Feline calicivirus vaccine

Abstract

To provide feline calicivirus vaccines.SOLUTION: Provided is an immunogenic composition comprising an alphavirus RNA replicon particle encoding a feline calicivirus (FCV) antigen.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a joint venture under 35 U.S.C. § 119(e) with U.S. Pat. No. 6,629,492 filed on December 8, 2017. U.S. Provisional Patent Application No. 62 / 596,508, filed November 6, 2017 Application No. 62 / 582,050, filed November 6, 2017, U.S. Provisional Patent Application No. 6 No. 2 / 581,955 and U.S. Provisional Patent Application No. 62, filed December 15, 2017 / 599,401, the entire contents of which are incorporated herein by reference. .

[0002] The present invention relates to a new vaccine for feline calicivirus. Methods for making and using the composition or in combination with other protective agents are also provided. [Background technology]

[0003] Feline calicivirus (FCV) typically causes upper respiratory tract disease in cats. Together with the coherpesvirus, it is thought to be the cause of approximately 80% of all feline respiratory diseases. The most common characteristic and clinical sign of FCV infection is the presence of erythrocytic leukemia on the tongue and oral mucosa. These vesicles begin as small, individual ulcers and grow over most of the tongue. The vesicles do not usually interfere with eating or drinking and generally resolve without any problems. Fever is also often observed in infected cats.

[0004] Certain strains of FCV are also known as limping syndrome. The lameness syndrome is characterized by fever, joint and muscle pain (lameness) ), and sometimes tongue / oral ulcers. In addition, some strains of FCV Other less common clinical signs include conjunctivitis, nasal congestion, and stomatitis. FCV-infected cats can be persistently infected and can cause long-term may shed infectious virus for a period of time.

[0005] FCV is a single-stranded positive control consisting of three open reading frames (ORFs). The genome is polyadenylated at the 3' end and encoded by the virus. The first open reading frame is bound at the 5' end by a transduced protein. The rHEMA is a single polypeptide containing a viral protease and an RNA-dependent RNA polymerase. This polypeptide is then transduced by the viral protease. The second open reading frame is divided into six regions, designated A to F. The major capsid protein (i.e., FCV capsid protein) having the [Scott et al., 60 Am J Vet Res.:652-658(199 9)]. Region A is cleaved to generate the mature capsid protein. Region B of ORF2 , D, and F are relatively conserved among FCV isolates, whereas regions C and E are variable. , region E of ORF2 contains the major B cell epitope [Radford et al., 38(2) Vet Res.:319-335(2007)]. ORF3 is a minor structure Protein-encoding [Sosnovtsev and Green, 277 Vir ology:193-203(2000)].

[0006] Several vector strategies have been used for many years to protect against certain pathogens. One such vector strategy is the Venezuelan equine encephalitis virus (VEE). (VEE) [Pushko et al., Virology 239:389-401 (1997) ], Sindbis (SIN) [Bredenbeek et al., Journal of Viro logy 67:6439-6446(1993)] and Semliki Forest virus (SF V)[Liljestrom and Garoff,Biotechnology(N Several different alphaviruses, including [Y) 9:1356-1361 (1991)] Alphavirus-derived replicon RNA particles (RP) [Vander Ve et al. en et al., Anim Health Res Rev.13(1):1-9(2012)d oi:10.1017 / S1466252312000011;Kamrud et al., J.G. en Virol 91(Pt 7):1723-1727(2010)] RP vaccines deliver a replication-deficient alphavirus RNA replicon into host cells. , resulting in expression of the desired antigen transgene(s) in vivo [Pushko et al., Virology 239(2):389-401(1997)]. It has an attractive safety and efficacy profile when compared to other conventional vaccine formulations. [Vander Veen et al., Anim Health Res Rev. 13(1 ):1-9(2012)]. The RP platform is used to encode pathogen antigens. and is the basis for several USDA-licensed vaccines for swine and poultry. .

[0007] FCV isolates have long been characterized as belonging to a single serotype, but are antigenically very different. are highly diverse and are vaccinated using relatively old vaccine strains of FCV, such as FCV F9. Antibodies from inoculated cats do not efficiently neutralize any current field isolates. New FCV strains causing systemic disease and high mortality have been identified [e.g., US See US Patent No. 7,449,323. These "highly virulent systemic" (VS-FCV) isolates These strains are responsible for local outbreaks and current vaccines are also susceptible to the This is likely because the current vaccine strains do not protect cats from the disease. Cats vaccinated with FCV may develop the disease caused by such "antigenically heterologous" FCV strains. Even vaccinated cats are not completely protected from the disease. This has led to concerns that these variant strains could be the cause of disease outbreaks. This stimulates more broadly reactive virus-neutralizing (VN) antibodies, thereby promoting new It is desirable to develop new vaccines that provide superior protection against exogenous isolates.

[0008] The citation of any reference herein does not imply that such reference is an "incorporated" application. This disclosure is not to be construed as an admission that any of the subject matter is available as "prior art." [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 7,449,323 [Non-patent literature]

[0010] [Non-Patent Document 1] Scott et al., 60 Am J Vet Res.:652-658(1999) [Non-patent document 2] Radford et al., 38(2)Vet Res.:319-335(2007) [Non-patent document 3] Sosnovtsev and Green,277 Virology:193-203(2000) [Non-patent document 4] Pushko et al., Virology 239:389-401 (1997) [Non-Patent Document 5] Bredenbeek et al., Journal of Virology 67:6439-6446 (1993) [Non-patent document 6] Liljestrom and Garoff,Biotechnology(NY)9:1356-1361(1991) [Non-Patent Document 7] Vander Veen et al., Anim Health Res Rev.13(1):1-9(2012)doi:10.1017 / S1466252312000011 [Non-patent document 8] Kamrud et al., J Gen Virol 91(Pt 7):1723-1727(2010) [Non-Patent Document 9] Pushko et al., Virology 239(2):389-401 (1997) Summary of the Invention [Means for solving the problem]

[0011] Thus, the present invention provides vectors encoding one or more feline calicivirus (FCV) antigens. Such vectors can be used in immunogenic compositions containing these vectors. The immunogenic composition of the present invention can be used in a vaccine. In embodiments, the vaccine protects a vaccinated subject (e.g., a mammal) from FCV. In particular embodiments of this type, the vaccinated subject is a cat. In this embodiment, the vaccinated subject is a domestic cat. and other diseases, e.g., combination vaccines to induce protective immunity against other infectious diseases in cats. Methods for making and using the immunogenic compositions and vaccines of the invention are also provided. It is served.

[0012] In a specific embodiment, the vector encodes one or more antigens derived from a feline pathogen. In certain embodiments, the feline pathogen is an alphavirus RNA replicon particle. In a specific embodiment of this type, the virus is an alphavirus. The RNA replicon particles encode FCV capsid proteins. In this study, alphavirus RNA replicon particles express the antigenicity of FCV capsid proteins. In certain embodiments, the FCV capsid protein encodes the FC In another embodiment, the alphavirus RNA fragment is a VF9-like capsid protein. The plicon particles encode antigenic fragments of the FCV F9-like capsid protein. In yet another embodiment, the FCV capsid protein is a virulent systemic FCV (VS In yet another embodiment, the alphavirus RNase I (FCV) capsid protein is A replicon particle encodes an antigenic fragment of the VS-FCV capsid protein. In yet other embodiments, the alphavirus RNA replicon particles comprise FCV F 9-like capsid protein or its antigenic fragment and VS-FCV capsid It encodes both the protein or an antigenic fragment thereof.

[0013] In an even more particular embodiment, the alphavirus RNA replicon particles are Equine encephalitis (VEE) alphavirus RNA replicon particle. In embodiments, the VEE alphavirus RNA replicon particles are TC-83 VEE alphavirus RNA replicon particles. In another embodiment, the alphavirus RNA replicon particle is a lufavirus RNA replicon particle. A replicon particle is a Sindbis (SIN) alphavirus RNA replicon particle. In yet other embodiments, the alphavirus RNA replicon particles are derived from Semliki Forest In an alternative embodiment, the SFV alphavirus RNA replicon particle is Naked DNA vectors contain nucleic acid constructs encoding one or more antigens derived from feline pathogens. In certain embodiments of this type, the naked DNA vector comprises an FCV capsid protein. The present invention also includes nucleic acid constructs encoding the protein or antigenic fragments thereof.

[0014] In certain embodiments, the alphavirus RNA replicon particles of the present invention comprise one or more In a particular embodiment of this type, the FCV antigen or antigenic fragment thereof is encoded by: Alphavirus RNA replicon particles contain 2–4 FCV antigens or their antigenic fragments. In a related embodiment, the alphavirus RNA replica of the invention encodes a fragment thereof. The conjugate particle contains one or more FCV antigens or antigenic fragments thereof and one or more non-FCV antigens. In a specific embodiment of this type, the CV antigen or an antigenic fragment thereof is encoded by: Alphavirus RNA replicon particles contain one or more FCV capsid proteins or an antigenic fragment thereof and 1 to 3 non-FCV antigens or antigenic fragments thereof In a more specific embodiment, the alphavirus RNA replicon particle encodes , VS-FCV capsid protein or its antigenic fragment and FCV-F9 FCV-like capsid protein or an antigenic fragment thereof and 1 to 3 non-FCV antigens or or an antigenic fragment thereof.

[0015] In another aspect, the present invention provides a method for producing a medicament comprising: The present invention provides immunogenic compositions comprising alphavirus RNA replicon particles that encode the alphavirus. In some embodiments, the immunogenic composition comprises two to four FCV antigens or antigenic fragments thereof. Specific embodiments of this type include alphavirus RNA replicon particles encoding the In , alphavirus RNA replicon particles encode FCV capsid proteins. In another embodiment, the alphavirus RNA replicon particle comprises an FCV capsid. In certain embodiments, the immunogenic composition encodes an antigenic fragment of a protein. , an alphavirus RNA replicon encoding an FCV F9-like capsid protein In another embodiment, the immunogenic composition comprises an FCV F9-like capsid protein. The alphavirus RNA replicon particles encode antigenic fragments of various In yet another embodiment, the immunogenic composition comprises a virulent systemic FCV (VS-FCV) capsid. The alphavirus RNA replicon particles encode the alphavirus RNA protein. In one embodiment, the immunogenic composition comprises an antigenic fragment of a VS-FCV capsid protein. In yet another embodiment, the alphavirus RNA replicon particle encodes: The immunogenic composition comprises an FCV F9-like capsid protein or an antigenic fragment thereof. and VS-FCV capsid protein or an antigenic fragment thereof In a more particular embodiment, the immunogen comprises an alphavirus RNA replicon particle. The composition is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle. In an even more specific embodiment, the immunoglobulin comprises an alphavirus RNA replicon particle. The epigenetic composition is the TC-83 VEE alphavirus RNA replicon particle, VE Contains E alphavirus RNA replicon particles.

[0016] In yet other embodiments, the immunogenic composition comprises an alphavirus RNA replicon particle. In certain embodiments of this type, the alphavirus RNA replicas comprise two or more sets. One set of replicon particles encodes the first antigen, and the other set encodes the alphavirus RNA replicon particles. The other set of molecules encodes a second antigen. In certain embodiments of this type, The first set of viral RNA replicon particles comprises one or more FCV antigens or antigenic fragments thereof. The second set of alphavirus RNA replicon particles encodes the fragment 1 In certain embodiments, the gene encoding one or more FeLV antigens or antigenic fragments thereof is The FCV antigen is derived from a virulent systemic feline calicivirus (VS-FCV) isolate. In one embodiment, the FCV antigen is derived from a classical (F9-like) feline calicivirus isolate. In yet another embodiment, the second set of alphavirus RNA replicon particles comprises: Two FCV antibodies, one derived from a virulent systemic FCV isolate and the other from an F9-like FCV, were identified. In yet other embodiments, the immunogenic composition encodes an FCV F Alphaviruses encoding 9-like capsid proteins or antigenic fragments thereof a first set of RNA replicon particles and a VS-FCV capsid protein or A second set of alphavirus RNA replicon particles encoding antigenic fragments In a related embodiment, the immunogenic composition comprises a VS-FCV capsid protein. Alphavirus RNA replicon particles encoding proteins or antigenic fragments thereof The first set and the FeLV glycoprotein (e.g., gp85) or an antigenic fragment thereof an antigen encoding a protein (e.g., FeLV glycoprotein gp70 and / or gp45) and a second set of lufa virus RNA replicon particles.

[0017] In yet other embodiments, the immunogenic composition comprises an alphavirus encoding a first antigen. one set of RNA replicon particles and one set of alphavirus R encoding a second antigen. Another set of alphavirus RNA replicon particles and another set of alphavirus RNA replicon particles encoding a third antigen and a third set of plicon particles. In certain embodiments of this type, the alphavirus The first set of RNA replicon particles was derived from classical (F9-like) feline caliciviruses. and a vector encoding an FCV antigen (e.g., a capsid protein) or an antigenic fragment thereof. The second set of alphavirus RNA replicon particles was used to express the highly virulent systemic feline calicivirus. FCV antigens (e.g., capsid proteins) derived from the virus or their antigenic flags and a third set of alphavirus RNA replicon particles encoding A gene encoding an FeLV antigen (e.g., FeLV gp85) or an antigenic fragment thereof. do.

[0018] Thus, the immunogenic composition may comprise multiple sets of alphavirus RNA replicon particles. In certain embodiments, the alphavirus RNA replicon The first set of particles contains the FCV F9-like capsid protein or its antigenic fragments. and / or VS-FCV capsid protein or an antigenic fragment thereof and one or more other sets of alphavirus RNA replicon particles encoding one or more Encodes non-FCV antigens.

[0019] In certain such embodiments, the non-FCV antigen or antigenic fragment thereof is administered to a cat. In another embodiment, the antigen is a protein antigen derived from a FHV. The V antigen is a protein antigen derived from the feline leukemia virus (FeLV). In this embodiment, the non-FCV antigen is a protein derived from feline pneumovirus (FPN). In yet other embodiments, the non-FCV antigen is a feline parvovirus (FPV) antigen. In yet another embodiment, the non-FCV antigen is a protein antigen derived from rabies virus. In yet other embodiments, the non-FCV antigen is a protein antigen derived from a feline virus. It is a protein antigen derived from infectious peritonitis virus (FIPV). In some embodiments, the non-FCV antigen is a protein antigen derived from the feline immunodeficiency virus. In other embodiments, the non-FCV antigen is a protein derived from Borna disease virus (BDV). In yet another embodiment, the non-FCV antigen is an antigen specific to a feline influenza virus. In yet other embodiments, the non-FCV antigen is a protein antigen derived from feline pancytopenia. In yet another embodiment, the antigen is a protein antigen derived from the FPV virus (FPLV). Non-FCV antigens are protein antigens derived from feline coronavirus (FCoV). In another embodiment, the non-FCV antigen is a antigen derived from feline rhinotracheitis virus (FVR). In yet another embodiment, the non-FCV antigen is a protein antigen. It is a protein antigen derived from the squirrel (Chlamydophila felis).

[0020] The present invention also provides all alphavirus RNA replicon particles of the present invention, naked DNA Nucleic acid constructs of the invention, including vectors, synthetic messenger RNAs and RNA replicons and all nucleic acid constructs of the present invention (e.g., synthetic messenger RNA, RNase A, A replicon), alphavirus RNA replicon particles and / or naked DNA vectors The present invention also includes immunogenic compositions and / or vaccines containing the target.

[0021] In certain embodiments, the nucleic acid construct of the invention comprises one or more FCV antigens or antigenic fragments thereof. In related embodiments of this type, the nucleic acid construct encodes two to four In another embodiment, the alpha-antigen encodes an FCV antigen or an antigenic fragment thereof. The viral RNA replicon particles contain one or more FCV antigens or antigenic fragments thereof. In certain embodiments, the nucleic acid construct includes an alphavirus RNA replicon. The particles contain nucleic acid constructs encoding 2 to 4 FCV antigens or antigenic fragments thereof. include.

[0022] In still other embodiments, the immunogenic composition comprises two to four FCV antigens or antigenic fragments thereof. Alphavirus RNA replicon particles containing a nucleic acid construct encoding a fragment, and In certain embodiments of this type, the vector comprises an alphavirus R NA replicon particles contain FCV F9-like capsid proteins or their antigenic fragments. and / or VS-FCV capsid protein or an antigenic fragment thereof and and FeLV glycoprotein (gp85) or an antigenic fragment thereof. In particular embodiments of the species, the antigenic fragment of gp85 is FeLV glycoprotein g In another related embodiment, the antigenic fragment of gp85 is FeLV p70. In a more particular embodiment, the immunogenic composition is a Venezuelan glycoprotein gp45. Alphavirus R, the ewe encephalitis (VEE) alphavirus RNA replicon particle In an even more specific embodiment, the NA replicon particle comprises a VEE alphavirus. The RNA replicon particles are TC-83 VEE alphavirus RNA replicon particles. be.

[0023] In yet other embodiments, the immunogenic composition comprises an alphavirus RNA replicon particle. and / or two or more sets of naked DNA vectors. In this study, either alphavirus RNA replicon particles and / or naked DNA vectors were used. The set comprises a first nucleic acid construct, an alphavirus RNA replicon particle and / or The other set of vectors, either naked or non-naked, contains a second nucleic acid construct. In embodiments, the first nucleic acid construct encodes an FCV antigen or an antigenic fragment thereof; The second nucleic acid construct comprises a feline calicivirus (FCV) antigen or an antigenic fragment thereof. In certain embodiments of this type, the FCV antigen encodes a virulent systemic feline calicivirus. In other embodiments, the FCV antigen is derived from a classical (F9) FCV isolate. In yet another embodiment, the second nucleic acid construct is derived from a feline calicivirus isolate. One is derived from a virulent systemic feline calicivirus isolate, and the other is a classical (F9-like) feline It encodes two FCV antigens derived from calicivirus isolates.

[0024] In yet other embodiments, the immunogenic composition comprises an alphavirus comprising a first nucleic acid construct. one set of RNA replicon particles and / or naked DNA vectors and a second nucleic acid Alphavirus RNA replicon particles and / or naked DNA vectors containing the constructs and another set of alphavirus RNA replicon particles comprising a third nucleic acid construct, and and / or a third set of naked DNA vectors. One nucleic acid construct encodes a FeLV antigen or an antigenic fragment thereof, and the second nucleic acid construct encodes a The construct contains feline calicivirus (FCV) antigens derived from the virulent systemic feline calicivirus. or an antigenic fragment thereof, and the third nucleic acid construct encodes a classical (F9-like) feline mosquito mosquito. Feline calicivirus (FCV) antigens or antigenic fragments thereof derived from caliciviruses Code the

[0025] In yet other embodiments, the immunogenic composition comprises an alphavirus comprising a first nucleic acid construct. one set of RNA replicon particles and / or naked DNA vectors and a second set of Alphavirus RNA replicon particles and / or naked DNA vectors containing the acid constructs and another set of alphavirus RNA replicon particles containing a third nucleic acid construct. a third set of naked DNA vectors and / or an alphavirus containing a fourth nucleic acid construct; and a fourth set of RNA replicon particles and / or naked DNA vectors. In certain embodiments of this type, the first nucleic acid construct is derived from a virulent systemic feline calicivirus. Feline calicivirus (FCV) antigens or antigenic fragments thereof, and classical (F9-like) feline calicivirus (FCV) antigen derived from feline calicivirus or The nucleotide sequence encodes both the nucleotide sequence and the antigenic fragment.

[0026] In yet other embodiments, the immunogenic composition comprises an alphavirus comprising a first nucleic acid construct. a set of RNA replicon particles and / or naked DNA vectors and a second nucleic acid construct Another example of an alphavirus RNA replicon particle and / or naked DNA vector containing and a third nucleic acid construct comprising an alphavirus RNA replicon particle and / or a third set of naked DNA vectors and a fourth set of alphavirus RNA containing nucleic acid constructs. A. A fourth set of replicon particles and / or naked DNA vectors and a fifth nucleic acid construct Alphavirus RNA replicon particles and / or naked DNA vectors containing In such an embodiment, the first nucleic acid construct, the second nucleic acid construct, The nucleotide sequences of the third nucleic acid construct, the fourth nucleic acid construct and the fifth nucleic acid construct are In certain embodiments of this type, the first nucleic acid construct is a vector for the highly virulent systemic feline calicivirus. a feline calicivirus (FCV) antigen or an antigenic fragment thereof derived from the virus; and feline calicivirus (FCV) antibodies derived from classical (F9-like) feline caliciviruses. The antigen-binding fragment encodes both the antigen or an antigenic fragment thereof.

[0027] Thus, the immunogenic compositions of the present invention induce protective immunity against non-FCV pathogens. Alphavirus R containing a nucleic acid construct encoding at least one non-FCV antigen for use in the The vector may contain NA replicon particles and / or naked DNA vectors. In certain embodiments, the non-FCV antigen is a tag derived from feline herpesvirus (FHV). In another embodiment, the non-FCV antigen is a feline leukemia virus (FeL) protein antigen. In yet another embodiment, the non-FCV antigen is a protein antigen derived from feline FCV. In yet another embodiment, the antigen is a protein antigen derived from a pneumovirus (FPN). Non-FCV antigens are protein antigens derived from feline parvovirus (FPV). In yet another embodiment, the non-FCV antigen is derived from feline infectious peritonitis virus (FIPV). In yet other embodiments, the non-FCV antigen is a protein antigen. In yet another embodiment, the non-FCV antigen is a protein antigen derived from rabies. In yet other embodiments, the non-FCV antigen is a protein antigen derived from a virus. In yet another embodiment, the antigen is a protein antigen derived from Luna Disease Virus (BDV). Non-FCV antigens are protein antigens derived from feline influenza viruses. In this embodiment, the non-FCV antigen is a antigen derived from feline pancytopenia virus (FPLV). In yet another embodiment, the non-FCV antigen is a feline coronavirus ( In yet other embodiments, the non-FCV antigen is a protein antigen derived from FCoV. The protein antigen is derived from feline rhinotracheitis virus (FVR). In this study, non-FCV antigens were protein antigens derived from Chlamydophila felis.

[0028] The present invention further provides a method for the production of a medicament for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of a medicament for the treatment of cancer, the method ... Alphavirus RNA replicas encoding one or more antigens or antigenic fragments thereof A composition comprising a granule containing one or more modified live / attenuated or killed feline pathogens. Combined immunogenic compositions and / or vaccines (multivalent vaccines) are provided. In this embodiment, the immunogenic composition comprises an antigen derived from FeLV or an antigenic fragment thereof. Modified live or dead alphavirus RNA replicon particles combined with In another embodiment, the immunogenic composition further comprises an anti-Chlamydophila felis antibody. Alphavirus RNA encoding antigens or antigenic fragments thereof derived from LV Modified live or killed feline rhinotracheitis virus (FV) combined with replicon particles In yet another embodiment, the immunogenic composition further comprises an antibody derived from FeLV. Alphavirus RNA replicon particles encoding the antigen or its antigenic fragments Further comprising a combination of modified live or killed feline pancytopenia virus (FPL). In certain embodiments, the vaccine comprises an immunization with one or more of these immunogenic compositions. The therapeutically effective amount is included.

[0029] In a more specific embodiment, the immunogenic composition comprises a capsid derived from a VS-FCV. Alphavirus RNA replicas encoding proteins or antigenic fragments thereof The present invention also includes modified live or dead F9-like FCVs. In this embodiment, the immunogenic composition comprises a capsid protein or a capsid protein derived from VS-FCV. comprises an alphavirus RNA replicon particle encoding an antigenic fragment thereof, and further, modified live or dead F9-like FCV, modified live or dead Chlamydovum. Ira Feliz, Modified Live or Dead FVR, and Modified Live or Dead FPL In a related embodiment, the immunogenic composition also comprises an antigen derived from FeLV. or an antigenic fragment thereof. In certain embodiments of this type, the FeLV feline antigen is a FeLV viral glycoprotein. In particular embodiments, the present invention provides immunogenic compositions comprising: The present invention provides a vaccine comprising an immunologically effective amount of one or more of the following:

[0030] In certain embodiments, the alphavirus RNA replicon particles of the present invention comprise a VS-FC V capsid protein or an antigenic fragment thereof. In embodiments, the VS-FCV capsid protein has the amino acid sequence of SEQ ID NO: 2 and 95 In a more specific embodiment of this type, the amino acid sequence includes an amino acid sequence having an identity of at least V The S-FCV capsid protein has 98% or more identity with the amino acid sequence of SEQ ID NO:2. In a further more specific embodiment of this type, the VS-FCV protein comprises an amino acid sequence having the amino acid sequence The capsid protein comprises the amino acid sequence of SEQ ID NO: 2. In a specific embodiment of this type The VS-FCV capsid protein contains the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 12. It is encoded by the code sequence.

[0031] In a related embodiment, the alphavirus RNA replicon particles of the invention are FCV Encoding an F9-like capsid protein or an antigenic fragment thereof. In a preferred embodiment, the FCV F9-like capsid protein has the amino acid sequence of SEQ ID NO:4. In a more specific embodiment of this type, the amino acid sequence has an amino acid sequence having 95% or more identity to The FCV F9-like capsid protein has a 98% or higher identity with the amino acid sequence of SEQ ID NO:4. In a further specific embodiment of this type, the FCV comprises an amino acid sequence having identity thereto. The F9-like capsid protein comprises the amino acid sequence of SEQ ID NO: 4. A specific example of this type In embodiments, the FCV F9-like capsid protein is SEQ ID NO: 3 or SEQ ID NO: 13 It is encoded by the nucleotide sequence

[0032] In certain embodiments, the alphavirus RNA replicon particles of the invention comprise FeLV glycoproteins. In a specific embodiment of this type, the gene encodes the FeLV glycoprotein (gp85). Protein gp85 has an amino acid sequence having 95% or more identity with the amino acid sequence of SEQ ID NO:6. In a more specific embodiment of this type, the FeLV glycoprotein (gp85) comprises , comprising the amino acid sequence of SEQ ID NO: 6. In an even more specific embodiment of this type, FeL The V glycoprotein (gp85) is identified by the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:14. It is coded as:

[0033] In a related embodiment, the FeLV glycoprotein gp70 has the amino acid sequence of SEQ ID NO:8 In a more specific embodiment of this type, the amino acid sequence has an amino acid sequence having 95% or more identity to The FeLV glycoprotein (gp85) comprises the amino acid sequence of SEQ ID NO: 8. In an even more specific embodiment, the FeLV glycoprotein (gp70) is SEQ ID NO: 7 or or encoded by the nucleotide sequence of SEQ ID NO:15.

[0034] In yet another embodiment, the alphavirus RNA replicon particles of the present invention are In a specific embodiment of this type, the rabies virus glycoprotein (G) is encoded by the rabies virus glycoprotein (G). The glycoprotein has an amino acid sequence having 95% or more identity with the amino acid sequence of SEQ ID NO: 10. In a more specific embodiment of this type, the rabies virus glycoprotein (G ) comprises the amino acid sequence of SEQ ID NO: 10. In a further more specific embodiment of this type, The rabies virus glycoprotein (G) has the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:16. Coded by columns.

[0035] The present invention further includes vaccines, including multivalent vaccines, that comprise the immunogenic compositions of the present invention. In certain embodiments, the vaccine is a non-adjuvanted vaccine. The vaccine helps prevent disease caused by FCV. Immunization with cutin induces antibodies in feline subjects.

[0036] The present invention also provides a method of immunizing a cat against a feline pathogen, such as FCV, comprising administering to the cat a therapeutically effective amount of the present invention. The method includes administering to a cat an immunologically effective amount of the vaccine or polyvalent vaccine. In certain embodiments, the vaccine is administered via intramuscular injection. The vaccine is administered via subcutaneous injection. In other embodiments, the vaccine is administered via intravenous injection. In yet other embodiments, the vaccine is administered via intradermal injection. In other embodiments, the vaccine is administered via oral administration. The vaccine is administered via intranasal administration. In a specific embodiment, the cat is a domestic cat. do.

[0037] The vaccines and multivalent vaccines of the present invention may be used as a primer vaccine and / or a booster vaccine. In a specific embodiment, the vaccine of the present invention can be administered as a It is administered as a single-dose vaccine without the need for subsequent administration. In embodiments, in the case of administration of both a primer vaccine and a booster vaccine, Immediate and booster vaccines can be administered by the same route. In certain such embodiments, the primer vaccine and the booster vaccine are both In an alternative embodiment, the primer vaccine and booster vaccine are administered by subcutaneous injection. - In the case of administration of both vaccines, the administration of the primer vaccine is by one route, The booster vaccine can be administered by another route. In this condition, the primer vaccine can be administered by subcutaneous injection, and the booster vaccine The compound can be administered orally.

[0038] The present invention further provides a method for immunizing a cat against FCV, comprising administering to the cat a vaccine as described above. In certain embodiments, the vaccine is administered to a cat in a manner that is effective against the disease. For example, about 1 x 10 4 ~Approx. 1×10 10It can contain RPs or more. In a more particular embodiment, the vaccine comprises about 1 x 10 5 ~Approx. 1×10 9 May contain RP In an even more specific embodiment, the vaccine comprises about 1 x 10 6 ~Approx. 1×10 8 R P. In certain embodiments, the cat is a domestic cat.

[0039] In certain embodiments, the vaccines of the invention are administered in doses of 0.05 mL to 3 mL. In a more particular embodiment, the administered dose is between 0.1 mL and 2 mL. In certain embodiments, the administered dose is between 0.2 mL and 1.5 mL. In some embodiments, the administered dose is between 0.5 mL and 2.0 mL. In an even more particular embodiment, the administered dose is 0.3 to 1.0 mL. The recommended dose is 0.4 mL to 0.8 mL.

[0040] These and other aspects of the present invention are better understood by reference to the following detailed description. It will be well understood. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention provides an effective and safe FCV vaccine. In certain embodiments, the vaccine In one aspect, the vaccine of the present invention is used to treat feline injection site sarcoma. Although it does not induce upper respiratory tract disease and / or In certain embodiments, the FCV vaccine helps protect the vaccinee from lameness or lameness syndrome. The capsid protein is derived from virulent systemic FCV (VS-FCV). In this state, the FCV capsid protein is expressed in more specific forms, such as the FCV F9 strain (F9-like FCV). It originates from an old stock.

[0042] Thus, the vaccine compositions of the present invention enhance protective immunity in recipient vaccinated animals. Immunoglobulins encoding antigens from one or more strains of feline calicivirus that aid in induction. Furthermore, the present invention improves the reliability of vaccination and provides effective doses of FCV vectors. Helps reduce feline upper respiratory tract disease in infected cats, thereby making the disease relatively transient or To provide new immunogenic compositions that result in milder disease and / or reduced infections. In certain embodiments of the invention, the vaccine comprises, for example, VS-FCV, or alternatively, F Alphaviruses encoding FCV capsids derived from classical strains such as CV F9-like strains Contains RNA replicon particles (RP).

[0043] In a more specific embodiment, the vaccine is a vaccine for Venezuelan equine encephalitis virus (VEE). It contains capsid proteins and glycoproteins, and the FCV capsid proteins and / or an alphavirus RNA replicon encoding an antigenic fragment thereof In an even more specific embodiment, the vaccine comprises avirulent T cells of VEE. C-83 strain capsid protein and glycoprotein, and one or more FC Encoding the V capsid protein and / or one or more antigenic fragments thereof Contains alphavirus RNA replicon particles (RP).

[0044] In another embodiment of the invention, the vaccine comprises one or more FCV capsid proteins and / or or one or more antigenic fragments thereof. The vaccine was administered to cats in the absence of adjuvant, and the vaccinated cats were then treated with F It can still effectively help protect against CV.

[0045] In order to provide a more complete understanding of the present invention, the following definitions are provided.

[0046] The use of singular terms for convenience of description is in no way intended to be limiting. Thus, for example, reference to a composition containing a "polypeptide" does not include reference to such a composition. Furthermore, the term "alphavirus RNA" includes reference to one or more of the polypeptides. Reference to "replicon particles" refers to multiple such alphaviruses unless otherwise indicated. Includes reference to RNA replicon particles.

[0047] As used herein, the term "approximately" is used interchangeably with the term "about" and means The value must be within 50% of the stated value, i.e., "approximately" 1 per milliliter. x10 8 1 ml of a composition containing alphavirus RNA replicon particles 0.5 x 10 8 ~1.5×10 8 Contains alphavirus RNA replicon particles It means to do.

[0048] As used herein, the term "cat" refers to any member of the Felidae family. Purebred and / or mixed breed pet cats, and wild or feral cats are both cats. do.

[0049] As used herein, the term "replicon" refers to a vector that, if present, is a vector that is expressed in a cell culture medium. one or more that may be able to successfully propagate the parent virus to a food or animal host. A modified RNA virus genome lacking the above elements (e.g., coding sequences for structural proteins) In the context of a suitable cell, the replicon amplifies itself and produces one or more subgenomes. can produce mu RNA species.

[0050] As used herein, the term "alphavirus RNA replicator" abbreviated as "RP" refers to The "con particle" is described, for example, in the article by Pushko et al. [Virology 239(2):389-4 01 (1997)], structural proteins, such as Alphavirus-derived capsid and glycoprotein-enclosed alphavirus-derived The replicon is an RNA replicon. The replicon is a structural component of the alphavirus (e.g., capsid). RPs do not encode amino acids and glycoproteins, and therefore cannot be used in cell culture or animal hosts ( They are unable to propagate (without the aid of a helper plasmid or similar component).

[0051] The terms "FCV F9-like" and "F9-like FCV" are used interchangeably with each other and with the term "classical FCV." FCV strain F9 is a typical representative when used herein. It can be characterized as a relatively old, pre-universal vaccine strain of FCV, which is thought to be In contrast, highly virulent systemic "VS-FCV" or as defined herein FCVs called "(VS)FCVs" are a relatively new class of FCVs. It is highly virulent and can be neutralized by antibodies raised against FCV F9-like strains. [U.S. Patent No. 7,449,323; Radford et al., 38(2) Vet res 319-335(2007)].

[0052] The term "non-FCV" refers to the respective pathogen and / or antigen (or immunogen) of a FCV. Neither the pathogen nor the FCV antigen (or immunogen), but a non-FCV protein antigen ( or immunogen) is not derived from FCV. (or immunogen).

[0053] The terms "originate from" and "originat" "es from" and "originating from" are given The classification is based on the protein antigens and the pathogens or strains of pathogens that naturally encode them. As used herein, the term "antigen" refers to the unmodified and unmodified forms of a given protein antigen. and / or the truncated amino acid sequence is encoded by the pathogen or a strain of the pathogen. In the nucleic acid construct of the present invention, a protein antigen derived from a pathogen is encoded. The sequence of a gene is determined by its mutation in the pathogen or strain of the pathogen from which it is derived (including naturally attenuated strains). Alteration of the amino acid sequence of the expressed protein antigen relative to the corresponding sequence of the protein antigen. and / or may be genetically engineered to result in truncation.

[0054] As used herein, the terms "protect" or "provide protection" or " "Induces protective immunity," "helps prevent disease," and "helps protect" are used to describe For example, "helps protect" does not require complete protection from any symptoms of In addition, the symptoms of the underlying infection are at least reduced and / or the underlying One or more of the cellular, physiological, or biochemical causes or mechanisms underlying the reduction and It can also mean that the protection is sufficient so that the protection is / is excluded. When used in a clinical setting, "reduced" refers to the degree of sensitivity, including not only the physiological state of infection but also the molecular state of infection. It should be understood to mean relating to the symptoms of infection.

[0055] As used herein, a "vaccine" refers to a vaccine administered to an animal, e.g., a cat (in certain embodiments). and a water-containing composition suitable for application to a human (including humans, but in other embodiments not specific to humans). The formulation contains one or more antigens, typically combined with a pharmaceutically acceptable carrier, such as a liquid containing and a composition comprising, when administered to an animal, a compound selected from the group consisting of: a compound of formula (I) and a medicament for the treatment of a disease caused by an infection with a wild-type microorganism; potent enough to minimally assist in the prevention of disease and / or the prognosis of disease Induce a strong enough immune response to aid in prevention, amelioration or cure.

[0056] As used herein, a multivalent vaccine is a vaccine that contains two or more different antigens. In certain embodiments of this type, the multivalent vaccine is directed against two or more different pathogens. stimulates the recipient's immune system.

[0057] The terms "adjuvant" and "immunostimulant" are used interchangeably herein and refer to In this context, adjuvants are defined as one or more substances that cause stimulation of the system. is used to enhance the immune response to one or more vaccine antigens / isolates. Therefore, an "adjuvant" nonspecifically increases the immune response to a specific antigen, In this case, the amount of antigen required for any given vaccine and / or the suitability for the antigen of interest may be It is a drug that reduces the frequency of injections required to produce an adequate immune response. Adjuvants are used to enhance the immune response to one or more vaccine antigens / isolates. For example, the American Association of Felin e Practitioners Feline Vaccination Guide lines suggest the use of a non-adjuvanted FeLV vaccine [AAFP Fe line Advisory Panel,15:785-808(2013)].

[0058] As used herein, a "non-adjuvanted vaccine" is a vaccine that does not contain an adjuvant. It is a single or multivalent vaccine.

[0059] As used herein, the term "pharmaceutically acceptable" means that the modified noun is Used adjectively to mean suitable for use in medicine. "Publicly acceptable" may be used to describe an excipient in, for example, a pharmaceutical vaccine. If so, it must be compatible with the other components of the composition and not be too inconvenient for the intended recipient animal, e.g., a cat. Characterize excipients as not harmful in any way.

[0060] Parenteral administration includes subcutaneous injection, submucosal injection, intravenous injection, intramuscular injection, intradermal injection, and Injection included.

[0061] As used herein, the term "antigen" refers to a specific protein (e.g., a protein antigen). The term "antigenic fragment" refers to a fragment that is antigenic, i.e., that recognizes an antigen of the immune system, e.g. that can specifically interact with globulins (antibodies) or T-cell antigen receptors A fragment of a protein, such as the antigenic flag of the FCV capsid protein. A fragment is a fragment of the capsid protein that is antigenic. Therefore, the antigenic fragments of the FCV capsid protein contain major B cell epitopes. Preferably, the antigenic fragment of the present invention is a fragment that can be used to treat antibodies and In certain embodiments, a given tandem For protein antigens, an antigenic fragment has at least 25% of the antigenicity of the full-length protein. In a preferred embodiment, the fragment is a fragment of the protein that retains the antigenic fragment. The fragment retains at least 50% of the antigenicity of the full-length protein. In embodiments, the antigenic fragment retains at least 75% of the antigenicity of the full-length protein. Antigenic fragments can be as small as 20 amino acids or as large as In some cases, it is a large fragment of the full-length protein that is missing only one amino acid. In certain embodiments, the antigenic fragment comprises 25 to 150 amino acid residues. In another embodiment, the antigenic fragment contains 50 to 250 amino acid residues. For example, in the case of FeLV, the FeLV gp45 glycoprotein and the FeLV gp7 The 0 glycoprotein is an antigenic fragment of the FeLV gp85 glycoprotein.

[0062] As used herein, when the amino acid residues of two sequences are identical, one amino acid is A sequence of amino acids is 100% "identical" to a second sequence of amino acids, or Therefore, if 50% of the amino acid residues in two amino acid sequences are identical, If the amino acid sequence is 50% "identical" to the second amino acid sequence, the amino acid sequence is 50% "identical" to the second amino acid sequence. Proteins, for example, amino acids contained in a portion of a protein or polypeptide to be compared In certain embodiments, the cleavage is performed on a contiguous block of amino acid residues that would normally be separated by two amino acid residues. Selected deletions or insertions that may alter the correspondence between amino acid sequences are taken into consideration. do.

[0063] As used herein, percent identity of nucleotide and amino acid sequences is Default parameters for alignment and identity Together with C, MacVector (MacVector, Inc. Cary, NC 2 7519), Vector NTI(Informax, Inc MD), Oxford Molecular Group PLC (1996) and Clustal Work These commercially available programs may be used to determine the same or similar It can also be used to determine sequence similarity using the default parameters. Alternatively, for example, GCG (Genetics Group Carbohydrate Genetics Group) can be used with default parameters. Computer Group,Program Manual for the GC G Package, Version 7, Madison, Wisconsin Use the install program to set up the Advanced A Blast search can be used.

[0064] As used herein, the term "inactivated" microorganisms is used interchangeably with the term "killed" microorganisms. For purposes of the present invention, an "inactivated" microorganism is one that is capable of inducing an immune response in an animal. The antigens of the present invention (e.g., inactivated Feline calicivirus) is killed by binary ethyleneimine, formalin, and beta-propiolactone. may be inactivated by an agent selected from the group consisting of thimerosal, thimerosal, or heat. In a specific embodiment, the inactivated feline calicivirus isolate combined with the RP of the present invention The strain is inactivated by binary ethyleneimine.

[0065] The alphavirus RNA replicon particles of the present invention may be lyophilized and diluted with sterile water. Alternatively, alphavirus RNA replicon particles may be stored separately. However, if it is intended to be mixed with other vaccine components before administration, the alphavirus RN A replicon particle is stored in a stabilizing solution of these components, e.g., a high sucrose solution. It is possible.

[0066] The vaccines of the present invention may be administered intravenously, intramuscularly, subcutaneously, orally, intranasally, intradermally and / or intraperitoneally. It can be readily administered by any standard route, including intravenous vaccination. If so, the vaccine composition is preferably compatible with a variety of recipient animals and administration routes. It will be understood that the formulation will be appropriate.

[0067] Thus, the present invention also provides a method for immunizing cats against FCV and / or other feline pathogens. One such method involves administering an immunologically effective amount of the vaccine of the present invention to a mammal. This involves injecting the cat with FCV vaccine, so that the cat produces the appropriate FCV antibodies.

[0068] Multivalent vaccines The present invention also provides multivalent vaccines. For example, proteins useful in feline vaccines The coding sequence of the antigen or antigenic fragment thereof, or such a protein antigen The combination of coding sequences may be used to encode FCV [e.g., FCV capsid protein] in a vaccine. The feline antigens were encoded by alphavirus RNA replicon particles (RPs). In a specific embodiment, alpha- The viral RNA replicon particles contain FCV F9-like capsid proteins or their antigenic fragments and VS-FCV capsid proteins or antigenic fragments thereof Such a multivalent vaccine would therefore be suitable for use in the present invention. Included in the invention.

[0069] Examples of pathogens from which one or more of such protein antigens may be derived include cat nasal Feline leukemia virus (FVR), feline leukemia virus (FeLV), feline pancytopenia virus (FPL) Feline herpesvirus (FHV), other FCV strains, feline parvovirus (FP V), feline infectious peritonitis virus (FIPV), feline immunodeficiency virus, Borna disease virus rabies virus, feline influenza virus, canine influenza virus flu, avian influenza, canine pneumovirus, feline pneumovirus, chlamydovitis Ira Feliz (FKA Chlamydia psit taci), Bordetella bronchiseptica septica) and Bartonella spp. (e.g., B. henselae) In certain embodiments, these feline pathogens or of capsid proteins or similar proteins from one or more canine pathogens. The coding sequence can be inserted into the same RP as the FCV antigen, or in combination with it. In combination, capsids derived from one or more of these feline or canine pathogens The coding sequence for the RP or a similar protein can be inserted into one or more other RPs. and the FCV F9-like capsid protein or its antigenic fragments can be used in a vaccine. fragment and / or VS-FCV capsid protein or antigenic fragment thereof The RP can be combined with an RP encoding the target gene.

[0070] Additionally, one or more other live attenuated virus isolates, e.g., live attenuated FCV F9-like viruses. live attenuated feline herpesvirus (e.g., modified live FCV F9) and / or live attenuated feline herpesvirus and and / or live attenuated feline parvovirus and / or live attenuated feline leukemia virus and and / or live attenuated feline infectious peritonitis virus and / or live attenuated feline immunodeficiency virus and and / or live attenuated Borna disease virus and / or live attenuated rabies virus and / or is a live attenuated feline influenza virus and / or a live attenuated canine influenza virus and / or live attenuated avian influenza and / or live attenuated canine pneumovirus and and / or live attenuated feline pneumovirus together with one or more antigens of FCV [e.g., FCV F9-like capsid protein or an antigenic fragment thereof and / or VS - an alpha-antibody encoding an FCV capsid protein or an antigenic fragment thereof In addition, live attenuated chlamydofumis can be added. Bordetella felis and / or live attenuated Bordetella bronchiseptica and / or live attenuated Toxic Bartonella species (e.g., B. henselae) could also be included in such multivalent vaccines. Cut.

[0071] Additionally, one or more other killed virus isolates, e.g., killed FCV strains and / or killed Killed feline herpesvirus and / or killed feline parvovirus and / or killed cat Leukemia virus and / or killed feline infectious peritonitis virus and / or killed feline immunoglobulin Epidemic-deficient virus and / or killed Borna disease virus and / or killed rabies virus and / or killed feline influenza virus and / or killed canine influenza Virus and / or killed avian influenza virus and / or killed canine influenza virus Feline pneumovirus and / or killed feline pneumovirus, along with one or more antigens of FCV [e.g., FCV F9-like capsid protein or an antigenic fragment thereof and / or or VS-FCV capsid protein or an antigenic fragment thereof] Alphavirus RNA replicon particles (RP) can be added. Dophylla felis and / or Bordetella bronchiseptica and / or Baltic Bacterins (or subfractions of bacterins, e.g., fimbriae) of the genus B. hensela (e.g., B. henselae) Subfractions) can also be included in such multivalent vaccines.

[0072] Additionally, the present invention is not limited to the specific configurations, process steps, and materials disclosed herein. It is understood that the compositions, process steps, and materials may vary somewhat. The scope of the present invention is to be limited only by the appended claims and their equivalents. Therefore, the terminology used herein is used only for the purpose of describing particular embodiments. It should further be understood that no limitation is intended. [Table 1]

[0073] array Feline calicivirus capsid (VS-FCV) SEQ ID NO: 1 atggctgacgacggatctgtgaccaccccagaacaagga acaatggtcggaggagtgatt gccgaacccagcgctcagatgtcaactgcggcggacatg gcctccggaaagtcggtggac tccgagtgggaagccttcttctcgttccacacgtccgtg aactggagcacctccgaaacc caaggaaagatcctcttcaagcagtccctgggtcccctg ctgaacccgtacctggagcac atcagcaagctgtacgtcgcttggagcgggtcgatcgaa gtgcgattttccatctcggga agcggcgtgttcggtggtaaactggccgccatcgtcgtg ccgcctggtgtcgaccctgtc cagtcaacctccatgctgcagtacccgcacgtcctgttc gacgcaagacaagtggagcca gtgatcttctccatcccggacctccgcaacagcctgtat cacttgatgtccgataccgat accacttccctcgtgatcatggtgtacaacgatctgatc aacccgtacgccaatgactcc aacagctcgggttgcatcgtgaccgtcgaaacgaagcct ggcatcgatttcaagtttcat ctgctgaaaccgcccggatccatgcttactcacgggtcc atcccttccgatctgatcccc aagagctcctccctgtggattgggaaccgccactggacc gatattaccgatttcgtgatt cggcctttcgtgttccaagccaaccggcacttcgacttc aaccaggagactgccggctgg tcaactccacggttccgcccattggccgtgactgtgtcg cagtcaaagggagccaagctc gggaacggcatcgccaccgactacattgtgcctggaatc cccgacggatggcctgatact accatccccaccaagctgacccctaccggagattacgcc atcacctcctccgacggcaat gatattgaaaccaagctggaatacgagaacgcggacgtg attaagaacaacaccaacttc cgctccatgtatatctgcggaagcctccagagggcttgg ggcgacaagaagatcagcaac accgggttcatcactaccggagtgatttctgacaactcc atcagcccttcgaacacaatt gaccagtccaagatcgtggtgtaccaggacaaccatgtc aattcggaggtccagactagc gacatcactcttgccatcctgggctacaccggaattgga gaagaggccataggcgccaac cgggactccgtcgtgagaatttccgtgcttccggaaact ggagcaaggggcggaaatcac cccatcttctacaaaaattccatgaagctgggctacgtg atctcctccattgacgtgttc aactcccaaatcctccacacctcgcgccagctgtcactg aacaactacttgttgccccct gactccttcgcggtgtaccggattattgacagcaacgga tcatggttcgacattgggatt gacagcgatgggttttcattcgtgggcgtgtcgtcattt ccaaagctggagtttccgctg tccgcctcatacatgggcatccagctcgcaaagatccgg ctggcgtccaacatccggtca tccatgactaagctgtga

[0074] Feline calicivirus capsid (VS-FCV) SEQ ID NO: 2 MADDGSVTTPEQGTMVGGVIAEPSAQMSTAADMASGKSV DSEWEAFFSFHTSVNWSTSET QGKILFKQSLGPLLNPYLEHISKLYVAWSGSIEVRFSIS GSGVFGGKLAAIVVPPGVDPV QSTSMLQYPHVLFDARQVEPVIFSIPDLRNSLYHLMSDT DTTSLVIMVYNDLINPYANDS NSSGCIVTVETKPGIDFKFHLLKPPGSMLTHGSIPSDLI PKSSSLWIGNRHWTDITDFVI RPFVFQANRHFDFNQETAGWSTPRFRPLAVTVSQSKGAK LGNGIATDYIVPGIPDGWPDT TIPTKLTPTGDYAITSSDGNDIETKLEYENADVIKNNTN FRSMYICGSLQRAWGDKKISN TGFITTGVISDNSISPSNTIDQSKIVVYQDNHVNSEVQT SDITLAILGYTGIGEEAIGAN RDSVVRISVLPETGARGGNHPIFYKNSMKLGYVISSIDV FNSQILHTSRQLSLNNYLLPP DSFAVYRIIDSNGSWFDIGIDSDGFSFVGVSSFPKLEFP LSASYMGIQLAKIRLASNIRS SMTKL

[0075] Feline calicivirus (VS-FCV) capsid (SEQ ID NO: 12) auggcugacgacggaucugugaccacccgaacaagga acaauggucggaggagugauu gccgaacccagcgcucagaugucaacugcggcggacaug gccuccggaaagucgguggac uccgagugggaagccuucuucucguuccacacguccgug aacuggagcaccuccgaaacc caaggaaagauccucuucaagcagucccuggguccccug cugaacccguaccuggagcac aucagcaagcuguacgucgcuuggagcgggucgaucgaa gugcgauuuuccaucucggga agcggcguguucggugguaaacuggccgccaucgucgug ccgccuggugucgacccuguc cagucaaccuccaugcugcaguacccgcacguccuguuc gacgcaagacaaguggagcca gugaucuucuccaucccggaccuccgcaacagccuguau cacuugauguccgauaccgau accacuucccucgugaucaugguguacaacgaucugauc aacccguacgccaaugacucc aacagcucggguugcaucgugaccgucgaaacgaagccu ggcaucgauuucaaguuucau cugcugaaaccgcccggauccaugcuuacucacgggucc aucccuuccgaucugaucccc aagagcuccucccuguggauugggaaccgccacuggacc gauauuaccgauuucgugauu cggccuuucguguuccaagccaaccggcacuucgacuuc aaccaggagacugccggcugg ucaacuccacgguuccgcccauuggccgugacugugucg cagucaaagggagccaagcuc gggaacggcaucgccaccgacuacauugugccuggaauc cccgacggauggccugauacu accauccccaccaagcugaccccuaccggagauuacgcc aucaccuccuccgacggcaau gauauugaaaccaagcuggaauacgagaacgcggacgug auuaagaacaacaccaacuuc cgcuccauguauaucugcggaagccuccagagggcuugg ggcgacaagaagaucagcaac accggguucaucacuaccggagugauuucugacaacucc aucagcccuucgaacacaauu gaccaguccaagaucgugguguaccaggacaaccauguc aauucggagguccagacuagc gacaucacucuugccauccugggcuacaccggaauugga gaagaggccauaggcgccaac cgggacuccgucgugagaauuuccgugcuuccggaaacu ggagcaaggggcggaaaucac cccaucuucuacaaaaauuccaugaagcugggcuacgug aucuccuccauugacguguuc aacucccaaauccuccacaccucgcgccagcugucacug aacaacuacuuguugcccccu gacuccuucgcgguguaccggauuauugacagcaacgga ucaugguucgacauugggauu gacagcgauggguuuucauucgugggcgugucgucauuu ccaaagcuggaguuuccgcug uccgccucauacaugggcauccagcucgcaaagauccgg cuggcguccaacauccgguca uccaugacuaagcuguga

[0076] Feline calicivirus (F9-like) capsid (SEQ ID NO: 3) atgactgccccggaacaaggaacgatggtcggaggagtg attgcagaaccgtcagcacag atgtccaccgctgccgacatggccactggaaagagcgtg gactccgaatgggaagccttc ttctccttccacacttcggtcaactggtcgactagcgaa acccaggggaagattttgttc aagcaatccctcggccctctgctgaacccctacctggag catctggccaagctgtacgtg gcatggtcgggcagcatcgaagtgcgctttagcatttcc ggctccggagtgttcggggga aagcttgctgccattgtcgtgccgccaggagtggacccg gtgcagtccacttctatgctc caatacccgcatgtcctgttcgacgccagacaggtggag cctgtgatcttttgcctgccg gatctcaggtccaccctgtatcacctcatgtccgacacc gacaccacctcgctcgtgatc atggtgtacaacgacctgatcaacccctacgctaacgac gccaacagctcaggttgcatt gtgactgtcgaaaccaagccaggccctgacttcaagttt catttgctgaagccgcccggt tccatgctgacccacggctcgatcccatccgacctgatc cccaagacgagctccctgtgg atcggaaaccgctactggtccgatattaccgacttcgtg atcagaccattcgtgttccaa gccaaccgccatttcgacttcaaccaggaaaccgcagga tggtcgacccctcgattccgc ccgatttcagtgaccatcaccgaacagaacggcgcgaag ctgggaattggcgtggcgacc gactacatcgtgccgggaatcccggatggatggcctgat acgaccattcccggggagctg atccctgccggggactacgccatcaccaacggtactgga aacgacatcaccactgccacc ggttacgacaccgccgacatcataaagaacaacaccaac ttcagaggaatgtacatttgc ggctccctgcaacgcgcttggggtgacaaaaagatctcg aacactgccttcatcacaaca gcgactctggacggcgataacaacaacaagatcaatcct tgtaataccatcgaccagtcc aaaatcgtggtgttccaggataaccacgtgggaaagaag gcgcagacctccgacgacact ctggcgctgcttggctacaccgggatcggcgagcaggcc attggaagcgatcgggatcgg gtcgtgcggatctccaccctccccgagactggagcaagg ggaggcaaccaccccatcttt tacaaaaacagcattaagctcggatacgtcatccgctcc atcgatgtgttcaactctcaa atcctgcacacttcgcggcagctgtccctgaaccactac ctcttgccgcccgactccttc gccgtctaccggatcattgattcgaacgggagctggttc gacatcggcattgatagcgat ggcttctcgtttgtgggcgtgtcgggcttcgggaagctg gagttcccactgagcgcctca tacatgggtatccagctggccaagatcaggctggcctcc aacatccgctcacctatgact aagctgtga

[0077] Feline calicivirus (F9-like) capsid (SEQ ID NO: 4) MTAPEQGTMVGGVIAEPSAQMSTAADMATGKSVDSEWEA FFSFHTSVNWSTSETQGKILF KQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFG GKLAAIVVPPGVDPVQSTSML QYPHVLFDARQVEPVIFCLPDLRSTLYHLMSDTDTTSLV IMVYNDLINPYANDANSSGCI VTVETKPGPDFKFHLLKPPGSMLTHGSIPSDLIPKTSSL WIGNRYWSDITDFVIRPFVFQ ANRHFDFNQETAGWSTPRFRPISVTITEQNGAKLGIGVA TDYIVPGIPDGWPDTTIPGEL IPAGDYAITNGTGNDITTATGYDTADIIKNNTNFRGMYI CGSLQRAWGDKKISNTAFITT ATLDGDNNNKINPCNTIDQSKIVVFQDNHVGKKAQTSDD TLALLGYTGIGEQAIGSDRDR VVRISTLPETGARGGNHPIFYKNSIKLGYVIRSIDVFNS QILHTSRQLSLNHYLLPPDSF AVYRIIDSNGSWFDIGIDSDGFSFVGVSGFGKLEFPLSA SYMGIQLAKIRLASNIRSPMT KL

[0078] Feline calicivirus (F9-like) capsid (SEQ ID NO: 13) augacugccccggaacaaggaacgauggucggaggagug auugcagaaccgucagcacag auguccaccgcugccgacauggccacuggaaagagcgug gacuccgaaugggaagccuuc uucuccuuccacacuucggucaacuggucgacuagcgaa acccaggggaagauuuuguuc aagcaaucccucggcccucugcugaaccccuaccuggag caucuggccaagcuguacgug gcauggucgggcagcaucgaagugcgcuuuagcauuucc ggcuccggaguguucggggga aagcuugcugccauugucgugccgccaggaguggacccg gugcaguccacuucuaugcuc caauacccgcauguccuguucgacgccagacagguggag ccugugaucuuuugccugccg gaucucagguccacccuguaucaccucauguccgacacc gacaccaccucgcucgugauc augguguacaacgaccugaucaaccccuacgcuaacgac gccaacagcucagguugcauu gugacugucgaaaccaagccaggcccugacuucaaguuu cauuugcugaagccgccggu uccaugcugacccacggcucgaucccauccgaccuugauc cccaagacgagcuccugugg aucggaaccgcuacuggugccgauauuaccgacuucggug aucagaccauucguuguccaa gccaaccgccauuucgacuacaaccgaaacgcagga uggucgaccccucgauuccgc ccgauuucagugaccaucaccgaacagaaccggcgcgaag cugggaauuggcguggcgacc gacuacaucgugccgggaaucccgguggauggccugau acgaccauucccggggagcug aucccugccgggggacuacgccaucaccaacgguacugga aacgacaucaccacugccacc gguuacgacaccgccgacaucauaaagaacaacaccaac uucagagaauguacauuugc ggcuccugcaacgcgcuuggggugacaaaaagaucucg aacacugcccuucaucacaaca gcgacucuggacggcggcauaacaacaacaagaucaauccu uguaauaccaucgaccagucc aaaaucgugguguuccagguaaaccacgugggaaagaag gcgcagaccuccgacgacacu cuggcgcugcuuggcuacaccgggaucggcgagcaggcc auuggaagcgaucgggaucgg gucgugcggaucuccacccuccccgagacuggagcaagg ggaggcaaccaccccaucuuu uacaaaaacagcauuaagcucggauacgucauccgcucc aucgauguguucaacucucaa auccugcacacuucgcggcagcugucccugaaccacuac cucuugccgcccgacuccuuc gccgucuaccggaucauugauucgaacgggagcugguuc gacaucggcauugauagcgau ggcuucucguuugugggcgugucgggcuucgggaagcug gaguucccacugagcgccuca uacauggguauccagcuggccaagaucaggcuggccucc aacauccgcucaccuaugacu aagcuguga

[0079] Feline leukemia virus envelope glycoprotein (gp85) SEQ ID NO: 5 atggagtcaccaacacaccctaaaccttctaaagacaaa accctctcgtggaatctcgccttccttgt gggcatcctgttcacaatcgacatcggcatggccaaccc ttcgccgcatcagatctacaatgtgacat gggtcattactaatgtgcagacaaacacccaggcaaatg ctacttctatgcttggtactctgactgat gcttatccaaccctgcacgtcgacctttgcgatctcgtc ggtgacacatgggagcccatcgtgctgaa tccaactaatgtcaaacatggtgccaggtattcttctag caaatacgggtgtaagaccactgatcgga agaaaacagcaacaaacctacccattctacgtgtgcccgg gtcacgcaccgtccctgggtccgaaggga acacattgtgggggagcccaagacggtttttgcgctgct tggggttgtgaaacaaccggagaagcctg gtggaagcctacctcatcttgggactacattactgtgaa aagaggctctagccaggataacagctgcg aagtaagtgtaatcccctggtgcttcaattcacccaga aaggccggcaggcatcatgggatggaccg aaaatgtggggacttagactctatcgcaccggatacgac cccatcgctctgtttactgtgtcacgcca agtctccaccattactccgccacaggccatgggggggaa tctggtcctccccgatcagaagccaccct cacggcaaagtcaaaccggctcaaaagtggccacccaac ggccccagacaaatgagtccgcacctagg tcagtggcacctacaacaatgggtccaaagcggatcgga accggagacaggctcattaacctcgtgca aggacttatctggcccttaacgctactgaccccaaaaa gaccaaggattgctggctctgccttgtga gcagacctccttactatgaggggatcgccattctcggaa actactcaaatcagaccaacccccctccg tcgtgtctgagcaccccccagcacaagcttactatttca gaagtcagtggacagggaatgtgcatcgg aaccgtgccaaagactcatcaagccctttgcaacaaaac tcaacaagggcacactggagctcattatc tcgccgcacctaacgggacctactgggcttgcaatactg gattgaccccgtgtatctctatggccgtg ctgaattggacttccgacttctgcgtgcttattgagctt tggcctagagtgacataccatcagcctga gtacgtctatacccatttcgccaaggcagtcagattccg gcgggagcctatctccctgactgtggcct tgatgctcggtggactgacagtgggaggaattgcagctg gagtcggaactggaaccaaggccctgctc gaaactgctcagttccggcagctgcagatggccatgcac actgacatccaggctctggaggaatcaat ttcagcccttgagaaaagcttgacctcgctgtctgaagt ggtcctccaaaacaggcgcggtttggaca tcctgttccttcaagagggtggtctgtgcgccgctctca aggaggaatgctgtttctacgctgaccat accgggctggtgcgcgataacatggcaaagctgcgggaa cgcttgaaacagaggcagcaactgttcga ctctcagcagggatggttcgagggctggtttaacaagag cccatggtttaccactctgatctcttcaa tcatgggtccactgctcatcctgcttctgattcttctct tcggaccgtgtattctcaacaggctggtg cagtttgtcaaggacagaatctcggtggtccaggccctg attcttactcagcagtatcagcagattaa gcagtacgaccccgatcggccttga

[0080] Feline leukemia virus envelope glycoprotein (gp85) SEQ ID NO: 6 MESPTHPKPSKDKTLSWNLAFLVGILFTIDIGMANPSPH QIYNVTWVITNVQTNTQANAT SMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGA RYSSSKYGCKTTDRKKQQQTY PFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAW WKPTSSWDYITVKRGSSQDNS CEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPI ALFTVSRQVSTITPPQAMGPN LVLPDQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTM GPKRIGTGDRLINLVQGTYLA LNATDPNKTKDCWLCLVSRPPYYEGIAILGNYSNQTNPP PSCLSTPQHKLTISEVSGQGM CIGTVPKTHQALCNKTQQGHTGAHYLAAPNGTYWACNTG LTPCISMAVLNWTSDFCVLIE LWPRVTYHQPEYVYTHFAKAVRFRREPISLTVALMLGGL TVGGIAAGVGTGTKALLETAQ FRQLQMAMHTDIQALEESISALEKSLTSLSEVVLQNRRG LDILFLQEGGLCAALKEECCF YADHTGLVRDNMAKLRERLKQRQQLFDSQQGWFEGWFNK SPWFTTLISSIMGPLLILLLI LLFGPCILNRLVQFVKDRISVVQALILTQQYQQIKQYDP DRP*

[0081] Feline leukemia virus envelope glycoprotein (gp85) SEQ ID NO: 14 auggagucaccaacacacccuaaaccuucuaaagacaaa acccucucguggaaucucgccuuccuugu gggcauccuguucacaaucgacaucggcauggccaaccc uucgccgcaucagaucuacaaugugacau gggucauuacuaauugugcagacaaacaccccaggcaaaug cuacuucuaugcuugguacucugacugau gcuuauccaacccugcacgucgaccuuugcgaucucguc ggugacacaugggagcccaucgugcugaa uccaacuaaugucaaacauggugccagguauucuucuag caaauacggguguaagaccacugaucgga agaaacagcaacaaaccuacccauucuacgugugcccgg guacgcaccguccuggguccgaagggga acacauugugggggagcccaagacgguuuuugcgcugcu ugggguguugugaaacaaccggagaagccug guggaagccuaccucaucuugggacuacauuacugugaa aagaggcucuaggcagguaacagcugcg areaaaguguaauccccuggugcuucaauucacccaga aaggccggcaggcaucaugggauggaccg aaaaugggggacuuagacucuaucgcaccggauacgac cccaucgcucuguuuacugugucaccca agucuccaccauuuacuccgccacaggccaugggggggaa ucugguccuccccgaucagaagccacccu cacggcaagucaaccggcucaaaaguggccacccaac ggccccagacaaaugaguccgcaccuagg ucaguggcacuacaacaauggguccaaagcggaucgga accggagacaggcucauuaaccucgugca agggacuuaucuggcccuuaacgcuacugaccccaacaa gaccaaggauuugcuggcuccugcuugugu gcagaccuccuuacuaugagggggaucgccauuucucggaa acuacucaaaucagaccaaccccccuccg ucgugucugagcaccccccagcacaagcuuacuauuuuca gaagucaguggacagggaaugugcaucgg aaccgugccaaagacucaucaagcccuuugcaacaaaac ucaacaagggcacacuggagcucauuauc ucgccgcaccuaacgggaccuacugggcuugcaauacug gauugaccccguguaucucuauggccgug cugaauuggacuuccgacuucugcgugcuuauugagcuu uggccuagagugacauaccaucagccuga guacgucuauacccauuucgccaaggcagucagauuccg gcgggagccuaucucccugacuguggccu ugaugcucgguggacugacagugggaggaauugcagcug gagucggaacuggaaccaaggcccugcuc gaaacugcucaguuccggcagcugcagauggccaugcac acugacauccaggcucuggaggaaucaau uucagcccuugagaaaagcuugaccucgcugucugaagu gguccuccaaaacaggcgcgguuuggaca uccuguuccuucaagaggguggucugugcgccgcucuca aggaggaaugcuguuucuacgcugaccau accgggcuggugcgcgauaacauggcaaagcugcgggaa cgcuugaaacagaggcagcaacuguucga cucucagcagggaugguucgagggcugguuuaacaagag cccaugguuuaccacucugaucucuucaa ucauggguccacugcucauccugcuucugauucuucucu ucggaccguguauucucaacaggcuggug caguuugucaaggacagaaucucggugguccaggcccug auucuuacucagcaguaucagcagauuaa gcaguacgaccccgaucggccuuga

[0082] Feline leukemia virus envelope glycoprotein (gp70) SEQ ID NO: 7 aatcctagtccacaccaaatatataatgtaacttgggta ataaccaatgtacaaactaacacc caagctaacgccacctctatgttaggaaccttaaccgat gcctaccctaccctacatgttgac ttatgtgacctagtgggagacacctgggaacctatagtc ctaaacccaaccaatgtaaaacac ggggcacgttactcctcctcaaaatatggatgtaaaact acagatagaaaaaaacagcaacag acataccccttttacgtctgccccggacatgccccctcg<00012�3>ttggggccaaagggaacacattgt ggaggggcacaagatgggttttgtgccgcatggggatgt gagaccaccggagaagcttggtgg aagcccacctcctcatgggactatatcacagtaaaaaga gggagtagtcaggacaatagctgt gagggaaaatgcaaccccctggttttgcagttcacccag aagggaagacaagcctcttgggac ggacctaagatgtggggattgcgactataccgtacagga It should be noted that there may be an error in the tag "ID=29" in the original text, which is likely " " instead of "<00012�3>". This has been corrected in the translation for consistency. tatgaccctatcgctttattcacg gtgtcccggcaggtatcaaccattacgccgcctcaggca atgggaccaaacctagtcttacct gatcaaaaacccccatcccgacaatctcaaacagggtcc aaagtggcgacccagaggccccaa acgaatgaaagcgccccaaggtctgttgcccccaccacc atgggtcccaaacggattgggacc ggagataggttaataaatttagtacaagggacataccta gccttaaatgccaccgaccccaac aaaactaaagactgttggctctgcctggtttctcgacca ccctattacgaagggattgcaatc ttaggtaactacagcaaccaaacaaacccccccccatcc tgcctatctactccgcaacacaaa ctaactatatctgaagtatcagggcaaggaatgtgcata gggactgttcctaaaacccaccag gctttgtgcaataagacacaacagggacatacaggggcg cactatctagccgcccccaacggc acctattgggcctgtaacactggactcaccccatgcatt tccatggcggtgctcaattggacc tctgatttttgtgtcttaatcgaattatggcccagagtg acttaccatcaacccgaatatgtg tacacacattttgccaaagctgtcaggttccgaaga

[0083] Feline leukemia virus envelope glycoprotein (gp70) SEQ ID NO: 8 NPSPHQIYNVTWVITNVQTNTQANATSMLGTLTDAYPTL HVDLCDLVGDTWEPIVLNPTNVKHGARYSSS KYGCKTTDRKKQQQTYPFYVCPGHAPSLGPKGTHCGGAQ DGFCAAWGCETTGEAWWKPTSSWDYITVKRG SSQDNSCEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYR TGYDPIALFTVSRQVSTITPPQAMGPNLVLP DQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTMGPKR IGTGDRLINLVQGTYLALNATDPNKTKDCWL CLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTIS EVSGQGMCIGTVPKTHQALCNKTQQGHTGAH YLAAPNGTYWACNTGLTPCISMAVLNWTSDFCCVLIELWP RVTYHQPEYVYTHFAKAVRFRR

[0084] Feline leukemia virus envelope glycoprotein (gp70) SEQ ID NO: 15 aauccuaguccacaccaaauauauaauguaacuugggua auaaccaauguacaaacuaacacc caagcuaacgccaccucuauguuaggaaccuuaaccgau gccuacccuacccuacauguugac uuaugugaccuagugggagacaccugggaaccuauaguc cuaaacccaaccaauguaaaacac ggggcacguuacuccuccucaaaauauggauguaaaacu acagauagaaaaaacagcaacag acauaccccuuuuaacgucugccccggcaugcccccucg uugggggccaaaggggaacacauugu ggaggggcacaagauggguuuugugccgcaugggggaugu gagaccaccggagaagcuuggugg aagccccaccuccucaugggacuauaucacaguaaaaaaga gggaguagucaggacauagcu gagggaaaugcaacccccugguuugcaguucacccag aagggaacaacccuugggac ggaccuaagaugugggggauugcgacuauaccguaacgga uaugacccuaucgcuuuauucacg gugucccggcagguaucaaccauuacgccgccucaggca augggaccaaaccuagucuuaccu gaucaaaaacccccaucccgacaaucucaaacagggucc aaaguggcgacccagaggccccaa acgaaugaaagcgccccaaggucuguugcccccaccacc augggucccaaacggauugggacc ggagauagguuaauaaauuuaguacaaggggacauaccua gccuuaaugccaccgaccccaac aaaacuaaagacuguuggcucugccugguuucucgacca cccuauuacgaagggauugcaauc uuagguaacuacagcaaccaaacaaccccccccccaucc ugccuaucuacuccgcaacacaaa cuaacuauaucugaaguaucagggcaaggaaugugcaua gggacuguuccuaaaacccaccag gcuuugugcaauaagacacaacagggacauacaggggcg cacuaucuagccgcccccaacggc accuauugggccuguaacacuggacucaccccaugcauu uccauggcggugcucaauuggacc ucugauuuuugugucuuaaucgaauuauggcccagagug acuuaccaucaacccgaauaugug uacacacauuuugccaaagcugucagguuccgaaga

[0085] Rabies virus G (SEQ ID NO: 9) atggtgccgcaggctctcctgtttgtcccccttctggtc tttccattgtgttttgggaaattccctatctacacaattc cggacaagttgggaccctggagcccaattgacattcatc atctcagctgcccgaacaatttggtcgtggaggacgaagg atgcaccaacctgtcggggttctcctacatggaattgaa agtcggatacatcagtgccattaagatgaacgggttcact tgcacaggcgtcgtgactgaagctgagacatacactaac ttcgtgggatatgtcactaccactttcaaaagaaagcatt tccgccctactcctgatgcttgtagggccgcatacaact ggaagatggccggtgaccccagatatgaggaatcacttca caatccgtaccctgactaccactggcttcggactgtcaa aaccaccaaggagtcactcgtgatcattagtccaagtgtg gctgatcttgacccatacgaccggtcacttcactcacgg gtgttcccgggggggaattgctctggtgtcgcagtgtcgt caacctactgctccacaaaccacgattacaccatttgga tgccagaaaatcctcggcttggtatgtcatgtgacatttt caccaattctcgggggaagagggcttccaaagggtctga aacttgcggctttgtcgatgagcggggcttgtataagtca cttaaaggtgcttgcaaactcaagctttgtggtgtcttg ggattgagattgatggatggaacttgggtcgcaatgcaga cttctaacgaaaccaaatggtgccctcccggacagcttg tgaatttgcatgactttcgctctgacgaaattgagcatct tgtcgtcgaggagttggtcaagaagcgggaagagtgtct ggatgctttggaatcaatcatgaccaccaagtcagtgtct ttcagacggctctcacatcttaggaaattggtgccaggt tttggaaaagcatataccattttcaacaagacccttatgg aagccgatgctcactacaagtctgtcaggacttggaatg agatcatcccgtctaaagggtgtcttagggtcggagggag atgtcatcctcatgtcaacggagtctttttcaatggtat cattcttggacctgacggaaatgtccttatccctgagatg caatcttccctctccagcaacacatggaacttcttgtc tcatcggtcatcccccttatgcacccctggctgacccat caaccgtgttcaagaacggtgacgaggcagaggattttg tcgaggtccaccttccgatgtgcatgaacggatctctgg tgtcgaccttggactccctaactggggaaagtatgtcct tctgtcggcaggagccctgactgccttgatgttgattatc ttcctgatgacttgttggaggagagtcaatcggtcggag ccaacacaacataatctcagaggaacaggaagggaggtgt cagtcacaccccaaagcgggaagatcatttcgtcttggg agtcatacaagagcggaggtgaaaccggactgtga

[0086] VirusG MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIH HLSCPNNLVVEDEGCTNLSGF SYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTT TFKRKHFRPTPDACRAAYNWK MAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVA DLDPYDRSLHSRVFPGGNCSG VAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRA SKGSETCGFVDERGLYKSLKG ACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNL HDFRSDEIEHLVVEELVKKRE ECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNK TLMEADAHYKSVRTWNEIIPS KGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSL LQQHMELLVSSVIPLMHPLAD PSTVFKNGDEAEEDFVEVHLPDVHERISGVDLGLPNWGKY VLLSAGALTALMLIIFLMTCW RRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESYKSG GETGL*

[0087] Rabies virus G (SEQ ID NO: 16) auggugccgcaggcucuccuguuugucccccuucugguc uuuccauuguguuuugggaaauucccuaucuacacaauuc cggacaaguugggacccuggagcccaauugacaucauc aucucagcugcccgaacaauuuggucguggaggacgaagg augcaccaaccugucgggguucuccuacauggaauugaa agucggauacaucagugccauuaagaugaacggguucacu ugcacaggcgucgugacugaagcugagacauacacuaac uucgugggauaugucacuaccacuuucaaaagaaagcauu uccgcccuacuccugaugcuuguagggccgcauacaacu ggaagauggccggugaccccagauaugaggaaucacuuca caauccguacccugacuaccacuggcuucggacugucaa aaccaccaaggagucacucgugaucauuaguccaagugug gcugaucuugacccauacgaccggucacuucacucacgg guguucccgggggggaauugcucuggugucgcagugucgu caaccuacugcuccacaaaccacgauuacaccauuugga ugccagaaaauccucggcuugguaugucaugugacauuuu caccaauucucgggggaagagggcuuccaaagggucuga aacuugcggcuuugucgaugagcggggcuuguauaaguca cuuaaaggugcuugcaaacucaagcuuuguggugucuug ggauugagauugauggauggaacuugggucgcaaugcaga cuucuaacgaaaccaaauggugcccucccggacagcuug ugaauuugcaugacuuucgcucugacgaaauugagcaucu ugucgucgaggaguuggucaagaagcgggaagagugucu ggaugcuuuggaaucaaucaugaccaccaagucagugucu uucagacggcucucacaucuuaggaaauuggugccaggu uuuggaaaagcauauaccauuuucaacaagacccuuaugg aagccgaugcucacuacaagucugucaggacuuggaaug agaucaucccgucuaaagggugucuuagggucggagggag augucauccucaugucaacggagucuuuuucaauguau cauucuuggaccugacggaaauguccuuaucccugagaug caaucuucccuccuccagcaacacauggaacuucuuguc ucaucggucaucccccuuaugcacccccuggcugacccau caaccguguucaagaacggugacgaggcagaggauuuug ucgagguccaccuucccgaugugcaugaacggaucucugg ugucgaccuuggacucccuaacuggggaaaguauguccu ucugucggcaggagccugacugccuugauguugauuauc uuccugagacuuguuggaggagagucaaucggucggag ccaacacaacauaaucucagaggaacaggaagggaggugu cagucacaccccaaagcgggaagaucauuucgucuuggg agucauacaagagcggagggugaaaccggacuguga

[0088] The following examples serve to provide further appreciation of the invention, but do not limit the scope of the invention. is in no way meant to limit

[0089] [Example] [Example 1] Incorporation of the coding sequence for FCV capsid proteins into alphavirus RNA replicon particles Incorporation of Introduction RNA viruses are vectors for introducing genetically engineered vaccine antigens into their genomes. However, their use to date has been limited to RNA viruses. Primarily limited to incorporating viral antigens and then introducing the virus into the recipient host As a result, protective antibodies are induced against the incorporated viral antigens. Virus RNA replicon particles are used to encode pathogen antigens. Alphavirus replicon platforms such as Venezuelan equine encephalitis virus (VEE) (VEE) [Pushko et al., Virology 239:389-401 (1997) ], Sindbis (SIN) [Bred enbeek et al., Journal of Virology 67:6439-6446 (1993)] and Semliki Forest virus (SFV) [which is hereby incorporated in its entirety]. Liljestrom and Garoff, Biotechno, incorporated herein by reference. logy (NY) 9:1356-1361 (1991)]. Furthermore, alphavirus RNA replicon particles have been developed from It is the basis for several USDA-licensed vaccines for cats and poultry. These include: Porcine Epidemic Diarrhea Vaccine (RNA Particles) (Product Code 19U5.P1), Swine Flu Avian influenza vaccine (RNA) (product code 19A5.D0), Avian influenza vaccine ( RNA) (product code 19O5.D0) and formulation (RNA particles) (product code 9PP 0.00).

[0090] Construction of alphavirus RNA replicon particles The amino acid sequence of the FCV capsid protein was used to perform in silico codon optimization ( The optimized sequences were generated by a commercial supplier (ATU The VS-F was prepared as synthetic DNA by the National Institute of Standards and Technology (NIS), Newark, CA. The amino acid sequences of CV capsid protein and FCV F9-like capsid protein Based on the above, synthetic genes for VS-FCV capsid protein [SEQ ID NO: No. 2] or the amino acid sequence of FCV F9-like capsid protein [SEQ ID NO: 4]. The construct to be loaded is then cloned into a suitable flag for cloning into an alphavirus replicon plasmid. The gene was codon-optimized for cats to have a linking sequence.

[0091] As previously described [see U.S. Patent No. 9,441,247; the contents of which are incorporated by reference]. [incorporated herein by reference thereto], with the following modifications to express the FCV capsid protein: A VEE replicon vector was constructed that was designed to express the AscI and Using PacI, the TC-83-derived replicon vector "pVEK" [U.S. Patent No. 9,444,445] was cloned. 41,247] was digested with the restriction enzymes AscI and Pac I was used to clone one of the FCV capsid protein genes (FCV F9-like or V Codon-optimized open reading frame nucleotide sequence of either S-FCV and a 5' flanking sequence (5'-GGCGCGCCGCACC-3') [SEQ ID NO: 11] and a DNA probe containing the 3' flanking sequence (5'-TTAATTAA-3'). The plasmid was then similarly digested. The synthetic gene fragment was then inserted into a similarly digested pVEK vector. The sets were ligated and synthesized to form FCV F9-like and VS-FCV capsid proteins, respectively. The resulting clones encoding the protein were designated "pVHV-F9" and "pVHV-Kal The "pVHV" vector was named after the multiple cloning site of pVEK. The transgene cassette was cloned via the AscI and PacI sites in was chosen to refer to a pVEK-derived replicon vector containing

[0092] To generate the dual construct, the VEE subgenomic promoter and the FCV Kale The pVHV vector region encoding the m(VS-FCV) capsid sequence was synthesized by PCR. The pV between the 3' end of the F9 FCV capsid sequence and the VEE 3'UTR sequence was removed. The subgenomic promoter sequence was overlapped and ligated into the HV-F9 vector. Confirmation of the FCV Kalem capsid sequence was performed using a plasmid designated "pVHV-F9-Kalem." This was achieved by sequencing the final vector clones.

[0093] TC-83 RNA replicon particles (RP) were produced as previously described [U.S. Pat. No. 6,233,166]. Nos. 9,441,247 and 8,460,913; the entire contents of which are incorporated herein by reference.

[0043] Briefly, the pVHV replicon vector was After linearization of the target DNA and helper DNA plasmids using NotI restriction enzyme, , MegaScript T7 RNA polymerase and cap analogue (Prome In vitro transcription was performed using a genomic DNA library (Genomic DNA Library, Madison, WI). The helper RNA used for production was derived from the VEE subgenomic promoter, as previously described. It lacks a promoter sequence [Kamrud et al., J Gen Virol 91 (Pt 7):1723-1727(2010)]. Purified RNA of replicon and helper components were combined, mixed with a suspension of Vero cells, electroporated in a 4 mm cuvette, and Opt iPro® SFM cell culture medium (Thermo Fisher, Waltham After overnight incubation, the ZetaPlus BioCap was The suspension was passed through a depth filter (3M, Maplewood, MN) and filtered with 5% sucrose. (w / v) with phosphate-buffered saline containing 400 mM NaCl buffer. The alphavirus was removed from the cells and medium by eluting the retained RP with PBS. The eluted RNA replicon particles were purified by centrifugation in a final 5% sucrose (w / v), filtered through a 0.22 micron membrane filter, and then aliquoted for storage. The titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers. It was decided that:

[0094] [Example 2] Evaluation of the efficacy and safety of a dual-construct FCV vaccine in cats Capsid protein of the avirulent TC-83 strain of Venezuelan equine encephalitis virus (VEE) and glycoproteins from two different FCV strains, a highly virulent systemic strain (VS-FCV) and and growth factor receptors encoding capsid proteins derived from classical vaccine strains (FCV F9-like) Dual-construct vaccine containing defective RNA particles (RP) is formulated in 5% sucrose and stored frozen. As shown in Table 1 below, this dual construct vaccine was used to protect against two FCV strains. Two groups of 10 cats each were administered at 13-14 weeks of age. Then, 21 days later, they were vaccinated with the dual construct FCV vaccine in a prime / boost regimen. Two groups of control cats received the same regimen of cell culture medium (minimum essential oils with Earle's salts). They were given a placebo vaccine consisting of EMEM (enhanced medium). [Table 2]

[0095] After vaccination, any local or systemic reactions to the vaccine and FCV sensitivity Observing cats for clinical signs of infection will help identify any adverse reactions to the vaccine. No adverse reactions were observed in any of the vaccinated cats.

[0096] Three weeks after the booster vaccination, cats in groups 1 and 2 were vaccinated with FCV strain 255( The vaccine was administered intranasally with a virulent culture of the classical FCV-causing strain. Three weeks after the vaccination, cats in groups 3 and 4 were infected with a virulent systemic FCV-inducing strain (FCV strain Kale). The mice were challenged by intranasal administration of a virulent culture of B. tumefaciens (B. tumefaciens).

[0097] Cats were observed for clinical signs of FCV infection for 14 days after challenge as follows: Cats were observed for mortality, depression / lethargy, body temperature, nasal and oral ulcers, nasal and ocular discharge, Clinical signs, including lameness, dehydration, and sneezing, were scored daily. Each clinical sign observed was recorded with a score of severity and clinical signs. Each cat was then assigned a weighted numerical score based on the number of days the condition was observed. A total weighted score was assigned based on the sum of the weighted scores. The mean and mean scores for each treatment group were then calculated. A median weighted score was calculated. For a challenge to be considered effective, 80% of the control cats had to be immunized. Clinical signs of FCV infection (other than fever) must be observed. The results of challenge are shown in the table below. Summarized in 2. [Table 3]

[0098] 100% of placebo-vaccinated control cats showed clinical signs of FCV infection (other than fever) Therefore, challenge was considered to be effective against both strains.

[0099] Dual-construct RP-FCV vaccine encoding virulent systemic and classical vaccine strains of FCV. Chin tested two different FCV strains, namely, a classical FCV strain and a highly virulent systemic FCV strain. The experimental vaccine was found to be safe for cats.

[0100] [Example 3] Evaluation of inhibition by administration of two different RNA particle vaccines Alphavirus RNA replication, including serological response, efficacy against challenge, and inhibition Studies were conducted to evaluate multiple aspects of the conjugated FCV vaccine. The RP-FCV construct vaccine encoding the capsid protein of the Cutin strain (F9) was It was formulated in a stabilizer consisting of gelatin, NZ amine, and sucrose and freeze-dried. Two groups of mice were vaccinated with RP-FCV vaccine at 17 weeks of age. After 21 days, the mice in the first group Cats in the second group received only a booster dose of the RP-FCV vaccine, while cats in the third group received only a booster dose of the RP-FCV vaccine. A booster dose of CV vaccine was administered simultaneously with a fixed dose of RP-rabies vaccine as shown in Table 3. The RP-rabies virus vaccine was administered to the same TC-83 VEE alphavirus. A construct encoding the rabies virus glycoprotein (G) in a genomic DNA platform. do. [Table 4]

[0101] After each vaccination, the cat should be monitored for any local or systemic reactions to the vaccine. Cats were observed for any adverse reactions to the vaccine. No adverse reactions were observed in any of the cats.

[0102] First vaccination date (study day 0), booster vaccination date (study day 21), and Cats were bled and serum was collected 6 weeks after the first vaccination (study day 42). Sera were tested for antibody titers against FCV F9 by serum neutralization assay. Rapid fluorescent focus inhibition test (RFFIT) was used to measure antibody titers against rabies virus. Serum was also tested. RIFFT results are reported as International Units / mL (IU / mL). The serological results are summarized in Tables 4 and 5 below. [Table 5]

[0103] Based on a comparison of FCV (F9) antibody titers (to serum samples collected after the booster) Simultaneous administration of RP-rabies virus vaccine has been shown to have a protective effect against RP-FCV (F9) vaccine. It does not inhibit the antibody response. [Table 6]

[0104] The study did not include a control group that received only the RP-rabies virus vaccine. To compare the post-booster rabies titers of the second group, previous data from other studies were used. The data is shown in Table 6 below. [Table 7]

[0105] RP - Based on rabies virus vaccine efficacy and post-vaccination rabies antibody titers Prior vaccination with RP-FCV vaccine increases antibody response to RP-rabies virus vaccine. Vector immunity is a concern for platform-based vaccines. However, the results of this study suggest that multiple RP-based vaccines can be used in animals without compromising efficacy. This suggests that it can be used.

[0106] Co-vaccination with RP-rabies virus inhibits the efficacy of RP-FCV(F9). The study was amended and continued to ensure that no unvaccinated controls were To help with this, an additional group of five age-matched cats was added to the study. Seventy-nine days after vaccination, all cats were challenged intranasally with a highly virulent classical FCV strain (FCV 255). This was given and triggered.

[0107] Over a 14-day period after challenge, the following clinical signs of FCV infection were observed: death, depression / lethargy, , fever, nasal and oral ulcers, nasal and ocular discharge, lameness, dehydration and sneezing The cats were observed and scored daily. After challenge, weight was measured over 4 days at intervals of 14 days. Each clinical sign observed was graded based on severity and the number of days the clinical sign was observed. Each cat was then given a weighted numerical score based on the sum of the weighted scores for each day. A total weighted score was assigned. The mean and median weighted scores for each treatment group were then calculated. For challenge to be considered effective, 80% of control cats had to show clinical signs of FCV infection (fever). The results of the challenge are summarized in the table below. [Table 8]

[0108] 100% of unvaccinated control cats showed clinical signs of FCV infection (other than fever). Both vaccinated groups (Group 1 and Group 2) showed a significant improvement in the immunization efficacy. , were significantly protected from virulent FCV challenge (p value 0.012 for both groups).

[0109] Based on a comparison of clinical scores, simultaneous vaccination with RP-rabies virus vaccine This experimental vaccine is not effective against cats. It was found to be safe.

[0110] [Example 4] Vaccination of cats with RP constructs encoding a single FCV F9-like capsid protein Evaluation of Cutin Efficacy Al, encoding a single FCV F9-like capsid protein (RP-FCV F9) To further evaluate the efficacy of a feline vaccine containing fa virus RNA replicon particles This study was conducted to compare the vaccine with a placebo control group against classical FCV challenge. Cats were vaccinated with either this monovalent vaccine or a placebo and then tested for classical FCV Clinical scores were determined based on typical signs of FCV infection, primarily oral and external ulcers. The results were scored based on the severity of ulcers and rhinitis for 14 days after FCV challenge. The administration system was the same as that described in Examples 2 and 3 above. The mean clinical score for the controls was 92, whereas the mean clinical score for the RP-FCV F9 vaccine was 1. The score for the RP-FCV F9 vaccine was only 2. Scores obtained with two vaccines containing one live attenuated FCV F9-like virus separately This study further demonstrated that the R gene encoding the classical vaccine strain of FCV was significantly lower than that of the FCV gene. We demonstrate that the P-FCV vaccine protects cats against FCV F9-like viruses.

[0111] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein may be made in accordance with the foregoing description. Such modifications are intended to be within the scope of the appended claims. Something is intended to be.

[0112] Furthermore, all base sizes or amino acid sizes given for nucleic acids or polypeptides Size, and all molecular weights or molecular weight values ​​are approximate and are provided for illustrative purposes. I want you to understand that.

Claims

1. Alphavirus RNA replicon particles encoding feline calicivirus (FCV) antigens An immunogenic composition comprising the antibody.

2. The FCV antigen is a capsid protein or an antigenic fragment thereof. Item 1. The immunogenic composition according to item 1.

3. The alphavirus RNA replicon particles are Venezuelan equine encephalitis (VEE) alphavirus. The immunogenic composition according to claim 1 or 2, which is an antiviral RNA replicon particle.

4. The capsid protein is an FCV F9-like capsid protein, Antigenic fragment of 9-like capsid protein, highly virulent systemic FCV (VS-FCV) capsid protein, and antigenic fragments of said VS-FCV capsid protein The immunogenic composition of claim 2 or 3, wherein the immunogenic composition is selected from the group consisting of:

5. The capsid protein is a VS-FCV capsid protein or an antigenic fragment thereof. The immunogenic composition of claim 4, which is a fragment.

6. The capsid protein is an FCV F9-like capsid protein or an antigenic The immunogenic composition of claim 4, which is a fragment.

7. an additional alphavirus RNA replicon particle encoding a second FCV antigen; wherein the second FCV antigen is an FCV strain different from that from which the FCV antigen is derived.

7. The immunogenic composition of claim 1, 2, 3, 4, 5 or 6, derived from

8. Additional antibodies encoding FCV F9-like capsid proteins or antigenic fragments thereof 6. The immunogenic composition of claim 5, comprising an alphavirus RNA replicon particle of

9. The additional alphavirus RNA replicon particles are VEE alphavirus RNA replicon particles. The immunogenic composition of claim 7 or 8, which is a plicon particle.

10. The alphavirus RNA replicon particle comprises an FCV F9-like capsid protein or an antigenic fragment of said FCV F9-like capsid protein. The immunogenic composition of claim 5.

11. The VS-FCV capsid protein has at least 9 amino acid sequences identical to those of SEQ ID NO:

2.

11. The method of claim 4, 5, 7, 8, 9 or 10, comprising an amino acid sequence having 5% identity with the Immunogenic compositions of the present invention.

12. The FCV F9-like capsid protein has at least the amino acid sequence of SEQ ID NO:

4. 95% identical amino acid sequence to claim 4, 6, 7, 8, 9, 10 or 1 1. The immunogenic composition described in 1.

13. The immunogenic composition according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. and a pharmaceutically acceptable carrier. hmm.

14. At least one non-FCV antigen for eliciting protective immunity against non-FCV feline pathogens 14. The vaccine composition of claim 13, further comprising:

15. The non-FCV feline pathogens include feline herpesvirus (FHV), feline leukemia virus (FCV), and FeLV), feline pneumovirus (FPN), feline parvovirus (FPV), feline infectious Feline infectious peritonitis virus (FIPV), feline immunodeficiency virus, Borna disease virus (BDV) , feline influenza virus, feline pancytopenia virus (FPLV), feline coronavirus virus (FCoV), feline rhinotracheitis virus (FVR), Chlamydophila felis ( Chlamydophila felis) and any combination thereof The vaccine of claim 14, wherein the vaccine is selected.

16. The non-FCV antigen is feline herpesvirus (FHV), feline leukemia virus (FeL), or V), feline pneumovirus (FPN), feline parvovirus (FPV), feline infectious gastroenteritis Feline immunodeficiency virus (FIPV), Borna disease virus (BDV), cat Influenza virus, feline pancytopenia virus (FPLV), feline coronavirus (FCoV), feline rhinotracheitis virus (FVR), Chlamydophila felis and its a killed or attenuated non-FCV antigen selected from the group consisting of any combination of the above killed or attenuated non-FCV antigens; The vaccine of claim 14, wherein is an attenuated non-FCV antigen.

17. The attenuated non-FCV antigen is a modified live Chlamydophila felis, a modified live Neisseria gonorrhoeae, or a modified live Neisseria gonorrhoeae. Feline rhinotracheitis virus (FVR), modified live feline leukemia virus (FeLV), modified Live modified feline pancytopenia virus (FPL), live modified feline herpesvirus (F HV), modified live feline pneumovirus (FPN), modified live feline parvovirus feline infectious peritonitis virus (FPV), modified live feline infectious peritonitis virus (FIPV), modified live feline Immunodeficiency virus, modified live Borna disease virus (BDV), modified live feline coronavirus virus (FCoV) and modified live feline influenza virus.

17. The vaccine of claim 16, wherein the live modified feline pathogen is selected from the group consisting of:

18. At least one protein antigen or antigenic fragment thereof derived from a non-FCV antigen. alphavirus RNA replicon particles containing a nucleotide sequence encoding the 18. The vaccine composition of claim 13, 14, 15, 16 or 17, comprising

19. The protein antigen or antigenic fragment thereof derived from a non-FCV feline pathogen is Feline herpesvirus (FHV), feline leukemia virus (FeLV), feline pneumovirus feline parvovirus (FPN), feline parvovirus (FPV), feline infectious peritonitis virus (FIPV) ), feline immunodeficiency virus, Borna disease virus (BDV), feline influenza virus , feline pancytopenia virus (FPLV), feline coronavirus (FCoV), feline nasal tracheitis virus (FVR), Chlamydophila felis, and any combination thereof 19. The vaccine of claim 18, selected from the group consisting of:

20. 13, 14, 15, 16, 17, 18 or 19, which is a non-adjuvanted vaccine.

10. The vaccine composition according to claim 9.

21. A method for immunizing a cat against pathogenic FCV, comprising the steps of: Administering an immunologically effective amount of the vaccine described in 17, 18, 19 or 20 to the cat. and a method comprising:

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