Feline leukemia virus vaccine

Abstract

The present invention provides vaccines for feline leukemia virus, as well as methods for making and using the vaccines alone or in combination with other protective agents.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 582,050, filed November 6, 2017, U.S. Provisional Patent Application No. 62 / 596,508, filed December 8, 2017, and U.S. Provisional Patent Application No. 62 / 599,401, filed December 15, 2017, the entire contents of all of which are incorporated herein by reference.

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

[0003] Feline leukemia virus (FeLV) is a retrovirus that infects domestic cats and causes significant morbidity and mortality worldwide. FeLV is primarily transmitted via saliva, but transmission via contact with bodily fluids has also been reported [Pacitti et al., Vet Rec 118:381-384(1986)doi:10.1136 / vr.118.14.381; Levy et al., J Feline Med Surg 10:300-316(2008)doi:10.1016 / j.jfms2008.03.002]. Clinical signs observed in cats during FeLV infection include cytoproliferative disorders (lymphoid or myeloid tumors), cytosuppressive disorders (immunosuppression, anemia, infectious diseases associated with bone marrow suppression), inflammatory disorders, neurological disorders, abortion, and enteritis [Hoover et al., J Am Vet Med Assoc 199:1287-1297 (1991); Levy and Crawford, Textbook of Veterinary Internal Medicine, 6th ed. (Ettinger SJ, Feldman EC., eds.) WB Saunders, Philadelphia, PA (2005)]. The prevalence of antigenemia can vary from 1-5% in healthy cats to 15-30% in affected cats [Hosie et al. Veterinary Records, 128:293-297 (1989); Braley, Feline Practice 22:25-29 (1994); Malik et al., Australian Veterinary Journal 75:323-327 (1997); Arjona et al., Journal of Clinical Microbiology 38:3448-3449 (2000)]. FeLV often establishes persistent infection with persistent viremia, often leading to death in the host cat.

[0004] The FeLV RNA genome encodes only three genes: (i) the ENV gene, which encodes the envelope glycoprotein; (ii) the GAG ​​gene, which encodes the major structural components of the virus; and (iii) the POL gene, which encodes the RNA polymerase [Thomsen et al., Journal of General Virology 73:1819-1824 (1992)]. The FeLV envelope (ENV) gene encodes the gp85 precursor protein, which is proteolytically processed by one or more cellular enzymes to generate the major envelope glycoprotein gp70 and the associated transmembrane protein p15E [DeNoronha et al., Virology 85:617-621 (1978); Nunberg et al., PNAS 81:3675-3679 (1983)]. The transmembrane protein p15E contains a conserved sequence among gammaretroviruses with immunosuppressive properties [Mathes et al., Nature 274:687-689 (1978)]. Recently, the European Medicines Agency's Committee for Veterinary Medicinal Products (CVMP) adopted a positive opinion for a vaccine containing a recombinant p45 FeLV envelope antigen derived from the gp70 surface glycoprotein of FeLV subgroup A, expressed as the active substance in Escherichia coli. The FeLV envelope glycoprotein is the target of FeLV-specific cytotoxic T cell responses and neutralizing antibodies and is therefore one of the major immunogens of FeLV [Flynn et al. J Virol 76(5):2306-2315 (2002)].

[0005] Various factors, including the host's immune status, host age, the infecting FeLV strain, viral load, and route of exposure to FeLV, can all affect the ultimate outcome of that exposure. Veterinarians and researchers previously classified FeLV infections based on the relative persistence of associated antigenemia, with relatively fortunate cases resulting in transient antigenemia and / or resolution of the infection. Antigenemia assays include p27 enzyme-linked immunosorbent assay (ELISA), virus isolation, and immunofluorescence assays [Hoover et al., J Am Vet Med Assoc 199:1287-1297 (1991); Rojko and Kociba, J Am Vet Med Assoc 199:1305-1310 (1991)]. These tests remain useful not only for clinical applications but also for determining whether clinical disease is the result of actively circulating virus.

[0006] Currently, four vaccines for FeLV are available in the United States, including two whole-virus adjuvanted killed vaccines, a dual-adjuvanted, multiple-antigen vaccine, and a nonadjuvanted, canarypox virus-vectored vaccine. Notably, the various vaccines have been shown to have varying degrees of efficacy [Sparkes, J Small Anim Pract 38:187-194 (1997) doi:10.1111 / j.1748-5827.1997.tb03339.x.] Previous studies have demonstrated the efficacy of whole virus adjuvant inactivated vaccines after challenge, including testing vaccinated and unvaccinated cats for viral RNA, proviral DNA, FeLV antibodies, and p27 antigen [Hines et al., J Am Vet Med Assoc 199:1428-1430(1991); Pedersen, J Vet Intern Med 7:34-39(1993)doi:10.1111 / j.1939-1676.1993.tb03166.x.]; Torres et al., Vet Immunol Immunopathol 134:122-131(2010)doi:10.1016 / j.vetimm.2009.10.017]. There are limited data evaluating the efficacy of available non-adjuvanted recombinant FeLV vaccines [Stuke et al., Vaccine 32:2599-2603(2014)doi:10.1016 / j.vaccine.2014.03.016].

[0007] More recently, the efficacy of two commercially available feline leukemia vaccines, one inactivated whole virus vaccine and the other a live canarypox virus vector vaccine, was compared after challenge with virulent feline leukemia virus [Patel et al., Clinical and Vaccine Immunology 22(7):798-805(2015)]. This study again found that the whole virus adjuvanted inactivated vaccine provided superior protection against FeLV infection. However, the use of the whole virus inactivated adjuvanted FeLV vaccine has been implicated as a factor leading to the development of feline injection site sarcomas [Kass et al., J. AM Vet Med Assoc 203(3):396-405(1993)]. Subsequent studies have not established a direct link between the inactivated adjuvanted vaccine and feline injection site sarcomas, but the perception remains that non-adjuvanted vaccines are safer. In fact, the American Association of Feline Practitioners Feline Vaccination Guidelines suggest using a non-adjuvanted FeLV vaccine to reduce the risk of feline injection site sarcoma and decrease local inflammation [AAFP Feline Advisory Panel, 15:785-808(2013)].

[0008] Several vector strategies have been employed in vaccines over the years to protect against certain pathogens. One such vector strategy involves alphavirus-derived replicon RNA particles (RPs) developed from several different alphaviruses, including Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of Virology 67:6439-6446 (1993)], and Semliki Forest virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9:1356-1361 (1991)] [Vander Veen, et al. Anim Health Res Rev. 13(1):1-9 (2012) doi:10.1017 / S1466252312000011; Kamrud et al., J Gen 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)]. RP has an attractive safety and efficacy profile when compared to some conventional vaccine formulations [Vander Veen, et al. Anim Health Res Rev. 13(1):1-9 (2012)]. RP platforms have been used to encode pathogen antigens and are the basis for several USDA-licensed vaccines for swine and poultry.

[0009] Unfortunately, until now, pet owners have been forced to choose between (i) non-adjuvanted FeLV vaccines, which are considered safe but have been found to be significantly less effective than inactivated adjuvanted vaccines [see Stuke et al., Vaccine 32:2599-2603 (2014); Patel et al., Clin Vaccine Immunol 22(7):798-808 (2015)], and (ii) adjuvanted FeLV vaccines, which are highly effective but are recognized by some as causing injection site sarcomas. Thus, there is an ongoing need for improved, safe, non-adjuvanted FeLV vaccines that do not induce feline injection site sarcomas but still protect vaccinees from the debilitating disease state caused by FeLV infection as effectively as their inactivated whole virus adjuvanted vaccine counterparts.

[0010] The citation of any reference herein shall not be construed as an admission that such reference is available as "Prior Art" to the instant application. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Pacitti et al., Vet Rec 118:381-384(1986)doi:10.1136 / vr.118.14.381 [Non-patent document 2] Levy et al.,J Feline Med Surg 10:300-316(2008)doi:10.1016 / j.jfms2008.03.002 [Non-patent document 3] Hoover et al., J Am Vet Med Assoc 199:1287-1297(1991) [Non-patent document 4] Levy and Crawford, Textbook of Veterinary Internal Medicine, 6th ed (Ettinger SJ, Feldman EC., eds.) WB Saunders, Philadelphia, PA (2005)

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[0012] Thus, the present invention provides vectors encoding one or more feline leukemia virus (FeLV) antigens. Such vectors can be used in immunogenic compositions comprising these vectors. The immunogenic compositions of the present invention can be used in vaccines. In one aspect of the present invention, the vaccine protects a vaccinated subject (e.g., a mammal) against FeLV. In a specific embodiment of this type, the vaccinated subject is a cat. In a more specific embodiment, the vaccinated subject is a domestic cat. The present invention further provides combination vaccines for inducing protective immunity against FeLV and other diseases, such as other feline infectious diseases. Methods of making and using the immunogenic compositions and vaccines of the present invention are also provided.

[0013] In specific embodiments, the vector is an alphavirus RNA replicon particle encoding one or more antigens derived from a feline pathogen. In specific embodiments, the feline pathogen is FeLV. In even more specific embodiments, the alphavirus RNA replicon particle encodes the FeLV glycoprotein (gp85). In related embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of gp85. In particular such embodiments, the antigenic fragment of gp85 is FeLV glycoprotein gp70. In certain related embodiments, the antigenic fragment of gp85 is FeLV glycoprotein gp45. In even more specific embodiments, the alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle. In even more specific embodiments, the VEE alphavirus RNA replicon particle is a TC-83 VEE alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Sindbis (SIN) alphavirus RNA replicon particle. In still other embodiments, the alphavirus RNA replicon particle is a Semliki Forest virus (SFV) alphavirus RNA replicon particle. In alternative embodiments, the naked DNA vector comprises a nucleic acid construct encoding one or more antigens derived from a feline pathogen. In particular embodiments of this type, the naked DNA vector comprises a nucleic acid construct encoding FeLV gp85 or an antigenic fragment thereof.

[0014] In certain embodiments, the alphavirus RNA replicon particles of the present invention encode one or more FeLV antigens or antigenic fragments thereof. In certain embodiments of this type, the alphavirus RNA replicon particles encode two to four FeLV antigens or antigenic fragments thereof. In other embodiments, the immunogenic composition comprises alphavirus RNA replicon particles encoding one or more FeLV antigens or antigenic fragments thereof. In related embodiments, the immunogenic composition comprises alphavirus RNA replicon particles encoding two to four FeLV antigens or antigenic fragments thereof. In certain embodiments of this type, the alphavirus RNA replicon particles encode FeLV glycoprotein (gp85) or an antigenic fragment thereof. In even more specific embodiments of this type, the antigenic fragment of gp85 is FeLV glycoprotein gp70. In certain related embodiments, the antigenic fragment of gp85 is FeLV glycoprotein gp45. In even more specific embodiments, the immunogenic composition comprises alphavirus RNA replicon particles that are Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles. In an even more specific embodiment, the VEE alphavirus RNA replicon particles are TC-83 VEE alphavirus RNA replicon particles.

[0015] In still other embodiments, the immunogenic composition comprises two or more sets of alphavirus RNA replicon particles. In certain such embodiments, one set of alphavirus RNA replicon particles encodes a particular antigen, and the other set of alphavirus RNA replicon particles encodes a second antigen. In a specific such embodiment, a first set of alphavirus RNA replicon particles encodes a FeLV antigen or an antigenic fragment thereof, and a second set of alphavirus RNA replicon particles encodes a feline calicivirus (FCV) antigen or an antigenic fragment thereof. In certain such embodiments, the FCV antigen is derived from a virulent systemic feline calicivirus (VS-FCV) isolate. In other embodiments, the FCV antigen is derived from a classical (F9-like) feline calicivirus isolate. In yet other embodiments, the second set of alphavirus RNA replicon particles encodes two FCV antigens, one derived from a virulent systemic feline calicivirus isolate and the other derived from a classical (F9-like) feline calicivirus isolate.

[0016] In yet other embodiments, the immunogenic composition comprises one set of alphavirus RNA replicon particles encoding a first antigen, another set of alphavirus RNA replicon particles encoding a second antigen, and a third set of alphavirus RNA replicon particles encoding a third antigen. In particular embodiments of this type, the first set of alphavirus RNA replicon particles encodes a FeLV antigen or an antigenic fragment thereof, the second set of alphavirus RNA replicon particles encodes a feline calicivirus (FCV) antigen or an antigenic fragment thereof derived from a virulent systemic feline calicivirus, and the third set of alphavirus RNA replicon particles encodes a feline calicivirus (FCV) antigen or an antigenic fragment thereof derived from a classical (F9-like) feline calicivirus.

[0017] Thus, in certain embodiments in which the immunogenic composition comprises multiple sets (e.g., 2-10) of alphavirus RNA replicon particles, a first set of alphavirus RNA replicon particles can encode a FeLV antigen or antigenic fragment thereof, and one or more other sets of alphavirus RNA replicon particles can encode one or more non-FeLV antigens. In certain such embodiments, the non-FeLV antigen is a protein antigen derived from feline herpesvirus (FHV). In other embodiments, the non-FeLV antigen is a protein antigen derived from feline calicivirus (FCV). In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline pneumovirus (FPN). In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline parvovirus (FPV). In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline infectious peritonitis virus (FIPV). In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline immunodeficiency virus. In still other embodiments, the non-FeLV antigen is a protein antigen derived from Borna disease virus (BDV). In yet other embodiments, the non-FeLV antigen is a protein antigen derived from feline influenza virus. In yet other embodiments, the non-FeLV antigen is a protein antigen derived from feline pancytopenia virus (FPLV). In yet other embodiments, the non-FeLV antigen is a protein antigen derived from feline coronavirus (FCoV). In yet other embodiments, the non-FeLV antigen is a protein antigen derived from feline rhinotracheitis virus (FVR). In yet other embodiments, the non-FeLV antigen is a protein antigen derived from Chlamydophila felis.

[0018] The present invention also includes any nucleic acid construct of the present invention, including synthetic messenger RNA, RNA replicons, as well as any alphavirus RNA replicon particles, naked DNA vectors of the present invention, and immunogenic compositions and / or vaccines comprising any nucleic acid construct of the present invention (e.g., synthetic messenger RNA, RNA replicon), alphavirus RNA replicon particles and / or naked DNA vectors.

[0019] In certain embodiments, the nucleic acid constructs of the invention encode one or more FeLV antigens or antigenic fragments thereof. In related embodiments of this type, the nucleic acid construct encodes two to four FeLV antigens or antigenic fragments thereof. In other embodiments, the alphavirus RNA replicon particle comprises a nucleic acid construct encoding one or more FeLV antigens or antigenic fragments thereof. In certain embodiments, the alphavirus RNA replicon particle comprises a nucleic acid construct encoding two to four FeLV antigens or antigenic fragments thereof.

[0020] In still other embodiments, the immunogenic composition comprises an alphavirus RNA replicon particle comprising a nucleic acid construct encoding two to four FeLV antigens or antigenic fragments thereof. In particular embodiments of this type, the alphavirus RNA replicon particle comprises a nucleic acid construct encoding FeLV glycoprotein (gp85) or an antigenic fragment thereof. In particular embodiments of this type, the antigenic fragment of gp85 is FeLV glycoprotein gp70. In other related embodiments, the antigenic fragment of gp85 is FeLV glycoprotein gp45. In even more particular embodiments, the immunogenic composition comprises an alphavirus RNA replicon particle that is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle. In an even more specific embodiment, the VEE alphavirus RNA replicon particle is a TC-83 VEE alphavirus RNA replicon particle.

[0021] In yet other embodiments, the immunogenic composition comprises two or more sets of alphavirus RNA replicon particles. In certain embodiments of this type, one set of alphavirus RNA replicon particles comprises a first nucleic acid construct, and the other set of alphavirus RNA replicon particles comprises a second nucleic acid construct. In specific embodiments of this type, the first nucleic acid construct encodes a FeLV antigen or an antigenic fragment thereof, and the second nucleic acid construct encodes a feline calicivirus (FCV) antigen or an antigenic fragment thereof. In certain embodiments of this type, the FCV antigen is derived from a virulent systemic feline calicivirus (VS-FCV) isolate. In other embodiments, the FCV antigen is derived from a classical (F9-like) feline calicivirus isolate. In yet other embodiments, the second nucleic acid construct encodes two FCV antigens, one derived from a virulent systemic feline calicivirus isolate and the other derived from a classical (F9-like) feline calicivirus isolate.

[0022] In still other embodiments, the immunogenic composition comprises one set of alphavirus RNA replicon particles comprising a first nucleic acid construct, another set of alphavirus RNA replicon particles comprising a second nucleic acid construct, and a third set of alphavirus RNA replicon particles comprising a third nucleic acid construct. In particular embodiments of this type, the first nucleic acid construct encodes a FeLV antigen or an antigenic fragment thereof, the second nucleic acid construct encodes a feline calicivirus (FCV) antigen or an antigenic fragment thereof derived from a virulent systemic feline calicivirus, and the third nucleic acid construct encodes a feline calicivirus (FCV) antigen or an antigenic fragment thereof derived from a classical (F9-like) feline calicivirus.

[0023] In yet other embodiments, the immunogenic composition comprises one set of alphavirus RNA replicon particles comprising a first nucleic acid construct, another set of alphavirus RNA replicon particles comprising a second nucleic acid construct, a third set of alphavirus RNA replicon particles comprising a third nucleic acid construct, and a fourth set of alphavirus RNA replicon particles comprising a fourth nucleic acid construct. In yet other embodiments, the immunogenic composition comprises a set of alphavirus RNA replicon particles comprising the first nucleic acid construct, another set of alphavirus RNA replicon particles comprising the second nucleic acid construct, the third set of alphavirus RNA replicon particles comprising the third nucleic acid construct, the fourth set of alphavirus RNA replicon particles comprising the fourth nucleic acid construct, and a fifth set of alphavirus RNA replicon particles comprising a fifth nucleic acid construct. In such embodiments, the nucleotide sequences of the first nucleic acid construct, the second nucleic acid construct, the third nucleic acid construct, the fourth nucleic acid construct, and the fifth nucleic acid construct are all different.

[0024] Thus, the immunogenic compositions of the present invention can contain alphavirus RNA replicon particles comprising a nucleic acid construct encoding at least one non-FeLV antigen for eliciting protective immunity against non-FeLV pathogens. In certain such embodiments, the non-FeLV antigen is a protein antigen derived from feline herpesvirus (FHV). In other embodiments, the non-FeLV antigen is a protein antigen derived from feline calicivirus (FCV). In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline pneumovirus (FPN). In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline parvovirus (FPV). In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline infectious peritonitis virus (FIPV). In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline immunodeficiency virus. In still other embodiments, the non-FeLV antigen is a protein antigen derived from Borna disease virus (BDV). In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline influenza virus. In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline pancytopenia virus (FPLV). In yet other embodiments, the non-FeLV antigen is a protein antigen derived from feline coronavirus (FCoV). In still other embodiments, the non-FeLV antigen is a protein antigen derived from feline rhinotracheitis virus (FVR). In still other embodiments, the non-FeLV antigen is a protein antigen derived from Chlamydophila felis.

[0025] The present invention further provides combination immunogenic compositions and / or vaccines (multivalent vaccines) comprising alphavirus RNA replicon particles encoding an antigen or antigenic fragment thereof derived from FeLV, along with one or more modified live (e.g., attenuated) or killed feline pathogens. In certain embodiments, the immunogenic composition comprises modified live or killed Chlamydophila felis combined with alphavirus RNA replicon particles encoding an antigen or antigenic fragment thereof derived from FeLV. In other embodiments, the immunogenic composition comprises modified live or killed feline rhinotracheitis virus (FVR) combined with alphavirus RNA replicon particles encoding an antigen or antigenic fragment thereof derived from FeLV. In yet other embodiments, the immunogenic composition comprises modified live or killed feline calicivirus (FCV) combined with alphavirus RNA replicon particles encoding an antigen or antigenic fragment thereof derived from FeLV. In yet other embodiments, the immunogenic composition comprises modified live or killed feline pancytopenia virus (FPL) combined with alphavirus RNA replicon particles encoding an antigen or antigenic fragment thereof derived from FeLV. In yet other embodiments, the immunogenic composition comprises a modified live or dead Chlamydophila felis, a modified live or dead FVR, a modified live or dead FCV, a modified live or dead FPL, and an alphavirus RNA replicon particle encoding an antigen or antigenic fragment thereof derived from FeLV. In a particular embodiment of this type, the feline antigen of FeLV is FeLV viral glycoprotein (gp85). In certain embodiments, the vaccine comprises an immunologically effective amount of one or more of these immunogenic compositions.

[0026] In certain embodiments, the FeLV antigen is FeLV glycoprotein (gp85). In specific embodiments of this type, the FeLV glycoprotein gp85 comprises an amino acid sequence having 95% or greater identity to the amino acid sequence of SEQ ID NO:2. In even more specific embodiments of this type, the FeLV glycoprotein (gp85) comprises the amino acid sequence of SEQ ID NO:2. In even more specific embodiments of this type, the FeLV glycoprotein (gp85) is encoded by the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:10. In a related embodiment, the FeLV glycoprotein gp70 comprises an amino acid sequence having 95% or greater identity to the amino acid sequence of SEQ ID NO:4. In even more specific embodiments of this type, the FeLV glycoprotein (gp85) comprises the amino acid sequence of SEQ ID NO:4. In even more specific embodiments of this type, the FeLV glycoprotein (gp70) is encoded by the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:11.

[0027] The present invention further includes vaccines and multivalent vaccines comprising the immunogenic compositions of the present invention. In certain embodiments, the vaccines are non-adjuvanted vaccines. In certain embodiments, the vaccines are useful for preventing diseases caused by FeLV. In related embodiments, immunization of cats with the vaccine induces antibodies in the feline subject.

[0028] The present invention also provides a method of immunizing a cat against a feline pathogen, e.g., FeLV, comprising administering to the cat an immunologically effective amount of a vaccine or multivalent vaccine of the present invention. In certain embodiments, the vaccine is administered via intramuscular injection. In alternative embodiments, 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 yet other embodiments, the vaccine is administered via oral administration. In still other embodiments, the vaccine is administered via intranasal administration. In a specific embodiment, the cat is a domestic cat.

[0029] The vaccines and multivalent vaccines of the present invention can be administered as primer vaccines and / or booster vaccines. In a specific embodiment, the vaccines of the present invention are administered as single-injection vaccines (single doses) without the need for subsequent administration. In certain embodiments, when both primer and booster vaccines are administered, the primer and booster vaccines can be administered by the same route. In certain such embodiments, both primer and booster vaccines are administered by subcutaneous injection. In an alternative embodiment, when both primer and booster vaccines are administered, the primer vaccine can be administered by one route and the booster vaccine can be administered by another route. In certain such embodiments, the primer vaccine can be administered by subcutaneous injection, and the booster vaccine can be administered orally.

[0030] The present invention further provides a method of immunizing a cat against FeLV, comprising injecting the cat with an immunologically effective amount of the vaccine of the present invention described above. In certain embodiments, the vaccine is administered in a dose of, for example, about 1 x 10 4 ~Approx. 1×10 10 In a more particular embodiment, the vaccine may comprise about 1 x 10 RP or more. 5 ~Approx. 1×10 9 In an even more particular embodiment, the vaccine may comprise about 1 x 10 RP. 6 ~Approx. 1×10 8 In certain embodiments, the cat is a domestic cat.

[0031] In a specific embodiment, the vaccine of the present invention is administered in a dose of 0.05 mL to 3 mL. In an even more specific embodiment, the administered dose is 0.1 mL to 2 mL. In an even more specific embodiment, the administered dose is 0.2 mL to 1.5 mL. In an even more specific embodiment, the administered dose is 0.3 mL to 1.0 mL. In an even more specific embodiment, the administered dose is 0.4 mL to 0.8 mL. In an even more specific embodiment, the administered dose is 0.5 mL to 1.5 mL.

[0032] These and other aspects of the present invention will be better understood by reference to the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention provides an improved, safe, non-adjuvanted FeLV vaccine. In one aspect, the vaccine of the present invention does not induce feline injection site sarcomas, yet still protects vaccinated subjects from the debilitating disease state caused by FeLV infection as effectively as an inactivated whole virus adjuvanted vaccine.

[0034] Thus, the vaccine compositions of the present invention comprise an immunologically effective amount of a vector encoding an antigen from one or more strains of feline leukemia virus that is useful for inducing protective immunity in recipient vaccinated animals. Furthermore, the present invention provides novel immunological compositions that improve the reliability of vaccination and help reduce antigenemia in FeLV-infected cats, thereby rendering the antigenemia transient and / or resulting in resolution of the infection. In certain aspects of the present invention, the vaccine comprises an alphavirus RNA replicon particle (RP) encoding the FeLV viral glycoprotein (gp85). In a more specific embodiment, the vaccine comprises an alphavirus RNA replicon particle (RP) comprising the capsid protein and glycoprotein of Venezuelan equine encephalitis virus (VEE), encoding the FeLV viral glycoprotein (gp85) and / or an antigenic fragment thereof (e.g., gp70 or gp45). In an even more specific embodiment, the vaccine comprises the capsid protein and glycoprotein of the avirulent TC-83 strain of VEE and includes an alphavirus RNA replicon particle (RP) encoding the FeLV viral glycoprotein (gp85) and / or an antigenic fragment thereof (e.g., gp70 or gp45).

[0035] In another embodiment of the present invention, the vaccine comprises a naked DNA vector encoding the FeLV viral glycoprotein (gp85) and / or an antigenic fragment thereof (e.g., gp70 or gp45). The vaccine of the present invention can be administered to cats in the absence of an adjuvant and still effectively serve to protect vaccinated cats against FeLV.

[0036] In order to provide a more complete understanding of the present invention, the following definitions are provided.

[0037] The use of singular terms for convenience of description is in no way intended to be limiting. Thus, for example, reference to a composition comprising a "polypeptide" includes reference to one or more such polypeptides. Further, reference to an "alphavirus RNA replicon particle" includes reference to a plurality of such alphavirus RNA replicon particles, unless otherwise indicated.

[0038] As used herein, the term "approximately" is used interchangeably with the term "about" and means that a value is within 50% of the stated value, i.e., "approximately" 1 x 10 per milliliter. 8 A composition containing 0.5 x 10 alphavirus RNA replicon particles per milliliter 8 ~1.5×10 8 This means that the vector contains alphavirus RNA replicon particles of the present invention.

[0039] As used herein, the term "cat" refers to any member of the Felidae family. Domestic cats, purebred and / or mixed breed pet cats, and wild cats are all cats.

[0040] As used herein, the term "replicon" refers to a modified RNA viral genome that lacks one or more elements (e.g., coding sequences for structural proteins) that, if present, would allow successful propagation of the parent virus in cell culture or an animal host. In the appropriate cellular context, the replicon can amplify itself and generate one or more subgenomic RNA species.

[0041] As used herein, the term "alphavirus RNA replicon particle," abbreviated as "RP," is an alphavirus-derived RNA replicon encased in structural proteins, e.g., capsid and glycoproteins, also derived from an alphavirus, as described, for example, by Pushko et al. [Virology 239(2):389-401 (1997)]. Because the replicon does not encode the structural components (e.g., capsid and glycoproteins) of an alphavirus, the RP cannot be propagated in cell culture or in an animal host (without the use of a helper plasmid or similar components).

[0042] The term "non-FeLV" is used to modify terms such as pathogen and / or antigen (or immunogen) to mean that the respective pathogen and / or antigen (or immunogen) is neither a FeLV pathogen nor a FeLV antigen (or immunogen), and that the non-FeLV protein antigen (or immunogen) is not derived from FeLV.

[0043] The terms "originate from," "originates from," and "originating from" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it, and as used herein, mean that the unmodified and / or truncated amino acid sequence of the given protein antigen is encoded by that pathogen or strain of pathogen. Within the nucleic acid constructs of the present invention, the coding sequence of a protein antigen derived from a pathogen can be genetically engineered to result in modification and / or truncation of the amino acid sequence of the expressed protein antigen relative to the corresponding sequence of that protein antigen in the pathogen or strain of pathogen (including naturally attenuated strains) from which it is derived.

[0044] As used herein, the terms "protect," "provide protection," "induce protective immunity," "help prevent disease," and "help protect" do not require complete protection from any manifestation of infection. For example, "help protect" can mean that protection is sufficient such that, after challenge, the underlying symptoms of infection are at least reduced and / or one or more of the underlying cellular, physiological, or biochemical causes or mechanisms that cause the symptoms are reduced and / or eliminated. When used in this context, "reduced" should be understood to mean related to the state of infection, including not only the physiological state of the infection, but also the molecular state of the infection.

[0045] As used herein, a "vaccine" is a composition comprising one or more antigens, typically combined with a pharmaceutically acceptable carrier, such as a liquid containing water, suitable for application to animals such as cats (including humans in certain embodiments, but not specifically to humans in other embodiments), which, upon administration to an animal, induces an immune response strong enough to minimally aid in protection from disease resulting from infection with a wild-type microorganism, i.e., strong enough to aid in the prevention of disease, and / or to aid in the prevention, amelioration, or cure of disease.

[0046] 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 stimulates the recipient's immune system against two or more different pathogens.

[0047] The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance the immune response to one or more vaccine antigens / isolates. Thus, an "adjuvant" is an agent that nonspecifically increases the immune response to a specific antigen, thereby reducing the amount of antigen required for any given vaccine and / or the frequency of injections required to produce an adequate immune response to the antigen of interest. In this context, an adjuvant is used to enhance the immune response to one or more vaccine antigens / isolates. The American Association of Feline Practitioners Feline Vaccination Guidelines suggest the use of a non-adjuvanted FeLV vaccine [AAFP Feline Advisory Panel, 15:785-808 (2013)].

[0048] As used herein, a "non-adjuvanted vaccine" is a vaccine or multivalent vaccine that does not contain an adjuvant.

[0049] As used herein, the term "pharmaceutically acceptable" is used adjectively to mean that the modified noun is appropriate for use in a pharmaceutical product. For example, when used to describe an excipient in a pharmaceutical vaccine, the term "pharmaceutically acceptable" characterizes the excipient as being compatible with the other components of the composition and not injuriously harmful to the intended recipient animal, e.g., a cat.

[0050] Parenteral administration" includes subcutaneous injection, submucosal injection, intravenous injection, intramuscular injection, intradermal injection and infusion.

[0051] As used herein, the term "antigenic fragment" with respect to a particular protein (e.g., a protein antigen) refers to a fragment of that protein that is antigenic, i.e., capable of specifically interacting with an antigen-recognition molecule of the immune system, e.g., an immunoglobulin (antibody) or a T-cell antigen receptor. For example, an antigenic fragment of the FeLV viral glycoprotein (gp85) is a fragment of the gp85 protein that is antigenic. Preferably, antigenic fragments of the present invention are immunodominant for antibody and / or T-cell receptor recognition. In certain embodiments, an antigenic fragment with respect to a given protein antigen is a fragment of that protein that retains at least 25% of the antigenicity of the full-length protein. In preferred embodiments, the antigenic fragment retains at least 50% of the antigenicity of the full-length protein. In even more preferred 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, in extreme cases, larger fragments, with only a single amino acid missing from the full-length protein. In certain embodiments, the antigenic fragment comprises 25 to 150 amino acid residues. In another embodiment, the antigenic fragment comprises between 50 and 250 amino acid residues. The gp45 glycoprotein and the gp70 glycoprotein are antigenic fragments of the gp85 glycoprotein.

[0052] As used herein, one amino acid sequence is 100% "identical" or has 100% "identity" to a second amino acid sequence if the amino acid residues in both sequences are identical. Thus, an amino acid sequence is 50% "identical" to a second amino acid sequence if 50% of the amino acid residues in the two amino acid sequences are identical. Sequence comparisons are made for contiguous blocks of amino acid residues contained within a given protein, e.g., a portion of a protein or polypeptide being compared. In certain embodiments, selected deletions or insertions that might otherwise alter the correspondence between the two amino acid sequences are taken into account.

[0053] As used herein, percent identity of nucleotide and amino acid sequences can be determined using the C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996), and Clustal W algorithms with default parameters for alignment and identity. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, an Advanced Blast search can be performed under default filter conditions using, for example, the pileup program of GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) with default parameters.

[0054] As used herein, the term "inactivated" microorganisms is used interchangeably with the term "dead" microorganisms. For purposes of the present invention, an "inactivated" microorganism is an organism that is capable of inducing an immune response in an animal but is unable to infect the animal. The antigens of the present invention (e.g., inactivated feline pancytopenia virus) may be inactivated by an agent selected from the group consisting of binary ethyleneimine, formalin, betapropiolactone, thimerosal, or heat. In a specific embodiment, the inactivated feline calicivirus isolate combined with the RP of the present invention is inactivated by binary ethyleneimine.

[0055] The alphavirus RNA replicon particles of the present invention may be lyophilized and rehydrated with a sterile water diluent, or, if the alphavirus RNA replicon particles are intended to be stored separately but mixed with other vaccine components prior to administration, the alphavirus RNA replicon particles can be stored in a stabilizing solution of these components, such as a high sucrose solution.

[0056] The vaccines of the present invention can be readily administered by any standard route, including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal, and / or intraperitoneal vaccination. Those skilled in the art will appreciate that vaccine compositions are preferably formulated appropriately for a variety of recipient animals and routes of administration.

[0057] Thus, the present invention also provides methods for immunizing cats against FeLV and / or other feline pathogens, one such method comprising injecting a cat with an immunologically effective amount of a vaccine of the present invention, such that the cat produces appropriate FeLV antibodies.

[0058] Multivalent vaccines The present invention also provides multivalent vaccines. For example, coding sequences for protein antigens or antigenic fragments thereof useful in feline vaccines, or combinations of such coding sequences for protein antigens, can be attached to or combined in the same alphavirus RNA replicon particle (RP) as those encoding feline antigens of FeLV (e.g., FeLV viral glycoprotein (gp85)) in the vaccine. Thus, such multivalent vaccines are encompassed by the present invention.

[0059] Examples of pathogens from which one or more of such protein antigens may be derived include feline rhinotracheitis virus (FVR), feline calicivirus (FCV), feline pancytopenia virus (FPL), feline herpesvirus (FHV), other FeLV strains, feline parvovirus (FPV), feline infectious peritonitis virus (FIPV), feline immunodeficiency virus, Borna disease virus (BDV), rabies virus, feline influenza virus, canine influenza virus, avian influenza, canine pneumovirus, feline pneumovirus, Chlamydophila felis (FKA Chlamydia psittaci), Bordetella bronchiseptica, and Bartonella species (e.g., B. henselae). In certain embodiments, the coding sequence for a capsid protein or similar protein from one or more of these feline or canine pathogens can be inserted into the same RP as a FeLV antigen. Alternatively, or in combination, the coding sequence for a capsid protein or similar protein from one or more of these feline or canine pathogens can be inserted into one or more other RPs, which can be combined with the RP encoding the FeLV antigen in a vaccine.

[0060] Additionally, alphavirus RNA replicon particles (RP) encoding a feline antigen of FeLV (e.g., FeLV viral glycoprotein (gp85)) can be added along with one or more other live attenuated viral isolates, such as other live attenuated FCV strains, live attenuated feline herpesvirus and / or live attenuated feline parvovirus and / or live attenuated feline leukemia virus and / or live attenuated feline infectious peritonitis virus and / or live attenuated feline immunodeficiency virus and / or live attenuated Borna disease virus and / or live attenuated rabies virus and / or live attenuated feline influenza virus and / or live attenuated canine influenza virus and / or live attenuated avian influenza and / or live attenuated canine pneumovirus and / or live attenuated feline pneumovirus. Additionally, live attenuated Chlamydophila felis and / or live attenuated Bordetella bronchiseptica and / or live attenuated Bartonella (e.g., B. henselae) can also be included in such a multivalent vaccine.

[0061] Additionally, alphavirus RNA replicon particles (RP) encoding feline antigens of FeLV (e.g., FeLV viral glycoprotein (gp85)) can be added along with one or more other killed virus isolates, such as killed FCV strains, killed feline herpesvirus, killed feline parvovirus, killed feline leukemia virus, killed feline infectious peritonitis virus, killed feline immunodeficiency virus, killed Borna disease virus, killed rabies virus, killed feline influenza virus, killed canine influenza virus, killed avian influenza virus, killed canine pneumovirus, and / or killed feline pneumovirus. Furthermore, bacterins of Chlamydophila felis, Bordetella bronchiseptica, and / or Bartonella (e.g., B. henselae) can also be included in such multivalent vaccines.

[0062] It is also to be understood that the present invention is not limited to the particular configurations, process steps, and materials disclosed herein, and as such, the configurations, process steps, and materials may vary somewhat.

[0063] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and their equivalents. [Table 1]

[0064] array Feline leukemia virus envelope glycoprotein (gp85) SEQ ID NO: 1 atggagtcaccaacacaccctaaaccttctaaagacaaaaccctctcgtggaatctcgccttccttgt gggcatcctgttcacaatcgacatcggcatggccaaccccttcgccgcatcagatctacaatgtgacat gggtcattactaatgtgcagacaaacaccccaggcaaatgctacttctatgcttggtactctgactgat gcttatccaaccctgcacgtcgacctttgcgatctcgtcggtgacacatgggagcccatcgtgctgaa tccaactaatgtcaaacatggtgccaggtattcttctagcaaatacgggtgtaagaccactgatcgga agaaacagcaacaaacctacccattctacgtgtgcccgggtcacgcaccgtccctgggtccgaaggga acacattgtgggggagcccaagacggtttttgcgctgcttggggttgtgaaacaaccggagaagcctg gtggaagcctacctcatcttgggactacattactgtgaaaagaggctctaggccaggataacagctgcg aaggaaagtgtaatcccctggtgcttcaattcacccagaaaggccggcaggcatcatgggatggaccg aaaatgtggggacttagactctatcgcaccggatacgaccccatcgctctgtttactgtgtcacgcca agtctccaccattactccgccacaggccatggggccgaatctggtcctccccgatcagaagccaccct cacggcaaagtcaaaccggctcaaaagtggccacccaacggccccagacaaatgagtccgcacctagg tcagtggcacctacaacaatgggtccaaaagcggatcggaaccggagacaggctcattaacctcgtgca agggacttatctggcccttaacgctactgaccccaacaagaccaaggattgctggctctgccttgtga gcagacctccttactatgaggggatcgccattctgggaaactactcaaatcagaccaacccccctccg tcgtgtctgagcacccccagcacaagcttactatttcagaagtcagtggacagggaatgtgcatcgg aaccgtgccaaagactcatcaagccctttgcaacaaaactcaacaagggcacactggagctcattatc tcgccgcacctaacgggacctactgggcttgcaatactggattgaccccgtgtatctctatggccgtg ctgaattggacttccgacttctgcgtgcttattgagctttggcctagagtgacataccatcagcctga gtacgtctatacccatttcgccaaggcagtcagattccggcgggagcctatctccctgactgtggcct tgatgctcggtggactgacagtgggaggaattgcagctggagtcggaactggaaccaaggccctgctc gaaactgctcagttccggcagctgcagatggccatgcacactgacatccaggctctggaggaatcaat ttcagcccttgagaaaagcttgacctcgctgtctgaagtggtcctccaaaacaggcgcggtttggaca tcctgttccttcaagagggtggtctgtgcgccgctctcaaggaggaatgctgtttctacgctgaccat accgggctggtgcgcgataacatggcaaagctgcgggaacgcttgaaacagaggcagcaactgttcga ctctcagcagggatggttcgagggctggtttaacaagagcccatggtttaccactctgatctcttcaa tcatgggtccactgctcatcctgcttctgattcttctcttcggaccgtgtattctcaacaggctggtg cagtttgtcaaggacagaatctcggtggtccaggccctgattcttactcagcagtatcagcagattaa gcagtacgaccccgatcggccttga Feline leukemia virus envelope glycoprotein (gp85) SEQ ID NO: 2 MESPTHPKPSKDKTLSWNLAFLVGILFTIDIGMANPSPHQIYNVTWVITNVQTNTQANAT SMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGARYSSSKYGCKTTDRKKQQQTY PFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAWWKPTSSWDYITVKRGSSQDNS CEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPN LVLPDQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTMGPKRIGTGDRLINLVQGTYLA LNATDPNKTKDCWLCLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGM CIGTVPKTHQALCNKTQQGHTGAHYLAAPNGTYWACNTGLTPCISMAVLNWTSDFCCVLIE LWPRVTYHQPEYVYTHFAKAVRFRREPISLTVALMLGGLTVGGIAAGVGTGTKALLETAQ FRQLQMAMHTDIQALEESISALEKSLTSLSEVVLQNRRGLDILFLQEGGLCAALKEECCF YADHTGLVRDNMAKLRERLKQRQQLFDSQQGWFEGWFNKSPWFTTLISSIMGPLLILLLI LLFGPCILNRLVQFVKDRISVVQALILTQQYQQIKQYDPDRP* Feline leukemia virus envelope glycoprotein (gp85) SEQ ID NO: 10 auggagucaccaacacacccuaaaccuucuaaagacaaaacccucucguggaaucucgccuuccuugu gggcauccuguucacaaucgacaucggcauggccaacccuucgccgcaucagaucuacaaugugacau gggucauuacuaauugugcagacaaacaccccaggcaaaugcuacuucuaugcuugguacucugacugau gcuuauccaacccugcacgucgaccuuugcgaucucgucggugacacaugggagcccaucgugcugaa uccaacuaaugucaaacauggugccagguauucuucuagcaaauacggguugaagaccacugaucgga agaaacagcaacaaaccuacccauucuacgugcccgggucacgcaccguccuggguccgaaggga acacauugugggggagcccaagacgguuuuugcgcuuggguugugaaacaaccggagaagccug spring aaagugaguaauccccuggugcuucaauucacccagaaaggccggcaggcaucaugggauggaccg aaaauugugggacuuagacucuaucgcaccggauacgaccccaucgcucuguuuacugucacgcca agucuccaccauuacuccgccacaggccauggggccgaaucuggucccccccgaucagaagccacccu cacggcaagucaaacccggcucaaaaguggccacccaacggcccagacaaugaguccgcaccuagg ucaguggcaaccuacaacaauggguccaaaagcggaucggaaccggagacaggcucauuaaccucgugca agggacuuaucuggcccuuaacgcuacugaccccaacaagaccaaggauugcuggcucugccuuuguga gcagaccuccuuacuaugagggaucgccauuucucggaacuaacucaaaucagaccaacccccccuccg ucgugucugagcaccccccagcacaagcuuacuauuuucagaagucaguggacagggaaugugcaucgg aaccgugccaaagacucaucaagcccuuugcaacaaaacucaacaagggcacacuggagcucauuauc ucgccgccaccuaacgggaccuacugggcuugcaauacuggauugaccccguguaucucuauggccgug cuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu guacgucuauacccauuucgccaaggcagucagauuccggcgggagccuaucucccugacuguggccu ugaugcucgguggacugacagugggaaauugcagcuggagucggaacugcaaagcccugcuc gaacugcucaguucggcagcugcagaugggccaugcacacugacauccaggcucuggaggaaucaau uucagcccuugagaaaagcuugaccucgcugucugaagugguccuccaaaacaggcgcgguuuggaca uccuguuccuucaagaggguggucugugcgccgcucucaaggagaaugcuguuucuacgcugaccau accgggcuggugcgcgauaacauggcaaagcugcgggaacgcuugaaacagaggcagcaacuguucga cucucagcagggaugguucgagggcugguuuaacaagagcccaugguuuaccacucugaucucuucaa ucauggguccacugcucaucaucccugcuucugauuucucucgggccguguauucucaacaggcuggug caguuuugucaaggacagaaucucggugguccaggcccugauucuuacucagcaguaucagcagauuaa gcaguacgaccccgaucggcuuga Feline leukemia virus envelope glycoprotein (gp70) SEQ ID NO: 3 aatcctagtccacaccaaatatataatgtaacttgggtaataaccaatgtacaaactaacacc caagctaacgccacctctatgttaggaaccttaaccgatgcctaccctaccctacatgttgac ttatgtgacctagtgggagacacctgggaacctatagtcctaaacccaaccaatgtaaaacac ggggcacgttactcctcctcaaaatatggatgtaaaactacagatagaaaaaaacagcaacag acataccccttttacgtctgccccggacatgccccctcgttggggccaaagggaacacattgt ggaggggcacaagatgggttttgtgccgcatggggatgtgagaccaccggagaagcttggtgg aagcccacctcctcatgggactatatcacagtaaaaagagggagtagtcaggacaatagctgt gagggaaaatgcaaccccctggttttgcagttcacccagaagggaagacaagcctcttgggac ggacctaagatgtggggattgcgactataccgtacaggatatgaccctatcgctttattcacg gtgtcccggcaggtatcaaccattacgccgcctcaggcaatgggaccaaacctagtcttacct gatcaaaaacccccatcccgacaatctcaaacagggtccaaagtggcgacccagaggccccaa acgaatgaaagcgccccaaggtctgttgcccccaccaccatgggtcccaaacggattgggacc ggagataggttaataaatttagtacaagggacatacctagccttaaatgccaccgaccccaac aaaactaaagactgttggctctgcctggtttctcgaccaccctattacgaagggattgcaatc ttaggtaactacagcaaccaaacaaaccccccccccatcctgcctatctactccgcaacacaaa ctaactatatctgaagtatcagggcaaggaatgtgcatagggactgttcctaaaacccaccag gctttgtgcaataagacacaacagggacatacaggggcgcactatctagccgcccccaacggc acctattgggcctgtaacactggactcaccccatgcatttccatggcggtgctcaattggacc tctgatttttgtgtcttaatcgaattatggcccagagtgacttaccatcaacccgaatatgtg tacacacattttgccaaagctgtcaggttccgaaga Feline leukemia virus envelope glycoprotein (gp70) SEQ ID NO: 4 NPSPHQIYNVTWVITNVQTNTQANATSMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGARYSSS KYGCKTTDRKKQQQTYPFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAWWKPTSSWDYITVKRG SSQDNSCEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPNLVLP DQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTMGPKRIGTGDRLINLVQGTYLALNATDPNKTKDCWL CLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGMCIGTVPKTHQALCNKTQQGHTGAH YLAAPNGTYWACNTGLTPCISMAVLNWTSDFCVLIELWPRVTYHQPEYVYTHFAKAVRFRR Feline leukemia virus envelope glycoprotein (gp70) SEQ ID NO: 11 aauccuaguccacaccaaauauauaauguaacuuggguaauaaccaauguacaaacuaacacc caagcuaacgccaccucuauguuaggaaccuuaaccgaugccuacccuacccuacauguugac uuaugugaccuagugggagacaccugggaaccuauaguccuaaacccaaccaauguaaaacac ggggcacguuacuccuccucaaaauauggauguaaaacuacagauagaaaaaaacagcaacag acauaccccuuuuacgucugccccggacaugcccccucguuggggccaaagggaacacauugu ggaggggcacaagauggguuuugugccgcauggggaugugagaccaccggagaagcuuggugg aagcccaccuccucaugggacuauaucacaguaaaaagagggaguagucaggacaauagcugu gagggaaaaugcaacccccugguuuugcaguucacccagaagggaagacaagccucuugggac ggaccuaagauguggggauugcgacuauaccguacaggauaugacccuaucgcuuuauucacg gugucccggcagguaucaaccauuacgccgccucaggcaaugggaccaaaccuagucuuaccu gaucaaaaacccccaucccgacaaucucaaacaggguccaaaguggcgacccagaggccccaa acgaaugaaagcgccccaaggucuguugcccccaccaccaugggucccaaacggauugggacc ggagauagguuaauaaauuuaguacaagggacauaccuagccuuaaaugccaccgaccccaac aaaacuaaagacuguuggcucugccugguuucucgaccacccuauuacgaagggauugcaauc uuagguaacuacagcaaccaaacaaacccccccccauccugccuaucuacuccgcaacacaaa cuaacuauaucugaaguaucagggcaaggaaugugcauagggacuguuccuaaaacccaccag gcuuugugcaauaagacacaacagggacauacaggggcgcacuaucuagccgcccccaacggc accuauugggccuguaacacuggacucaccccaugcauuuccauggcggugcucaauuggacc ucugauuuuugugucuuaaucgaauuauggcccagagugacuuaccaucaacccgaauaugug uacacacauuuugccaaagcugucagguuccgaaga Feline calicivirus (VS-FCV) capsid (SEQ ID NO: 5) atggctgacgacggatctgtgaccaccccagaacaaggaacaatggtcggaggagtgatt gccgaacccagcgctcagatgtcaactgcggcggacatggcctccggaaagtcggtggac tccgagtgggaagccttcttctcgttccacacgtccgtgaactggagcacctccgaaacc caaggaaagatccctcttcaagcagtccctgggtcccctgctgaacccgtacctggagcac atcagcaagctgtacgtcgcttggagcgggtcgatcgaagtgcgattttccatctcggga agcggcgtgttcggtggtaaactggccgccatcgtcgtgccgcctggtgtcgaccctgtc cagtcaacctccatgctgcagtacccgcacgtcctgttcgacgcaagacaagtggagcca gtgatcttctccatcccggacctccgcaacagcctgtatcacttgatgtccgataccgat accacttccctcgtgatcatggtgtacaacgatctgatcaacccgtacgccaatgactcc aacagctcgggttgcatcgtgaccgtcgaaacgaagcctggcatcgatttcaagtttcat ctgctgaaaccgcccggatccatgcttactcacgggtccatcccttccgatctgatcccc aagagctcctccctgtggattgggaaccgccactggaccgatattaccgatttcgtgatt cggcctttcgtgttccaagccaaccggcacttcgacttcaaccaggagactgccggctgg tcaactccacggttccgcccattggccgtgactgtgtcgcagtcaaagggagccaagctc gggaacggcatcgccaccgactacattgtgcctggaatccccgacggatggcctgatact accatccccaccaagctgacccctaccggagattacgccatcacctcctccgacggcaat gatattgaaaccaagctggaatacgagaacgcggacgtgattaagaacaacaccaacttc cgctccatgtatatctgcggaagcctccagagggcttggggcgacaagaagatcagcaac accgggttcatcactaccggagtgatttctgacaactccatcagcccttcgaacacaatt gaccagtccaagatcgtggtgtaccaggacaaccatgtcaattcggaggtccagactagc gacatcactcttgccatcctgggctacaccggaattggagaagaggccataggcgccaac cgggactccgtcgtgagaatttccgtgcttccggaaactggagcaaggggcggaaatcac cccatcttctacaaaaattccatgaagctgggctacgtgatctcctccattgacgtgttc aactcccaaatcctccacacctcgcgccagctgtcactgaacaactacttgttgccccct gactccttcgcggtgtaccggattattgacagcaacggatcatggttcgacattgggatt gacagcgatgggttttcattcgtgggcgtgtcgtcatttccaaagctggagtttccgctg tccgcctcatacatgggcatccagctcgcaaagatccggctggcgtccaacatccggtca tccatgactaagctgtga Feline calicivirus (VS-FCV) capsid (SEQ ID NO:6) MADDGSVTTPEQGTMVGGVIAEPSAQMSTAADMASGKSVDSEWEAFFSFHTSVNWSTSET QGKILFKQSLGPLLNPYLEHISKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPV QSTSMLQYPHVLFDARQVEPVIFSIPDLRNSLYHLMSDTTDTTSLVIMVYNDLINPYANDS NSSGCIVTVETKPGIDFKFHLLKPPGSMLTHGSIPSDLIPKSSSLWIGNRHWTDITDFVI RPFVFQANRHFDFNQETAGWSTPRFRPLAVTVSQSKGAKLGNGIATDYIVPGIPDGWPDT TIPTKLTPTGDYAITSSDGNDIETKLEYENADVIKNNTNFRSMYICGSLQRAWGDKKISN TGFITTGVISDNSISPSNTIDQSKIVVYQDNHVNSEVQTSDITLAILGYTGIGEAIGAN RDSVVRISVLPETGARGGNHPIFYKNSMKLGYVISSIDVFNSQILHTSRQLSLNNYLLPP DSFAVYRIIDSNGSWFDIGIDSDGFSFVGVSSFPKLEFPLSASYMGIQLAKIRLASNIRS SMTKL Feline calicivirus (VS-FCV) capsid (SEQ ID NO: 12) auggcugacgacggaucugugaccacccgaacaaggaacaauggucggaggagugauu gccgaacccagcgcucagaugucaacugcggcggacauggccuccggaaagucgguggac uccgagugggaagccuucuucucguuccacacguccgugaacuggagcaccuccgaaacc caaggaaagauccucuucaagcagucccuggguccccugcugaacccguaccuggagcac aucagcaagcuguacgucgcuuggagcgggucgaucgaagugcgauuuuccaucucggga agcggcguguucggugguaaacuggccgccaucgucgugccgccuggugucgacccuguc cagucaaccuccaugcugcaguacccgcacguccuguucgacgcaagacaaguggagcca gugaucuucuccaucccggaccuccgcaacagccuguaucacuugauguccgauaccgau accacuucccucgugaucaugguguacaacgaucugaucaacccguacgccaaugacucc aacagcucggguugcaucgugaccgucgaaacgaagccuggcaucgauuucaaguuucau cugcugaaaccgcccggauccaugcuuacucacggguccaucccuuccgaucugaucccc aagagcuccucccuguggauugggaaccgccacuggaccgauauuaccgauuucgugauu cggccuuucguguuccaagccaaccggcacuucgacuucaaccaggagacugccggcugg ucaacuccacgguuccgcccauuggccgugacugugucgcagucaaagggagccaagcuc gggaacggcaucgccaccgacuacauugugccuggaauccccgacggauggccugauacu accauccccaccaagcugaccccuaccggagauuacgccaucaccuccuccgacggcaau gauauugaaaccaagcuggaauacgagaacgcggacgugauuaagaacaacaccaacuuc cgcuccauguauaucugcggaagccuccagagggcuuggggcgacaagaagaucagcaac accggguucaucacuaccggagugauuucugacaacuccaucagcccuucgaacacaauu gaccaguccaagaucgugguguaccaggacaaccaugucaauucggagguccagacuagc gacaucacucuugccauccugggcuacaccggaauuggagaagaggccauaggcgccaac cgggacuccgucgugagaauuuccgugcuuccggaaacuggagcaaggggcggaaaucac cccaucuucuacaaaaauuccaugaagcugggcuacgugaucuccuccauugacguguuc aacucccaaauccuccacaccucgcgccagcugucacugaacaacuacuuguugcccccu gacuccuucgcgguguaccggauuauugacagcaacggaucaugguucgacauugggauu gacagcgauggguuuucauucgugggcgugucgucauuuccaaagcuggaguuuccgcug uccgccucauacaugggcauccagcucgcaaagauccggcuggcguccaacauccgguca uccaugacuaagcuguga Feline calicivirus (F9-like) capsid (SEQ ID NO: 7) atgactgccccggaacaaggaacgatggtcggaggagtgattgcagaaccgtcagcacag atgtccaccgctgccgacatggccactggaaagagcgtggactccgaatgggaagccttc ttctccttccacacttcggtcaactggtcgactagcgaaacccaggggaagattttgttc aagcaatccctcggccctctgctgaacccctacctggagcatctggccaagctgtacgtg gcatggtcgggcagcatcgaagtgcgctttagcatttccggctccggagtgttcggggga aagcttgctgccattgtcgtgccgccaggagtggacccggtgcagtccacttctatgctc caatacccgcatgtcctgttcgacgccagacaggtggagcctgtgatcttttgcctgccg gatctcaggtccaccctgtatcacctcatgtccgacaccgacaccacctcgctcgtgatc atggtgtacaacgacctgatcaacccctacgctaacgacgccaacagctcaggttgcatt gtgactgtcgaaaccaagccaggccctgacttcaagtttcatttgctgaagccgcccggt tccatgctgacccacggctcgatcccatccgacctgatccccaagacgagctccctgtgg atcggaaaccgctactggtccgatattaccgacttcgtgatcagaccattcgtgttccaa gccaaccgccatttcgacttcaaccaggaaaccgcaggatggtcgacccctcgattccgc ccgatttcagtgaccatcaccgaacagaacggcgcgaagctgggaattggcgtggcgacc gactacatcgtgccgggaatcccggatggatggcctgatacgaccattcccggggagctg atccctgccggggactacgccatcaccaacggtactggaaacgacatcaccactgccacc ggttacgacaccgccgacatcataaagaacaacaccaacttcagaggaatgtacatttgc ggctccctgcaacgcgcttggggtgacaaaaagatctcgaacactgccttcatcacaaca gcgactctggacggcgataacaacaacaagatcaatccttgtaataccatcgaccagtcc aaaatcgtggtgttccaggataaccacgtgggaaagaaggcgcagacctccgacgacact ctggcgctgcttggctacaccgggatcggcgagcaggccattggaagcgatcgggatcgg gtcgtgcggatctccaccctccccgagactggagcaaggggaggcaaccaccccatcttt tacaaaaacagcattaagctcggatacgtcatccgctccatcgatgtgttcaactctcaa atcctgcacacttcgcggcagctgtccctgaaccactacctcttgccgcccgactccttc gccgtctaccggatcattgattcgaacgggagctggttcgacatcggcattgatagcgat ggcttctcgtttgtgggcgtgtcgggcttcgggaagctggagttcccactgagcgcctca tacatgggtatccagctggccaagatcaggctggcctccaacatccgctcacctatgact aagctgtga Feline calicivirus (F9-like) capsid (SEQ ID NO: 8) MTAPEQGTMVGGVIAEPSAQMSTAADMATGKSVDSEWEAFFSFHTSVNWSTSETQGKILF KQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPVQSTSML QYPHVLFDARQVEPVIFCLPDLRSTLYHLMSDTTDTTSLVIMVYNDLINPYANDANSSGCI VTVETKPGPDFKFHLLKPPGSMLTHGSIPSDLIPKTSSLWIGNRYWSDITDFVIRPFVFQ ANRHFDFNQETAGWSTPRFRPISVTITEQNGAKLGIGVATDYIVPGIPDGWPDTTIPGEL IPAGDYAITNGTGNDITTATGYDTADIIKNNTNFRGMYICGSLQRAWGDKCISNTAFITT ATLDGDNNNKINPCNTIDQSKIVVFQDNHVGKKAQTSDDTLALLGYTGIGEQAIGSDRDR VVRISTLPETGARGGNHPIFYKNSIKLGYVIRSIDVFNSQILHTSRQLSLNHYLLPPDSF AVYRIIDSNGSWFDIGIDSDGFSFVGVSGFGKLEFPLSASYMGIQLAKIRLASNIRSPMT KL Feline calicivirus (F9-like) capsid (SEQ ID NO: 13) augacugccccggaacaaggaacgauggucggaggagugauugcagaaccgucagcacag auguccaccgcugccgacauggccacuggaaagagcguggacuccgaaugggaagccuuc uucuccuuccacacuucggucaacuggucgacuagcgaaacccaggggaagauuuuguuc aagcaauccccucggccucugcugaaccccuaccuggagcaucuggccaagcuguacgug gcauggucgggcagcaucgaagugcgcuuuagcauuuccggcuccggaguguucggggga aagcuugcugccauugucgugccgccaggaguggacccggugcaguccacuucuaugcuc caauacccgcauguccuguucgacgccagacagguggagccugugaucuuuugccugccg gaucucagguccacccuguaucaccucauguccgacaccgacaccaccucgcucgugauc augguguacaacgaccugaucaaccccuacgcuaacgacgccaacagcucagguugcauu gugacugucgaaaccaagccaggcccugacuucaaguuucauuugcugaagccgcccggu uccaugcugacccacggcucgaucccauccgaccugauccccaagacgagcucccugugg aucggaaaccgcuacugguccgauauuaccgacuucgugaucagaccauucguguuccaa gccaaccgccauuucgacuucaaccaggaaaccgcaggauggucgaccccucgauuccgc ccgauuucagugaccaucaccgaacagaacggcgcgaagcugggaauuggcguggcgacc gacuacaucgugccgggaaucccggauggauggccugauacgaccauucccggggagcug aucccugccggggacuacgccaucaccaacgguacuggaaacgacaucaccacugccacc gguuacgacaccgccgacaucauaaagaacaacaccaacuucagaggaauguacauuugc ggcucccugcaacgcgcuuggggugacaaaaagaucucgaacacugccuucaucacaaca gcgacucuggacggcgauaacaacaacaagaucaauccuuguaauaccaucgaccagucc aaaaucgugguguuccaggauaaccacgugggaaagaaggcgcagaccuccgacgacacu cuggcgcugcuuggcuacaccgggaucggcgagcaggccauuggaagcgaucgggaucgg gucgugcggaucuccacccuccccgagacuggagcaaggggaggcaaccaccccaucuuu uacaaaaacagcauuaagcucggauacgucauccgcuccaucgauguguucaacucucaa auccugcacacuucgcggcagcugucccugaaccacuaccucuugccgcccgacuccuuc gccgucuacgggaucauugauucgaacgggagcugguucgacaucggcauugauagcgau ggcuucucguuuguggggcgugucgggcuucgggaagcuggaguucccacugagcgccuca uacauggguauccagcuggccaagaucaggcuggccuccaacauccgcucaccuaugacu aagcuguga The following examples serve to provide further appreciation of the invention, but are in no way meant to limit the effective scope of the invention.

[0065] [Example] [Example 1] Integration of the coding sequence of FELV GP85 into alphavirus RNA replicon particles. Introduction RNA viruses have been used as vector vehicles to introduce genetically engineered vaccine antigens into the genome. However, their use to date has been limited primarily to incorporating viral antigens into the RNA virus and then introducing the virus into a recipient host. This results in the induction of protective antibodies against the incorporated viral antigens. Alphavirus RNA replicon particles have been used to encode pathogenic antigens. Such alphavirus replicon platforms have been developed from several different alphaviruses, including Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of Virology 67:6439-6446 (1993)], and Semliki Forest virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9:1356-1361 (1991)]. Additionally, alphavirus RNA replicon particles are the basis of several USDA-licensed vaccines for swine and poultry, including the porcine epidemic diarrhea vaccine (RNA particles) (product code 19U5.P1), the swine influenza vaccine (RNA) (product code 19A5.D0), the avian influenza vaccine (RNA) (product code 19O5.D0), and the formulation (RNA particles) (product code 9PP0.00).

[0066] Construction of alphavirus RNA replicon particles The amino acid sequence of FeLV gp85 was used to generate a codon-optimized (feline codon-using) nucleotide sequence in silico. The optimized sequence was prepared as synthetic DNA by a commercial supplier (ATUM, Newark, CA). Therefore, a synthetic gene was designed based on the amino acid sequence of gp85. The construct (gp85_wt) had the wild-type amino acid sequence [SEQ ID NO: 2] and was codon-optimized for cats to have suitable flanking sequences for cloning into an alphavirus replicon plasmid.

[0067] A VEE replicon vector designed to express FeLV gp85 was constructed as previously described [see U.S. Pat. No. 9,441,247; the entire contents of which are incorporated herein by reference], with the following modifications: The TC-83-derived replicon vector "pVEK" [disclosed and described in U.S. Pat. No. 9,441,247] was digested with the restriction enzymes AscI and PacI. A DNA plasmid containing the codon-optimized open reading frame nucleotide sequence of the FeLV gp85 gene, with the 5' flanking sequence (5'-GGCGCGCCGCACC-3') [SEQ ID NO: 9] and the 3' flanking sequence (5'-TTAATTAA-3'), was similarly digested with the restriction enzymes AscI and PacI. The synthetic gene cassette was then ligated into the digested pVEK vector, and the resulting clone was renamed "pVHV-FeLV gp85." The "pVHV" vector designation was chosen to refer to a pVEK-derived replicon vector containing a transgene cassette cloned via the AscI and PacI sites in the multiple cloning site of pVEK.

[0068] TC-83 RNA replicon particles (RPs) were produced according to previously described methods [U.S. Patent Nos. 9,441,247 and 8,460,913, the contents of which are incorporated herein by reference]. Briefly, pVHV replicon vector DNA and helper DNA plasmids were linearized with NotI restriction enzyme, followed by in vitro transcription using MegaScript T7 RNA polymerase and cap analog (Promega, Madison, WI). Importantly, the helper RNA used for this production lacks the VEE subgenomic promoter sequence, as previously described [Kamrud et al., J Gen Virol 91(Pt 7):1723-1727(2010)]. Purified RNAs of the replicon and helper components were combined, mixed with a suspension of Vero cells, electroporated in a 4 mm cuvette, and reconstituted in OptiPro® SFM cell culture medium (Thermo Fisher, Waltham MA). After overnight incubation, alphavirus RNA replicon particles were purified from the cells and medium by passing the suspension through a ZetaPlus BioCap depth filter (3M, Maplewood, MN), washing with phosphate-buffered saline containing 5% sucrose (w / v), and finally eluting the retained RP with 400 mM NaCl buffer. The eluted RP was formulated to a final 5% sucrose (w / v), passed through a 0.22 micron membrane filter, and aliquoted for storage. The titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers.

[0069] [Example 2] Comparison of the efficacy and safety of FELV vaccines in cats A vaccine containing alphavirus RNA replicon particles (RP) containing the capsid protein and glycoprotein of the avirulent TC-83 strain of Venezuelan equine encephalitis virus (VEE) and encoding the FeLV viral glycoprotein (gp85) was formulated in 5% sucrose. The liquid vaccine was frozen for storage prior to use. This vaccine was compared to a commercially available vaccine containing recombinant canarypox encoding FeLV, as shown in Table 1 below. Five groups of eight feline subjects were vaccinated with either a single dose at 8-9 weeks of age or a prime / boost regimen at 8-9 weeks of age followed by a second dose 21 days later. The doses for each experimental vaccination group are shown in Table 1 below. [Table 2]

[0070] All cats were administered 1.0 mL of the respective vaccine regimen subcutaneously. Cats were 8-9 weeks old at the time of their first vaccination (including cats in Group 3). Cats in Group 4 were vaccinated when the amount of vaccine indicated on the commercial vaccine label was provided. After vaccination, cats were observed daily for general health and for any adverse reactions to the vaccine by palpation of the injection site for two days after each vaccination and twice weekly for two weeks after each vaccination. No adverse reactions were observed with any of the vaccines.

[0071] Four weeks after the booster vaccination (four weeks after the single vaccination for cats in Group 3), all cats were challenged with a virulent culture of FeLV. Cats were challenged on four separate days over a one-week period (study days 49, 52, 54, and 56) by administering 1.0 mL of challenge virus via the oronasal route (0.3 mL in each nostril and 0.4 mL orally). Three weeks after challenge, serum samples were collected weekly for 10 weeks postchallenge. Serum samples were tested for the presence of FeLV p27 antigen by ELISA. Animals were considered infected with FeLV if they exhibited persistent antigenemia. Antigenemia was defined as a positive p27 ELISA result for three consecutive weeks or five or more times during the eight-week study period. For USDA approval, a FeLV vaccine must protect 75% of cats vaccinated with the test product. Furthermore, for a challenge to be considered effective, 80% of the control cats had to exhibit persistent antigenemia (see Shipley et al., JAVMA, Vol. 199, No. 10 (Nov. 15, 1991)). The challenge results are summarized in Table 2 below. [Table 3]

[0072] As shown in Table 2, when administered in a two-dose regimen (i.e., primary and booster vaccinations), the RP-FeLV vaccine protected 100% of cats at both doses tested. Furthermore, when administered in a single dose, the RP-FeLV vaccine protected 87% of cats. In contrast, the commercial vaccine only protected 57% of cats in a two-dose regimen. Furthermore, challenge appears to be effective, as over 80% of control cats exhibited persistent antigenemia [see Table 2]. Finally, all RP-FeLV vaccine formulations were found to be safe for cats.

[0073] [Example 3] Determination of dose-response of RP-FELV vaccine by vaccination and challenge The RP-FeLV vaccine of Example 2 was formulated into a vaccine formulation that included enzymatically hydrolyzed casein (NZ-Amine®), gelatin, and sucrose. The vaccine was then lyophilized. Four groups of 10 cats each were vaccinated, as summarized in Table 3 below. [Table 4]

[0074] All cats were vaccinated subcutaneously with 1.0 mL of each test product and were 8-9 weeks of age at the time of their first vaccination.

[0075] After vaccination, cats were observed for general daily health and for any adverse reactions to the vaccine by palpation of the injection site for two days after each vaccination and twice weekly for two weeks after each vaccination. No adverse reactions to any of the vaccines were observed.

[0076] Three weeks after the booster vaccination, all cats were challenged with a virulent culture of FeLV. Cats were challenged on four separate days over a one-week period (study days 42, 45, 47, and 49) by administering 1.0 mL of challenge virus via the oronasal route (0.3 mL in each nostril and 0.4 mL orally). Three weeks after challenge, serum samples were collected weekly for 12 weeks after challenge. Serum samples were tested for the presence of FeLV p27 antigen by ELISA. Animals were considered infected with FeLV if they were found to exhibit persistent antigenemia. Antigenemia was defined as a positive p27 ELISA result for three consecutive weeks or five or more times during the eight-week study period. For USDA approval, FeLV vaccines must protect 75% of cats vaccinated with the test product. For a challenge to be considered effective, 80% of the control cats had to exhibit persistent antigenemia [Shipley et al., JAVMA, Vol. 199, No. 10, November 15, 1991]. The challenge results are summarized in Table 4 below. [Table 5]

[0077] In this short-term immunity study, the minimum protective dose of RP-FeLV vaccine for 100% protection in cats was approximately 1.0 × 10 when administered in a two-dose (primary and booster) regimen. 5 ~Approx. 2.0×10 6 The RP-FeLV vaccines tested were safe for cats. Challenge was effective, as at least 80% of control cats exhibited persistent antigenemia.

[0078] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims.

[0079] Furthermore, it is to be understood that all base sizes or amino acid sizes, and all molecular weights or molecular weight values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes.

Claims

1. A vaccine for use in preventing disease caused by feline leukemia virus (FeLV) in cats, comprising an alphavirus RNA replicon particle encoding FeLV glycoprotein (gp85) or an antigenic fragment thereof and a pharmaceutically acceptable carrier, wherein antibodies are induced in the cat when the cat is immunized with the vaccine, and the vaccine is a non-adjuvanted vaccine.

2. 2. The vaccine of claim 1, wherein said alphavirus RNA replicon particles are Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles.

3. The vaccine of claim 1 or 2, comprising one or more additional alphavirus RNA replicon particles encoding a second FeLV gp85 or an antigenic fragment thereof derived from a different FeLV strain than the one from which the FeLV gp85 is derived.

4. 4. The vaccine of claim 3, wherein said one or more additional alphavirus RNA replicon particles are VEE alphavirus RNA replicon particles.

5. 5. The vaccine of claim 1, 2, 3 or 4, wherein the FeLV glycoprotein (gp85) comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

2.

6. 5. The vaccine of claim 1, 2, 3 or 4, wherein the antigenic fragment of the FeLV glycoprotein (gp85) is FeLV gp70.

7. 7. The vaccine of claim 6, wherein the FeLV gp70 comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

4.

8. 8. The vaccine of claim 5, 6 or 7, further comprising at least one non-FeLV antigen for eliciting protective immunity against a non-FeLV feline pathogen.

9. 9. The vaccine of claim 8, wherein the non-FeLV feline pathogen is selected from the group consisting of feline herpesvirus (FHV), feline calicivirus (FCV), feline pneumovirus (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 rhinotracheitis virus (FVR), Chlamydophila felis, and any combination thereof.

10. 10. The vaccine of claim 1, 2, 3, 4, 5, 6, 7, 8 or 9, further comprising an alphavirus RNA replicon particle comprising a nucleotide sequence encoding at least one protein antigen or an antigenic fragment thereof derived from a non-FeLV antigen.

11. 11. A method of immunizing a cat against pathogenic FeLV, comprising administering to said cat an immunologically effective amount of the vaccine of claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

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