such as aluminum hydroxide.
  • a pharmaceutically acceptable carrier such as a saline solution and/or an optionally adjuvant, such as aluminum hydroxide.
  • the live vaccine can further include, in addition to the FCV- Diva strain, at least one other feline calicivirus strain, preferably selected from the group consisting of FCV-F9, FCV-M8, FCV-255and FCV-2280.
  • the vaccine further includes a vaccine for immunizing a cat against at least one or more other feline pathogens, preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica.
  • a vaccine for immunizing a cat against at least one or more other feline pathogens preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica.
  • a vaccine for immunizing a cat against at least one or more other feline pathogens preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and fel
  • Groups of 4-5 cats were inoculated intranasally (IN) as well as subcutaneously (SQ) with 1 ml of 10 6 TCIDso/ml per cat.
  • One group (TS25, H) was also inoculated with a 10-fold higher dose of TS25 (2xl0 7 TCIDso/ml cat).
  • Minimal clinical signs were observed for all the groups, and were comparable to those obtained with the widely used vaccine strain, F9. All cats were re-inoculated with 10 6 TCIDso/ml of TS25 or p61.
  • Serum neutralization data were analyzed with cut-off titers of >23 and >15.
  • Table 7A Neutralization titers for Attenuated FCV-Diva 33585 following IN vaccination. % Positive % Positive (Cut-off % Increase (Cut-off % Increase Strain Titer >23) (vs. F9 Titer) Titer >15) (vs. F9 Titer) 33585 TS25 26 0 37 16 p47 N 27 4 31 p49 F 22 29 p61 N 15 25 F9 26 32
  • Tables 7A-7B contains the serum neutralization data for serum samples obtained from cats infected by the intranasal (IN) and subcutaneous (SQ) routes of inoculation.
  • various passages of attenuated 33585 p61, p47 NLFK, p49 Fc3Tg, and TS-25
  • the cross neutralization coverage of passage 61 virus is less than that of lower passages of 33585 (p47, p49 and TS25).
  • SQ subcutaneously
  • FCV-33585 TS25 when administered sub-cutaneously, offers superior cross neutralization coverage compared with the widely used vaccine strain F9.
  • the vaccine comprises an inactivated or killed vaccine comprising FCV-Diva.
  • the inactivated vaccine is made by methods well known in the art. For example, once the virus is propagated to high titers, it would be readily apparent by those skilled in the art that the virus antigenic mass could be obtained by methods well known in the art. For example, the virus antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate viral antigenic mass to produce vaccines.
  • the calicivirus is inactivated by treatment with formalin, betapropriolactone (BPL), or with binary ethyleneimine (BEI), or other methods known to those skilled in the art.
  • Inactivation by formalin is performed by mixing the calicivirus suspension with 37% formaldehyde to a final formaldehyde concentration of 0.05%.
  • the calicivirus-formaldehyde mixture is mixed by constant stirring for approximately 24 hours at room temperature.
  • the inactivated calicivirus mixture is then tested for residual live virus by assaying for growth on a suitable feline cell line such as CRFK cells.
  • Inactivation by BEI is performed by mixing the calicivirus suspension of the present invention with 0.1 M BEI (2-bromo-ethylamine in 0.175 N NaOH) to a final BEI concentration of 1 mM.
  • the calicivirus-BEI mixture is mixed by constant stirring for approximately 48 hours at room temperature, followed by the addition of 1.0 M sodium thiosulfate to a final concentration of 0.1 mM. Mixing is continued for an additional two hours.
  • the inactivated calicivirus mixture is tested for residual live calicivirus by assaying for growth on a suitable feline cell line such as NLFK cells.
  • the aforementioned inactivated calicivirus of the present invention is mixed with any one of the pharmaceutically carriers for formulating inactivated virus vaccines to the appropriate dosage level.
  • the inactivated vaccine further can include, in addition to FCV-Diva, atleast one other feline calicivirus strain, preferably selected from the group consisting of FCV-F9, FCV-M8, FCV-255, and FCV-2280.
  • the vaccine further includes a vaccine for immunizing a cat against one or more other feline pathogens, preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus.
  • the vaccine comprises a recombinant virus vector containing a nucleic acid encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof, comprising the amino acid sequence of SEQ ID NO:2, or comprising the nucleotide sequence selected from SEQ ID NO:l.
  • the recombinant virus vector is a feline herpesvirus that immunizes a cat against both feline calicivirus and feline herpesvirus.
  • the recombinant virus vector comprises one or more antigens preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica.
  • a recombinant virus vector that expresses the FCV-Diva capsid protein or a specific immunogenic fragment thereof a cDNA encoding the capsid protein or a specific immunogenic fragment thereof is inserted into the genome of a virus vector such as herpesvirus, poxvirus, or adenovirus.
  • recombinant virus vector vaccines include but are not limited to, adenovirus, adeno-associated virus, parvovirus, and various poxvirus vectors to express the FCV-Diva capsid protein or a specific immunogenic fragment thereof.
  • the present invention includes recombinant poxvirus vector vaccines that express the FCV-Diva capsid protein or a specific immunogenic fragment thereof made according to the methods taught in any one of U.S. Pat. Nos.
  • the cDNA encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof is operably linked to a eukaryote promoter at the 5' end of the cDNA encoding the antigen and a eukaryote termination signal and poly(A) signal at the 3' end of the cDNA encoding the antigen.
  • operably linked means that the nucleic acids of the present invention (as a cDNA molecule) and a nucleic acid (DNA) containing an expression control sequence, e.g., transcription promoter and termination sequences, are situated in a vector or cell such that expression of the antigen encoded by the cDNA is regulated by the expression control sequence.
  • an expression control sequence e.g., transcription promoter and termination sequences
  • promoters suitable for expressing the FCV- Diva capsid protein or a specific immunogenic fragment thereof in the recombinant virus vectors are the cytomegalovirus immediate-early (CMV) promoter, the Rous sarcoma virus long terminal repeat (RSN-LTR) promoter, the simian virus 40 (SN40) immediate-early promoter, and inducible promoters such as the metallothionein promoter.
  • CMV cytomegalovirus immediate-early
  • RSN-LTR Rous sarcoma virus long terminal repeat
  • SN40 simian virus 40 immediate-early promoter
  • inducible promoters such as the metallothionein promoter.
  • An example of a D ⁇ A having a termination and poly(A) signal is the SN40 late poly(A) region.
  • Another example of a viral expression system suitable for producing the antigen is the Sindbis Expression system available from Invitrogen. The use of these commercially available expression vectors
  • the vaccine is provided as a nucleic acid or D ⁇ A molecule vaccine that elicits an active immune response in the cat.
  • the D ⁇ A molecule vaccine consists of D ⁇ A having a nucleic acid sequence which encodes the capsid protein or a specific immunogenic fragment thereof of FCN- Diva.
  • the D ⁇ A molecule vaccine comprises the nucleic acid sequence of SEQ ID NO: 1 , or a fragment thereof encoding a specific immunogenic fragment of SEQ ID NO:2.
  • the nucleic acid encoding the capsid protein or a specific immunogenic fragment thereof is operably linked at or near a transcriptional promoter. This enables transcription of the capsid protein, or a specific immunogenic fragment thereof, from the nucleic acid when the nucleic acid is inoculated into the cells of the cat.
  • the DNA molecule is a plasmid.
  • Promoters that are useful for DNA vaccines are well known in the art and include, but are not limited to, the RSV LTR promoter, the CMV immediate early promoter, and the SV40 T antigen promoter. It is further preferred that the nucleic acid be operably linked at or near the termination codon of the sequence encoding the capsid protein or a specific immunogenic fragment thereof to a nucleic acid fragment comprising a transcription termination signal and poly(A) recognition signal.
  • the DNA vaccine is provided to the cat in an accepted pharmaceutical carrier or in a lipid or liposome carrier similar to those disclosed in U.S. Pat. No. 5,703,055 to Feigner.
  • the DNA vaccine can be provided to the cat by a variety of methods such as intramuscular injection, intrajet injection, or biolistic bombardment. Making DNA vaccines and methods for their use are provided in U.S. Pat. Nos. 5,589, 466 and 5,580,859, both to Feigner. Finally, a method for producing pharmaceutical grade plasmid DNA is taught in U.S. Pat. No. 5,561,064 to Marquet et al. Therefore, using the abovementioned methods, DNA vaccines that express the FCV-Diva capsid protein or a specific immunogenic fragment thereof are used to immunize cats against virulent feline calicivirus.
  • the DNA vaccine is that the DNA molecule is conveniently propagated as a plasmid which is a simple and inexpensive means for producing a vaccine, and since the vaccine is not live, many of the regulatory issues associated with live recombinant virus vector vaccines are not an issue with DNA vaccines.
  • the DNA vaccine of the present invention can comprise synthetically produced nucleic acids that are made by chemical synthesis methods well known in the art.
  • the vaccine consists of the isolated and purified FCV-Diva capsid protein or a specific immunogenic fragment thereof.
  • a vaccine wherein the FCV-Diva capsid protein or a specific immunogenic fragment thereof comprises the amino acid sequence of SEQ ID NO:2.
  • the capsid protein or a specific immunogenic fragment thereof is produced in a recombinant bacterium or eukaryote expression vector that produces the antigen that can be isolated and purified to make the vaccine.
  • the FCV- Diva capsid protein or a specific immunogenic fragment thereof is produced in a microorganism such as bacteria, yeast, or fungi, in a eukaryote cell such as a mammalian or an insect cell, or via a recombinant virus vector such as adenovirus, poxvirus, herpesvirus, Semlicki Forest virus, baculovirus, bacteriophage, Sindbis virus, or Sendai virus.
  • Suitable bacteria for producing the FCV-Diva capsid protein or a specific immunogenic fragment thereof include Escherichia coli, Bacillus subt ⁇ lis, or any other bacterium that is capable of expressing heterologous polypeptides.
  • Suitable yeast types for expressing the FCV-Diva capsid protein or a specific immunogenic fragment thereof include Saccharomyces cerevisiae,
  • the nucleic acid encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof is in a plasmid, and the nucleic acid is operably linked to a promoter which effects the expression of the capsid protein or a specific immunogenic fragment thereof in a microorganism.
  • Suitable promoters include, but are not limited to, T7 phage promoter, T3 phage promoter, ⁇ - galactosidase promoter, and the Sp6 phage promoter.
  • Expression of the FCV-Diva capsid protein or a specific immunogenic fragment thereof in a microorganism enables the capsid protein to be produced using fermentation technologies that are used commercially for producing large quantities of recombinant antigenic polypeptides.
  • Methods for isolating and purifying antigens are well known in the art and include methods such as gel filtration, affinity chromatography, ion exchange chromatography, or centrifugation.
  • a fusion polypeptide is made wherein the capsid protein or a specific immunogenic fragment thereof is linked to another polypeptide which enables isolation by affinity chromatography.
  • a fusion polypeptide is made using one of the expression systems infra.
  • the cDNA nucleic acid sequence encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof is linked at either the 5' end or 3' end to a nucleic acid encoding a polypeptide.
  • the nucleic acids are linked in the proper codon reading frame to enable production of a fusion polypeptide wherein the amino and/or carboxyl terminus of the capsid protein or portion thereof is fused to a polypeptide which allows for the simplified recovery of the antigen as a fusion polypeptide.
  • the fusion polypeptide can also prevent the antigen from being degraded during purification. While a vaccine comprising the fusion polypeptide is efficacious, in some instances it can be desirable to remove the second polypeptide after purification. Therefore, it is also contemplated that the fusion polypeptide contains a cleavage site at the junction between the antigen and the polypeptide.
  • the cleavage site consists of an amino acid sequence that is cleaved with an enzyme specific for the amino acid sequence at the site.
  • cleavage sites include the enterokinase cleavage site which is cleaved by enterokinase, the factor Xa cleavage site which is cleaved by factor Xa, and the GENENASE cleavage site which is cleaved by GENENASE (GENENASE is a trademark of New England Biolabs, Beverly, Mass.).
  • the following are methods for producing the capsid protein or a specific immunogenic fragment thereof as a fusion polypeptide or as an isolated antigen free of the polypeptide.
  • Glutathione S-transferase GST
  • the cDNA encoding the capsid protein or a specific immunogenic fragment thereof is fused in the proper codon reading frame with the DNA encoding GST.
  • the GST part of the fusion polypeptide allows the rapid purification of the fusion polypeptide using glutathione Sepharose 4B affinity chromatography.
  • the GST portion of the fusion polypeptide can be removed by cleavage with a site-specific protease such as thrombin or factor Xa to produce an antigen free of the GST polypeptide.
  • a site-specific protease such as thrombin or factor Xa
  • the capsid protein or a specific immunogenic fragment thereof, free of the GST polypeptide is produced by a second round of glutathione Sepharose 4B affinity chromatography.
  • Another method for producing a vaccine comprising the FCV-Diva capsid protein or a specific immunogenic fragment thereof is a method which links in-frame the cDNA encoding the antigen and DNA codons that encode polyhistidine.
  • the polyhistidine preferably comprises six histidine residues which allows purification of the fusion polypeptide by metal affinity chromatography, preferably nickel affinity chromatography.
  • a cleavage site such as an enterokinase cleavage site is fused in the proper reading frame between the codons encoding the polyhistidine and the codons encoding the antigen.
  • the antigen, free of the polyhistidine is made by removing the polyhistidine by cleavage with enterokinase followed by a second round of metal affinity chromatography which binds the free polyhistidine.
  • a DNA encoding a fusion polypeptide consisting of the DNA encoding an antigen linked to DNA encoding a portion of protein A wherein DNA encoding an enterokinase cleavage site is interposed in the proper codon reading frame between the DNA encoding protein A and the antigen.
  • the protein A enables the fusion polypeptide to be isolated by IgG affinity chromatography, and the capsid protein free of the protein A is produced by cleavage with enterokinase. The protein A is then removed by a second round of IgG affinity chromatography.
  • Another method for producing a vaccine comprising the FCV-Diva capsid protein or a specific immunogenic fragment thereof is based on methods disclosed in U.S. Pat. No.
  • the Daniels et al. method can be used to make the FCV-Diva vaccine which consists of enterotoxin molecule wherein each molecule has inserted therein upwards of 100 amino acid residues of the FCV-Diva capsid protein.
  • Other methods for making fusion polypeptide vaccines which can be used to make the vaccines of the present invention is disclosed in U.S. Pat. No. 5,585, 100 to Mond et al. and U.S. Pat. No. 5,589,384 to Liscombe.
  • the pMAL Fusion and Purification System available from New England Biolabs is another example of a method for making a fusion polypeptide wherein a maltose binding protein is fused to the capsid protein or a specific immunogenic fragment thereof.
  • the maltose binding protein facilitates isolation of the fusion polypeptide by amylose affinity chromatography.
  • the maltose binding protein can be linked to the antigen by one of the above mentioned cleavage sites which enables the antigen to be made free of the maltose binding protein.
  • FCV-Diva capsid protein or a specific immunogenic fragment thereof for vaccines
  • a particularly useful system is the baculovirus expression system that is disclosed in U.S. Pat. No. 5,229, 293 to Matsuura et al..
  • Baculovirus expression vectors suitable to produce the capsid protein or a specific immunogenic fragment thereof are the pPbac and pMbac vectors from Stratagene; and the Bac-N-Blue vector, the pBlueBac4.5 vector, pBlueBacHis2-A,B,C, and the pMelBac available from Invitrogen, Carlsbad, Calif.
  • Another eukaryote system useful for expressing the FCV-Diva capsid protein or a specific immunogenic fragment thereof for vaccines is a yeast expression system such as the ESP Yeast Protein Expression and Purification System available from Stratagene.
  • Another yeast expression system is any one of the Pichia-based Expression systems from Invitrogen.
  • Mammalian expression systems are also embraced by the present invention.
  • Examples of mammalian expression systems are the LacSwitch II system, the pBK Phagemid, pXTl vector system, and the pSG5 vector system from Stratagene; the pTargeT mammalian expression vector system, the pSI mammalian expression vector, pCI mammalian expression vector, and pAdVantage vectors available from Promega Corporation, Madison, Wis.; and the Ecdysone-Inducible Mammalian Expression System, pCDM8, pcDNAl.l, and pcDNAl.l/Amp available from Invitrogen.
  • the present invention further includes an embodiment consisting of vaccines that comprise the FCV-Diva capsid protein or particular epitopes of the capsid protein as components of a heat-stable spore delivery system made according to the method taught in U.S. Pat. No. 5, 800,821 to Acheson et al. Therefore, the present invention provides a genetically engineered bacterial cell containing a nucleic acid encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof.
  • the spores germinate in the gastrointestinal tract of the cat and the bacteria expresses the capsid protein or a specific immunogenic fragment thereof which comes into contact with the cat's immune system and elicits an immune response.
  • the vaccine has the advantage of being heat stable; therefore, it can be stored at room temperature for an indefinite period of time.
  • the present invention further comprises vaccines that provide passive immunity to feline calicivirus.
  • a vaccine that elicits passive immunity against feline calicivirus consists of polyclonal antibodies or monoclonal antibodies that are against the FCV-Diva capsid protein, a specific immunogenic fragment thereof, or the whole FCV-Diva virus.
  • the FCV-Diva, capsid protein thereof, or a specific immunogenic fragment thereof is injected into a suitable host for preparing the antibodies, preferably the host is a horse, swine, rabbit, sheep, or goat.
  • the capsid protein or a specific immunogenic fragment thereof or whole calicivirus FCV-Diva is mixed with an adjuvant such as Freund's complete or the less toxic TiterMax available from CytRx Corp., Norcross, Ga., which then administered to the host by methods well known in the art.
  • an adjuvant such as Freund's complete or the less toxic TiterMax available from CytRx Corp., Norcross, Ga.
  • Antibody production is monitored and when sufficient antibody has been produced, the serum is removed from the host and preferably;the antibody is recovered from the serum.
  • the passive immunity vaccine can comprise one or more monoclonal antibodies against one or more epitopes of the FCV-Diva capsid protein or whole virus FCV-Diva, or a specific immunogenic fragment thereof.
  • monoclonal antibodies can be made using hybridoma technologies well known in the art, the monoclonal antibodies against the antigen can also be made according to phage display methods such as that disclosed in U.S. Pat. No. 5,977,322 to Marks et al. Felinized antibodies against the capsid protein or portion thereof can be made according to methods which have been used for humanizing antibodies such as those disclosed in U.S. Pat. Nos. 5,693,762 and 5,693,761 both to Queen et al.
  • a phage display kit that is useful for making monoclonal antibodies is the Recombinant Phage Antibody System available from Amersham Pharmacia Biotech. Methods to Determine Whether an Animal has been Vaccinated with a Diva stain An important aspect of any vaccination program is to be able to distinguish animals vaccinated against a disease from animals infected with the disease.
  • the present invention further includes methods or assays that distinguish cats vaccinated with the vaccine of the present invention from cats infected with a field strain of feline calicivirus, cats vaccinated with another commercial vaccine preparation, or cats never exposed to feline calicivirus.
  • the assays are either the immunologically based antibody-capture or an antigen-capture assays.
  • a biological sample from the cat is tested for the presence of antibodies against FCV-Diva and optionally for the presence of antibodies against other feline calicivirus strains. The presence of antibodies against FCV-Diva indicates the cat was vaccinated with the vaccine of the present invention.
  • the presence of antibodies in a biological sample against a feline calicivirus other than the FCV-Diva indicates the cat is infected with a feline calicivirus other than FCV- Diva.
  • the assay can further include controls that comprise capsid proteins from other calicivirus strains such as the F-9 strain.
  • a serum sample from a cat that had been vaccinated with the vaccine of the present invention will react with FCV-Diva antigens whereas a sample from a cat that is infected with, or exposed to, another feline calicivirus will not contain antibodies against the capsid protein of the FCV-Diva.
  • the antibody-detection or antigen detection assays are performed using an enzyme-linked immunosorbent assay (ELISA) embodiment.
  • ELISA enzyme-linked immunosorbent assay
  • a microtiter plate is provided containing a plurality of wells wherein a first well or series of wells contains the capsid protein of the FCV-Diva immobilized to the surface therein and a second well or series of wells contains antigens from another feline calicivirus strain (or capsid protein from a commercially available calicivirus vaccine) immobilized to the surface therein.
  • the biological sample is added to the wells containing the bound antigens and antibodies against feline calicivirus are allowed to bind to form an antigen-antibody complex.
  • the biological sample can be provided neat or in a limiting dilution series in a physiological solution. Unbound material in the sample is removed from the antibody-antigen complex by washing. The complex is then reacted with a labeled anti-species antibody or labeled monoclonal antibody that binds to feline antibodies to form an antigen-antibody-anti-species-antibody complex. This complex can be detected when the labeled monoclonal or polyclonal antibody is conjugated to a reporter ligand such as horseradish-peroxidase or alkaline phosphatase.
  • a reporter ligand such as horseradish-peroxidase or alkaline phosphatase.
  • the second monoclonal or polyclonal antibody can be conjugated to reporter ligands such as a fluorescing ligand, biotin/avidin colored latex, colloidal gold magnetic beads, radioisotopes or the like. Detection is by methods well known in the art for detecting the particular reporter ligand. Therefore, a sample from a cat that had been vaccinated with the vaccine of the present invention will produce antibodies against only the FCV-Diva capsid protein whereas a sample from a cat that is infected with, or exposed to, feline calicivirus will not contain antibodies against the capsid protein of the FCV-Diva.
  • reporter ligands such as a fluorescing ligand, biotin/avidin colored latex, colloidal gold magnetic beads, radioisotopes or the like. Detection is by methods well known in the art for detecting the particular reporter ligand. Therefore, a sample from a cat that had been vaccinated with the vaccine of the present invention
  • a microtiter plate containing a plurality of wells wherein a first well or series of wells contains polyclonal or monoclonal antibodies against the capsid protein or a specific immunogenic fragment thereof of the FCV-Diva immobilized to the surface therein and a second well or series of wells contains antibodies against antigens of another feline calicivirus strain (or capsid protein or a specific immunogenic fragment thereof from a commercially available calicivirus vaccine) immobilized to the surface therein.
  • the biological sample is added to the wells containing the bound antibodies and antigens against feline calicivirus are allowed to bind to form an antibody-antigen complex.
  • the biological sample can be provided neat or in a limiting dilution series in a physiological solution. Unbound material in the sample is removed from the antibody-antigen complex by washing. The complex is then reacted with a labeled polyclonal antibody or labeled monoclonal antibody against FCV-Diva or a specific immunogenic fragment thereof to form an antibody-antigen-antibody complex. This complex can be detected when the labeled monoclonal or polyclonal antibody is conjugated to a reporter ligand such as horseradish-peroxidase or alkaline phosphatase.
  • a reporter ligand such as horseradish-peroxidase or alkaline phosphatase.
  • the labeled monoclonal or polyclonal antibody can be conjugated to reporter ligands such as a fluorescing ligand, , biotin/avidin colored latex, colloidal gold magnetic beads, radioisotopes or the like. Detection is by methods well known in the art for detecting the particular reporter ligand.
  • ELISA and its variations are well known in the art. The ELISA can be provided as a kit for distinguishing cats vaccinated with the vaccine of the present invention from unvaccinated cats, and from cats infected with feline calicivirus or vaccinated with a commercially available feline calicivirus vaccine.
  • the present invention further includes rapid immunodiffusion-based methods, their devices, and kits comprising the same. Therefore, the present invention can be provided with a kit that comprises any one of the methods described in U.S. Pat. No. 5,620,845 to Gould et al., U.S. Pat. No. 5,559,041 to Kang et al., U. S. Pat. No. 5,656,448 to Kang et al., U.S. Pat. No. 5,728,587 to Kang et al., U.S. Pat. No.
  • the aforementioned fusion polypeptide wherein the polypeptide comprises a marker epitope that elicits an antibody in the vaccinated cat that would not normally be present in the feline.
  • the marker epitope could be from a pathogen that does not infect cats or a synthetic polypeptide that elicits an antibody in cats that would not normally occur in cats. Therefore, if a sample from a cat contained antibodies against the marker epitope and the capsid protein, the cat was vaccinated with the vaccine of the present invention. The sample is tested according to any of the aforementioned diagnostic methods.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

The present invention relates to a vaccine for immunizing a cat against feline calicivirus. The present invention also relates to a nucleic acid clone that encodes the capsid protein of the isolated feline calicivirus. The present invention further relates to a live or killed vaccine comprising the isolated feline calicivirus, a subunit vaccine comprising the capsid protein of the isolated feline calicivirus, a nucleic acid vaccine comprising a nucleic acid clone of the isolated feline calicivirus, and a recombinant virus vector vaccine comprising nucleic acid encoding the capsid protein of the isolated feline calicivirus. The present invention also relates to a method for identifying a feline calicivirus useful for producing a vaccine composition and for assays for diagnosing cats infected with feline calicivirus.

Description

FELINE CALICIVIRUS VACCINES Field of the Invention The present invention relates to a vaccine for immunizing a cat against feline calicivirus. The present invention also relates to a nucleic acid clone that encodes the capsid protein of the isolated feline calicivirus. The present invention further relates to a live or killed vaccine comprising the isolated feline calicivirus, a subunit vaccine comprising the capsid protein of the isolated feline calicivirus, a nucleic acid vaccine comprising a nucleic acid clone of the isolated feline calicivirus, and a recombinant virus vector vaccine comprising nucleic acid encoding the capsid protein of the isolated feline calicivirus. The present invention also relates to a method for identifying a feline calicivirus useful for producing a vaccine composition and for assays for diagnosing cats infected with feline calicivirus. Background of the Invention The caliciviruses are a family of small viruses that possess a plus-strand, non- segmented, polyadenylated RNA genome. The buoyant densities of these non- enveloped viruses range from 1.36 to 1.39 g/ml (Oglesby, A.S., et al., Biochemical and biophysical properties of vesicular exanthema of swine virus, Virology 44, pp. 329-341 (1971); Burroughs, J.N and Brown, F., Physico-chemical evidence for re- classification of the caliciviruses, J. Gen. Virol, 22, pp. 281-285 (1974); Soergel,
M.E., et al., Biophysical comparisons of calicivirus serotypes isolated from pinnipeds, Intervirology, 5, pp 239-244 (1975)). Members of this family include feline calicivirus (FCV), San Miguel sea lion virus (SMSV), vesicular exanthema virus of swine (VEV) and human calicivirus (Schaffer, F.L., Caliciviruses In: Comparative Virology, 14 (Fraenkel-Conrat, H. and Wagner, R., eds), pp. 249-284 (1979)). The signs of illness produced by FCV infections in the cat vary markedly, depending on the strain involved, the severity of the exposure and the resistance of the host. Some strains apparently cause few or no signs of illness. Strains of low virulence produce no or only moderate pyrexia but do produce ulcers of the tongue, hard palate and nose. Strains of high virulence produce pyrexia, anorexia, depression, dyspnea or polypnea and frequently death, especially in neonatal animals. Ulcers of the tongue, hard palate and nose may, but do not always, occur in addition to pneumonic signs produced by the strains of high virulence. A FCV strain has also been implicated to some extent in causation of the urethritis-cystitis complex. FCV has been shown to be shed from the throat and in the feces of carrier cats for at least a year. Because recovered cats often become carriers, shedding accounts for the persistence of the virus in nature. Morbidity from FCV infection tends to be high, and mortality in young kittens with severe signs of pneumonic disease may approach 100%. The mild form of the disease, characterized by lingual ulcers, is seldom fatal. The disease caused by highly virulent or pneumonic FCV is first manifested as fever (104-105° F) and anorexia. The fever tends to fluctuate after the initial elevation, but the temperature tends to remain elevated through the course of the disease. Polypnea or dyspnea appears shortly after the initial fever rise, and moist rales may be ausculated. Depression tends to be marked and, except for the obvious respiratory distress, the infected cat may appear to be suffering from acute feline panleukopenia. Death occurs within days of the onset of signs. The pure pneumonic form of FCV infection appears to be most prevalent among neonatal (14-21 days old) and weaned kittens up to 4 months of age. Older susceptible cats exposed to the highly virulent FCVs may show initial signs suggestive of exudative pneumonia, which then becomes proliferative and interstitial as the cat recovers. In this age group, oral and nasal ulcers may occur noncurrently with the pneumonia. FCV could also cause polyarthritis and limping syndrome. L addition, the more recent highly virulent strains cause facial edema. Despite the availability of vaccines for immunizing cats against FCV, none of the present vaccines have been shown to be completely protective against all strains and isolates of FCV. In some cats, some of the present vaccines may even cause disease. Therefore, there is a need for an efficacious vaccine that affords protection to cats against FCV-related diseases. Literature Cited U.S. PATENT DOCUMENTS
3937812 Feb. /1976 Bittle et al. 3944469 Mar. / 1976 Bittle et al. 4486530 Dec. /1984 David et al. 4786589 Nov. /1988 Rounds et al. 5169789 Dec /1992 Bernstein et al. 5229293 Jul. /1993 Matsuura et al. 5266313 Nov. /1993 Esposito et al. 5338683 Aug. /1994 Paoletti et al. 5494807 Feb. /1996 Paoletti et al. 5559041 Sep. /1996 Kang et al. 5561064 Oct. /1996 Marquet et al. 5580859 Dec. /1996 Feigner 5585100 Dec. /1996 Mond et al. 5589384 Dec /1996 Liscombe 5589466 Dec /1996 Feigner 5620845 Apr. /1997 Gould et al. 5656448 Aug. /1997 Kang et al. 5693761 Dec /1997 Queen et al. 5693762 Dec /1997 Queen et al. 5695928 Dec /1997 Stewart et al. 5703055 Dec /1997 Feigner 5716784 Feb. /1998 DiCesare 5716822 Feb. /1998 Wardley 5718901 Feb. /1998 Wardley 5725863 Mar. /1998 Daniels et al 5728587 Mar, /1998 Kang et al. 5800821 Sep. /1998 Acheson et al. 5977322 NovN1999 Marks et al. 6010703 Jan. /2000 Maes et al. 6355246 Mar/2002 Kruger et al.
FOREIGN PATENT DOCUMENTS 0484382 Mar. /1995 EP 2004/083390 WO OTHER PUBLICATIONS Burroughs, J.N and Brown, F., J. Gen. Virol., 22, pp. 281-285 (1974); Clarke and Lambden in J. Gen. Virol. 78: 291-301 (1997).
Motin et al., Infect. Immun. 64: 4313-4318 (1996).
Oglesby, A.S., et al., Virology 44, pp. 329-341 (1971);
Soergel, M.E., et al., Intervirology, 5, pp 239-244 (1975)
Yokoyama, N., et al., Vaccine, vol. 14, No. 17/18, pp. 1657-1663 (1996). Summary of the Invention The invention includes a vaccine for immunizing cats against feline calicivirus comprising an FCV-Diva strain, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier. The invention also provides a vaccine wherein the Diva strain comprises a nucleic acid encoding SEQ ID NO:2 which is disclosed below. The invention also provides a vaccine wherein the Diva strain comprises a nucleic acid comprising SEQ ID NO:l which is also disclosed below. Optionally the vaccine may contain an adjuvant. The FCV-Diva strain of the vaccine may be inactivated or it may be live or live attenuated. Optionally the vaccine may contain at least one other feline calicivirus strain selected from the group consisting of FCV-F9, FCV-M8, FCV-255, and FCV-2280. Optionally the vaccine may contain at least one other feline pathogen selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica. The invention also comprises a vaccine to immunize cats against feline calicivirus which comprises: a nucleic acid encoding an FCV-Diva capsid protein or a specific immunogenic fragment thereof, wherein the nucleic acid is operably linked to a heterologous promoter sequence, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier. The nucleic acid may optionally be inserted in a plasmid or in a recombinant virus vector. The recombinant viruses used may include but are not limited to feline herpesvirus, raccoon poxvirus, canary poxvirus, adenovirus, Semlicki Forest virus, sindbis virus, and vaccinia virus. In a preferred embodiment, the nucleic acid encodes the capsid protein comprising the amino acid of SEQ ID NO: 2 or a specific immunogenic fragment thereof. In another preferred embodiment, the nucleic acid encoding the capsid protein is SEQ ID NO: 1 or a fragment thereof which encodes a specific immunogenic fragment.of SEQ ID NO:2. SEQ ID NO: 1 and the encoded amino acid sequence SEQ ID NO:2 are reproduced below.
M C S T C A N V L K Y Y N W D P H F R 1 ATGTGCTCAA CCTGCGCTAA CGTGCTTAAA TACTATAACT GGGATCCCCA CTTTAGGCTT V I N P N K F L S V G F C D N P M C C 61 GTTATCAACC CCAACAAGTT TCTTTCTGTT GGTTTCTGTG ATAATCCGCT TATGTGTTGT Y P E L P E F G T V D C D Q S P L Q 121 TATCCCGAAT TGCTTCCTGA ATTTGGAACG GTGTGGGACT GTGATCAGTC TCCACTTCAA I Y L E S I L G D D E S S T Y E A I D 181 ATTTACCTAG AATCTATCCT TGGTGATGAT GAATGGAGTT CCACATACGA GGCAATTGAC P C V P P H D E A G K I F Q P H P G 241 CCATGCGTGC CACCAATGCA CTGGGATGAA GCTGGCAAGA TCTTTCAGCC ACACCCTGGT V L M H H I I G E V A K A D P N P N 301 GTTTTGATGC ACCACATTAT TGGAGAAGTT GCTAAGGCTT GGGACCCGAA CCTTCCCAAC F R L E A D D G S I T T P E Q G T T V G 361 TTCCGTTTGG AAGCTGATGA TGGTTCCATC ACCACCCCCG AGCAGGGAAC TACAGTTGGC G V I A E P S V Q M S A A A D M A T G K 421 GGAGTCATTG CTGAGCCGAG TGTCCAAATG TCAGCGGCTG CTGACATGGC AACTGGCAAA S V D S E W E A F F S F H T S V N W S T 481 AGCGTTGACT CTGAGTGGGA AGCTTTCTTC TCTTTTCACA CCAGTGTCAA CTGGAGTACA S E T Q G K I L F K Q S L G P L L N P Y 541 TCCGAAACCC AAGGAAAGAT TCTATTCAAG CAAAGCTTGG GGCCTCTTCT CAACCCATAC T H A K L Y V A W S G S I E V R F S 601 CTCACGCATC TTGCTAAACT TTATGTCGCA TGGTCTGGCT CTATTGAAGT GAGATTCTCT I S G S G V F G G K L A A I V V P P G I 661 ATTTCTGGGT CTGGTGTTTT CGGAGGAAAA CTGGCTGCAA TTGTTGTGCC ACCTGGAATC E P I Q S T S M L Q Y P H V L F D A R Q 721 GAGCCGATCC AAAGCACTTC AATGCTTCAG TATCCTCACG TTCTATTTGA CGCTCGTCAG V E P V I F T I P D L R S T Y H L M S 781 GTGGAACCTG TGATCTTTAC TATCCCTGAT CTTAGAAGTA CCCTCTATCA CCTTATGTCT D T D T T S L V I M I Y N D L I N P Y A 841 GATACTGACA CTACTTCTTT AGTGATCATG ATATACAATG ATCTCATTAA CCCTTATGCT N D S N S S G C I V T V E T K P G P D F 901 AATGATTCTA ACTCATCTGG GTGCATTGTT ACTGTGGAAA CAAAACCGGG TCCTGACTTC K F H L L K P P G S M L T H G S V P C D 961 AAATTCCATT TATTGAAACC TCCTGGTTCT ATGTTAACTC ATGGCTCTGT CCCGTGTGAC I P K S S S L W I G N R F W S D I T D 1021 CTAATACCAA AGTCTTCTTC TCTTTGGATT GGAAATAGGT TCTGGTCTGA TATCACTGAT F V I R P F V F Q A N R H F D F N Q E T 1081 TTTGTTAT C GGCCATTTGT GTTCCAAGCT AACCGTCACT TTGATTTCAA CCAAGAGACG A G W S T P R F R P I T V T I S Q K E G 1141 GCAGGTTGGA GTACACCAAG ATTCAGACCA ATCACAGTCA CAATTAGTCA AAAAGAAGGT E M L G I G V A T D Y I V P G I P D G W 1201 GAAATGCTTG GGATTGGCGT AGCCACTGAT TATATTGTAC CAGGCATACC TGATGGATGG P D T T I P N E L I P A G D Y A I T N Q 1261 CCAGATACAA CAATACCTAA TGAGCTCATT CCGGCTGGTG ATTATGCTAT TACCAATCAG S G N D I Q T K E E Y E S A M i l S N N 1321 AGTGGCAATG ATATACAAAC AAAAGAGGAA TACGAATCTG CCATGATAAT CAGCAACAAC T N F K S M Y I C G S L Q R A W G N K K 1381 ACAAATTTCA AAAGCATGTA CATTTGTGGG TCCCTTCAAC GAGCGTGGGG TAATAAGAAA V S N T A F I T T A T V K E N K L I P S 1441 GTGTCTAACA CTGCTTTCAT TACTACTGCT ACGGTAAAGG AAAACAAATT AATTCCCAGC N T I D Q T K I A I F Q D N H V N R D V 1501 AATACCATTG ACCAAACAAA GATTGCAATT TTCCAAGACA ATCACGTCAA TCGCGATGTG Q T S D D T L A L L G Y T G I G E E A I 1561 CAAACATCTG ATGACACATT GGCTTTACTT GGGTACACAG GAATTGGTGA AGAAGCAATT G A D R E K V V R I G V L P E A G R G 1621 GGTGCTGATA GGGAGAAAGT TGTGCGCATT GGTGTCCTTC CAGAAGCTGG CGCGCGCGGT G N H P I F Y R N S M K L G Y V I K S I 1681 GGCAACCATC CAATCTTCTA TAGGAAT CT ATGAAATTAG GTTATGTTAT CAAATCTATA D V F N S Q I L H T S R Q L S L N N Y L 1741 GATGTGTTTA ACTCGCAGAT CTTGCACACC TCTAGACAAC TATCACTGAA CAACTACCTT L S P D S F A V Y R I I D S N G S W F D 1801 CTATCACCTG ACTCTTTTGC TGTTTATAGG ATAATTGATT CTAATGGATC TTGGTTTGAT I G I D S D G F S F Y G V S S I G K L E 1861 ATAGGCATTG ATAGTGATGG ATTCTCCTTT GTTGGTGTTT CTTCTATCGG TAAATTAGAA F P L T A S Y M G N Q L A K I R L A S N 1921 TTTCCTCTTA CTGCCTCCTA CATGGGAAAT CAATTGGCAA AGATTCGACT TGCCTCAAAC I R S T M T K L 1981 ATTAGGAGCA CAATGACAAA ATTA The invention also includes a vaccine to immunize cats against feline calicivirus comprising an isolated FCV-Diva capsid protein, or a specific immunogenic fragment thereof, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier. In a preferred embodiment the FCV-Diva capsid protein comprises the amino acid sequence of SEQ ID NO:2 or a specific immunogenic fragment thereof. The invention includes a method of immunizing a cat against feline calicivirus comprising administering to the cat an effective dose of any of the compositions described herein. The invention includes any of the substances or compositions described herein for use as vaccine. The invention also includes any of the compositions described herein for the manufacture of a vaccine for treating or preventing a disease or disorder in an animal caused by infection with feline calicivirus.. The invention also includes a method for immunizing a cat against feline calicivirus comprising administering to the cat an effective dose of a vaccine selected from the group consisting of a live FCV-Diva strain, a live attenuated FCV-Diva strain, a killed FCV-Diva strain, an isolated FCV-Diva capsid protein, and a nucleic acid comprising a a sequence encoding an FCV-Diva capsid protein or a specific immunogenic fragment thereof, in a pharmaceutically acceptable carrier. Optionally the method may include administration of the vaccine in combination with an effective dose of a vaccine derived from other feline pathogens selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica. The invention further provides a method which comprises the administration of an isolated FCV-Diva capsid protein comprising the amino acid sequence of SEQ ID NO:2. The invention provides a method which comprises the administration of a nucleic acid encoding an FCV-Diva capsid protein wherein the nucleic acid comprises SEQ ID NO: 1 or a fragment thereof which encodes a specific immunogenic fragment.of SEQ ID NO:2. The invention also provides a method which comprises the administration of a nucleic acid encoding an FCV-Diva capsid protein wherein the nucleic acid is operably linked to heterologous promoter. Optionally the nucleic acid may be contained within a plasmid or a recombinant virus vector. The invention also comprises an isolated FCV-Diva capsid protein comprising SEQ ID NO: 2 or a specific immunogenic fragment thereof.. The invention further comprises a nucleic acid encoding a FCV-Diva capsid protein comprising the nucleic acid sequence of SEQ ID NO: 1. In another embodiment the invention comprises an antibody specific for an FCV-Diva strain or capsid protein. The antibodies may be polyclonal or monoclonal. The invention also provides a self contained kit containing the antibodies of the invention. A specifically disclosed monoclonal antibody which is specific for Diva strains is identified herein as "1-4 mAb" and a hybridoma producing that antibody has been deposited with the American Type Culture Collection (ATCC) as described in Example 3.
A composition comprising the antibody specific for an FCV Diva strain or capsid protein optionally comprises an acceptable carrier or diluent. In another embodiment the invention also comprises a method of determining whether a cat has been vaccinated or infected with a Diva strain comprising determining whether the cat has produced antibodies specific for an FCV-Diva capsid protein. Such a method may include an antigen capture assay, an antibody capture assay or an ELISA assay. In a further embodiment the invention comprises a method of determining whether a particular FCV strain is a Diva strain which comprises determining what residues occupy positions 455 and 458 of the capsid protein wherein a the presence of a methionine at residue 455 and a serine at position 458 of the capsid protein indicates the strain is a Diva strain. In yet a further embodiment the invention includes a method of determining whether a particular FCV strain is a Diva strain which comprises determining whether the antibodies selected from the group consisting of FCV 8-1 A and 1-4 mAb bind specifically to said FCV strain wherein specific binding indicates the strain is a Diva strain Detailed Description of the Invention The present invention includes feline calicivirus vaccines derived from FCV-Diva strains. In certain embodiments, the vaccines comprise a modified live vaccine and/or a killed vaccine and a pharmaceutically acceptable carrier, the vaccine may also comprise an adjuvant. Nucleic acid vaccines are also contemplated. Subunit vaccines are additionally contemplated. For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections that describe or illustrate certain features, embodiments or applications of the invention. Definitions and Abbreviations The following definitions are provided herein to promote a better understanding of the present invention. The term "FCV-Diva" or "FCV-Diva strains" includes but is not limited to the two Diva feline calicivirus strains reported by Schorr-Evans at al J. Feline Medicine and Surg. 5:217-226 (2003). We have discovered that the FCV-Diva strains described by Schorr-Evans at al. contain unique amino acid residues at position 455 and 458 of the FCV capsid protein sequence. Position 455 of the capsid protein derived from the FCV-Diva strains is a methionine and position 458 is a serine, while the capsid proteins derived from the other 153 FCV strains we have compared them o (published and unpublished) have different amino acids at these positions. Methods of determining whether a particular FCV strain is a Diva strain are discussed extensively herein. The term " FCV-Diva capsid protein" refers to a capsid protein encoded by a FCV-Diva strain. For example, the capsid protein encoded by SEQ ID NO:l. The term "a specific immunogenic fragment thereof, when used in conjunction with the term FCV-Diva capsid protein, means a fragment of an FCV- Diva capsid protein which comprises the unique residues at position 455 and 458 and which stimulates production of antibody or sensitized cells during an immune response. The term "antibody" refers to an immunoglobulin molecule with the capacity to bind with a specific antigen as the result of a specific immune response. Immunoglobulins are serum proteins made up of light and heavy polypeptide chains and divisible into classes, which contain within them antibody activities toward a wide range of antigens. The term "polyclonal antibody" refers to a mixed population of antibodies made against a particular pathogen or antigen. In general, the population contains a variety of antibody groups, each group directed towards a particular epitope of the pathogen or antigen. To make polyclonal antibodies, the whole pathogen or an isolated antigen is introduced by inoculation or infection into a host that induced the host to make antibodies against the pathogen or antigen. The term "monoclonal antibody" refers to antibodies produced by a single line of hybridoma cells all directed towards one epitope on a particular antigen. The antigen used to make the monoclonal antibody can be provided as an isolated protein of the pathogen or the whole pathogen. A hybridoma is a clonal cell line that consists of hybrid cells formed by the fusion of a myeloma cell and a specific antibody- forming cell. In general, monoclonal antibodies are of mouse origin; however, monoclonal antibody also refers to a clonal population of an antibody made against a particular epitope of an antigen produced by phage display technology or method that is equivalent to phage display or hybrid cells of non-mouse origin. The term "specific for," when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind FCV-Diva capsid proteins or Diva strains exclusively (i.e., are able to distinguish FCV-Diva capsid protein from other known proteins or FCV strains from other FCV strains by virtue of measurable differences in binding affinity. It will be understood that specific antibodies may also interact non-specifically with other proteins and strains (for example, S. aureus protein A or other antibodies in ELIS A techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), Antibodies A Laboratory Manual: Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. Antibodies that recognize and bind fragments of the FCV-Diva capsid proteins of the invention are also contemplated, provided that the antibodies are specific to FCV-Diva capsid proteins. Antibodies of the invention can be produced using any method well known and routinely practiced in the art. Identification of antibodies specific for FCV Diva strains is discussed in Example 3. The term "active immunity" includes both humoral immunity and/or cell mediated immunity against feline calicivirus induced by vaccinating a cat with the vaccine of the present invention. The term "passive immunity" refers to the protection against feline calicivirus provided to a cat as a result of vaccinating the cat with a vaccine comprising antibodies against the FCV- Diva or an immunogenic component or fragment of a component thereof. An "immunologically protective amount" or "effective amount to produce an immune response" of an antigen is an amount effective to induce an immunogenic response in the recipient that is adequate to prevent or ameliorate signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with feline calicivirus. Either humoral immunity or cell-mediated immunity or both may be induced. The immunogenic response of an animal to a vaccine composition may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain. The protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number and overall physical condition and overall health and performance of the subject. The immune response may comprise, without limitation, induction of cellular and/or humoral immunity. The amount of a vaccine that is therapeutically effective may vary depending on the particular virus used, the condition of the cat and can be determined by a veterinary physician. "Isolated" as used herein and as understood in the art, whether referring to an "isolated" nucleic acid or polypeptide, is taken to mean separated from the original cellular environment in which the polypeptide or nucleic acid is normally found. As used herein therefore, by way of example only, a recombinant cell line constructed with a nucleic acid of the invention makes use of the "isolated" nucleic acid. In addition, the immunogenic and vaccine compositions of the present invention can include one or more pharmaceutically-acceptable carriers. As used herein, "a pharmaceutically-acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. The carrier(s) must be "acceptable" in the sense of being compatible with the components of the invention and not deleterious to the subject to be immunized. Typically, the carriers will be will be sterile and pyrogen free. The vaccine compositions optionally may include vaccine-compatible pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Any adjuvant known in the art may be used in the vaccine composition, including oil-based adjuvants such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, mycolate-based adjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (i.e., mureins, mucopeptides, or glycoproteins such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), Biostim™ (e.g., 01K2), the "Iscoms" of EP 109942, EP 180564 and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, Pluronic® polyols. Adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), alum, aluminum hydroxide gel, cholesterol, oil-in water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), AMPHIGEN® adjuvant, saponin, Quil A, QS-21 (Cambridge Biotech Inc.,
Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL) or other saponin fractions, monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat- labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, or muramyl dipeptide, among many others. The immunogenic compositions can further include one or more other immunomodulatory agents such as, e.g., interleukins, interferons, or other cytokines. The immunogenic compositions can also include gentamicin and Merthiolate. While the amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan, the present invention contemplates compositions comprising from about 50 μg to about 2000 μg of adjuvant and preferably about 500 μg/2 ml dose of the vaccine composition. In another preferred embodiment, the present invention contemplates vaccine compositions comprising from about 1 μg/ml to about 60 μg/ml of antibiotic, and more preferably less than about 30 μg/ml of antibiotic. The immunogenic compositions of the present invention can be made in various forms depending upon the route of administration. For example, the immunogenic compositions can be made in the form of sterile aqueous solutions or dispersions suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized immunogenic compositions are typically maintained at about
4°C, and can be reconstituted in a stabilizing solution, e.g., saline or/and HEPES, with or without adjuvant.
Growth of Viral Isolates and Sequence Determination of region E of the Capsid Gene of FCV-Diva Strain FCV-33585 Example 1, Growth of FCV-Diva in cell culture FCV isolate 33585 (Diva) was received from Cornell University Diagnostic
Lab (Ithaca, New York). The virus was purified once by limiting dilution on 96 a well plate and the purified viruses were amplified and frozen stocks were made. The cells used were NLFK (Norden Laboratories Feline Kidney) cells. The growth medium is OptiMEM (Life Technologies; Gaithersburg, MD), supplemented with antibiotics and
3% irradiated fetal bovine serum (Cambrex Bio Science Rockland, Inc; Rockland,
ME).
Example 2, RT-PCR and DNA sequence analysis Viral RNA was isolated from 140 ul of viral supernatant using an RNeasy Kit
(QIAGEN Inc.; Valencia, CA). Approximately 1 ug of viral RNA was used for RT- PCR (Ready-To-Go™ RT-PCR beads, Amersham Biosciences; Piscataway, NJ). The conditions used were: 30 min at 42°C; 7 min at 95°C; followed by 40 cycles of 1 min at 94°C, 1 min at 52°C and 1 min at 72°C; followed by a final incubation at 72°C for 7 min, and storage at 4°C. Three sets of gene-specific primers were used for each RNA sample. FCV-N1 (5'-TTCGGCCTTTTGTGTTCC-3' SEQ ID NO:3) with FCV-Cl (5'- TTGAGAATTGAACACATC-3' SEQ ID NO:4) generates a DNA fragment of 670 bp. FCV-N1 with FCV-C2 (5'-TCCTCGCCAATCCCAGTGTA-3' SEQ ID NO:5) generates a DNA fragment of 580 bp. FCV- N2 (5'-
CATTTCGACTTTAACCAAGA-3' SEQ ID NO:6) with FCV-C2 generates a DNA fragment of 478 bp. Depending on the quality of RT-PCR, different methods were used to purify the product. If there was a single band of the correct size, a QIAquick PCR Purification Kit (Qiagen) was used. If there were multiple bands, a Qiaquick Gel Extraction Kit (Qiagen) was used to purify the band of correct size. About 1-5 ng/ul of purified DNA was used for sequencing. Based on phylogenetic analysis of the hypervariable region of FCV capsid protein for all published and known sequences, FCV-33585 is very closed related to FCV-Diva 5 and FCV-Diva 24 as reported by Schorr-Evans et al (J. Feline Medicine and Surg. 5:217-226 (2003)). A ClustalW alignment comparing the amino acid sequence of region E from 83 known FCV capsid protein sequences contained within Genbank to Diva strains FCV-Diva 15, FCV-Diva 24 and FCV-Diva 33585, is shown in Figure 1. The sequences used for the comparison and their Genbank accession numbers from which they were derived are shown in Table 1. Table 1
FCV strain 103-1991 (Genbank Accession #: AP186244) :SEQ ID NO: 7
TLSNTQVPAGDYAIVNEKNNDITTRTGYESATTITN TNFKSMYICGSLQRAWGDKKISNTG
FCV 182cvs5A (AF031875) : SEQ ID NO:8
TIFGDQTPAGDYAITNDKDSDITTRSEYEAASEIKNNTNFKSMYICGSLQRAWGDKKISNTG
FCV strain 184cvsllA (AF184952) : SEQ ID Nθ:9
TISEI TPVGDYAITAGNNSDITTADEYDRANVIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 2024: SEQ ID NO: 10
TIPEKLVPAGNYAIANGTGNDITTAKDYDSATVIQNNTNFKGMYICGSLQRA GDKKISNTA
FCV 213-95 (AF283778) : SEQ ID Nθ:ll
TIPEKT-TPVGDYAITGPSGNDIITAVDYDSAGSIRNDTNFRG YICGSLQRAWGDKKISNTA
FCV strain 2280 (X99445) : SEQ ID Nθ:12
TIPEKLTPAGNYAITTGNNSDIATATEYDHADEIKNNTNFKSMYICGSLQRAWGDKKISNTA
FCV strain 254cvs465 (AF043227) : SEQ ID Nθ:13
TIPEKLTPAGDYAITTGNGTDIVTAAQFDAADVIKNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain 255 (U07130) : SEQ ID Nθ:14
TIPEKLVPAGHYAIANGTGNDITTA DYDSATVIQNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain 255 PI persistent infection clone (FCU83970) : SEQ ID Nθ:15
TIPEKLVPAGNYAIDNGTGNDITTAKDYDSATVIQNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain 255 (P5 persistant infection clone) (FCU83974) : SEQ ID Nθ:16
TIPEELVPAGDYAIANGTGNDITTAKDYDSATVIQNNTNFKGMYICGSLQRAWGDKKISNTA FCV strain 255 (P6 persistant infection clone) (FCU83975) : SEQ ID Nθ:17
TIPEKLVPAGDYAIANGTG DITTAKDYDSATVIQNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain 255 (P7 persistant infection clone) (FCU83976) : SEQ ID NO:18
TIPEKLVPAGNYAIANGTGNDITTAKDYDSGTVIQNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain 255 (P8 persistant infection clone) (FCU83977) : SEQ ID Nθ:19
TISEELIPAGDYAITKRHRVTTSPHPAGYDAADVIKNNTNFKGMYICGSLQRAWGDKKISKTA
FCV strain 255 (P9 persistant infection clone) (FCU83978) : SEQ ID Nθ:20
TIPEELVPVGDYAIANETGNDIATAKDYDSATVIQNNTNFKGMYFCGSLQRAWGDKKISNTA
F FCCVV ssttrraaiinn 225588ccvvss448844 ((AAFF003388338833)) :: SSEEQO IIDD NNθθ::2211
TIPDELVPAGDYAITSGNNNDITTPADYDAADVIKNNTNFRSMYICGSLQRAWGDKKISNTG
FCV strain 259cvs48A (AF184953) : SEQ ID NO:22
TIAEELIPAGDYAITDGRGITOIDTAVGYDTAQVIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 28043.87 (U06645) : SEQ ID NO:23
TIPEDLIPAGDYAITKGNGTDITTSADYDSADVIKNDTNFKSMYICGALERAWGDKKISNTA
FCV strain 431 capsid (AX078768) : SEQ ID NO:24
TIPEKLTPAGDYAITNGGNNDITTAADYDGASIIKNNTNFKG YICGALQRAWGDKKISNTA
FCV strain 63-1991 (AX186246) : SEQ ID NO:25
TIPEKLTPAGDYAITSSDGNDITSALGYDSADVIKNGTNFRSMYICGSLQRAWGDKKISNTA
FCV strain 82857-91 (U06648) : SEQ ID NO:26
TIPED IPVGDYAITKGNGTDITTSADYDSADVIKNDTNFKSMYICGPLQRAWGDKKISNPA
FCV strain 89243-91 (U06651) : SEQ ID NO:27
TIPEDLIPAGDYAITKGNGTDITTSADYDSADVIKNDTNFKSMYICGAFQRAWGDKKISNTA
FCV strain 91-1 (AB029581) : SEQ ID NO:28
TISETLIPAGDYAITDAIGNDIKTASGYDAAGIIKNNTNFRGMYICGSLQRAWGDTKISNTA
FCV strain 91-10 (AB029588) : SEQ ID NO:29
TIPGELIPAGDYAITNGTGNDITTATGYDTAD1 KNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 91-11 (AB029589) : SEQ ID Nθ:30
TIPEELTPAGDYAIVTTIGNDITTAAAYDAADVIVNNTNFKGMYICGSLQRAWGDKRISNTA
FCV strain 91-12 (AB029590) : SEQ ID NO:31 IPETLTPAGDYSITTADGNDITTATQYDAASVIKNTTNFRGMYICGSLQRAWGDKKISATA
FCV strain 91-13 (AB029591) : SEQ ID NO:32
TISEELTPAGDYAITTGSGNDITTATGYDSADVIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 91-14 (AB029592) : SEQ ID NO:33
TIPEELTPAGNYAITDGSGNDITTASGFDKADVIKNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain 91-16 (AB029593) : SEQ ID NO:34
TIDDKLTPAGDYAITTGAGNDITTAAAYDSADVIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 91-17 (AB029594) : SEQ ID NO:35
TIDDKLTPAGDYAITTGAGNDITTAAVYDSADVIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 91-18 (AB029595) : SEQ ID NO:36
TINDKLTPAGDYAITTGNGNDIATAAMYDSADVIQNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 91-19 (AB029596) : SEQ ID NO:37
TIPENLTPAGDYSITNQINNDITTSAEYDAATIIHNNTNFKSMYICGALQRAWGDKKISNTA
FCV strain 91-2 (AB029582) : SEQ ID NO:38
TIAEELVPAGDYAITKGNGNDITTATDYDTADVIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 91-20 (AB029597) : SEQ ID NO:39
TIPEDLIPAGDYAITNGTNNDIKTAAQYDSADVIINNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain 91-22 (AB029598) : SEQ ID Nθ:40
TIPGTLTPAGDYAIVDHTSNDIATAAKYDAAFKITNNTNFKGMYICGALQRAWGDKKISNTA
FCV strain 91-25 (AB029599) : SEQ ID Nθ:41
TIPET TPAGLYAIIDQTNSDIITAAGYDAATTITNNTNFKSMFICGALQRAWGDKKISNTA
FCV strain 91-26 (AB029600) : SEQ ID NO:42
TIPERLTPAGNYSITTDNGTDIVTADLYDAADVIKNTTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 91-27 (AB029601) : SEQ ID NO:43
TIAEELTPAGDYAITNSTGIDITTPTAYDSADVIRNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 91-3 (AB029583) : SEQ ID NO:44
TIAESLIPAGDYAITTGSGNDIITAAEYDAADVIKNNTNFRGMYICGSLQRAWGDKKISDAA
FCV strain 91-31 (AB029602) : SEQ ID NO:45
TIAEELTPAGDYSITTRDGNDIKTAADYDAADVIKNTTNFRGMYICGSLQRAWGDKKISNTA
FCV strain 91-33 (AB029603) : SEQ ID NO:46
TITEELTPAGDYSITTSKGNDIVTSRDYDSADEIKNATNFRGMYICGALQRAWGDKKISNTA
FCV strain 91-34 (AB029604) : SEQ ID NO:47
TIPGELTPAGDYAITDGGNNDITTAKGYDSANIIKNNTNFRGMYICGSLQRAWGDKKISNTA FCV strain 91-35 (AB029605) : SEQ ID NO:48
TIPDKLIPAGDYAIVNQTNQYITGPKEYDSAIKITNNTNFKSMYICGALQRAWGDKKISNTA FCV strain 91-36 (AB029606) : SEQ ID NO:49
TIPEELIPAGDYAITNETDNDIATRAAYESAIEIRNNTNFKSMYICGALHRAWGDKKISNTA
FCV strain 91-37 (AB029607) : SEQ ID Nθ:50
TIPDDLVPVGNYSITDQRNNDIQTKEQYDAATAIKNTTSFRGMYICGALQRAWGDKKISNTG
FCV strain 91-38 (AB029608) : SEQ ID NO:51
TIPDDLVPVGNYSITDQRNNDIQTKEQYDAATAIKNTTSFRGMYICGALQRAWGDKKISNTG
FCV strain 91-40 (AB029609) : SEQ ID NO:52
TIPGDLIPAGDYAIVNQLNNDITSAEGYDSAIEIRNNTNFKGMYICGALQRAWGDKKISNTA
FCV strain 91-41 (AB029610.1) : SEQ ID NO:53
TIPGRLIPAGNYAITNEADNDITTPSAYDAAVAIKNNTNFRGMYICGALQRAWGDKQISATA
FCV strain 91-5 (AB029584) : SEQ ID NO:54
TIPEVITPVGDYAIVNQNNDDITTAAGYDAALSITNNTNFKSMYICGALQRAWGDKKISNTA
FCV strain 91-6 (AB029585) : SEQ ID NO:55
TIPEVITPVGDYAIVNQNQDDITTAAEYDAALSITNNTNFKSMYICGALQRAWGDKKISNTA
FCV strain 91-7 (AB029586) : SEQ ID NO:56
TIPETITPTGDYTIVDQNQSDIVTAAGYDAAHSITNNTNFKSMYICGALQRAWGDKKISNTA
FCV strain 91-9 (AB029587) : SEQ ID NO:57
TIPGELIPAGNYAITNGAGNDITTATGYDTADIIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain A4 (AF109468) : SEQ ID NO:58
TISEELTPAGDYAITTGNGCDITTASVYDSANVIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain C01 (AF260320) : SEQ ID NO:59
TIAEELIPAGDYAITDGSNNDIITANGYDAADVIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain C28 (Af260319) : SEQ ID Nθ:60
TIDGELTPAGDYAITTGAGSDIASAAEYDSADVIRNNTNFRGMYICGALQRAWGDKKISATA
FCV strain C46 (AF259956) : SEQ ID Nθ:61
TIPDELTPAGDYAITDQRGNDITTANGYDAASEIMNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain C58 (AF259394) : SEQ ID NO:62
TIGSELTPAGDYSITTGDGNDIETAAVYDAANVIRNTTNFRGMYICGSLQRAWGDKKVSNTA
FCV strain CFI-68 (FCU13992) : SEQ ID NO:63
TIPGELVPVGDYAITNGTNNDITTAAQYDAATEIRNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain Fl-76-77 (AF184951) : SEQ ID NO:64
TIPGKLTPAGDYAITTANGNDIITAAGYDAADIIVNNTNFKSMYICGSLQRAWGDKKISNTA
FCV strain F4 (P27405) : SEQ ID NO:65
TIADK IPAGDYSITTGEGNDIKTAQAYDTAAWKNTTNFRGMYICGSLQRAWGDKKISNTA
FCV strain F65 (AF109465) : SEQ ID NO:66
TIPSKLVPAGGYAITAKNGNDITTAAQYDAAGEI NNTNFKSMYICGALQRAWGDKKVSNTA
FCV strain F9 (Z11536) : SEQ ID NO:67
TIPGE IPAGDYAITNGTGNDITTATGYDTAD11KNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain fcv255 (U06646) : SEQ ID NO:68
TIPEKLVPAGHYAIANGTGNDITTGKDYDSATVIQNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain fcvLLK (U06649) : SEQ ID NO:69
TIPEELTPAGDYAITDSTGGDITTAAKYDITDKVTNNTNFKSMYICGCLQRAWGDKKISNTA
FCV strain fpl-Bolin (U06652) : SEQ ID Nθ:70
TIAEKLTPTGNYSITTNSGTDIVTPSAYDTADVIRNTTEFRGMYICGALQRAWGDKKISATA
FCV strain fpl-nσi (U06653) : SEQ ID Nθ:71
TIPEELTPEGDYAITNRSGNDIATAEGFGSADIIKNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain fri-nci (U06647) : SEQ ID NO:72
TIAEKLTPTGNYSITTNSGTDIVTPSGYDTADVIRNTTEFRGMYICGALQRAWGDKKISATA
FCV strain FS (U06650) : SEQ ID NO:73
TISEELTPAGDYAITTRIGNDITTAEAYDSADVIKNNTNFRGMYICGSLRRAWGDKKISNTA
FCV strain FT (E12464) : SEQ ID NO:74
TIADKLIPAGDYSITTGEGND KTAQAYDTAAWKNTTNFRGMYICGSLQRAWGDKKISNTA
FCV strain Gl (AX078766) : SEQ ID NO:75
T GEELTPAGDYSITNGSGNDIATANAYDSADVITNTTNFRGMYICGALQRAWGDKKISSTA
FCV strain JOK63 (AF109466) : SEQ ID NO:76
TIPEELTPAGDYSITNNANNDIATAADYDSTGVIKNTTNFRGMYTCGSLQRAWGDKKISSTA
FCV strain KCD (L09719) : SEQ ID NO:77
TISEELIPAGDYAITNDIGNDITTPAGYDAADTIKNNTNFRGMYVCGSLQRAWGDKKISNTA
FCV strain KS109 (X99446) : SEQ ID NO:78
TIPEDLVPAGDYAITNGKGNDITTATGYDTADIIRNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain KS20 (X99447) : SEQ ID NO:79
TIPEVLTPAGNYSITTGSGNDIVTAKDYDGADVIKNTTNFRGMYICGSLQRAWGDKKISNTA FCV strain KS40 (X99448) : SEQ ID Nθ:80
TISERLVPAGDYAITNRVGNDITTAEGYDAADKIKNATNFKGMYICGSLQRAWGDKKISNTA
FCV strain KS8 (X99449) : SEQ ID Nθ:81
TIPEELIPAGNYAITNSTGADITTRAEYESADEIKNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain LS012 (AF109467) : SEQ ID Nθ:82
TIPEDLVPVGNYAITTGSGNDITTASEYDSADWRNNTNFRGMYICGALQRAWGDKQISSTA
FCV strain LS015 (AF109464) : SEQ ID Nθ:83
TIPSELTPAGNYAITSGSGNDIETAAEYDSADVIRMNTNFRSMYICGALQRAWGDKKISNTA
FCV strain NADC (L09718) : SEQ ID Nθ:84
TIAEDLTPAGDYAITSGNGNDITTGSEYDSTEVIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain ϋl (AF357012) : SEQ ID NO:85
TIPEQLTPAGIYSITASNGTDITTAAGYDAAETIVNTTNFKSMYICGSLQRAWDDKKISNTA
FCV strain U2 (AY053460) : SEQ ID NO:86
TIGEKLVPAGDYAITNSSGNDITTASQYDTADIIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV unkno n-1 (AR198747) : SEQ ID NO:87
TIPEQLTPAGIYSITASNGTDITTAAGYDAAETIVNTTNFKSMYICGSLQRAWDDKKISNTA
FCV unknown-2 capsid (AF486286) : SEQ ID NO:88
TIATELIPAGDYAITTSNGNDIVTAAGYDAASEIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV unknown-2 (AR198746) : SEQ ID NO:89
TIPEQLTPAGIYSITASNGTDITTAAGYDAAETIVNTTNFKSMYICGSLQRAWGDKKISNTA
FCV unkno n-3 (AR198745) : SEQ ID Nθ:90
TIPEQLTPAGIYSITASNGTVITTAAGYDAAETIVNTTNFKSMYICGSLQRAWGDKKISNTA
FCV strain ϋrbana (L40021) : SEQ ID Nθ:91
TIGEKLVPAGDYAITNGSGNDITTANQYDAADIIRNNTNFKGMYICGSLQRAWGDKKISNTA
FCV strain V16 (AF031869) : SEQ ID NO:92
TIAERLTPGGDYAITSGNNNDITTAADYDAADIIKNNTNFRGMYICGSLQRAWGDKKISNTG
FCV strain V179 (AF031870) : SEQ ID NO:93
TISEVLTPAGGYAITTGDNNDITTAA.EYDAASVIKNNTNFRGMYICGSLQRAWGDKKISSTG
FCV strain V20 (AF031872) : SEQ ID NO:94
TIPEKLTPAGKYAITTGNGNDITTAQGFDSADVIKNDTNFRGMYICGSLQRAWGDKDISNTA
FCV strain V25 (AF038382) : SEQ ID NO:95
TIPKKLTPAGDYAITSGDGNDITTAQQYDAADVIKNNTNFKGMYVCGSLQRAWGDKKISNTA
FCV strain V274 (AF031877) : SEQ ID NO:96
TIPEKLIPAGDYAITNMGGNDITTAAAYDAADVIKNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain V276 (AF032106) : SEQ ID NO:97
TISEKLIPAGDYAITNNRGNDITTAAAYDAADVI NNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain V280891 (AF186247) : SEQ ID NO:98
TISEELIPAGDYAISNSSGNDITTPSAYDTASVINNTTNFRGMYICGSLQRAWGDKKISNTA
FCV strain V290892 (AF184594) : SEQ ID NO:99
TIPNELIPAGDYAIVNESNNDIITKSGYESATKITNNTNFKSMYICGSLQRAWGDKKISNTA
FCV strain V3 (AF038381) : SEQ ID Nθ:100
TISEELIPTGDYAITNGTGNDISTSSEYDAAEVIRNNTNFRGMYICGSLQRAWGDKKISNTA
FCV strain V310792 (AF186245) : SEQ ID Nθ:101
TIPSELTPAGDYAIVNETNNDIVTRAGYESASKITNNTNFKSMYICGSLQRAWGDKKISNTA
FCV strain V61 (AF031871) : SEQ ID Nθ:102
TISEVLTPAGDYAITTGDNNDITTAAEYDAASVIKNNTNFRGMYICGSLQRAWGDKKISSTA
FCV strain V66-97 (FCA9721) : SEQ ID Nθ:103
TIPSKLTPAGDYAITRGDGNDITTAAEYDAANTIENNTNFRSMYICGALQRAWGDKKISNTA
FCV strain V77 (AF038126) : SEQ ID Nθ:104
TIPEELTPAGKYAITTSIGNDIITAAAYDAADVIKNDTNFRGMYICGSLQRAWGDKKISNTA
FCV strain V83 (AF031876) : SEQ ID Nθ:105
TISEKLTPAGDYAITTSNGNDITTAKAYDTADVIKNNTNFRGMYICGSLQRAWGDKKISNTA
The FCV-Diva strains (FCV-Diva 15, FCV-Diva 24 and FCV-Diva 33585) are shown in Figure 1 to have unique amino acid residues at positions 32 and 35 of the truncated sequence used for the comparison. These residues correspond to positions 455 and 458 of the full-length FCV capsid protein sequence. We have determined the complete sequence of the nucleic acid molecule encoding the capsid protein of FCV- Diva 33585, and report that nucleic acid sequence as SEQ ID NO:l and the encoded polypeptide sequence as SEQ ID NO:2. Identifying an FCV-Diva strain Identification of an FCV-Diva strain can be accomplished by determining what residues occupy positions 455 and 458 of the capsid protein. This can be accomplished at the protein or nucleic acid level. . It will be appreciated that as an RNA virus, calicivirus RNA can be analyzed directly, but that it is more convenient to create a DNA copy of the RNA by reverse transcription. DNA is more stable than RNA, and the methods for determining DNA sequence information are technically simpler. Hybridization methods are also easier to perform with DNA targets, primers and probes. A preferred method of preparing nucleic acid to determine whether a particular calicivirus isolate is a Diva strain is by reverse transcriptase- polymerase chain reaction (RT-PCR). In general, feline calicivirus RNA is extracted from a biological sample using methods well known in the art, such as the Qiagen QIAmp Viral RNA kit (Qiagen, Valencia, CA). The RT-PCR reaction is performed by any one of the methods well known in the art such as the BRL One-Step RT-PCR Kit (Life Technologies, Bethesda, Md.). As described in US Patent 6,355,246, a RT-PCR primer pair flanks a target sequence from within the region of the viral genome encoding the capsid protein. For example, US Patent 6,355,246 and 6,355,246 describe primer sequences suitable for capsid gene amplification. The primers described in Example 2 are preferred. The PCR amplified products are resolved on agarose gels containing ethidium bromide and visualized under UV light. 1. Nucleotide Sequence Analysis Analysis of the nucleotide sequence encoding region E of the FCV capsid protein as described in the Example above can be accomplished using either the dideoxy chain termination method or the Maxam Gilbert method (see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., CSHP, New York (1989); Zyskind et al., Recombinant DNA Laboratory Manual; Acad. Press, (1988)). Although the sequence determination performed above was done manually, it should be recognized that the field of DNA sequencing has advanced considerably in the past several years, and that the invention contemplates such advances. Most notably, within the past decade, there has been increasing reliance on automated DNA sequence analysis.
2. Diva Strain-Specific Probes The design and use of allele-specific probes is described by e.g., Saiki et al., (Nature 324, 163-166 (1986)); Dattagupta in EP 235,726; Saiki in WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target nucleic acids from FCV-Diva strains, but which do not hybridize to the corresponding segment from other known FCV strains, due to the presence of the unique sequence found in the FCV-Diva strains. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between FCV- Diva strains and other known strains. Some probes are designed to hybridize to a segment of target nucleic acid such that the site of interest aligns with a central position (e.g., in a 15 mer at the 7 position; in a 16 mer, at either the 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridization between different allelic forms. These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising the E region of FCV-Diva capsid to hybridize thereto. The G+C content in the probes of the invention usually ranges between 10 and 75 %, preferably between 35 and 60 %, and more preferably between 40 and 55 %. The length of these probes can range from 10, 15, 20, or 30 to at least 100 nucleotides, preferably from 10 to 50, more preferably from 18 to 35 nucleotides. A particularly preferred probe is 25 nucleotides in length. Preferably the Diva alleles are within 4 nucleotides of the center of the probe. In particularly preferred probes, the Diva marker is at the center of the probe. . Shorter probes may lack specificity for a target nucleic acid sequence, and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes are expensive to produce and can sometimes self-hybridize to form hairpin structures. Methods for the synthesis of oligonucleotide probes are well known and can be applied to the probes of the present invention. Often the probes of the present invention are labeled or immobilized on a solid support. Labels and solid supports are well known in the art. Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example, peptide nucleic acids which are disclosed in International Patent Application WO 92/20702 and Morpholino analogs which are described in U.S. Patents Numbered 5,185,444; 5,034,506 and 5,142,047. The probe may have to be rendered "non-extendable" in that additional dNTPs cannot be added to the probe. In and of themselves analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3' end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group. Alternatively, the 3' hydroxyl group simply can be cleaved, replaced or modified. The probes of the present invention are useful for a number of purposes. They can be used in Southern hybridization to DNA or Northern hybridization to RNA. The probes can also be used to detect PCR amplification products. By assaying the hybridization to an allele-specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample. High-throughput parallel hybridizations in array format are specifically encompassed within "hybridization assays" and are described below. Diva-specific probes are often used in pairs, one member of a pair showing a perfect match to a Diva strain and the other member showing a perfect match to another FCV strain. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple variants of the target sequence. 3. FCV-Diva Specific Primers An FCV-Diva specific primer hybridizes to a site on a target nucleic acid overlapping a Diva specific allele and only primes amplification of an allelic form to which the primer exhibits perfect complementarily (See Gibbs, Nucleic Acid Res. 17, 2427-2448,1989). This primer is used in conjunction with a second primer that hybridizes at a distal site. Amplification proceeds from the two primers leading to a detectable product signifying the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the Diva specific site and the other of which exhibits perfect complementarily to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is included in the 3 '-most position of the oligonucleotide aligned with the site in question, because this position is most destabilizing to elongation from the primer (See, e.g., WO 93/22456). The invention of course, contemplates such primers with distal mismatches as well as primers that, because of chosen conditions, form unstable base pairing and thus prime inefficiently.
4. Denaturing Gradient Gel Electrophoresis Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. See Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co, New York, 1992), Chapter 7.
5. Single-Strand Conformation Analysis Alleles of target sequences can be differentiated using single-strand conformation analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single- stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures that are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences. Other modifications of the methods above exist, including FCV-Diva specific hybridization on filters, allele-specific PCR, PCR plus restriction enzyme digest (RFLP-PCR), denaturing capillary electrophoresis, primer extension and time-of- flight mass spectrometry, and the 5' nuclease (TAQMAN ™) assay. A method that is particularly useful in practicing the present invention is the
PCR-based TAQMAN technology (Heid et al., Genome Res. 6: 986-994 (1996)). TAQMAN is a registered trademark of Roche Molecular Systems, Inc. (Alameda, CA). Methods and apparatus for performing TAQMAN-based reactions and detecting the reaction products are available from Applied Biosystems Division, Perkin-Elmer (Foster City, CA). The TAQMAN methodology is based upon RT-PCR being carried out using an oligonucleotide probe labeled at the 5' end with an energy donor fluor, and at the 3' end with an energy quencher. The probe is complementary to a sequence within the target sequence which is generally 100 nucleotides or less from either the upstream primer or the downstream primer. During the RT-PCR reaction, if a sample contains an FCV-Diva strain, the probe is degraded by the 5' exonuclease activity of the DNA polymerase, releasing the fluor from the effect of the quencher, allowing for detection of its fluorescence. The use of dual-labeled oligonucleotide probes in PCR reactions is disclosed in U.S. Pat. No. 5,716,784 to DiCesare, which is incorporated herein by reference. In an alternative homogeneous hybridization-based procedure, molecular beacons are used for FCV-Diva specific allele discriminations. Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., Nature Biotechnology, 16:49-531 1998). Preferred techniques for FCV genotyping should allow for large scale, automated analysis. Examples of the later are DASH (Dynamic Allele-Specific hybridization) that is amenable to formatting in microtiter plates (Hybaid) and "single- stringency" DNA-chip hybridization (Affymetrix). It should be recognized of course, that this list is not inclusive. High-throughput parallel hybridizations in array format are specifically encompassed by the invention and are described below. Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants. Efficient access to FCV sequence information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (the chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime. The chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA I gene, in S. cerevisiae mutant strains, and in the protease gene of HIV- 1 virus (Hacia et al., Nature Genetics, 14(4): 441-447, 1996; Shoemaker et al., Nature Genetics,
14(4):450-456, 1996; Kozal et al., Nature Medicine, 2:753-759, 1996). Chips of various formats for use in detecting particular sequences can be produced on a customized basis by Affymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories. In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments. EP785280 describes a tiling strategy for the detection of various sequences. Briefly, arrays may generally be "tiled" for a large number of specific sequences. By "tiling" is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as pre-selected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e. nucleotides. Tiling strategies are further described in PCT application No. WO 95/11995. In a particular aspect, arrays are tiled for a number of specific, identified marker sequences. In particular the array is tiled to include a number of detection blocks, each detection block being specific for a specific sequence. For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes an area of interest. Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data from the scanned array is then analyzed to identify which allele or alleles are present in the sample. Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 and US patent No. 5,424,186. Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length. In further embodiments, the chip may comprise an array including at least one of the sequences comprising between 6-800 contiguous nucleotides of SEQ ID NO. 1 and the sequences complementary thereto, or a fragment thereof at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides, including at least one Diva-specific site. In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these nucleic acids of the invention. Other methods to determine whether an FCV strain is a Diva strain can be envisaged by one skilled in the art and include methods that include antibody-based detection methods. Such methods including ELISA are discussed below in the section relating to "Determining Whether an Animal has been Vaccinated with a Diva Strain". It will be recognized that the methods described there can easily be adapted once appropriate antisera have been obtained to the Diva strains. Identification of antibodies specific for Diva strains is discussed in the following examples. Example 3 Screening of Monoclonal Antibodies for Diva Strain Specificity Monoclonal antibodies were either obtained from commercial sources, or generated internally. Two assays were used to screen for their specificity with respect to FCV-33585 and F9. For the irnmunofluorescense assay (IFA), a stock of FCV- 33585 was used to infect a 24-well plate with NLFK cells of about 90% confluency. Approximately 20 hours post-infection, the plate was rinsed twice with lx PBS, and fixed with 80% acetone. Monoclonal antibodies were diluted to approximately 2 ug/ul and added to the plate (0.2 ml well). Following a 1 hour incubation (rocking) at RT, each well was washed twice with lx PBS. Secondary antibody (anti-mouse FITC, 10 ug/ml). After 30 min. rocking at RT, the plate covered with foil, each well was washed twice with lx PBS, and air dried. Each well was then observed under a microscope for FITC staining intensity.
Table 2. Immunofluoresence of FCV-33585 cand F9 with various monoclonal antibodies IFA Source Catalog/ID No. F9 33585 Accurate Chemical YVS7401 - - Accurate Chemical YVS7402 - - Accurate Chemical MEDCLA309 +++ - Chemicon MAB8962 - - Cortex Biochem CR1260M - - Custom Monoclonals, Int. S1-9 - + Custom Monoclonals, Int. FCV 1-43 + - Custom Monoclonals, Int. FCV8-1A + ++ Novocastra Lab NCL-1G9 +++ - In-house (Pfizer) 1-4 mAb + ++++ In-house (Pfizer) 1-12 mAb ++++ ++++ In-house (Pfizer) 3-3 mAb ++++ ++++ In- house (Pfizer) 3-5 mAb +++ ++++ In-house (Pfizer) rabbit serum ++++ +++++
The above results suggest that monoclonal antibody 1-4 mAb (Pfizer) and FCV 8-1 A (Custom Monoclonals, Int., .813 Harbor Blvd. #284, West Sacramento, CA 95091) specifically recognize epitopes present on FCV-33585 but not on F9, as very little staining was seen with F9 using these two antibodies.
Purification of FCV-33585 and F9
About 200 ml of cell culture supernatant from NLFK cells infected with FCV-33585 or F9, respectively, was centrifuged at 3,000 rpm for 30 minutes at 10°C. 25 ml of the supernatant was transferred into Beckman Ultraclear centrifuge tubes, and 10 ml of a 10% sucrose solution was underlayed into the bottom of the tube. The tubes were then centrifuged at 27,000 rpm for 2 hours at 15°C. Following centrifugation, supernatants were removed, and the pellets were resuspended in 250 ul of sterile water. The protein concentration was determined using the Micro BCA Protein Assay Kit (Pierce Chemical Co.; Rockford, IL). ELISA assay
For the ELISA assay, a 96-well ELISA plate was coated with 100 ul of purified FCV- 33585 or F9 virus at a concentration of 5 ug/ml in PBS buffer, pH 7.4. The plate was incubated (uncovered) at 37°C overnight in a non-humidified incubator. The following day, virus on the plates was fixed by applying 0.1 ml of methanol, followed by incubation at RT for 5 minutes. The plate was then washed six times with distilled water. Blocking followed by adding 200 ul of 10% of horse serum in lx PBS, and incubating overnight at 4°C. The plate was again washed six times with distilled water. Monoclonal antibodies were added to each well (100 ul/well) at a concentration of 1 ug/ml (Table 3). or diluted 1:100, 1:400, and 1:2000 in PBST (lx PBS with 0.5% Tween-20) (Table 4). Each antibody was tested in triplicate. Following incubation at 37°C for 1 hour, wells were washed 6 times with PBST. 100 ul of a 1:2000 dilution of peroxidase-conjugated AffiniPure goat anti-Mouse IgG (H+L; Jackson ImmunoResearch; cat. No.715-035- 150) was added, and the plate was incubated for 1 hour at 37°C. Wells were then washed 6 times with PBST, and lOOul of ABTS peroxidase substrate (KPL; Gaithersburg, Maryland; cat. No. 50-66-18) were added to each well. Following a 10 min incubation at RT, the plate was read at 405- 490 nm (dual wavelength) using an ELISA plate reader. Specific activity was calculated based on the signal/noise ratio. Table 3. ELISA Results for Monoclonal Antibodies reacted against FCV-33585 and F9 Viruses Specific Activity Source Catlogue/ID No. F9 33585 Accurate Chemical YVS7401 2.5 1.4 Accurate Chemical YVS7402 1 0 Accurate Chemical MEDCLA309 60 0.1 Chemicon MAB8962 1 8.6 Cortex Biochem CR1260M 1.5 7.1 Custom Monoclonals, Int. S1-9 0 5.7 Custom Monoclonals, Int. FCV 1-43 1 4.3 Custom Monoclonals, Int. FCV8-1A 5 20 Maine Biotech MAB790P 1 5.7 Maine Biotech MAB791 P 1.5 1 Novocastra Lab NCL-1G9 20 0 In-house (Pfizer) 1 -4 mAb 4 14.3 In-house (Pfizer) 1-12 mAb 80 8.6 In-house (Pfizer) 3-3 mAb 115 101.4 In-house (Pfizer) 3-5 mAb 80 102.9 In-house (Pfizer) rabbit serum 185 145.7
Table 4. ELISA Results for Monoclonal Antibodies reacted against FCV-33585 and F9 Viruses Specific Activity Source Catlogue/ID No. F9 33585 1:100 1:400 1:2000 1:100 1:400 1:2000 Accurate Chemical MEDCLA309 13 13 12 0.8 0.8 0.9 Chemicon MAB8962 1 1 ND 5 5 ND Custom Monoclonal S1-9 1 1 ND 3 4 ND Custom Monoclonal FCV 1-43 1 1 ND 2 1 ND Custom Monoclonal FCV8-1A 3 3.5 2 17 16 7 Novocastra Lab NCL-1G9 15 22 16 0.8 0.8 0.8 In-house (Pfizer) 1-4 mAb 1 1 1 7 10 8 In-house (Pfizer) 1-12 mAb 10 15 20 6 8.5 4 In-house (Pfizer) 3-3 mAb 11 16.5 25 20 28 38 In-house (Pfizer) 3-5 mAb 10.5 13 17 17 26 31 In-house (Pfizer) rabbit serum 6.5 8 4 5 7 6 Buffer 1 1 1 1 1 1
As was seen in the IFA, these results (Tables 3 and 4) suggest that monoclonal antibody 1-4 mAb (Pfizer) and FCV 8-1 A (Custom Monoclonals, Int.) specifically recognize epitopes present on FCV-33585 but not on F9. Thus, FCV-33585 and F9, a commonly used vaccine strain, have immunologically distinct epitopes. The antibodies described here therefore can be used to distinguish a Diva strain from other FCV strains such as F9. The hybridoma producing the antibody we have designated "1-4 mAb" has been deposited with the American Type Culture Collection, 10801 University Blvd, Manassass, VA, 20110-2209 and was given the ATCC accession number PTA-6312.
Vaccines of the Invention Example 4 Use of FCV-Diva 33585 as an Immunogen
Groups of test animals were challenged with FCV-Diva 33585 and samples of serum from the test animals was tested after infection to see if the serum was capable of neutralizing a panel of isolates often FCV strains (F9, 33585, J-1, 2280, 255NVSL, 100869-1, 89391, 88287, H and 94580). It is surprising that significant viral neutralization titers were present in the majority of samples analyzed, demonstrating that the challenge strain, 33585, is likely a good vaccine strain. The results are outlined in Table 5. Table 5 Neutralization Titers After Challenge with FCV-Diva 33585 F-9 33585 J-1 . 2280 255NVSL 100869-1 89391 88287 H 94580 2896 >289S 256 <12 64 181 23 45 16 64
>5792 >2895 181 <12 >2895 2048 45 32 362 1448
>5792 >2895 >2895 45 >2895 128 45 45 128 724
These results suggest that FCV Diva isolates are useful for developing efficacious vaccines for immunizing cats against disease caused by heterologous feline calicivirus strains. Therefore, the present invention provides vaccines that are based upon live or killed FCV-Diva strains, nucleic acid vaccines encoding for an FCV-Diva capsid protein or a specific immunogenic fragments thereof, and isolated capsid protein or a specific immunogenic fragment thereof. The vaccine of the present invention is generally intended to be a prophylactic treatment which immunizes cats against disease caused by virulent strains of feline calicivirus. However, the vaccine is also intended for the therapeutic treatment of cats already infected with a virulent strain of feline calicivirus. For example, a vaccine comprising antibodies produced by immunizing a heterologous host with FCV-Diva, or immunogenic component thereof, is used for the therapeutic treatment of a feline calicivirus-infected cat. However, even vaccines that provide active immunity, i.e., vaccines comprising FCV-Diva or mutants thereof, or a specific immunogenic fragments thereof, would be expected to be effective when given as a therapeutic treatment against various diseases. Thus, the immunity that is provided by the present invention can be either active immunity or passive immunity and the intended use of the vaccine can be either prophylactic or therapeutic. The route of administration for any one of the embodiments of the vaccine of the present invention includes, but is not limited to, oronasal, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral as well as transdermal or by inhalation or suppository. The preferred routes of administration include oronasal, intramuscular, intraperitoneal, intradermal, and subcutaneous injection. The vaccine can be administered by any means that includes, but is not limited to, syringes, nebulizers, misters, needleless injection devices, or microprojectile bombardment gene guns (Biolistic bombardment). The vaccine for any one of the embodiments of the present invention is formulated in a pharmaceutically accepted carrier according to the mode of administration to be used. One skilled in the art can readily formulate a vaccine that comprises a live or killed FCV-Diva, an FCV-Diva capsid protein or an a specific immunogenic fragment thereof, a recombinant virus vector encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof, or a DNA molecule encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof. In cases where intramuscular injection is preferred, an isotonic formulation is preferred. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In particular cases, isotonic solutions such as phosphate buffered saline are preferred. The formulations can further provide stabilizers such as gelatin and albumin. In some embodiments, a vaso-constrictive agent is added to the formulation. The pharmaceutical preparations according to the present invention are provided sterile and pyrogen free. However, it is well known by those skilled in the art that the preferred formulations for the pharmaceutically accepted carrier which comprise the vaccines of the present invention are those pharmaceutical carriers approved in the regulations promulgated by the United States Department of Agriculture, or equivalent government agency in a foreign country such as Canada or Mexico or any one of the European nations, for live feline calicivirus vaccines, killed feline calicivirus vaccines, polypeptide (antigen) subunit vaccines, recombinant virus vector vaccines, antibody vaccines, and DNA vaccines. Therefore, the pharmaceutically accepted carrier for commercial production of the vaccine of the present invention is a carrier that is already approved or will be approved by the appropriate government agency in the United States of America or foreign country. The vaccine can further be mixed with an adjuvant that is pharmaceutically acceptable. In certain formulations of the vaccine of the present invention, the vaccine is combined with other feline vaccines to produce a polyvalent vaccine product that can protect cats against a wide variety of diseases caused by other feline pathogens. Currently, commercial manufacturers of feline vaccines, as well as end users prefer polyvalent vaccine products. Therefore, in a preferred embodiment, the present invention provides a polyvalent vaccine which immunizes cats against feline calicivirus and at least one other feline pathogen, preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus. Inoculation of a cat is preferably by a single vaccination that produces a full, broad immunogenic response. In another embodiment of the present invention, the cat is subjected to a series of vaccinations to produce a full, broad immune response. When the vaccinations are provided in a series, the vaccinations can be provided between about one day to four weeks or longer apart. In particular embodiments, the cat is vaccinated at different sites simultaneously. Live Vaccines In one embodiment of the vaccine of the present invention, the vaccine comprises a live vaccine comprising FCV-Diva. It is well known in the art that a calicivirus strain which is virulent when administered to a cat by the oronasal route, the natural route of infection, is generally apathogenic when administered by another route such as the intraperitoneal, intramuscular, intradermal, or subcutaneous routes. However, in certain cats, e.g., immunocompromised cats, inoculated by these other routes, the virus causes disease. Therefore, it is desirable that particular live vaccines comprise an attenuated or mutated variant of FCV-Diva, which stimulates the cat's immune system without causing disease. Methods for making attenuated viruses are well known in the art and include such methods as serial passage in cell culture on a suitable cell line or ultraviolet or chemical mutagenesis. Another method of producing an attenuated strain is to produce a temperature sensitive variant and is illustrated below. Example 5 Generation of a Temperature-Sensitive FCV-Diva 33585 Clone
Ultraviolet light exposure:
Starting material was derived from FCV-33585 that had been passaged twenty times in Fc3Tg cells (feline tongue, ATCC CCL-176). A 250 microliter aliquot of virus- containing supernatant was spotted onto a culture dish and spread out to approximately 25 millimeter diameter. The virus-containing dish was placed 8.5 inches from the germicidal ultraviolet lamp in a laminar flow biosafety hood. The ultraviolet lamp was turned on for a total of 30-50 seconds (35 and 45 seconds in two separate experiments) in 10 second intervals. The supernatant was mixed by gentle pipetting between each exposure. Post UV exposure processing:
The UV light-treated virus-containing supernatant was directly plaqued on 24 hr old Fc3Tg cell monolayers, as were 1 ml dilutions of the supernatant. All were then allowed to incubate at 30°C for 90-120 minutes. Agarose overlays were applied to the monolayers and the plates incubated at 30°C for 3-4 days to allow for plaque development for the purpose of enumeration. The end result was a reduction in virus titers of approximately 3 logs. Selection of Temperature Sensitive Variants
Plaques were picked using a 1ml micropipette tip to transfer an agarose plug to 250 microliters of liquid culture media. The suspended agarose plug was stored at -80°C. The titer of virus in each picked plaque was determined by endpoint titration on Fc3Tg cells at both 30°C and 39°C. Plaques that contained virus which had significantly higher titers at 30°C compared to 39°C were used to prepare virus stocks. Results One of the selected temperature sensitive variants which we have designated FCV33585 TS25 was characterized further. This variant of FCV-33585 shows reduced viral replication at 39°C compared to 30°C as demonstrated by Table 6 Table 6
Figure imgf000029_0001
The parental virus, which had been passaged 20 times at 30°C in Fc3Tg cells, does not show reduced replication at 37°C compared to 30°C. Further, after UV light treatment the vast majority of plaques yield similar titers at both 30°C and 39°C. Example 6 Attenuation of FCV-Diva Strain 33585 Serial dilutions of FCV-Diva 33585 (10"2 to 10"7) were prepared and 0.1 ml of each dilution was added to monolayers of Fc3Tg and NLFK cells in 6-well plates. After 3-4 days incubation at 30°C, wells were examined under a microscope for CPE (cytopathic effect). Supernatants from the wells with the highest dilution and obvious CPE were harvested and used to re-inoculate wells containing fresh monolayers of cells for further passaging. At passages 7 (p7) and 17 ( i 7) for both Fc3Tg and NLFK cells, and later for p44 from NLFK and p46 from Fc3Tg cells, virus was plaque purified twice, followed by a single limited dilution plating in a 96-well plate. To assess the virulence phenotype of the passaged viruses, 1 ml of 106 TCIDso/ml of each purified virus was inoculated oronasally into cats (5/group). Both plO and p20 virus from Fc3Tg as well as NLFK cells were shown to retain a level of virulence equal to that of the parental virus, and resulted in death in all cats within 4 days of inoculation. On the contrary, inoculation with p47 (NLFK) and p49 (Fc3Tg) virus resulted in only one death. 13 out of 19 inoculated cats showed symptoms of lameness, depression, sneezing and nasal discharge, but all recovered and displayed few clinical symptoms afterwards. Surviving cats were re-inoculated with 106 TCIDso/ml of virus, and serum samples from these cats will be analyzed for cross- neutralization patterns in comparison with the virulent parent virus. Meanwhile, virus is being further passaged, and will be monitored for virulence and cross-neutralization patterns. As another example, attenuated variants of FCV-Diva are made by serial passage of the virus on a cell line such as Crandall-Reese Feline Kidney (CRFK) cells for between about 10 and 100 passages. During passage on the cell line, the virus loses its ability to cause disease in the cat, e.g., becomes avirulent or non-pathogenic, while maintaining its ability to replicate in the cat and produce a protective immune response. Therefore, to make the vaccine, feline calicivirus isolate FCV-Diva, or an attenuated or mutated variant thereof, is grown in cell culture on a suitable feline cell line, i.e., CRFK cells, to titers sufficient for producing a vaccine. The calicivirus is harvested according to methods well known in the art. The calicivirus is then concentrated, frozen, and stored at -70°C, or freeze-dried and stored at 4°C. Prior to vaccination the calicivirus is mixed to an appropriate dosage, which is from about 103 to 108 tissue culture infectious doses per ml (TC-D5o/ml)) with a pharmaceutically acceptable carrier, such as a saline solution and/or an optionally adjuvant, such as aluminum hydroxide. The live vaccine can further include, in addition to the FCV- Diva strain, at least one other feline calicivirus strain, preferably selected from the group consisting of FCV-F9, FCV-M8, FCV-255and FCV-2280. In a preferred embodiment, the vaccine further includes a vaccine for immunizing a cat against at least one or more other feline pathogens, preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica. Example 7 Use of attenuated FCV-Diva 33585 as an Immnunogen Both the temperature sensitive variant FCV-33585 TS25 and passage 61 (NLFK cells) were purified three times as described above. Groups of 4-5 cats were inoculated intranasally (IN) as well as subcutaneously (SQ) with 1 ml of 106 TCIDso/ml per cat. One group (TS25, H) was also inoculated with a 10-fold higher dose of TS25 (2xl07 TCIDso/ml cat). Minimal clinical signs were observed for all the groups, and were comparable to those obtained with the widely used vaccine strain, F9. All cats were re-inoculated with 106 TCIDso/ml of TS25 or p61. For the group that received 2xl07 TCIDso/ml of the TS25 virus initially (TS25, H), 5xl06 TCIDso/ml was used for the re-inoculation. Serum samples from each of the vaccinated cats were used in the serum neutralization assay against each of 26 FCV viral strains (F9, J-1, H, 2280, 255NVSL, 94580, 100869-1, 33585, 88287, 89391, 101920-1, 122217, 17932-17, 19306, 26391- 4, 27086, 30101-2, 32561-1, 32561-14, 32561-15, 32561-7, 36069-2, 41927-8, 84883-02, 21 and 49). Serum neutralization data were analyzed with cut-off titers of >23 and >15. Table 7A. Neutralization titers for Attenuated FCV-Diva 33585 following IN vaccination. % Positive % Positive (Cut-off % Increase (Cut-off % Increase Strain Titer >23) (vs. F9 Titer) Titer >15) (vs. F9 Titer) 33585 TS25 26 0 37 16 p47 N 27 4 31 p49 F 22 29 p61 N 15 25 F9 26 32
Table 7B. Neutralization titers for Attenuated FCV-Diva 33585 following SQ vaccination. % Positive % Positive (Cut-off % Increase (Cut-off % Increase Strain Titer >23) (vs. F9 Titer) Titer >15) (vs. F9 Titer) 33585 (p61) 17 6 23 35 TS25 30 88 35 106 TS25 H 30 88 39 129 F9 16 17
Tables 7A-7B) contains the serum neutralization data for serum samples obtained from cats infected by the intranasal (IN) and subcutaneous (SQ) routes of inoculation. When compared to F9, various passages of attenuated 33585 (p61, p47 NLFK, p49 Fc3Tg, and TS-25) did not provide broader cross neutralization (Table 7 A) .Furthermore, the cross neutralization coverage of passage 61 virus is less than that of lower passages of 33585 (p47, p49 and TS25). However, when virus was inoculated subcutaneously (SQ) the cross neutralization profile for TS25, as well as for TS25 H, was significantly improved that obtained with F9 (Table 7B). For TS25 , using the coverage titers of >23 and >15, there is an increase of 88% and 106%, respectively, over the coverage obtained with F9. Similar results are seen when the neutralization data follow SQ vaccination is reported either as the arithmetic mean (Table 7C) or geometric mean (Tables 7D) of titers for each group.
Table 7C. Neutralization titers for Attenuated FCV-Diva 33585 following SQ vaccination (Arithmetic means). % Positive % Positive (Cut-off % Increase (Cut-off % Increase Strain Titer >23) (vs. F9 Titer) Titer >15) (vs. F9 Titer) 33585 (p61) 19 27 27 17 TS25 42 180 46 100 TS25, H 39 160 46 100 F9 15 23
Table 7D. Neutralization titers for Attenuated FCV-Diva 33585 following SQ vaccination (Geometic means). % Positive % Positive (Cut-off % Increase (Cut-off % Increase Strain Titer >23) (vs. F9 Titer) Titer >15) (vs. F9 Titer) 33585 (p61) 12 19 27 TS25 27 80 39 160 TS25, H 31 107 35 133 F9 15 15
We conclude that the low passaged and attenuated FCV-33585 TS25, when administered sub-cutaneously, offers superior cross neutralization coverage compared with the widely used vaccine strain F9.
Inactivated Vaccines In another embodiment of the present invention, the vaccine comprises an inactivated or killed vaccine comprising FCV-Diva. The inactivated vaccine is made by methods well known in the art. For example, once the virus is propagated to high titers, it would be readily apparent by those skilled in the art that the virus antigenic mass could be obtained by methods well known in the art. For example, the virus antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate viral antigenic mass to produce vaccines. The calicivirus is inactivated by treatment with formalin, betapropriolactone (BPL), or with binary ethyleneimine (BEI), or other methods known to those skilled in the art. Inactivation by formalin is performed by mixing the calicivirus suspension with 37% formaldehyde to a final formaldehyde concentration of 0.05%. The calicivirus-formaldehyde mixture is mixed by constant stirring for approximately 24 hours at room temperature. The inactivated calicivirus mixture is then tested for residual live virus by assaying for growth on a suitable feline cell line such as CRFK cells. Inactivation by BEI is performed by mixing the calicivirus suspension of the present invention with 0.1 M BEI (2-bromo-ethylamine in 0.175 N NaOH) to a final BEI concentration of 1 mM. The calicivirus-BEI mixture is mixed by constant stirring for approximately 48 hours at room temperature, followed by the addition of 1.0 M sodium thiosulfate to a final concentration of 0.1 mM. Mixing is continued for an additional two hours. The inactivated calicivirus mixture is tested for residual live calicivirus by assaying for growth on a suitable feline cell line such as NLFK cells. The aforementioned inactivated calicivirus of the present invention is mixed with any one of the pharmaceutically carriers for formulating inactivated virus vaccines to the appropriate dosage level. The inactivated vaccine further can include, in addition to FCV-Diva, atleast one other feline calicivirus strain, preferably selected from the group consisting of FCV-F9, FCV-M8, FCV-255, and FCV-2280. In a preferred embodiment, the vaccine further includes a vaccine for immunizing a cat against one or more other feline pathogens, preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus. Recombinant Vaccines In a further embodiment of the present invention, the vaccine comprises a recombinant virus vector containing a nucleic acid encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof, comprising the amino acid sequence of SEQ ID NO:2, or comprising the nucleotide sequence selected from SEQ ID NO:l. hi one particular embodiment, the recombinant virus vector is a feline herpesvirus that immunizes a cat against both feline calicivirus and feline herpesvirus. Li another embodiment, the recombinant virus vector comprises one or more antigens preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica. To make a recombinant virus vector that expresses the FCV-Diva capsid protein or a specific immunogenic fragment thereof, a cDNA encoding the capsid protein or a specific immunogenic fragment thereof is inserted into the genome of a virus vector such as herpesvirus, poxvirus, or adenovirus. U. S. Pat. No. 5,716,822 to Wardley et al. describes a method for inserting DNA encoding the feline calicivirus strain CF1768 FJV capsid protein into the feline herpesvirus thymidine kinase gene. Other recombinant virus vector vaccines embraced by the present invention, include but are not limited to, adenovirus, adeno-associated virus, parvovirus, and various poxvirus vectors to express the FCV-Diva capsid protein or a specific immunogenic fragment thereof. In particular, the present invention includes recombinant poxvirus vector vaccines that express the FCV-Diva capsid protein or a specific immunogenic fragment thereof made according to the methods taught in any one of U.S. Pat. Nos. 5,338,683 and 5,494,807 to Paoletti et al., which teach recombinant virus vaccines consisting of either vaccinia virus or canary poxvirus expressing foreign antigens; U.S. Pat. No. 5,266,313 to Esposito et al., which teaches recombinant raccoon poxvirus vectors expressing rabies virus antigens; and U.S. Pat. No. 6, 010,703 to Maes et al., which teaches recombinant racoon poxvirus vectors that express the feline herpesvirus gD or gB antigens. For any of the aforementioned recombinant virus vectors, the cDNA encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof is operably linked to a eukaryote promoter at the 5' end of the cDNA encoding the antigen and a eukaryote termination signal and poly(A) signal at the 3' end of the cDNA encoding the antigen. As used herein, the term "operably linked" means that the nucleic acids of the present invention (as a cDNA molecule) and a nucleic acid (DNA) containing an expression control sequence, e.g., transcription promoter and termination sequences, are situated in a vector or cell such that expression of the antigen encoded by the cDNA is regulated by the expression control sequence. Methods for cloning DNA such as the cDNA encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof and operably linking DNA containing expression control sequences thereto are well known in the art. Examples of promoters suitable for expressing the FCV- Diva capsid protein or a specific immunogenic fragment thereof in the recombinant virus vectors are the cytomegalovirus immediate-early (CMV) promoter, the Rous sarcoma virus long terminal repeat (RSN-LTR) promoter, the simian virus 40 (SN40) immediate-early promoter, and inducible promoters such as the metallothionein promoter. An example of a DΝA having a termination and poly(A) signal is the SN40 late poly(A) region. Another example of a viral expression system suitable for producing the antigen is the Sindbis Expression system available from Invitrogen. The use of these commercially available expression vectors and systems are well known in the art. In an embodiment further still of the present invention, the vaccine is provided as a nucleic acid or DΝA molecule vaccine that elicits an active immune response in the cat. The DΝA molecule vaccine consists of DΝA having a nucleic acid sequence which encodes the capsid protein or a specific immunogenic fragment thereof of FCN- Diva. In a preferred embodiment, the DΝA molecule vaccine comprises the nucleic acid sequence of SEQ ID NO: 1 , or a fragment thereof encoding a specific immunogenic fragment of SEQ ID NO:2. The nucleic acid encoding the capsid protein or a specific immunogenic fragment thereof is operably linked at or near a transcriptional promoter. This enables transcription of the capsid protein, or a specific immunogenic fragment thereof, from the nucleic acid when the nucleic acid is inoculated into the cells of the cat. Preferably, the DNA molecule is a plasmid.
Promoters that are useful for DNA vaccines are well known in the art and include, but are not limited to, the RSV LTR promoter, the CMV immediate early promoter, and the SV40 T antigen promoter. It is further preferred that the nucleic acid be operably linked at or near the termination codon of the sequence encoding the capsid protein or a specific immunogenic fragment thereof to a nucleic acid fragment comprising a transcription termination signal and poly(A) recognition signal. The DNA vaccine is provided to the cat in an accepted pharmaceutical carrier or in a lipid or liposome carrier similar to those disclosed in U.S. Pat. No. 5,703,055 to Feigner. The DNA vaccine can be provided to the cat by a variety of methods such as intramuscular injection, intrajet injection, or biolistic bombardment. Making DNA vaccines and methods for their use are provided in U.S. Pat. Nos. 5,589, 466 and 5,580,859, both to Feigner. Finally, a method for producing pharmaceutical grade plasmid DNA is taught in U.S. Pat. No. 5,561,064 to Marquet et al. Therefore, using the abovementioned methods, DNA vaccines that express the FCV-Diva capsid protein or a specific immunogenic fragment thereof are used to immunize cats against virulent feline calicivirus. The advantage of the DNA vaccine is that the DNA molecule is conveniently propagated as a plasmid which is a simple and inexpensive means for producing a vaccine, and since the vaccine is not live, many of the regulatory issues associated with live recombinant virus vector vaccines are not an issue with DNA vaccines. One skilled in the art would appreciate that the DNA vaccine of the present invention can comprise synthetically produced nucleic acids that are made by chemical synthesis methods well known in the art. In an embodiment further still of the present invention, the vaccine consists of the isolated and purified FCV-Diva capsid protein or a specific immunogenic fragment thereof. In particular, a vaccine wherein the FCV-Diva capsid protein or a specific immunogenic fragment thereof comprises the amino acid sequence of SEQ ID NO:2. Preferably, the capsid protein or a specific immunogenic fragment thereof is produced in a recombinant bacterium or eukaryote expression vector that produces the antigen that can be isolated and purified to make the vaccine. For example, the FCV- Diva capsid protein or a specific immunogenic fragment thereof is produced in a microorganism such as bacteria, yeast, or fungi, in a eukaryote cell such as a mammalian or an insect cell, or via a recombinant virus vector such as adenovirus, poxvirus, herpesvirus, Semlicki Forest virus, baculovirus, bacteriophage, Sindbis virus, or Sendai virus. Suitable bacteria for producing the FCV-Diva capsid protein or a specific immunogenic fragment thereof include Escherichia coli, Bacillus subtϊlis, or any other bacterium that is capable of expressing heterologous polypeptides. Suitable yeast types for expressing the FCV-Diva capsid protein or a specific immunogenic fragment thereof include Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Candida, or any other yeast capable of expressing heterologous polypeptides. Methods for using the aforementioned bacteria, eukaryotic cells or recombinant virus vectors to produce antigens for vaccines are well known in the art. To produce the vaccine consisting of the capsid protein or a specific immunogenic fragment thereof, the nucleic acid encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof is in a plasmid, and the nucleic acid is operably linked to a promoter which effects the expression of the capsid protein or a specific immunogenic fragment thereof in a microorganism. Suitable promoters include, but are not limited to, T7 phage promoter, T3 phage promoter, β- galactosidase promoter, and the Sp6 phage promoter. Expression of the FCV-Diva capsid protein or a specific immunogenic fragment thereof in a microorganism enables the capsid protein to be produced using fermentation technologies that are used commercially for producing large quantities of recombinant antigenic polypeptides. Methods for isolating and purifying antigens are well known in the art and include methods such as gel filtration, affinity chromatography, ion exchange chromatography, or centrifugation. To facilitate isolation of the FCV-Diva capsid protein or a specific immunogenic fragment thereof, a fusion polypeptide is made wherein the capsid protein or a specific immunogenic fragment thereof is linked to another polypeptide which enables isolation by affinity chromatography. Preferably, a fusion polypeptide is made using one of the expression systems infra. For example, the cDNA nucleic acid sequence encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof is linked at either the 5' end or 3' end to a nucleic acid encoding a polypeptide. The nucleic acids are linked in the proper codon reading frame to enable production of a fusion polypeptide wherein the amino and/or carboxyl terminus of the capsid protein or portion thereof is fused to a polypeptide which allows for the simplified recovery of the antigen as a fusion polypeptide. The fusion polypeptide can also prevent the antigen from being degraded during purification. While a vaccine comprising the fusion polypeptide is efficacious, in some instances it can be desirable to remove the second polypeptide after purification. Therefore, it is also contemplated that the fusion polypeptide contains a cleavage site at the junction between the antigen and the polypeptide. The cleavage site consists of an amino acid sequence that is cleaved with an enzyme specific for the amino acid sequence at the site. Examples of such cleavage sites that are contemplated include the enterokinase cleavage site which is cleaved by enterokinase, the factor Xa cleavage site which is cleaved by factor Xa, and the GENENASE cleavage site which is cleaved by GENENASE (GENENASE is a trademark of New England Biolabs, Beverly, Mass.). The following are methods for producing the capsid protein or a specific immunogenic fragment thereof as a fusion polypeptide or as an isolated antigen free of the polypeptide. An example of a procaryote expression system for producing the FCV-Diva capsid protein or a specific immunogenic fragment thereof as a fusion polypeptide for use in vaccines is the Glutathione S-transferase (GST) Gene Fusion System available from Amersham Pharmacia Biotech, Piscataway, N.J., which uses the pGEX-4T-l expression vector plasmid. The cDNA encoding the capsid protein or a specific immunogenic fragment thereof is fused in the proper codon reading frame with the DNA encoding GST. The GST part of the fusion polypeptide allows the rapid purification of the fusion polypeptide using glutathione Sepharose 4B affinity chromatography. After purification, the GST portion of the fusion polypeptide can be removed by cleavage with a site-specific protease such as thrombin or factor Xa to produce an antigen free of the GST polypeptide. The capsid protein or a specific immunogenic fragment thereof, free of the GST polypeptide, is produced by a second round of glutathione Sepharose 4B affinity chromatography. Another method for producing a vaccine comprising the FCV-Diva capsid protein or a specific immunogenic fragment thereof is a method which links in-frame the cDNA encoding the antigen and DNA codons that encode polyhistidine. The polyhistidine preferably comprises six histidine residues which allows purification of the fusion polypeptide by metal affinity chromatography, preferably nickel affinity chromatography. To produce the capsid protein or a specific immunogenic fragment thereof free of the polyhistidine, a cleavage site such as an enterokinase cleavage site is fused in the proper reading frame between the codons encoding the polyhistidine and the codons encoding the antigen. The antigen, free of the polyhistidine, is made by removing the polyhistidine by cleavage with enterokinase followed by a second round of metal affinity chromatography which binds the free polyhistidine. This method was shown to be useful for preparing the LcrV antigen of Y. pestis which was disclosed in Motin et al. Infect, hnmun. 64: 4313-4318 (1996). The Xpress System, available from Invitrogen, Carlsbad, California, is an example of a commercial kit that is available for making and then isolating polyhistidine- polypeptide fusion protein. A method further still for producing a vaccine comprising the FCV-Diva capsid protein or a specific immunogenic fragment thereof uses a method disclosed by Motin et al., Infect, hnmun. 64: 3021-3029 (1995). Motin et al. disclosed a DNA encoding a fusion polypeptide consisting of the DNA encoding an antigen linked to DNA encoding a portion of protein A wherein DNA encoding an enterokinase cleavage site is interposed in the proper codon reading frame between the DNA encoding protein A and the antigen. The protein A enables the fusion polypeptide to be isolated by IgG affinity chromatography, and the capsid protein free of the protein A is produced by cleavage with enterokinase. The protein A is then removed by a second round of IgG affinity chromatography. Another method for producing a vaccine comprising the FCV-Diva capsid protein or a specific immunogenic fragment thereof is based on methods disclosed in U.S. Pat. No. 5,725,863 to Daniels et al. The Daniels et al. method can be used to make the FCV-Diva vaccine which consists of enterotoxin molecule wherein each molecule has inserted therein upwards of 100 amino acid residues of the FCV-Diva capsid protein. Other methods for making fusion polypeptide vaccines which can be used to make the vaccines of the present invention is disclosed in U.S. Pat. No. 5,585, 100 to Mond et al. and U.S. Pat. No. 5,589,384 to Liscombe. Finally, the pMAL Fusion and Purification System available from New England Biolabs is another example of a method for making a fusion polypeptide wherein a maltose binding protein is fused to the capsid protein or a specific immunogenic fragment thereof. The maltose binding protein facilitates isolation of the fusion polypeptide by amylose affinity chromatography. The maltose binding protein can be linked to the antigen by one of the above mentioned cleavage sites which enables the antigen to be made free of the maltose binding protein. While bacterial methods are used to produce the FCV-Diva capsid protein or a specific immunogenic fragment thereof for vaccines, it can be desirable to produce the capsid protein or a specific immunogenic fragment thereof in a eukaryote expression system. A particularly useful system is the baculovirus expression system that is disclosed in U.S. Pat. No. 5,229, 293 to Matsuura et al.. Baculovirus expression vectors suitable to produce the capsid protein or a specific immunogenic fragment thereof are the pPbac and pMbac vectors from Stratagene; and the Bac-N-Blue vector, the pBlueBac4.5 vector, pBlueBacHis2-A,B,C, and the pMelBac available from Invitrogen, Carlsbad, Calif. Another eukaryote system useful for expressing the FCV-Diva capsid protein or a specific immunogenic fragment thereof for vaccines is a yeast expression system such as the ESP Yeast Protein Expression and Purification System available from Stratagene. Another yeast expression system is any one of the Pichia-based Expression systems from Invitrogen. Mammalian expression systems are also embraced by the present invention. Examples of mammalian expression systems are the LacSwitch II system, the pBK Phagemid, pXTl vector system, and the pSG5 vector system from Stratagene; the pTargeT mammalian expression vector system, the pSI mammalian expression vector, pCI mammalian expression vector, and pAdVantage vectors available from Promega Corporation, Madison, Wis.; and the Ecdysone-Inducible Mammalian Expression System, pCDM8, pcDNAl.l, and pcDNAl.l/Amp available from Invitrogen. The present invention further includes an embodiment consisting of vaccines that comprise the FCV-Diva capsid protein or particular epitopes of the capsid protein as components of a heat-stable spore delivery system made according to the method taught in U.S. Pat. No. 5, 800,821 to Acheson et al. Therefore, the present invention provides a genetically engineered bacterial cell containing a nucleic acid encoding the FCV-Diva capsid protein or a specific immunogenic fragment thereof. When the recombinant bacterial spore vaccine is orally administered to the cat, the spores germinate in the gastrointestinal tract of the cat and the bacteria expresses the capsid protein or a specific immunogenic fragment thereof which comes into contact with the cat's immune system and elicits an immune response. The vaccine has the advantage of being heat stable; therefore, it can be stored at room temperature for an indefinite period of time.
Passive Immunity Vaccines While the above embodiments of the present invention provide active immunity against feline calicivirus, the present invention further comprises vaccines that provide passive immunity to feline calicivirus. A vaccine that elicits passive immunity against feline calicivirus consists of polyclonal antibodies or monoclonal antibodies that are against the FCV-Diva capsid protein, a specific immunogenic fragment thereof, or the whole FCV-Diva virus. To make a passive immunity vaccine comprising polyclonal antibodies, the FCV-Diva, capsid protein thereof, or a specific immunogenic fragment thereof is injected into a suitable host for preparing the antibodies, preferably the host is a horse, swine, rabbit, sheep, or goat. Methods for producing polyclonal antibody vaccines from these hosts are well known in the art. By way of example, the capsid protein or a specific immunogenic fragment thereof or whole calicivirus FCV-Diva is mixed with an adjuvant such as Freund's complete or the less toxic TiterMax available from CytRx Corp., Norcross, Ga., which then administered to the host by methods well known in the art. Antibody production is monitored and when sufficient antibody has been produced, the serum is removed from the host and preferably;the antibody is recovered from the serum. The passive immunity vaccine can comprise one or more monoclonal antibodies against one or more epitopes of the FCV-Diva capsid protein or whole virus FCV-Diva, or a specific immunogenic fragment thereof. Methods and hybridomas for producing monoclonal antibodies are well known in the art. While monoclonal antibodies can be made using hybridoma technologies well known in the art, the monoclonal antibodies against the antigen can also be made according to phage display methods such as that disclosed in U.S. Pat. No. 5,977,322 to Marks et al. Felinized antibodies against the capsid protein or portion thereof can be made according to methods which have been used for humanizing antibodies such as those disclosed in U.S. Pat. Nos. 5,693,762 and 5,693,761 both to Queen et al. A phage display kit that is useful for making monoclonal antibodies is the Recombinant Phage Antibody System available from Amersham Pharmacia Biotech. Methods to Determine Whether an Animal has been Vaccinated with a Diva stain An important aspect of any vaccination program is to be able to distinguish animals vaccinated against a disease from animals infected with the disease.
Therefore, the present invention further includes methods or assays that distinguish cats vaccinated with the vaccine of the present invention from cats infected with a field strain of feline calicivirus, cats vaccinated with another commercial vaccine preparation, or cats never exposed to feline calicivirus. In general, the assays are either the immunologically based antibody-capture or an antigen-capture assays. In either type of assay a biological sample from the cat is tested for the presence of antibodies against FCV-Diva and optionally for the presence of antibodies against other feline calicivirus strains. The presence of antibodies against FCV-Diva indicates the cat was vaccinated with the vaccine of the present invention. Conversely, the presence of antibodies in a biological sample against a feline calicivirus other than the FCV-Diva indicates the cat is infected with a feline calicivirus other than FCV- Diva. The assay can further include controls that comprise capsid proteins from other calicivirus strains such as the F-9 strain. Thus, in these assays, a serum sample from a cat that had been vaccinated with the vaccine of the present invention will react with FCV-Diva antigens whereas a sample from a cat that is infected with, or exposed to, another feline calicivirus will not contain antibodies against the capsid protein of the FCV-Diva. In general, the antibody-detection or antigen detection assays are performed using an enzyme-linked immunosorbent assay (ELISA) embodiment. For an antibody-capture ELISA, a microtiter plate is provided containing a plurality of wells wherein a first well or series of wells contains the capsid protein of the FCV-Diva immobilized to the surface therein and a second well or series of wells contains antigens from another feline calicivirus strain (or capsid protein from a commercially available calicivirus vaccine) immobilized to the surface therein. Next, the biological sample is added to the wells containing the bound antigens and antibodies against feline calicivirus are allowed to bind to form an antigen-antibody complex. The biological sample can be provided neat or in a limiting dilution series in a physiological solution. Unbound material in the sample is removed from the antibody-antigen complex by washing. The complex is then reacted with a labeled anti-species antibody or labeled monoclonal antibody that binds to feline antibodies to form an antigen-antibody-anti-species-antibody complex. This complex can be detected when the labeled monoclonal or polyclonal antibody is conjugated to a reporter ligand such as horseradish-peroxidase or alkaline phosphatase. Alternatively, the second monoclonal or polyclonal antibody can be conjugated to reporter ligands such as a fluorescing ligand, biotin/avidin colored latex, colloidal gold magnetic beads, radioisotopes or the like. Detection is by methods well known in the art for detecting the particular reporter ligand. Therefore, a sample from a cat that had been vaccinated with the vaccine of the present invention will produce antibodies against only the FCV-Diva capsid protein whereas a sample from a cat that is infected with, or exposed to, feline calicivirus will not contain antibodies against the capsid protein of the FCV-Diva. For an antigen-capture ELISA, a microtiter plate is provided containing a plurality of wells wherein a first well or series of wells contains polyclonal or monoclonal antibodies against the capsid protein or a specific immunogenic fragment thereof of the FCV-Diva immobilized to the surface therein and a second well or series of wells contains antibodies against antigens of another feline calicivirus strain (or capsid protein or a specific immunogenic fragment thereof from a commercially available calicivirus vaccine) immobilized to the surface therein. Next, the biological sample is added to the wells containing the bound antibodies and antigens against feline calicivirus are allowed to bind to form an antibody-antigen complex. The biological sample can be provided neat or in a limiting dilution series in a physiological solution. Unbound material in the sample is removed from the antibody-antigen complex by washing. The complex is then reacted with a labeled polyclonal antibody or labeled monoclonal antibody against FCV-Diva or a specific immunogenic fragment thereof to form an antibody-antigen-antibody complex. This complex can be detected when the labeled monoclonal or polyclonal antibody is conjugated to a reporter ligand such as horseradish-peroxidase or alkaline phosphatase. Alternatively, the labeled monoclonal or polyclonal antibody can be conjugated to reporter ligands such as a fluorescing ligand, , biotin/avidin colored latex, colloidal gold magnetic beads, radioisotopes or the like. Detection is by methods well known in the art for detecting the particular reporter ligand. ELISA and its variations are well known in the art. The ELISA can be provided as a kit for distinguishing cats vaccinated with the vaccine of the present invention from unvaccinated cats, and from cats infected with feline calicivirus or vaccinated with a commercially available feline calicivirus vaccine. Since it is important to be able to test samples in the field in order to distinguish cats infected with feline calicivirus from cats vaccinated with the vaccine of the present invention, the present invention further includes rapid immunodiffusion-based methods, their devices, and kits comprising the same. Therefore, the present invention can be provided with a kit that comprises any one of the methods described in U.S. Pat. No. 5,620,845 to Gould et al., U.S. Pat. No. 5,559,041 to Kang et al., U. S. Pat. No. 5,656,448 to Kang et al., U.S. Pat. No. 5,728,587 to Kang et al., U.S. Pat. No. 5,695,928 to Stewart et al., U.S. Pat. No. 5,169,789 to Bernstein et al. U.S. Pat. No. 4,486,530 to David et al., and U.S. Pat. No. 4,786,589 to Rounds et al. While the aforementioned disclose particular rapid immunodiffusion methods, the present invention is not to be construed to be limited to the aforementioned. It is within the scope of the present invention to embrace derivations and modifications of the aforementioned. In another method for distinguishing cats vaccinated with the vaccine of the present invention from cats infected with, or exposed to feline calicivirus is to provide as the vaccine the aforementioned fusion polypeptide wherein the polypeptide comprises a marker epitope that elicits an antibody in the vaccinated cat that would not normally be present in the feline. For example, the marker epitope could be from a pathogen that does not infect cats or a synthetic polypeptide that elicits an antibody in cats that would not normally occur in cats. Therefore, if a sample from a cat contained antibodies against the marker epitope and the capsid protein, the cat was vaccinated with the vaccine of the present invention. The sample is tested according to any of the aforementioned diagnostic methods. All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

What is claimed is:
I. A vaccine for immunizing cats against feline calicivirus comprising an FCV-Diva strain, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier.
2. The vaccine of claim 1 wherein said FCV-Diva strain comprises a nucleic acid encoding SEQ ID NO:2.
3. The vaccine of claim 1 wherein said FCV-Diva strain comprises the nucleic acid sequence of SEQ ID NO: 1.
4. The vaccine of claim 1 comprising an adjuvant.
5. The vaccine of claim 1, wherein the FCV-Diva strain is live attenuated.
6. The vaccine of claim 1, wherein the FCV-Diva strain is inactivated.
7. The vaccine of claim 1, which comprises at least one other feline calicivirus strain selected from the group consisting of FCV-F9, FCV-M8, FCV-255, and FCV-2280.
8. The vaccine of claim 1, which comprises at least one other feline pathogen, selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, feline Chlamydia, and feline panleukopenia virus, rabies virus and Bordetella bronchiseptica.
9. A vaccine to immunize cats against feline calicivirus which comprises: a nucleic acid encoding an FCV-Diva capsid protein or a specific immunogenic fragment thereof wherein the nucleic acid is operably linked to a heterologous promoter sequence, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier.
10. The vaccine of claim 9, wherein the nucleic acid is in a plasmid.
II. The vaccine of claim 9 wherein the nucleic acid is in a recombinant virus vector.
12. The vaccine of claim 11, wherein the recombinant virus vector is selected from the group consisting of feline herpesvirus, raccoon poxvirus, canary poxvirus, adenovirus, Semliki Forest virus, Sindbis virus, and vaccinia virus.
13. The vaccine of claim 9 wherein the nucleic acid encoding the FCV-Diva capsid protein encodes the amino acid sequence of SEQ ID NO:2 or a specific immunogenic fragment thereof.
14. The vaccine of claim 9 wherein the nucleic acid encoding the FCV-Diva capsid protein comprises the sequence of SEQ ID NO: 1.
15. A vaccine to immunize cats against feline calicivirus comprising: an isolated FCV-Diva capsid protein or a specific immunogenic fragment thereof in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier.
16. The vaccine of claim 15, wherein the FCV-Diva capsid protein comprises the amino acid sequence of SEQ ID NO:2 or a specific immunogenic fragment thereof.
17. A method for immunizing a cat against feline calicivirus comprising administering to the cat an effective dose of a vaccine as recited in any of claims 1 to 16. .
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WO2006078975A2 (en) * 2005-01-21 2006-07-27 Merial Limited Improved vaccine against feline calicivirus
WO2007012944A3 (en) * 2005-07-28 2007-04-26 Pfizer Prod Inc Methods of vaccine administration, new feline caliciviruses, and treatments for immunizing animals against feline paraovirus and feline herpes virus
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US10434165B2 (en) * 2015-08-20 2019-10-08 Boehringer Ingelheim Animal Health USA Inc. FCV recombinant vaccines and uses thereof
US10188725B2 (en) 2015-12-14 2019-01-29 Elanco Us Inc. Hybrid core feline vaccines
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CN108473539B (en) * 2015-12-23 2022-03-22 英特维特国际股份有限公司 Feline calicivirus vaccine
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CN110272488A (en) * 2018-03-16 2019-09-24 洛阳普莱柯万泰生物技术有限公司 Feline calicivirus monoclonal antibody and its application
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