Feline leukemia virus vaccine

Abstract

The present invention provides a vaccine for feline leukemia virus and methods of making and using the vaccine alone, or in combinations with other protective agents.

Claims

1. A vaccine to aid in the prevention of disease due to FeLV comprising an immunogenic composition comprising a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle that encodes a feline leukemia virus (FeLV) antigen, wherein the FeLV antigen is a FeLV glycoprotein (gp85) or an antigenic fragment thereof, and a pharmaceutically acceptable carrier.

2. The vaccine of claim 1, wherein an antibody is induced in a feline when said feline is immunized with the vaccine.

3. The vaccine composition of claim 1 that further comprises at least one non-FeLV antigen for eliciting protective immunity to a non-FeLV feline pathogen.

4. The vaccine of claim 3, 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 panleukopenia virus (FPLV), feline coronavirus (FCOV), feline rhinotracheitis virus (FVR), Chlamydophila felis, and any combination thereof.

5. The vaccine of claim 4, wherein the non-FeLV antigen is a killed or attenuated non-FeLV antigen selected from the group of killed or attenuated non-FeLV antigens 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 panleukopenia virus (FPLV), feline coronavirus (FCOV), feline rhinotracheitis virus (FVR), Chlamydophila felis, and any combination thereof.

6. The vaccine of claim 5, wherein the attenuated non-FeLV antigen is a modified live feline pathogen selected from the group consisting of a modified live Chlamydophila felis, a modified live feline rhinotracheitis Virus (FVR), a modified live feline calicivirus (FCV), a modified live feline panleukopenia virus (FPL), a modified live feline herpesvirus (FHV), a modified live feline pneumovirus (FPN), a modified live feline parvovirus (FPV), a modified live feline infectious peritonitis virus (FIPV), a modified live feline immunodeficiency virus, a modified live borna disease virus (BDV), a modified live feline coronavirus (FCOV), and a modified live feline influenza virus.

7. The vaccine composition of claim 1, that further comprises an alphavirus RNA replicon particle comprising a nucleotide sequence encoding at least one protein antigen or an antigenic fragment thereof that originates from a non-FeLV antigen.

8. The vaccine of claim 7, wherein the protein antigen or an antigenic fragment thereof that originates from a non-FeLV feline pathogen 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 panleukopenia virus (FPLV), feline coronavirus (FCOV), feline rhinotracheitis virus (FVR), Chlamydophila felis, and any combination thereof.

9. The vaccine composition of claim 1, that is a nonadjuvanted vaccine.

10. A method of immunizing a feline against a pathogenic FeLV comprising administering to the feline an immunologically effective amount of the vaccine of claim 9.

11. The vaccine of claim 1, further comprising a modified live feline calicivirus (FCV), a modified live feline panleukopenia virus (FPLV), a modified live feline rhinotracheitis virus (FVR), and an attenuated live strain of Chlamydophila felis.

12. The vaccine of claim 1, wherein the FeLV antigen is a FeLV glycoprotein (gp85) that comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO: 2.

13. The vaccine of claim 1, wherein the FeLV antigen is an antigenic fragment of the FeLV glycoprotein (gp85), and the antigenic fragment is FeLV (gp70).

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention provides an improved, safe nonadjuvanted FeLV vaccine. In one aspect, the vaccines of the present invention do not induce feline injection-site sarcomas, yet still provide protection to the vaccinates from the debilitating disease state caused by FeLV infection as efficaciously as an inactivated whole-virus adjuvanted vaccine.

(2) Accordingly, the vaccine compositions of the present invention include an immunologically effective amount of a vector encoding an antigen from one or more strains of feline leukemia virus that aids in eliciting protective immunity in the recipient vaccinated animal. Furthermore, the present invention provides new immunologic compositions to improve the reliability of vaccination to aid in the reduction of antigenemia in a feline infected by FeLV and to thereby yield a transient antigenemia and/or lead to the elimination of the infection. In a particular aspect of the present invention, the vaccines comprise an alphavirus RNA replicon particle (RP) encoding the FeLV viral glycoprotein (gp85). In more specific embodiments, the vaccines comprise alphavirus RNA replicon particles (RPs) that comprise the capsid protein and glycoproteins of Venezuelan Equine Encephalitis Virus (VEE) and encode the FeLV viral glycoprotein (gp85) and/or an antigenic fragment thereof (e.g., gp70 or gp45). In even more specific embodiments, the vaccines comprise alphavirus RNA replicon particles (RPs) that comprise the capsid protein and glycoproteins of the avirulent TC-83 strain of VEE and encode the FeLV viral glycoprotein (gp85) and/or an antigenic fragment thereof (e.g., gp70 or gp45). In another aspect of the present invention, the vaccines comprise naked DNA vectors that encode the FeLV viral glycoprotein (gp85) and/or an antigenic fragment thereof (e.g., gp70 or gp45). The vaccines of the present invention can be administered to a feline in the absence of an adjuvant and still effectively aid in the protection of the vaccinated feline against FeLV.

(3) In order to more fully appreciate the invention, the following definitions are provided.

(4) The use of singular terms for convenience in description is in no way intended to be so limiting. Thus, for example, reference to a composition comprising a polypeptide includes reference to one or more of such polypeptides. In addition, reference to an alphavirus RNA replicon particle includes reference to a plurality of such alphavirus RNA replicon particles, unless otherwise indicated.

(5) As used herein the term approximately is used interchangeably with the term about and signifies that a value is within fifty percent of the indicated value i.e., a composition containing approximately 1?10.sup.8 alphavirus RNA replicon particles per milliliter contains from 0.5?10.sup.8 to 1.5?10.sup.8 alphavirus RNA replicon particles per milliliter.

(6) As used herein, the term feline refers to any member of the Felidae family. Domestic cats, pure-bred and/or mongrel companion cats, and wild or feral cats are all felines.

(7) 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 they were present, would enable the successful propagation of the parental virus in cell cultures or animal hosts. In suitable cellular contexts, the replicon will amplify itself and may produce one or more sub-genomic RNA species.

(8) As used herein, the term alphavirus RNA replicon particle, abbreviated RP, is an alphavirus-derived RNA replicon packaged in structural proteins, e.g., the capsid and glycoproteins, which also are derived from an alphavirus, e.g., as described by Pushko et al., [Virology 239(2):389-401 (1997)]. An RP cannot propagate in cell cultures or animal hosts (without a helper plasmid or analogous component), because the replicon does not encode the alphavirus structural components (e.g., capsid and glycoproteins).

(9) The term non-FeLV, is used to modify terms such as pathogen, and/or antigen (or immunogen) to signify that the respective pathogen, and/or antigen (or immunogen) is neither an FeLV pathogen nor a FeLV antigen (or immunogen) and that a non-FeLV protein antigen (or immunogen) does not originate from an FeLV.

(10) 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 that pathogen that naturally encodes it, and as used herein signify that the unmodified and/or truncated amino acid sequence of that given protein antigen is encoded by that pathogen or strain of that pathogen. The coding sequence within a nucleic acid construct of the present invention for a protein antigen originating from a pathogen may have been genetically manipulated so as to result in a 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) it originates from.

(11) As used herein, the terms protecting, or providing protection to, or eliciting protective immunity to, aids in prevention of disease, and aids in the protection do not require complete protection from any indication of infection. For example, aids in the protection can mean that the protection is sufficient such that, after challenge, symptoms of the underlying infection are at least reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced and/or eliminated. It is understood that reduced, as used in this context, means relative to the state of the infection, including the molecular state of the infection, not just the physiological state of the infection.

(12) As used herein, a vaccine is a composition that is suitable for application to an animal, e.g., feline (including, in certain embodiments, humans, while in other embodiments being specifically not for humans) comprising one or more antigens typically combined with a pharmaceutically acceptable carrier such as a liquid containing water, which upon administration to the animal induces an immune response strong enough to minimally aid in the protection from a disease arising from an infection with a wild-type micro-organism, i.e., strong enough for aiding in the prevention of the disease, and/or preventing, ameliorating or curing the disease.

(13) As used herein, a multivalent vaccine is a vaccine that comprises two or more different antigens. In a particular embodiment of this type, the multivalent vaccine stimulates the immune system of the recipient against two or more different pathogens.

(14) The terms adjuvant and immune stimulant 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 an immune response to one or more vaccine antigens/isolates. Accordingly, adjuvants are agents that nonspecifically increase an immune response to a particular antigen, thus reducing the quantity of antigen necessary in any given vaccine, and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. In this context, an adjuvant is used to enhance an immune response to one or more vaccine antigens/isolates. The American Association of Feline Practitioners Feline Vaccination Guidelines suggest the use of nonadjuvanted FeLV vaccines [AAFP Feline Advisory Panel, 15: 785-808 (2013)].

(15) As used herein, a nonadjuvanted vaccine is a vaccine or a multivalent vaccine that does not contain an adjuvant.

(16) As used herein, the term pharmaceutically acceptable is used adjectivally to mean that the modified noun is appropriate for use in a pharmaceutical product. When it is used, for example, to describe an excipient in a pharmaceutical vaccine, it characterizes the excipient as being compatible with the other ingredients of the composition and not disadvantageously deleterious to the intended recipient animal, e.g., feline.

(17) Parenteral administration includes subcutaneous injections, submucosal injections, intravenous injections, intramuscular injections, intradermal injections, and infusion.

(18) As used herein the term antigenic fragment in regard to a particular protein (e.g., a protein antigen) is a fragment of that protein that is antigenic, i.e., capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. For example, an antigenic fragment of an FeLV viral glycoprotein (gp85) is a fragment of the gp85 protein that is antigenic. Preferably, an antigenic fragment of the present invention is immunodominant for antibody and/or T cell receptor recognition. In particular 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 an antigenic fragment retains at least 50% of the antigenicity of the full length protein. In more preferred embodiments, an 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 at the other extreme, be large fragments that are missing as little as a single amino acid from the full-length protein. In particular embodiments the antigenic fragment comprises 25 to 150 amino acid residues. In other embodiments, the antigenic fragment comprises 50 to 250 amino acid residues. The gp45 glycoprotein and the gp70 glycoprotein are antigenic fragments of the gp85 glycoprotein.

(19) As used herein one amino acid sequence is 100% identical or has 100% identity to a second amino acid sequence when the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is 50% identical to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In a particular embodiment, selected deletions or insertions that could otherwise alter the correspondence between the two amino acid sequences are taken into account.

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

(21) As used herein, the term inactivated microorganism is used interchangeably with the term killed microorganism. For the purposes of this invention, an inactivated microorganism is an organism which is capable of eliciting an immune response in an animal, but is not capable of infecting the animal. An antigen of the present invention (e.g., an inactivated feline panleukopenia virus) may be inactivated by an agent selected from the group consisting of binary ethyleneimine, formalin, beta-propiolactone, thimerosal, or heat. In a particular embodiment, inactivated feline calicivirus isolates combined with an RP of the present invention are inactivated by binary ethyleneimine.

(22) The alphavirus RNA replicon particles of the present invention may be lyophilized and rehydrated with a sterile water diluent. On the other hand, when the alphavirus RNA replicon particles are stored separately, but intend to be mixed with other vaccine components prior to administration, the alphavirus RNA replicon particles can be stored in the stabilizing solution of those components, e.g., a high sucrose solution.

(23) A vaccine of the present invention can be readily administered by any standard route including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal, and/or intraperitoneal vaccination. The skilled artisan will appreciate that the vaccine composition is preferably formulated appropriately for each type of recipient animal and route of administration.

(24) Thus, the present invention also provides methods of immunizing a feline against FeLV and/or other feline pathogens. One such method comprises injecting a feline with an immunologically effective amount of a vaccine of the present invention, so that the feline produces appropriate FeLV antibodies.

(25) Multivalent Vaccines:

(26) The present invention also provides multivalent vaccines. For example, the coding sequence of a protein antigen or antigenic fragment thereof, or combination of such coding sequences of protein antigens useful in a feline vaccine can be added to an alphavirus RNA replicon particle (RP) or combined in the same RP as one that encodes a feline antigen of the FeLV [e.g., the FeLV viral glycoprotein (gp85)] in the vaccine. Accordingly, such multivalent vaccines are included in the present invention.

(27) Examples of pathogens that one or more of such protein antigens can originate from include feline rhinotracheitis Virus (FVR), feline calicivirus (FCV), feline panleukopenia 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 spp. (e.g., B. henselae). In particular embodiments, a coding sequence for a capsid protein or analogous protein from one or more of these feline or canine pathogens can be inserted into the same RP as the FeLV antigen. Alternatively, or in combination therewith, a coding sequence for a capsid protein or analogous 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 that encodes the FeLV antigen in a vaccine.

(28) In addition, an alphavirus RNA replicon particle(RP) that encodes a feline antigen of the FeLV [e.g., the FeLV viral glycoprotein (gp85)] can be added together with one or more other live, attenuated virus isolates such as a live attenuated other FCV strain, a live attenuated feline herpesvirus and/or a live attenuated feline parvovirus and/or a live, attenuated feline leukemia virus, and/or a live, attenuated feline infectious peritonitis virus and/or a live, attenuated feline immunodeficiency virus and/or a live, attenuated borna disease virus and/or a live, attenuated rabies virus, and/or a live, attenuated feline influenza virus and/or a live, attenuated canine influenza virus, and/or a live, attenuated avian influenza, and/or a live, attenuated canine pneumovirus, and/or a live, attenuated feline pneumovirus. In addition, a live, attenuated Chlamydophila felis, and/or a live, attenuated Bordetella bronchiseptica and/or a live, attenuated Bartonella spp. (e.g., B. henselae) can also be included in such multivalent vaccines.

(29) Furthermore, an alphavirus RNA replicon particle (RP) that encodes a feline antigen of the FeLV [e.g., the FeLV viral glycoprotein (gp85)] can be added together with one or more other killed virus isolates such as a killed FCV strain, and/or a killed feline herpesvirus and/or a killed feline parvovirus and/or a killed feline leukemia virus, and/or a killed feline infectious peritonitis virus and/or a killed feline immunodeficiency virus and/or a killed borna disease virus and/or a killed rabies virus, and/or a killed feline influenza virus and/or a killed canine influenza virus, and/or a killed avian influenza virus, and/or a killed canine pneumovirus, and/or a killed feline pneumovirus. In addition, bacterins of Chlamydophila felis, and/or Bordetella bronchiseptica and/or Bartonella spp. (e.g., B. henselae) can also be included in such multivalent vaccines.

(30) It is also to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat.

(31) It is also to be understood that the terminology employed herein is used 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 equivalents thereof.

(32) TABLE-US-00001 SEQUENCE TABLE SEQ ID NO: Description Type 1 FeLV viral glycoprotein (gp85) nucleic acid DNA 2 FeLV viral glycoprotein (gp85) amino acid 3 FeLV viral glycoprotein (gp70) nucleic acid DNA 4 FeLV viral glycoprotein (gp70) amino acid 5 Feline Calicivirus (VS-FCV) nucleic acid DNA 6 Feline Calicivirus (VS-FCV) amino acid 7 Feline Calicivirus (F9-like) nucleic acid DNA 8 Feline Calicivirus (F9-like) amino acid 9 GGCGCGCCGCACC nucleic acid 10 FeLV viral glycoprotein (gp85) nucleic acid RNA 11 FeLV viral glycoprotein (gp70) nucleic acid RNA 12 Feline Calicivirus (VS-FCV) nucleic acid RNA 13 Feline Calicivirus (F9-like) nucleic acid RNA TTAATTAA nucleic acid

(33) TABLE-US-00002 SEQUENCES FelineLeukemiaVirusenvelopeglycoprotein(gp85)SEQIDNO:1 atggagtcaccaacacaccctaaaccttctaaagacaaaaccctctcgtggaatctcgccttccttgt gggcatcctgttcacaatcgacatcggcatggccaacccttcgccgcatcagatctacaatgtgacat gggtcattactaatgtgcagacaaacacccaggcaaatgctacttctatgcttggtactctgactgat gcttatccaaccctgcacgtcgacctttgcgatctcgtcggtgacacatgggagcccatcgtgctgaa tccaactaatgtcaaacatggtgccaggtattcttctagcaaatacgggtgtaagaccactgatcgga agaaacagcaacaaacctacccattctacgtgtgcccgggtcacgcaccgtccctgggtccgaaggga acacattgtgggggagcccaagacggtttttgcgctgcttggggttgtgaaacaaccggagaagcctg gtggaagcctacctcatcttgggactacattactgtgaaaagaggctctagccaggataacagctgcg aaggaaagtgtaatcccctggtgcttcaattcacccagaaaggccggcaggcatcatgggatggaccg aaaatgtggggacttagactctatcgcaccggatacgaccccatcgctctgtttactgtgtcacgcca agtctccaccattactccgccacaggccatggggccgaatctggtcctccccgatcagaagccaccct cacggcaaagtcaaaccggctcaaaagtggccacccaacggccccagacaaatgagtccgcacctagg tcagtggcacctacaacaatgggtccaaagcggatcggaaccggagacaggctcattaacctcgtgca agggacttatctggcccttaacgctactgaccccaacaagaccaaggattgctggctctgccttgtga gcagacctccttactatgaggggatcgccattctcggaaactactcaaatcagaccaacccccctccg tcgtgtctgagcaccccccagcacaagcttactatttcagaagtcagtggacagggaatgtgcatcgg aaccgtgccaaagactcatcaagccctttgcaacaaaactcaacaagggcacactggagctcattatc tcgccgcacctaacgggacctactgggcttgcaatactggattgaccccgtgtatctctatggccgtg ctgaattggacttccgacttctgcgtgcttattgagctttggcctagagtgacataccatcagcctga gtacgtctatacccatttcgccaaggcagtcagattccggcgggagcctatctccctgactgtggcct tgatgctcggtggactgacagtgggaggaattgcagctggagtcggaactggaaccaaggccctgctc gaaactgctcagttccggcagctgcagatggccatgcacactgacatccaggctctggaggaatcaat ttcagcccttgagaaaagcttgacctcgctgtctgaagtggtcctccaaaacaggcgcggtttggaca tcctgttccttcaagagggtggtctgtgcgccgctctcaaggaggaatgctgtttctacgctgaccat accgggctggtgcgcgataacatggcaaagctgcgggaacgcttgaaacagaggcagcaactgttcga ctctcagcagggatggttcgagggctggtttaacaagagcccatggtttaccactctgatctcttcaa tcatgggtccactgctcatcctgcttctgattcttctcttcggaccgtgtattctcaacaggctggtg cagtttgtcaaggacagaatctcggtggtccaggccctgattcttactcagcagtatcagcagattaa gcagtacgaccccgatcggccttga FelineLeukemiaVirusenvelopeglycoprotein(gp85)SEQIDNO:2 MESPTHPKPSKDKTLSWNLAFLVGILFTIDIGMANPSPHQIYNVTWVITNVQTNTQANAT SMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGARYSSSKYGCKTTDRKKQQQTY PFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAWWKPTSSWDYITVKRGSSQDNS CEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPN LVLPDQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTMGPKRIGTGDRLINLVQGTYLA LNATDPNKTKDCWLCLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGM CIGTVPKTHQALCNKTQQGHTGAHYLAAPNGTYWACNTGLTPCISMAVLNWTSDFCVLIE LWPRVTYHQPEYVYTHFAKAVRFRREPISLTVALMLGGLTVGGIAAGVGTGTKALLETAQ FRQLQMAMHTDIQALEESISALEKSLTSLSEVVLQNRRGLDILFLQEGGLCAALKEECCF YADHTGLVRDNMAKLRERLKQRQQLFDSQQGWFEGWFNKSPWFTTLISSIMGPLLILLLI LLFGPCILNRLVQFVKDRISVVQALILTQQYQQIKQYDPDRP* FelineLeukemiaVirusenvelopeglycoprotein(gp85)SEQIDNO:10 auggagucaccaacacacccuaaaccuucuaaagacaaaacccucucguggaaucucgccuuccuugu gggcauccuguucacaaucgacaucggcauggccaacccuucgccgcaucagaucuacaaugugacau gggucauuacuaaugugcagacaaacacccaggcaaaugcuacuucuaugcuugguacucugacugau gcuuauccaacccugcacgucgaccuuugcgaucucgucggugacacaugggagcccaucgugcugaa uccaacuaaugucaaacauggugccagguauucuucuagcaaauacggguguaagaccacugaucgga agaaacagcaacaaaccuacccauucuacgugugcccgggucacgcaccgucccuggguccgaaggga acacauugugggggagcccaagacgguuuuugcgcugcuugggguugugaaacaaccggagaagccug guggaagccuaccucaucuugggacuacauuacugugaaaagaggcucuagccaggauaacagcugcg aaggaaaguguaauccccuggugcuucaauucacccagaaaggccggcaggcaucaugggauggaccg aaaauguggggacuuagacucuaucgcaccggauacgaccccaucgcucuguuuacugugucacgcca agucuccaccauuacuccgccacaggccauggggccgaaucugguccuccccgaucagaagccacccu cacggcaaagucaaaccggcucaaaaguggccacccaacggccccagacaaaugaguccgcaccuagg ucaguggcaccuacaacaauggguccaaagcggaucggaaccggagacaggcucauuaaccucgugca agggacuuaucuggcccuuaacgcuacugaccccaacaagaccaaggauugcuggcucugccuuguga gcagaccuccuuacuaugaggggaucgccauucucggaaacuacucaaaucagaccaaccccccuccg ucgugucugagcaccccccagcacaagcuuacuauuucagaagucaguggacagggaaugugcaucgg aaccgugccaaagacucaucaagcccuuugcaacaaaacucaacaagggcacacuggagcucauuauc ucgccgcaccuaacgggaccuacugggcuugcaauacuggauugaccccguguaucucuauggccgug cugaauuggacuuccgacuucugcgugcuuauugagcuuuggccuagagugacauaccaucagccuga guacgucuauacccauuucgccaaggcagucagauuccggcgggagccuaucucccugacuguggccu ugaugcucgguggacugacagugggaggaauugcagcuggagucggaacuggaaccaaggcccugcuc gaaacugcucaguuccggcagcugcagauggccaugcacacugacauccaggcucuggaggaaucaau uucagcccuugagaaaagcuugaccucgcugucugaagugguccuccaaaacaggcgcgguuuggaca uccuguuccuucaagaggguggucugugcgccgcucucaaggaggaaugcuguuucuacgcugaccau accgggcuggugcgcgauaacauggcaaagcugcgggaacgcuugaaacagaggcagcaacuguucga cucucagcagggaugguucgagggcugguuuaacaagagcccaugguuuaccacucugaucucuucaa ucauggguccacugcucauccugcuucugauucuucucuucggaccguguauucucaacaggcuggug caguuugucaaggacagaaucucggugguccaggcccugauucuuacucagcaguaucagcagauuaa gcaguacgaccccgaucggccuuga FelineLeukemiaVirusenvelopeglycoprotein(gp70)SEQIDNO:3 aatcctagtccacaccaaatatataatgtaacttgggtaataaccaatgtacaaactaacacc caagctaacgccacctctatgttaggaaccttaaccgatgcctaccctaccctacatgttgac ttatgtgacctagtgggagacacctgggaacctatagtcctaaacccaaccaatgtaaaacac ggggcacgttactcctcctcaaaatatggatgtaaaactacagatagaaaaaaacagcaacag acataccccttttacgtctgccccggacatgccccctcgttggggccaaagggaacacattgt ggaggggcacaagatgggttttgtgccgcatggggatgtgagaccaccggagaagcttggtgg aagcccacctcctcatgggactatatcacagtaaaaagagggagtagtcaggacaatagctgt gagggaaaatgcaaccccctggttttgcagttcacccagaagggaagacaagcctcttgggac ggacctaagatgtggggattgcgactataccgtacaggatatgaccctatcgctttattcacg gtgtcccggcaggtatcaaccattacgccgcctcaggcaatgggaccaaacctagtcttacct gatcaaaaacccccatcccgacaatctcaaacagggtccaaagtggcgacccagaggccccaa acgaatgaaagcgccccaaggtctgttgcccccaccaccatgggtcccaaacggattgggacc ggagataggttaataaatttagtacaagggacatacctagccttaaatgccaccgaccccaac aaaactaaagactgttggctctgcctggtttctcgaccaccctattacgaagggattgcaatc ttaggtaactacagcaaccaaacaaacccccccccatcctgcctatctactccgcaacacaaa ctaactatatctgaagtatcagggcaaggaatgtgcatagggactgttcctaaaacccaccag gctttgtgcaataagacacaacagggacatacaggggcgcactatctagccgcccccaacggc acctattgggcctgtaacactggactcaccccatgcatttccatggcggtgctcaattggacc tctgatttttgtgtcttaatcgaattatggcccagagtgacttaccatcaacccgaatatgtg tacacacattttgccaaagctgtcaggttccgaaga FelineLeukemiaVirusenvelopeglycoprotein(gp70)SEQIDNO:4 NPSPHQIYNVTWVITNVQTNTQANATSMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGARYSSS KYGCKTTDRKKQQQTYPFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAWWKPTSSWDYITVKRG SSQDNSCEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPNLVLP DQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTMGPKRIGTGDRLINLVQGTYLALNATDPNKTKDCWL CLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGMCIGTVPKTHQALCNKTQQGHTGAH YLAAPNGTYWACNTGLTPCISMAVLNWTSDFCVLIELWPRVTYHQPEYVYTHFAKAVRFRR FelineLeukemiaVirusenvelopeglycoprotein(gp70)SEQIDNO:11 aauccuaguccacaccaaauauauaauguaacuuggguaauaaccaauguacaaacuaacacc caagcuaacgccaccucuauguuaggaaccuuaaccgaugccuacccuacccuacauguugac uuaugugaccuagugggagacaccugggaaccuauaguccuaaacccaaccaauguaaaacac ggggcacguuacuccuccucaaaauauggauguaaaacuacagauagaaaaaaacagcaacag acauaccccuuuuacgucugccccggacaugcccccucguuggggccaaagggaacacauugu ggaggggcacaagauggguuuugugccgcauggggaugugagaccaccggagaagcuuggugg aagcccaccuccucaugggacuauaucacaguaaaaagagggaguagucaggacaauagcugu gagggaaaaugcaacccccugguuuugcaguucacccagaagggaagacaagccucuugggac ggaccuaagauguggggauugcgacuauaccguacaggauaugacccuaucgcuuuauucacg gugucccggcagguaucaaccauuacgccgccucaggcaaugggaccaaaccuagucuuaccu gaucaaaaacccccaucccgacaaucucaaacaggguccaaaguggcgacccagaggccccaa acgaaugaaagcgccccaaggucuguugcccccaccaccaugggucccaaacggauugggacc ggagauagguuaauaaauuuaguacaagggacauaccuagccuuaaaugccaccgaccccaac aaaacuaaagacuguuggcucugccugguuucucgaccacccuauuacgaagggauugcaauc uuagguaacuacagcaaccaaacaaacccccccccauccugccuaucuacuccgcaacacaaa cuaacuauaucugaaguaucagggcaaggaaugugcauagggacuguuccuaaaacccaccag gcuuugugcaauaagacacaacagggacauacaggggcgcacuaucuagccgcccccaacggc accuauugggccuguaacacuggacucaccccaugcauuuccauggcggugcucaauuggacc ucugauuuuugugucuuaaucgaauuauggcccagagugacuuaccaucaacccgaauaugug uacacacauuuugccaaagcugucagguuccgaaga FelineCalicivirus(VS-FCV)capsid(SEQIDNO:5) atggctgacgacggatctgtgaccaccccagaacaaggaacaatggtcggaggagtgatt gccgaacccagcgctcagatgtcaactgcggcggacatggcctccggaaagtcggtggac tccgagtgggaagccttcttctcgttccacacgtccgtgaactggagcacctccgaaacc caaggaaagatcctcttcaagcagtccctgggtcccctgctgaacccgtacctggagcac atcagcaagctgtacgtcgcttggagcgggtcgatcgaagtgcgattttccatctcggga agcggcgtgttcggtggtaaactggccgccatcgtcgtgccgcctggtgtcgaccctgtc cagtcaacctccatgctgcagtacccgcacgtcctgttcgacgcaagacaagtggagcca gtgatcttctccatcccggacctccgcaacagcctgtatcacttgatgtccgataccgat accacttccctcgtgatcatggtgtacaacgatctgatcaacccgtacgccaatgactcc aacagctcgggttgcatcgtgaccgtcgaaacgaagcctggcatcgatttcaagtttcat ctgctgaaaccgcccggatccatgcttactcacgggtccatcccttccgatctgatcccc aagagctcctccctgtggattgggaaccgccactggaccgatattaccgatttcgtgatt cggcctttcgtgttccaagccaaccggcacttcgacttcaaccaggagactgccggctgg tcaactccacggttccgcccattggccgtgactgtgtcgcagtcaaagggagccaagctc gggaacggcatcgccaccgactacattgtgcctggaatccccgacggatggcctgatact accatccccaccaagctgacccctaccggagattacgccatcacctcctccgacggcaat gatattgaaaccaagctggaatacgagaacgcggacgtgattaagaacaacaccaacttc cgctccatgtatatctgcggaagcctccagagggcttggggcgacaagaagatcagcaac accgggttcatcactaccggagtgatttctgacaactccatcagcccttcgaacacaatt gaccagtccaagatcgtggtgtaccaggacaaccatgtcaattcggaggtccagactagc gacatcactcttgccatcctgggctacaccggaattggagaagaggccataggcgccaac cgggactccgtcgtgagaatttccgtgcttccggaaactggagcaaggggcggaaatcac cccatcttctacaaaaattccatgaagctgggctacgtgatctcctccattgacgtgttc aactcccaaatcctccacacctcgcgccagctgtcactgaacaactacttgttgccccct gactccttcgcggtgtaccggattattgacagcaacggatcatggttcgacattgggatt gacagcgatgggttttcattcgtgggcgtgtcgtcatttccaaagctggagtttccgctg tccgcctcatacatgggcatccagctcgcaaagatccggctggcgtccaacatccggtca tccatgactaagctgtga FelineCalicivirus(VS-FCV)capsid(SEQIDNO:6) MADDGSVTTPEQGTMVGGVIAEPSAQMSTAADMASGKSVDSEWEAFFSFHTSVNWSTSET QGKILFKQSLGPLLNPYLEHISKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPV QSTSMLQYPHVLFDARQVEPVIFSIPDLRNSLYHLMSDTDTTSLVIMVYNDLINPYANDS NSSGCIVTVETKPGIDFKFHLLKPPGSMLTHGSIPSDLIPKSSSLWIGNRHWTDITDFVI RPFVFQANRHFDFNQETAGWSTPRFRPLAVTVSQSKGAKLGNGIATDYIVPGIPDGWPDT TIPTKLTPTGDYAITSSDGNDIETKLEYENADVIKNNTNFRSMYICGSLQRAWGDKKISN TGFITTGVISDNSISPSNTIDQSKIVVYQDNHVNSEVQTSDITLAILGYTGIGEEAIGAN RDSVVRISVLPETGARGGNHPIFYKNSMKLGYVISSIDVFNSQILHTSRQLSLNNYLLPP DSFAVYRIIDSNGSWFDIGIDSDGFSFVGVSSFPKLEFPLSASYMGIQLAKIRLASNIRS SMTKL FelineCalicivirus(VS-FCV)capsid(SEQIDNO:12) auggcugacgacggaucugugaccaccccagaacaaggaacaauggucggaggagugauu 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 FelineCalicivirus(F9-like)capsid(SEQIDNO: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 FelineCalicivirus(F9-like)capsid(SEQIDNO:8) MTAPEQGTMVGGVIAEPSAQMSTAADMATGKSVDSEWEAFFSFHTSVNWSTSETQGKILF KQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPVQSTSML QYPHVLFDARQVEPVIFCLPDLRSTLYHLMSDTDTTSLVIMVYNDLINPYANDANSSGCI VTVETKPGPDFKFHLLKPPGSMLTHGSIPSDLIPKTSSLWIGNRYWSDITDFVIRPFVFQ ANRHFDFNQETAGWSTPRFRPISVTITEQNGAKLGIGVATDYIVPGIPDGWPDTTIPGEL IPAGDYAITNGTGNDITTATGYDTADIIKNNTNFRGMYICGSLQRAWGDKKISNTAFITT ATLDGDNNNKINPCNTIDQSKIVVFQDNHVGKKAQTSDDTLALLGYTGIGEQAIGSDRDR VVRISTLPETGARGGNHPIFYKNSIKLGYVIRSIDVFNSQILHTSRQLSLNHYLLPPDSF AVYRIIDSNGSWFDIGIDSDGFSFVGVSGFGKLEFPLSASYMGIQLAKIRLASNIRSPMT KL FelineCalicivirus(F9-like)capsid(SEQIDNO:13) augacugccccggaacaaggaacgauggucggaggagugauugcagaaccgucagcacag auguccaccgcugccgacauggccacuggaaagagcguggacuccgaaugggaagccuuc uucuccuuccacacuucggucaacuggucgacuagcgaaacccaggggaagauuuuguuc aagcaaucccucggcccucugcugaaccccuaccuggagcaucuggccaagcuguacgug gcauggucgggcagcaucgaagugcgcuuuagcauuuccggcuccggaguguucggggga aagcuugcugccauugucgugccgccaggaguggacccggugcaguccacuucuaugcuc caauacccgcauguccuguucgacgccagacagguggagccugugaucuuuugccugccg gaucucagguccacccuguaucaccucauguccgacaccgacaccaccucgcucgugauc augguguacaacgaccugaucaaccccuacgcuaacgacgccaacagcucagguugcauu gugacugucgaaaccaagccaggcccugacuucaaguuucauuugcugaagccgcccggu uccaugcugacccacggcucgaucccauccgaccugauccccaagacgagcucccugugg aucggaaaccgcuacugguccgauauuaccgacuucgugaucagaccauucguguuccaa gccaaccgccauuucgacuucaaccaggaaaccgcaggauggucgaccccucgauuccgc ccgauuucagugaccaucaccgaacagaacggcgcgaagcugggaauuggcguggcgacc gacuacaucgugccgggaaucccggauggauggccugauacgaccauucccggggagcug aucccugccggggacuacgccaucaccaacgguacuggaaacgacaucaccacugccacc gguuacgacaccgccgacaucauaaagaacaacaccaacuucagaggaauguacauuugc ggcucccugcaacgcgcuuggggugacaaaaagaucucgaacacugccuucaucacaaca gcgacucuggacggcgauaacaacaacaagaucaauccuuguaauaccaucgaccagucc aaaaucgugguguuccaggauaaccacgugggaaagaaggcgcagaccuccgacgacacu cuggcgcugcuuggcuacaccgggaucggcgagcaggccauuggaagcgaucgggaucgg gucgugcggaucuccacccuccccgagacuggagcaaggggaggcaaccaccccaucuuu uacaaaaacagcauuaagcucggauacgucauccgcuccaucgauguguucaacucucaa auccugcacacuucgcggcagcugucccugaaccacuaccucuugccgcccgacuccuuc gccgucuaccggaucauugauucgaacgggagcugguucgacaucggcauugauagcgau ggcuucucguuugugggcgugucgggcuucgggaagcuggaguucccacugagcgccuca uacauggguauccagcuggccaagaucaggcuggccuccaacauccgcucaccuaugacu aagcuguga
The following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention.

EXAMPLES

Example 1

(34) Incorporation of the Coding Sequences for FeLV GP85 into the Alphavirus RNA Replicon Particles

Introduction

(35) RNA viruses have been used as vector-vehicles for introducing vaccine antigens, which have been genetically engineered into their genomes. 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. The result is 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) the contents of which are hereby incorporated herein in their entireties], and Semliki Forest virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9:1356-1361 (1991), the contents of which are hereby incorporated herein in their entireties]. Moreover, alphavirus RNA replicon particles are the basis for several USDA-licensed vaccines for swine and poultry. These include: Porcine Epidemic Diarrhea Vaccine, RNA Particle (Product Code 19U5.P1), Swine Influenza Vaccine, RNA (Product Code 19A5.D0), Avian Influenza Vaccine, RNA (Product Code 1905.D0), and Prescription Product, RNA Particle (Product Code 9PP0.00).

(36) Alphavirus RNA Replicon Particle Construction

(37) An amino acid sequence for FeLV gp85 were used to generate codon-optimized (feline codon usage) nucleotide sequences in silica Optimized sequences were prepared as synthetic DNA by a commercial vendor (ATUM, Newark, CA). Accordingly, a synthetic gene was designed based on the amino acid sequence of gp85. The construct (gp85_wt) was wild-type amino acid sequence [SEQ ID NO: 2], codon-optimized for feline, with flanking sequence appropriate for cloning into the alphavirus replicon plasmid.

(38) The VEE replicon vectors designed to express FeLV gp85 were constructed as previously described [see, U.S. Pat. No. 9,441,247 B2; the contents of which are hereby incorporated herein by reference in their entireties], with the following modifications. The TC-83-derived replicon vector pVEK [disclosed and described in U.S. Pat. No. 9,441,247 B2] was digested with restriction enzymes AscI and PacI. A DNA plasmid containing the codon-optimized open reading frame nucleotide sequence of the FeLV gp85 genes, with 5 flanking sequence (5-GGCGCGCCGCACC-3) [SEQ ID NO: 9] and 3 flanking sequence (5-TTAATTAA-3), was similarly digested with restriction enzymes AscI and PacI. The synthetic gene cassette was then ligated into the digested pVEK vector, and the resulting clone was re-named pVHV-FeLV gp85. The pVHV vector nomenclature was chosen to refer to pVEK-derived replicon vectors containing transgene cassettes cloned via the AscI and PacI sites in the multiple cloning site of pVEK.

(39) Production of TC-83 RNA replicon particles (RP) was conducted according to methods previously described [U.S. Pat. No. 9,441,247 B2 and U.S. Pat. No. 8,460,913 B2; the contents of which are hereby incorporated herein by reference]. Briefly, pVHV replicon vector DNA and helper DNA plasmids were linearized with NotI restriction enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and cap analog (Promega, Madison, WI). Importantly, the helper RNAs used in the production lack the VEE subgenomic promoter sequence, as previously described [Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)]. Purified RNA for the replicon and helper components were combined and mixed with a suspension of Vero cells, electroporated in 4 mm cuvettes, and returned to OptiPro? SFM cell culture media (Thermo Fisher, Waltham MA). Following overnight incubation, alphavirus RNA replicon particles were purified from the cells and media 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. Eluted RP were formulated to a final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and dispensed into aliquots for storage. Titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers.

Example 2

(40) Comparative Efficacy and Safety of FeLV Vaccines in Cats

(41) A vaccine comprising an alphavirus RNA replicon particle (RP) comprising the capsid protein and glycoproteins 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 before use. This vaccine was compared with a commercially available vaccine comprising a recombinant canary pox encoding FeLV, as shown in Table 1 below. Five groups of eight feline subjects were vaccinated either with a single dose at 8-9 weeks, or in a prime/boost regimen of 8-9 weeks of age and then 21 days later. The doses for each experimental vaccinate group is provided in Table 1 below.

(42) TABLE-US-00003 TABLE 1 VACCINATION PROTOCOL Vaccinate No. of Vaccination Group Animals Vaccine RP/dose Days 1 8 RP-FeLV 4.35 ? 10.sup.8 0, 21 2 8 RP-FeLV 3.55 ? 10.sup.7 0, 21 3 8 RP-FeLV 1.5 ? 10.sup.8 21 (one shot) 4 8 PureVax? .sup.# Does not apply 0, 21 5 8 Placebo none 0, 21 .sup.# A vaccine containing a recombinant canary pox encoding FeLV sold by Merial?

(43) All cats were subcutaneously vaccinated with 1.0 mL of their respective vaccine regimen. Cats were 8-9 weeks of age at the time of the initial vaccination (including cats in Group 3). The cats of Group 4 were vaccinated at the times provided with the quantity of vaccine as directed on the label of the commercial vaccine. Following the vaccination the cats were observed for adverse reactions to the vaccines by observing the general health daily, as well as palpating the site of injection for the two days following each vaccination and twice per week for two weeks following each vaccination. No adverse reactions were observed for any of the vaccines.

(44) All cats were challenged with a virulent culture of FeLV four weeks after the booster vaccination (four weeks after the one-shot vaccination for the Group 3 cats). The cats were challenged on four separate days over one week (study days 49, 52, 54 and 56) by administering 1.0 mL of challenge virus by the oronasal route (0.3 mL in each nostril and 0.4 mL orally). Three weeks after challenge serum samples were collected each week through ten weeks post-challenge. Serum samples were tested by ELISA for the presence of FeLV p27 antigen. An animal is considered infected with FeLV if it is persistently antigenemic. Antigenemia is defined as a positive p27 ELISA result for three consecutive weeks or on five or more occasions during the eight week testing period. An FeLV vaccine must protect 75% of the cats vaccinated with the test product for USDA licensure. In addition, in order for the challenge to be regarded as valid, 80% of the control cats must be persistently antigenemic [see, Shipley et al., JAVMA, Vol. 199, No. 10, (Nov. 15, 1991)]. The results of the challenge are summarized in the Table 2 below.

(45) TABLE-US-00004 TABLE 2 VACCINATION AND CHALLENGE Treatment % Cats % Cats Group Vaccine RP dose Antigenemic Protected 1 RP-FeLV 4.35 ? 10.sup.8 0% 100% 2 RP-FeLV 3.55 ? 10.sup.7 0% 100% 3 RP-FeLV 1.5 ? 10.sup.8 13% 87% (one shot)* 4 PureVax? .sup.# Does not apply 43% 57% 5 Placebo Does not apply 88% 12% .sup.# A vaccine containing a recombinant canary pox encoding FeLV sold by Merial? *All other groups received a two-dose regimen, see, Table 1 above.

(46) As Table 2 demonstrates, the RP-FeLV vaccines protected 100% of the cats when administered in a two-dose regimen (i.e., primary and booster vaccination) at both doses tested. Moreover, the RP-FeLV vaccine protected 87% of the cats when administered as a single dose. In direct contrast, the commercially available vaccine only protected 57% of the cats, even with a two-dose regimen. In addition, the challenge is regarded as valid because greater than 80% of the control cats were persistently antigenemic [see, Table 2]. Finally, all of the RP-FeLV vaccine formulations were found safe in cats.

Example 3

(47) Determination of the Dose Dependence of an RP-FeLV Vaccine by Vaccination and Challenge

(48) The RP-FeLV vaccine of Example 2 was formulated in a vaccine formulation that included enzymatically hydrolyzed casein (NZ-Amine?), gelatin, and sucrose. The vaccine was then lyophilized. Four groups of ten cats each were vaccinated as summarized in Table 3 below:

(49) TABLE-US-00005 TABLE 3 VACCINATION PROTOCOL Treatment No. of Vaccination Group Animals Vaccine RP/dose Days 1 10 RP-FeLV 1.1 ? 10.sup.5 0, 21 2 10 RP-FeLV 2.1 ? 10.sup.6 0, 21 3 10 RP-FeLV 6.5 ? 10.sup.7 0, 21 4 10 Non-vaccinated None NA Controls

(50) All cats were vaccinated with 1.0 mL of respective test product, subcutaneously. The cats were 8-9 weeks of age at the time of initial vaccination. Following the vaccination the cats were observed for adverse reactions to the vaccines by observing their general daily health, as well as palpating the site of injection for the two days following each vaccination and twice per week for the two weeks following each vaccination. No adverse reactions to any of the vaccines were observed.

(51) All of the cats were challenged with a virulent culture of FeLV three weeks after the booster vaccination. Cats were challenged on four separate days over one week (study days 42, 45, 47 and 49) by administering 1.0 mL of challenge virus by the oronasal route (0.3 mL in each nostril and 0.4 mL orally). Three weeks after challenge serum samples were collected each week through twelve weeks post-challenge. Serum samples were tested by ELISA for the presence of FeLV p27 antigen. An animal is considered infected with FeLV if it is found to be persistently antigenemic. Antigenemia is defined as a positive p27 ELISA result for three consecutive weeks, or on five or more occasions during the eight-week testing period. For USDA licensure an FeLV vaccine must protect 75% of the cats vaccinated with the test product. For the challenge to be considered valid, 80% of the control cats must be persistently antigenemic [Shipley et al., JAVMA, Vol. 199, No. 10, Nov. 15, 1991]. The results of the challenge are summarized in the Table 4 below:

(52) TABLE-US-00006 TABLE 4 DOSE DEPENDENCE OF RP-FELV Treatment % Cats % Cats Group Vaccine RP/dose Antigenemic Protected 1 RP-FeLV 1.1 ? 10.sup.5 10% 90% 2 RP-FeLV 2.1 ? 10.sup.6 0% 100% 3 RP-FeLV 6.5 ? 10.sup.7 0% 100% 4 Non-vaccinated None 90% 10% Controls

(53) In this study of short term immunity, the minimum protective dose of the RP-FeLV vaccine for 100% protection of the cats was between about 1.0?10.sup.5 to about 2.0?10.sup.6 RPs, when administered in a two dose (primary and booster vaccination) regimen. The challenge was valid because at least 80% of the control cats were persistently antigenemic. All RP-FeLV vaccine formulations tested were safe in cats.

(54) 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 fall within the scope of the appended claims.

(55) It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.