Link:  Pharm/Biotech Resources

Title:  Feline calicivirus genes and vaccines in particular recombinant vaccines

United States Patent:  6,914,134

Issued:  July 5, 2005

Inventors:  Audonnet; Jean-Christophe Francis (Lyons, FR); Baudu; Philippe Guy Nicolas (Craponne, FR); Brunet; Sylvie Claudine (Lyons, FR)

Assignee:  Merial (Lyons, FR)

Appl. No.:  209507

Filed:  July 30, 2002

The invention relates to the sequence of the capsid gene and a corresponding cDNA sequence, of a dominant FCV strain called FCV 431. It also relates to the sequence of the capsid gene as well as the cDNA sequence of a complementary strain called G1. The cDNA sequences may be incorporated into expression vectors for the preparation of immunogenic preparations and of recombinant or subunit vaccines allowing vaccination against feline calicivirosis.

BRIEF SUMMARY OF THE INVENTION

The objective of the invention is in particular, starting with selected strains, the isolation and characterization of genes encoding immunogenic proteins which can be used for vaccination against feline calicivirosis.

Another objective of the invention is to provide recombinant in vitro and in vivo expression vectors containing and expressing at least one such nucleotide sequence.

Yet another objective of the invention is to provide immunogenic preparations or vaccines against feline calicivirosis.

Yet another objective of the invention is to provide multivalent immunogenic preparations and multivalent vaccines against feline calicivirosis and against at least one other feline pathogen.

The invention essentially relates to two FCV strains obtained by pharyngeal swabs taken in France and the United Kingdom on cats exhibiting signs of infection by feline caliciviruses. They are respectively strain G1 (deposited at the Collection Nationale de Cultures de Microorganismes (or CNCM) of Institut Pasteur, Paris, France, under the accession number I-2167) and strain 431 (deposited at the CNCM under the accession number I-2166), both deposited on Mar. 12, 1999. This FCV G1 strain isolated in France does not correspond to the FCV strain isolated in the United Kingdom in 1978 by Tohya (Tohya Y. et al. Jpn. J. Sci., 1990, 52, 955-961) and also called G1.

The selection of the FCV 431 and G1 strains was carried out by cross-serum neutralization tests with respect to the FCV isolates of a reference panel. This reference panel is composed of 18 current isolates of FCV taken from cats exhibiting signs of infection with feline calicivirus and coming from three distinct geographical regions. 7 isolates are American, these isolates are identified RMI1, RMI2, RMI3, RMI5, RMI6, RMI7 and RMI9. 7 isolates are French, they are designated A2, F1, G1, G3, F3031, H3-2 and H1-4. The last 4 isolates are English, they are designated 431, 388b, 337 and J5.

The panel strains are accessible from the Applicant simply on request. They have also been published in a review article "Archives of Virology" (Poulet et al. Arch. Virol. February 2000. 145(2). 243-261), available online on Internet on the date of filing with the editor.

During cross-serum neutralization tests between 18 FCV isolates of the reference panel, it was found, surprisingly, that the antiserum for isolate 431 neutralizes 14 of the 17 heterologous isolates of the reference panel (the homologous serum neutralization titer is not taken into account). By comparison, the antisera for the "historical" vaccine strains 255 and F9 neutralize only 2 of the 18 panel isolates each.

Unexpectedly, the Applicant has therefore found with the FCV 431 strain a dominant strain which can be used for the protection of the Felidae and in particular of cats against most FCV strains. By virtue of the panel of FCV strains disclosed here, it is possible for persons skilled in the art to select other dominant FCV strains. By way of equivalence, the invention also covers through the FCV 431 strain the FCV strains which are equivalent thereto, which have antibodies with broad cross-neutralization spectrum.

Equivalence exists when the antiserum for an FCV strain seroneutralizes at least 13 of the 18 heterologous isolates of the reference panel (that is to say including FCV 431), preferably when it seroneutralizes at least 14 of the 18 heterologous isolates of the reference panel, still more preferably when it seroneutralizes at least 15 of the 18 heterologous isolates of the reference panel.

It is generally considered that an FCV strain seroneutralizes another FCV strain when the heterologous serum neutralization titer is greater than or equal to 1.2 log₁₀ VN₅₀ (Povey C. and Ingersoll J., Infection and Immunity, 1975, 11, 877-885). The Applicant took this value as the positivity threshold. However, the cross-serum neutralization results obtained with an FCV isolate having a homologous serum neutralization titer of less than or equal to 2 log₁₀ VN₅₀ cannot be interpreted.

A second method for establishing the equivalence of an FCV strain with respect to the FCV 431 strain is to use monoclonal antibodies specific for the FCV 431 strain and to test the candidate FCV strain by indirect immunofluorescence (IIF). The Applicant has thus succeeded in producing several monoclonal antibodies which have proved specific for the 431 strain. One of them was called 44. There is equivalence if there is reactivity in immunofluorescence with monoclonal antibodies specific for 431, for example with the monoclonal antibody 44. This monoclonal antibody and the corresponding hybridoma are available from the Applicant upon simple request and are also disclosed in the article by Poulet et al., supra. The corresponding hybridoma was also deposited on 11 Aug. 1999 at the CNCM under the accession number I-2282. It goes without saying, however, that persons skilled in the art are perfectly capable of producing monoclonal antibodies by conventional techniques and of selecting, relative to the panel, those which are specific for the 431 strain.

The other FCV G1 strain was chosen for its complementarity to the FCV 431 strain, namely that the combination of the antisera for 431 and for G1 seroneutralize 100% of the isolates of the reference panel, that is to say that the FCV G1 strain has a homologous serum neutralization titer greater than or equal to 2 log₁₀ VN₅₀ and heterologous serum neutralization titers greater than or equal to 1.2 log₁₀ VN₅₀ with respect to the FCV isolates of the reference panel against which the 431 antiserum does not seroneutralize or seroneutralizes weakly (value less than 1.2 log₁₀ VN₅₀ ) The invention also covers the equivalent FCV strains having the same complementarity with respect to the FCV 431 strain. It is also possible to produce and select antibodies specific for this strain, which makes it possible to determine equivalents on this other basis.

The Applicant has, in addition, succeeded in isolating, characterizing and sequencing the gene for the capsid of FCV 431 and FCV G1, the capsid protein, and has determined the corresponding cDNA (complementary DNA) sequences.

The subject matter of the invention is therfore a nucleic acid fragment comprising all or part of the nucleotide sequence encoding the capsid protein of the 431 virus whose amino acid sequence is represented in SEQ ID NO: 4 or in FIG. 2, or an immunologically active fragment of this protein, that is to say an epitope, peptide or polypeptide of the capsid protein.

The subject matter of the invention is in particular a DNA fragment comprising the cDNA sequence of SEQ ID NO: 3 or a fragment conserving the essential properties of the complete sequence, that is to say encoding a peptide, polypeptide or epitope substantially conserving the immunogenic activity of the capsid protein. The subject of the invention is in particular a DNA fragment comprising this cDNA sequence, which is in particular coupled with elements for the regualtion of transcription.

The subject of the invention is in particular a DNA fragment comprising the cDNA sequence of SEQ ID NO: 6 or a fragment conserving the essential properties of the complete sequence, that is to say encoding a peptide, polypeptide or epitope substantially conserving the immunogenic activity of the capsid protein. The subject of the invention is in particular a DNA fragment comprising this cDNA sequence, which is in particular coupled with elements for the regulation of transcription.

It goes without saying that the invention automatically covers the nucleic acid fragments, DNA fragments and cDNA sequences which are equivalent, that is to say the nucleotide fragments and sequences specific for the FCV capsid which do not change the functionality or the strain specificity of the described sequence or of the polypeptides encoded by this sequence. The sequences which differ by degeneracy of the code will of course be included.

The invention also automatically covers the nucleotide sequences (RNA, DNA, cDNA) which are equivalent in the sense that they encode an FCV capsid protein, or a specific peptide, polypeptide or epitope of FCV capsid protein, which is capable of inducing in vivo in the feline species, in particular in cats, antibodies having substantially the same cross-neutralization as the antiserum for the FCV 431 strain. They are in particular the sequences obtained from equivalent FCV strains according to the definition given above with respect to the panel and/or the monoclonal antibody 44.

The subject of the invention is also a nucleic acid fragment comprising all or part of the nucleotide sequence encoding the G1 virus capsid protein as represented in SEQ ID NO: 2 or in FIG. 1, or an immunologically active fragment of this protein, that is to say an epitope, peptide or polypeptide substantially conserving the immunogenic activity of the capsid protein.

The subject of the invention is in particular a DNA fragment comprising the cDNA sequence of SEQ ID NO: 1 or a fragment conserving the essential properties of the complete sequence, that is to say encoding a peptide, polypeptide or epitope substantially conserving the immunogenic activity of the capsid protein. The subject of the invention is in particular a DNA fragment comprising this cDNA sequence, in particular coupled with elements for the regulation of transcription. It goes without saying that the invention automatically covers the nucleic acid fragments, DNA fragments and cDNA sequences which are equivalent, that is to say the nucleotide fragments and sequences which do not change the functionality or the strain specificity of the sequence described or of the polypeptides encoded by this sequence. The sequences which differ by degeneracy of the code will of course be included.

The invention also automatically covers the nucleotide sequences (RNA, DNA, cDNA) which are equivalent in the sense that they encode a peptide, polypeptide or epitope capable of inducing in vivo in the feline species, in particular in cats, antibodies having substantially the same cross-neutralization spectrum as the antiserum for the FCV G1 strain. They are in particular nucleotide sequences obtained from FCV strains which are complementary in the sense given above.

The gene encoding the capsid protein of the FCV G1 and FCV 431 viruses has a size of 2007 nucleotides for FCV 431 and 2010 nucleotides for FCV G1. The capsid protein has a size of 668 amino acids for FCV 431 and of 669 amino acids for FCV G1, and a mass of 60-65 kDa (protein p65).

The subject of the invention is also an expression vector comprising at least one DNA fragment according to the invention, in particular a type 431 cDNA or a type G1 cDNA, under conditions allowing its expression in vivo. According to a specific feature, the expression vector comprises a type 431 cDNA and a type G1 cDNA. "Type" should be understood to mean that the cDNA is complementary to an RNA sequence of the strain considered.

These expression vectors may be poxviruses, for example the vaccinia virus, avipoxes (canarypox, fowlpox), including the species-specific poxviruses (swine pox, raccoonpox and camelpox), adenoviruses and herpesviruses, such as the feline herpesviruses (e.g. FR-A-2 741 806). For the poxviruses, persons skilled in the art can refer to WO-A-9215672, WO-A-9526751, WO-A-9012882 and WO-A-9527780; U.S. Pat. Nos. 6,004,777; 5,990,091; 5,989,561; 5,843,456.

Several insertion strategies may be used for the expression of several heterologous nucleotide sequences from the same expression vector in vivo. These insertion strategies are in particular the use of a double expression cassette having an opposite orientation, or the use of a double expression cassette having an identical orientation, or alternatively a multiple expression cassette having an "IRES" (Internal Ribosome Entry Site) element situated between each insert (Patent EP-A1-0803573).

The heterologous nucleotide sequences are inserted under the control of signals for the regulation of transcription and in particular of promoters, which are preferably brought during the insertion. It is not however excluded to cause these heterologous nucleotide sequences to be expressed under the control of signals which are specific to the expression vector used. For the canarypox expression vectors, one of the preferred promoters is the H6 vaccinia promoter (Taylor J. et al. Vaccine, 1988, 6, 504-508; Guo P. et al. J. Virol., 1989, 63, 4189-4198; Perkus M. et al. J. Virol., 1989, 63, 3829-3836).

The in vivo expression vectors may also be plasmids. The term plasmid is intended to cover any DNA transcription unit in the form of a polynucleotide sequence comprising a cDNA sequence according to the invention and the elements necessary for its expression in vivo. The circular plasmid form, supercoiled or otherwise, is preferred. The linear form also falls within the scope of this invention.

Each plasmid comprises a promoter capable of ensuring, in the host cells, the expression of the cDNA inserted under its control. It is in general a strong eukaryotic promoter in particular an early cytomegalovirus promoter CMV-IE, of human or murine origin, or possibly of another origin such as rats or guinea pigs. More generally, the promoter is either of viral origin or of cellular origin. As viral promoter other than CMV-IE, there may be mentioned the early or late promoter of the SV40 virus or the LTR promoter of the Rous Sarcoma virus. As cellular promoter, there may be mentioned the promoter of a cytoskeletal gene, such as for example the desmin promoter or alternatively the actin promoter. A subfragment of these promoters, which conserves the same promoter activity is included in the present invention, e.g. the truncated CMV-IE promoters according to WO-A-98/00166.

When several heterologous sequences (cDNA and/or genes of FCV or of other feline pathogens) are present in the same plasmid, these may be present in the same transcription unit or in two different units.

The plasmids may also comprise other elements for regulation of transcription, such as for example stabilizing sequences of the intron type, preferably intron II of the rabbit β-globin gene (van Ooyen et al. Science, 1979, 206: 337-344), signal sequence of the protein encoded by the gene for tissue plasminogen activator (tPA; Montgomery et al. Cell. Mol. Biol. 1997, 43: 285-292), and polyadenylation signal (polyA), in particular of the gene for bovine growth hormone (bGH) (U.S. Pat. No. 5,122,458) or of the rabbit β-globin gene.

The subject of the invention is also the use of the cDNAs according to the invention for the in vitro production of capsid proteins or of their fragments and immunologically active epitopes and their incorporation into immunogenic preparations and subunit vaccines.

The subject of the invention is also an immunogenic preparation or vaccine against feline calicivirosis comprising at least one recombinant in vivo expression vector according to the invention and a veterinarily acceptable vehicle or excipient, and optionally an adjuvant.

The notion of immunogenic preparation covers any preparation capable, once administered to cats, of inducing at least an immune response directed against the feline pathogen considered. Vaccine is understood to mean a preparation capable of inducing effective protection.

Preferably, this immunogenic preparation or this vaccine comprises an in vivo expression vector into which is inserted a type FCV 431 cDNA, which includes its equivalents or an FCV G1 cDNA, which includes the equivalents of the latter.

According to a first very advantageous characteristic feature, this immunological preparation or this vaccine comprises an expression vector into which is inserted an FCV 431 type cDNA, which includes its equivalents, and an FCV G1 type cDNA, which also includes the equivalents of the latter.

According to a second very advantageous specific feature, this immunological preparation or this vaccine comprises at least two expression vectors: in the first is inserted an FCV 431 type cDNA, which includes its equivalents, and in the second a cDNA of the FCV G1 strain, which also includes the equivalents of the latter.

To supplement the preparations and vaccines in accordance with the invention with adjuvants, it is possible to use any appropriate adjuvant known to persons skilled in the art. However, it is preferable either to formulate them in the form of oil-in-water emulsions, or to add to them polymers of acrylic or methacrylic acid or copolymers of maleic anhydride and of alkenyl derivative, or alternatively a cationic lipid containing a quaternary ammonium salt.

Among the polymers, the polymers of acrylic or methacrylic acid which are crosslinked, in particular with polyalkenyl ethers of sugars or polyalcohols, are preferred. These compounds are known under the term carbomer (Pharmeuropa vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462 (incorporated by way of reference) describing such acrylic polymers crosslinked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 3, the hydrogen atoms of at least three hydroxyls being replaced with unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol® (BF Goodrich, Ohio, USA) are particularly appropriate. They are crosslinked with an allyl sucrose or with allylpentaerythritol. Among them, there may be mentioned Carbopol® 974P, 934P and 971P.

Among the copolymers of maleic anhydride and of alkenyl derivative, the EMA® copolymers (Monsanto) which are copolymers of maleic anhydride and of ethylene, which are linear or crosslinked, for example crosslinked with divinyl ether, are preferred. Reference may be made to J. Fields et al., Nature, 186: 778-740, 4 Jun. 1960 (incorporated by way of reference).   These polymers are dissolved in water or in physiological saline (NaCl at 20 g/l) and the pH is adjusted to 7.3-7.4 with sodium hydroxide, to give the adjuvant solution into which the expression vector or the subunits will be incorporated.

The concentration of polymer in the final vaccine composition will be from 0.01% to 1.5% W/V, more particularly from 0.05 to 1% W/V, preferably from 0.1 to 0.4% W/V.

The cationic lipids containing a quaternary ammonium salt, which are particularly but not exclusively suitable for the plasmid expression vectors.

DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanammonium; WO-A-9634109), preferably coupled with a neutral lipid, in particular DOPE (dioleoyl-phosphatidyl-ethanolamine), to form DMRIE-DOPE, is preferred. Preferably, the plasmid is mixed with this adjuvant immediately before use and it is preferable, before its administration to the animal, to allow the mixture thus prepared time to form a complex, for example for a period ranging from 10 to 60 minutes, in particular of the order of 30 minutes.

When DOPE is present, the DMRIE:DOPE molar ratio ranges preferably from 95:5 to 5:95, more particularly 1:1.

The plasmid:adjuvant DMRIE or DMRIE-DOPE weight ratio may range in particular from 50:1 to 1:10, in particular from 10:1 to 1:5, preferably from 1:1 to 1:2.

The in vivo expression vectors encoding an FCV type cDNA according to the invention may also encode feline GM-CSF or may be combined with a second vector encoding feline GM-CSF (e.g. the plasmids pJP089 and pJP090, FIG. 5 and FIG. 6 respectively). In the case of plasmids, a mixture of two plasmids is preferred. In the case of viral vectors, a single vector is preferred. This expression vector or this mixture of expression vectors may also be supplemented with adjuvant as described above.

The subject of the invention is also a multivalent immunogenic preparation or a multivalent vaccine against feline calicivirosis and against at least one other feline pathogen, using the same recombinant in vivo expression vector containing and expressing at least one FCV type cDNA according to the invention and at least one nucleotide sequence of an immunogen or another feline pathogen or of an immunologically active fragment of this immunogen.

The subject of the invention is also a multivalent immunogenic preparation or a multivalent vaccine comprising at least one in vivo expression vector into which is inserted at least one FCV type cDNA according to the invention and at least a second expression vector into which is inserted a sequence encoding an immunogen, or an immunologically active fragment, of another feline pathogen. Appropriate plasmids into which is inserted a sequence encoding an immunogen, or an immunologically active fragment, of another feline pathogen, may be in particular those described in Examples 7 to 15 and 17 to 19 of Patent Application WO-A-9803660 (pPB179, pPB180, pPB181, pAB009, pAB053, pAB052, pAB056, pAB058, pAB029, pAB030, pAB083, pAB041).

The monovalent or multivalent recombinant vaccines as described above may also be combined with at least one conventional vaccine (inactivated, attenuated live, subunit) directed against at least one feline pathogen which is identical or different.

Said other feline pathogens are in particular chosen from the group comprising the feline rhinotrachitis virus or the feline herpesvirus (FHV), the feline leukemia virus (FeLV), the feline parvoviruses (FPV), the feline infectious peritonitis virus (FIPV), the feline immunodeficiency virus (FIV), the rabies virus, Chlamydia.

The subject of the invention is also the isolated, purified or synthetic capsid proteins of the FCV G1 strain and of the FCV 431 strain, having an amino acid sequence represented in SEQ ID Nos: 2 and 4 respectively. This automatically covers the equivalent proteins, that is to say the proteins derived from strains which are equivalent to the FCV 431 and FCV G1 strains according to the definitions given above (use of the panel and/or of a monoclonal antibody, in particular the monoclonal antibody 44). Advantageously, these capsid proteins may be assembled in the form of empty capsids.

The subject of the invention is also the fragments and epitopes (at least about 8 to 10 amino acids) of these proteins, which conserve the specificity and immunogenicity of the whole protein.

The capsid proteins, optionally assembled in the form of empty capsids, and their fragments and epitopes, may be produced by expression in vitro. The corresponding nucleotide sequence is inserted into an in vitro expression system and expressed by this system, and the product of expression harvested and optionally purified, as is known per se.

The expression system may be of viral origin, in particular the baculovirus (U.S. Pat. No. 4,745,051). The coding sequence or a fragment (in the case of the epitope or of the fragment) is integrated into the baculovirus genome (e.g. the baculovirus Autographa californica Nuclear Polyhedrosis Virus AcNPV) and the latter is then propagated, in particular in insect cells, e.g. Spodoptera frugiperda Sf9 (deposit ATCC CRL 1711).

The in vitro expression system may be of prokaryotic origin, e.g. Escherichia coli, or of eukaryotic origin, in particular yeasts, e.g. Saccharomyces cerevisiae, or mammalian eukaryotic cells, in particular cell lines such as CHO (hamster ovary cells), HeLa, BHK or insect cells, e.g. Spodoptera frugiperda (supra), or alternatively feline cells.

As promoters which can be used in these cellular constructs, there may be mentioned the strong viral promoters such as those of the SV40 virus (Fiers et al., Nature, (1978) 273:113) and the early promoter (CMV-IE) of the human CMV virus or cytomegalovirus (McGregor and Caskey, Nucleic Acids Res. 17:2365, 1989) or of murine or other origin, or alternatively that of the polyhedrin gene of the baculovirus AcNPV (Hooft van Iddekinge et al., 1983, Virology 131:561-565).

Persons skilled in the art know how to purify and/or isolate the proteins, assembled or otherwise in the form of empty capsids, their fragments and epitopes from the product of the techniques described above. By way of example, it can be recalled that persons skilled in the art have at their disposal various methods which comprise in particular: precipitation based on the solubility of the proteins, fragments and epitopes of interest according to the saline conditions of the medium, precipitation with organic solvents, polymers or other materials, affinity precipitation and selective denaturation, column chromatography, including high-performance liquid chromatography (HPLC), ion-exchange chromatography, affinity chromatography, immunoaffinity chromatography, chromatography using ligands, immunoprecipitation, gel filtration, electrophoresis, filtration methods, in particular ultrafiltration, and gradient ultracentrifugation.

Persons skilled in the art can refer by way of example to K. Y. Green et al., J. Clin. Microb., July 1997, Vol 35, 7:1909-1914, for the production of capsids in baculovirus propagated on Sf9 cells and harvested by ultracentrifugation on sucrose gradients (10 to 50%).

The capsid proteins, and their fragments and epitopes, may also be produced by chemical synthesis by the methods available to persons skilled in the art.

The subject of the invention is also the immunogenic preparations and vaccines comprising at least one subunit antigen formed of a capsid protein, preferably assembled in the form of empty capsids, of FCV 431 and/or FCV G1, or of a corresponding fragment or epitope, in a veterinarily acceptable vehicle or excipient, and preferably an adjuvant. Preferably, the preparations and vaccines according to the invention comprise subunit antigens derived from the two strains FCV 431 and FCV G1. Likewise, the preparations and vaccines may comprise nonassembled capsid proteins and proteins assembled in the form of empty capsids.

To supplement the subunit vaccines and immunogenic preparations according to the invention with adjuvants, it is possible to use as adjuvant (1) aluminum hydroxide, (2) a polymer of acrylic or methacrylic acid, a polymer of maleic anhydride and of alkenyl derivative (which are described above), or (3) to formulate the immunogenic preparation or vaccine in the form of an oil-in-water emulsion, in particular the emulsion SPT described p 147 "Vaccine Design, The Subunit and Adjuvant Approach" edited by M. Powell, M. Newman, Plenum Press 1995, and the emulsion MF59 described p 183 in the same book.

The oil-in-water emulsion may in particular be based on light liquid paraffin oil (European Pharmacopeia type), isoprenoid oil such as squalane, squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or of decene; esters of acids or alcohols containing a linear alkyl group, more particularly vegetable oils, ethyl oleate, propylene glycol di(caprylate/caprate), glyceryl tri(caprylate/caprate), propylene glycol dioleate; esters of branched fatty alcohols or acids, in particular esters of isostearic acid. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular the esters of sorbitan, mannide, glycerol, polyglycerol, propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, the polyoxypropylene-polyoxyethylene block copolymers, in particular the Pluronic® copolymers, especially L121.

The subject of the invention is also the multivalent vaccines and immunogenic preparations in which the FCV valency is a subunit valency as described above.

The subject of the present invention is also a method of immunizing cats against diseases caused by the feline calicivirosis viruses.

This method comprises the administration of an immunological preparation or of a vaccine according to the invention to cats. This administration may be made in particular by the parenteral route, by subcutaneous, intradermal, intramuscular or intraperitoneal administration. Preferably, the administration is made by the subcutaneous or intramuscular route.

Various means of administration may be used for the plasmid vaccines and immunogenic preparations, in particular gold particles coated with DNA and discharged so as to penetrate into the cells of the skin of the subject to be immunized (Tang et al. Nature 1992. 356. 152-154) and liquid-jet injectors which make it possible to transfect both skin cells and cells of the underlying tissues (Furth et al. Analytical Bioch. 1992. 205, 365-368).

Persons skilled in the art possess the necessary competence to precisely define the number of administration and the doses to be used for each immunization protocol.

Claim 1 of 23 Claims

1. An isolated nucleic acid molecule comprising the nucleotide sequence represented in SEQ ID NO: 1 (FCV G1 capsid).

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