Multivalent feline vaccine

Abstract

To provide novel multivalent vaccines for felines, and to provide methods of making and using the multivalent vaccines alone or in combinations with other protective agents.SOLUTION: Provided is an immunogenic composition, comprising Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles encoding one or more antigens from a feline pathogen and a modified live feline pathogen. Also provided is a vaccine to aid in the prevention of disease due to feline calicivirus (FCV) and / or feline leukemia virus (FeLV), comprising the immunogenic composition and a pharmaceutically acceptable carrier.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on December 15, 2017, the entire contents of which are incorporated herein by reference. U.S. Provisional Patent Application No. 62 / 599,401, filed December 8, 2017; U.S. Provisional Patent Application No. 62 / 596,508 filed November 6, 2017; Patent application Ser. No. 62 / 582050 and U.S. Provisional Patent No. 62 / 582050 filed November 6, 2017 Priority under 35 U.S.C. §119(e) of application Ser. No. 62 / 581,955 is claimed.

[0002] The present invention relates to a novel multivalent vaccine for cats. Methods for making and using the compositions in combination with other agents are also provided. [Background technology]

[0003] Feline respiratory disease is typified by sinusitis, conjunctivitis, lacrimation, salivation, and oral ulcers. The two most common pathogens associated with upper respiratory tract disease in cats are feline calicivirus and feline calicivirus. Also known as feline herpesvirus (FCV) and feline herpesvirus type 1 (FHV-1) The two feline viruses are feline viral rhinotracheitis virus (FVR). is thought to be the cause of approximately 80% of all feline respiratory diseases worldwide. Chlamydophila felis is a cat-like insect. A third pathogen that may also play a role in haustorial disease. Therefore, these three pathogens A vaccine against this is now commercially available.

[0004] FVR is an alphaherpesvirus related to canine herpesvirus 1 and may FVR is the most important respiratory pathogen in cats. It is highly contagious and can affect kittens and cats. It is a large enveloped DNA virus that can cause severe disease in cats. Therefore, most cats will be exposed to FVR during their lifetime. The Feline-1™ vaccine contains a modified live feline viral rhinotracheitis virus. It has.

[0005] The most common characteristic and clinical sign of FCV infection is vesicles (ulcers) on the tongue and oral mucosa. The development of ulcers, which begin as small, isolated sores but spread and affect most of the tongue. Fever is also often seen in infected cats. Certain strains of FCV can cause lymptoms. It also causes a disease in cats known as limping syndrome. It is characterized by fever, joint and muscle pain (limping), and occasional tongue / mouth ulcers. Additionally, some strains of FCV have been associated with chronic stomatitis in infected cats. Cats infected with FCV may become persistently infected, transmitting infectious virus for a long period of time. It can emit gas.

[0006] FCV isolates are antigenically highly variable, and older vaccine strains of FCV, such as FCV F9, Antibodies from cats vaccinated with IFN-γ do not effectively neutralize all current field isolates. Furthermore, new FCV strains associated with systemic disease and high mortality have been identified. See U.S. Patent No. 7,449,323. These "virulent systemic" The (VS-FCV) isolates are responsible for local outbreaks and are not currently used in vaccines. Chin also does not appear to protect cats from disease caused by these strains. .

[0007] FCV is a single-stranded positive-sense nucleic acid consisting of three open reading frames (ORFs). It contains an RNA genome that is polyadenylated at the 3' end and terminates at the 5' end with a viral code. The first open reading frame is a single polypeptide chain. Viral protease and RNA-dependent RNA polymerase expressed in the polypeptide This polypeptide is then post-translationally cleaved by the viral protease The second open reading frame encodes the major capsid protein (i.e., FC V capsid protein), which has six regions denoted as AF [ Scott et al.,60 Am.J.Vet.Res.:652-658(19 99)]. Region A is cleaved to produce the mature capsid protein. Region of ORF2 Regions B, D, and F are relatively conserved among FCV isolates, while regions C and E are variable. Region E of ORF2 contains the major B cell epitope [Radford et al. al., 38(2) Vet Res.: 319-335 (2007)]. ORF3 encodes a minor structural protein [Sosnovtsev and Gree n,277 Virology:193-203(2000)].

[0008] Chlamydophila felis is a species found worldwide. Chlamydophila felis (C. felis) is a bacterium specific to domestic cats. It can cause conjunctival inflammation, rhinitis and respiratory problems in cats. The squirrel (C. felis) has a relatively small genome encoding only about 1000 proteins. The bacterium also contains a plasmid containing 75,000 base pairs. ivac® Feline-1 vaccine is a modified live Chlamydophila feline vaccine. Contains the squirrel (Chlamydophila felis).

[0009] In addition to the three pathogens associated with feline respiratory disease mentioned above, cats commonly infect themselves with three other viruses: Feline leukemia virus (FeLV), feline panleukopenia virus (FPV or FPLV) and rabies virus. It is a retrovirus that infects humans and causes significant morbidity and mortality worldwide. FeLV is primarily transmitted through saliva, but has also been reported to spread through contact with bodily fluids. Pacitti et al., Vet Rec 118:381-384( 1986)doi:10.1136 / vr.118.14.381;Levy et a l., J Feline Med Surg 10:300-316(2008)doi :10.1016 / j.jfms.2008.03.002]. Clinical signs in cats include cell proliferative disorders (lymphoid or myeloid tumors), cytostatic disorders, and Harms (immunosuppression, anemia, infectious diseases related to bone marrow suppression), inflammatory disorders, neurological disorders, miscarriage, and enteritis [Hoover et al., J Am Vet Med As soc 199:1287-1297(1991);Levy and Crawfor d,Textbook of Veterinary Internal Medicine ne,6th ed (Ettinger SJ, Feldman EC., eds .) W.B. Saunders, Philadelphia, PA. (2005)]. The prevalence of antigenemia varies 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 Ve Terinary Journal 75:323-327(1997);Arjona et al., Journal of Clinical Microbiology 38:3448-3449 (2000)]. FeLV is often associated with persistent viruses. It establishes a persistent infection with rhusemia, often leading to death of the host cat.

[0010] The single-stranded RNA genome of FeLV contains only three genes: (i) envelope glycoprotein (ii) the ENV gene, which encodes the protein; and (iii) the GAG ​​gene, which encodes the major structural elements of the virus. and (iii) the POL gene encoding the RNA polymerase [T homsen et al., Journal of General Virolog y 73:1819-1824(1992)]. FeLV envelope (ENV) gene is proteolytically processed by one or more cellular enzymes to produce the major envelope glycoprotein. The gp85 precursor that produces the protein gp70 and the related transmembrane protein p15E Encoding proteins [DeNoronha, et al., Virology 8 5:617-621(1978);Nunberg et al.,PNAS 81:3 675-3679 (1983)]. The transmembrane protein p15E has immunosuppressive properties. It contains sequences conserved among gammaretroviruses [Mathes et al., Nature 274:687-689(1978)]. In recent years, the European Medicines Agency's veterinary medicine The Committee on Medicinal Products for People with Malignant Breast Cancer (CVMP) has approved the use of Escherichia coli as an active ingredient. ) recombinant p derived from the gp70 surface glycoprotein of FeLV subgroup A expressed in 45 A positive opinion was adopted for vaccines containing FeLV envelope antigens. LV envelope glycoproteins are targets of FeLV-specific cytotoxic T cell responses. It is sometimes a neutralizing antibody and therefore one of the major immunogens of FeLV [Flynn et al., J. Virol. 76(5):2306-2315(2002)].

[0011] Feline panleukopenia (FPV or FPLV) is an extremely contagious, often fatal disease in cats. Panleukopenia is a viral disease that affects animals. FPLV is caused by a lack of white blood cells (leukocytes). In very young kittens, it infects and destroys the actively dividing cells of the retina and cerebellum. The virus can also spread transplacentally in pregnant cats, causing embryonic resorption, fetal mummification, and stillbirth. Infected cats shed the virus in their urine, faeces and nasal secretions. When susceptible cats come into contact with these secretions or infected cat fleas, they become infected. Clinical signs of FPLV infection are also known as feline distemper or feline parvo. It is classified.

[0012] Feline panleukopenia virus is a virus that belongs to the family Parvoviridae. FPLV is a member of the Parvovirus genus. It is closely related to canine enteritis virus and canine parvovirus type 2 (CPV-2). FPLV has a sequenced single-stranded DNA genome [Liu et al., Ge nome Announc.Mar-Apr;3(2)(2015):e01556-1 4]. The commercially available Nobivac® Feline-1 vaccine is a modified Contains live feline panleukopenia virus.

[0013] Rabies is a preventable zoonotic disease that causes inflammation of the brain in humans and other mammals. Clinical rabies is typically classified as either violent rabies or paralytic rabies. Violent rabies is characterized by restlessness, irritability, and aggression. Rabies is characterized by excessive salivation, deep, labored breathing, paralysis, and eventually coma. The causative agent of rabies is the rabies virus, which infects most mammals, including cats. can be contaminated and maintain reservoirs of wild and susceptible domestic animals.

[0014] The rabies virus consists of five structural proteins: nucleoprotein (N), phosphoprotein (P), ), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polynucleotides Dietzschold e, an enveloped RNA virus that encodes a methylase t al., Crit Rev Immunol 10:427-439(1991)] Glycoprotein (G) is considered to be a protective antigen that induces virus-neutralizing antibodies [Co x et al., Infect Immun 16:754-759 (1977)] Several rabies vaccines are produced to combat the disease. The animal-derived whole virus killed rabies virus vaccine is the most commonly used in the United States. These whole virus killed rabies virus vaccines require high levels of antigen. Unfortunately, the use of this adjuvant is associated with a perceived risk of injection site reactivity, hypersensitivity, and even injection site sarcoma in cats. Recently, modified live vaccines have been used as oral vaccine baits for immunization of wild animals. It is widely used [Mahl et al., Vet Res 45(1):77(2 014)]. In addition, a recombinant vaccine expressing glycoprotein (G) is currently available for use in cats. Nucleic acid vaccines are also being used in laboratory studies, but currently None are licensed in the United States.

[0015] Some vector strategies have long been used with vaccines in an effort to protect against certain pathogens. One such vector strategy involves the use of alphavirus-derived vectors. This includes the use of replicon RNA particles (RP) [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)], this Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virol ogy 239:389-401(1997)], Sindbis (SIN) [Breden beek et al., Journal of Virology 67:6439- 6446 (1993)] and Semliki Forest virus (SFV) [Liljestrom and Garoff,Biotechnology(NY)9:1356-1361 (1991)]. delivers a replication-deficient alphavirus RNA replicon to host cells and allows for in vivo (1 or resulting in expression of a desired antigenic transgene (or genes) [Pushko et al., Virology 239(2):389-401(1997)]. RP has several Possesses an attractive safety and efficacy profile when compared to conventional vaccine formulations [Vander Veen,et al.Anim Health Res Rev.1 3(1):1-9.(2012)]. The RP platform encodes pathogenic antigens. and is the basis for several USDA-licensed vaccines for swine and poultry. It is.

[0016] In particular, alphavirus RNA replicon particles, particularly Venezuelan equine encephalitis (VEE) alphaviruses, The lufavirus RNA replicon particle catalyzes a systemic antiviral state, preventing the deadly virus Protects against virulence attacks [Konopka et al., J. Virol., 83 (2 9):12432-12442(2009)], especially for rapid protection against foot-and-mouth disease virus [Segundo et al., J. Virol., 87(10):544 7-5460(2013)]. Therefore, alphavirus RN A replicon particles enhance the innate immune response to live viruses, and are therefore detrimental to the immune response. Therefore, the alphavirus RNA replicon particles were used in the vaccine to It seems that it should not be combined with Irus.

[0017] Citation of any reference herein is not intended to be an admission that such reference is "Prior Art" to the present application. This document should not be construed as an endorsement that the technology is available as a "technical" technology. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] U.S. Patent No. 7,449,323 [Non-patent literature]

[0019] [Non-Patent Document 1] Scott et al.,60 Am.J.Vet.Res.:652-658(1999) [Non-patent document 2] Radford et al.,38(2)Vet Res.:319-335(2007) [Non-patent document 3] Sosnovtsev and Green,277 Virology:193-203(2000) [Non-patent document 4] Pacitti et al., Vet Rec 118:381-384(1986)doi:10.1136 / vr.118.14.381 [Non-Patent Document 5] Levy et al.,J Feline Med Surg 10:300-316(2008)doi:10.1016 / j.jfms.2008.03.002 [Non-patent document 6] Hoover et al., J Am Vet Med Assoc 199:1287-1297(1991) [Non-Patent Document 7] Levy and Crawford, Textbook of Veterinary Internal Medicine, 6th ed (Ettinger SJ, Feldman EC., eds.) WB Saunders, Philadelphia, PA. (2005) [Non-licensed document 8] Hosie et al.Veterinary Records,128:293-297(1989)

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[0020] Thus, the present invention provides a method for producing a medicament containing one or more antigens from one or more feline pathogens. The alphavirus RNA replicon particles that encode the alphavirus are delivered to one or more modified live cat replicon vectors. All of the immunogenic compositions of the present invention include multivalent vaccines. In a particular embodiment of this type, the subject to be vaccinated is a cat. In a more particular embodiment, the subject to be vaccinated is a domestic cat. Methods of making and using the immunogenic compositions and vaccines are also provided.

[0021] In certain embodiments, the immunogenic composition comprises one or more feline caliciviruses (FC V) Alphavirus RNA replicon particles encoding antigens and modified live feline pathogens In another embodiment, the immunogenic composition comprises one or more feline leukemia viruses. Alphavirus RNA replicon particles encoding FeLV antigens and modified In yet another embodiment, the immunogenic composition comprises one or more Alphavirus RNA replicon particles encoding rabies virus antigens and modified In certain embodiments, the modified live feline pathogen is a feline virus. In another embodiment, the modified live feline pathogen is: In yet another embodiment, the modified Live feline pathogen Chlamydophila felis (Live) felis). In yet another embodiment, the modified live feline pathogen is a modified In yet another embodiment, the modified F9-like feline calicivirus (FCV F9-like) is The modified live feline pathogen was modified live Bordetella bronchiseptica (Bordete The present invention further provides these alpha- Immunogens containing any combination of viral RNA replicon particles and modified live feline pathogens In a specific embodiment of this type, the immunogenic composition comprises an FCV antibody. Alphavirus RNA replicon particles encoding the antigen, and alphavirus RNA replicon particles encoding the FeLV antigen F-virus RNA replicon particles, modified live FVR, modified live FPLV, and and modified live Chlamydophila felis Includes:

[0022] In certain embodiments of this type, the immunogenic composition contains an FCV capsid protein. In a more particular embodiment, the alphavirus RNA replicon particles include those encoding F In another embodiment, the CV capsid protein is an FCV F9-like capsid protein. Alphavirus RNA replicon particles express antigens of FCV F9-like capsid proteins In yet another embodiment, the FCV capsid protein encodes a highly virulent systemic fragment. In yet another embodiment, the capsid protein is a virulent FCV (VS-FCV) capsid protein. The viral RNA replicon particle encodes an antigenic fragment of the VS-FCV capsid protein. In yet another embodiment, the alphavirus RNA replicon particles are F9-like capsid protein or its antigenic fragment and VS-FCV capsid protein or The nucleic acid encodes both the nucleotide sequence or an antigenic fragment thereof.

[0023] In other embodiments, the immunogenic composition comprises a VS-FCV capsid protein or an antibody thereof. The alphavirus RNA replicon particles encode the virulent fragment, and the VS-FCV capsule The amino acid sequence of the nucleotide sequence of SEQ ID NO:2 is 95% or more identical to the amino acid sequence of SEQ ID NO:2. In a more specific embodiment, the VS-FCV capsid protein comprises the sequence set forth in SEQ ID NO:2. In an even more specific embodiment, the VS-FCV capsid protein comprises the amino acid sequence The protein is encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 12. In certain embodiments, the alphavirus RNA replicon particles of the invention are FCV F9-like Encoding a capsid protein or an antigenic fragment thereof. Specific embodiments of this type In this case, the FCV F9-like capsid protein has a sequence identity of 95% or more with the amino acid sequence of SEQ ID NO: 4. In a more specific embodiment, the FCV F9-like capsid comprises an amino acid sequence comprising the identity of The dode protein comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the FCV F9-like capsid protein is a nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 13. It is encoded by a nucleotide sequence.

[0024] In a related embodiment, the immunogenic composition comprises one or more feline leukemia viruses (F alphavirus RNA replicon particles encoding (eLV) antigens. In some embodiments, the FeLV antigen is a FeLV glycoprotein (e.g., gp85). In this state, alphavirus RNA replicon particles express antigenic fragments of FeLV gp85. In an even more specific embodiment of this type, the antibody encodes an FeLV glycoprotein. In a related embodiment, the proteolytic fragment is FeLV gp70. In a more specific embodiment of this type, the antigenic fragment is FeLV gp45. LV gp85 has an amino acid sequence that contains 95% or more identity with the amino acid sequence of SEQ ID NO:6. In a more specific embodiment of this type, FeLV gp85 comprises the sequence SEQ ID NO:6 In an even more specific embodiment of this type, the FeLV gp 85 is encoded by the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:14. In some embodiments, the FeLV gp70 has a sequence identity of 95% or more with the amino acid sequence of SEQ ID NO:8. In a more specific embodiment of this type, the amino acid sequence comprises an amino acid sequence that is identical to FeLV. gp70 comprises the amino acid sequence of SEQ ID NO: 8. An even more specific embodiment of this type In this embodiment, FeLV gp70 is expressed by the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 15. It is coded as:

[0025] In yet another embodiment, the immunogenic composition comprises rabies virus glycoprotein (G). In yet another embodiment, the alphavirus RNA replicon particles include The rabies virus RNA replicon particle encodes an antigenic fragment of rabies virus G. In a more specific embodiment of this type, the rabies virus G has the amino acid sequence of SEQ ID NO: 10. An even more specific example of this type is an amino acid sequence containing 95% or more identity to the sequence. In an embodiment, the rabies virus G comprises the amino acid sequence of SEQ ID NO: 10. In an even more specific embodiment, the rabies virus G is SEQ ID NO: 9 or SEQ ID NO: 16 It is encoded by the nucleotide sequence

[0026] In certain embodiments, the alphavirus RNA replicon particles are (VEE) Alphavirus RNA Replicon Particles. In an embodiment, the VEE alphavirus RNA replicon particles are TC-83 VEE alphavirus RNA replicon particles. In another embodiment, the alphavirus RNA replicon particle is an alphavirus RNA replicon particle. The replicon particle is a Sindbis (SIN) alphavirus RNA replicon particle. In yet another embodiment, the alphavirus RNA replicon particles are isolated from the Semliki Forest In an alternative embodiment, the naked virus is a SFV alphavirus RNA replicon particle. The DNA vector encodes one or more antigens derived from one or more feline pathogens. In a particular embodiment of this type, the naked DNA vector is In a specific embodiment of this type, the vector encodes a naked protein or an antigenic fragment thereof. The DNA vector encodes the VS-FCV capsid protein or an antigenic fragment thereof. In other embodiments of this type, the naked DNA vector is FeLV gp85 or Encodes an antigenic fragment.

[0027] In certain embodiments, the immunogenic composition comprises at least one modified live feline pathogen and , one or more FCV capsid proteins or antigenic fragments thereof, one or more FeLV glycoprotein or an antigenic fragment thereof, and / or one or more rabies Alphavirus RNA replicas encoding the disease virus G protein or its antigenic fragments In a particular embodiment of this type, the alphavirus may comprise a recombinant particle. The RNA replicon particles contain VS-FCV capsid protein or its antigenic fragment and FC The VF9-like capsid protein or an antigenic fragment thereof is encoded by the In this form, the alphavirus RNA replicon particle contains the VS-FCV capsid protein or an antigenic fragment thereof and FeLV gp85 or an antigenic fragment thereof. In other embodiments, the alphavirus RNA replicon particle comprises an FCV F9-like capsid. The protein or its antigenic fragment and FeLV gp85 or its antigenic fragment are both co-expressed. In another embodiment, the alphavirus RNA replicon particle is VS-FCV. Capsid protein or its antigenic fragment and rabies virus G protein or its antigen In yet another embodiment, the alphavirus RNA replicon encodes both the alphavirus RNA fragment and the alphavirus RNA fragment. The virus particles contain FCV F9-like capsid protein or its antigenic fragment and rabies virus G In yet another embodiment, the alpha-codon encodes both the alpha-codon and the alpha-codon. The virus RNA replicon particle contains the VS-FCV capsid protein or its antigenic fragment. fragments thereof, FeLV gp85 or antigenic fragments thereof, and rabies virus G protein or or an antigenic fragment thereof. The VS-FCV capsid protein or an antigenic fragment thereof, FCV F9-like capsid, cid protein or an antigenic fragment thereof, and FeLV gp85 or an antigenic fragment thereof In yet other embodiments, the alphavirus RNA replicon particle encodes V S-FCV capsid protein or an antigenic fragment thereof, FeLV gp85 or an antigen rabies virus G protein or its antigenic fragment, and FCV F9-like virus In yet another embodiment, the nucleic acid sequence encodes an alpha-peptide protein or an antigenic fragment thereof. the feLV RNA replicon particle comprises FeLV gp85 or an antigenic fragment thereof, and In yet another embodiment, the rabies virus G protein or an antigenic fragment thereof is encoded by the rabies virus G protein. The present study demonstrates that alphavirus RNA replicon particles express FeLV gp85 or its antigenic fragments. fragment, FCV F9-like capsid protein or its antigenic fragment, and rabies virus G It encodes a protein or an antigenic fragment thereof.

[0028] Thus, the present invention provides a method for producing at least one modified live feline pathogen and a method for producing a plurality of feline pathogens. Any one or more alphavirus RNA replicas of the invention encoding the somatic antigen. Any one or more of the antigens of the present invention encoding antigen particles and / or single feline pathogen antigens. and an alphavirus RNA replicon particle of the present invention. In one embodiment, the modified live feline pathogen is a modified live FVR. In yet another embodiment, the modified live feline pathogen is a modified live FPLV. The modified live feline pathogen is modified live Chlamydophila felis (Chlamydo In yet another embodiment, the modified live feline pathogen is , modified live FCV F9-like. In yet another embodiment, modified live feline pathogen The body is a modified live Bordetella bronchiseptica (Bordetella bronc hiseptica). In certain embodiments, two or more modified live feline pathogens In a specific embodiment of this type, the immunogenic composition comprises: Modified Live FVR and Modified Live Chlamydophila In other embodiments, three or more modified live feline pathogens are included in the immunogenic composition. In a specific embodiment of this type, the immunogenic composition comprises a modified live FVR, a modified Contains modified live Chlamydophila and modified live feline disease In yet another embodiment, the agent is FPLV. In a specific embodiment of this type, the immunogenic composition comprises a modified Modified Live FVR, Modified Live Chlamydophila, Modified In certain embodiments, the immunogenicity of the immunized FCV includes live modified FPLV, and live modified F9-like FCV. All of the alphavirus RNA replicon particles in the original composition were found to be resistant to Venezuelan equine encephalitis ( In an even more specific embodiment, the alphavirus is an alphavirus RNA replicon particle (VEE). indicates that all of the VEE alphavirus RNA replicon particles in the immunogenic composition are TC- 83 VEE alphavirus RNA replicon particle.

[0029] In a further embodiment, the alphavirus RNA replicon particles are capable of binding to other feline pathogens. This type of specific antigen can encode a protein antigen (or an antigenic fragment thereof) derived from the antigen. In an embodiment, the protein antigen is derived from feline pneumovirus (FPN). In another embodiment, the protein antigen is derived from feline parvovirus (FPV). In another embodiment, the protein antigen is derived from feline infectious peritonitis virus (FIPV). In yet another embodiment, the protein antigen is derived from feline immunodeficiency virus. In yet other embodiments, the protein antigen is derived from Borna Disease Virus (BDV). In yet other embodiments, the protein antigen is derived from a feline influenza virus. In yet another embodiment, the protein antigen is derived from feline coronavirus (FCoV). do.

[0030] The present invention further provides an alphavirus RNA replicon particle of the present invention, comprising one or more of the alphavirus RNA replicon particles of the present invention. or multiple modified live (e.g., attenuated) feline pathogens together with killed feline pathogens. Immunogenic compositions and / or vaccines containing the antibodies (multivalent vaccines) are provided. In the present invention, the immunogenic composition comprises killed Chlamydophila felis. felis), and / or dead FVR, and / or dead F9-like FCV, and and / or killed VS-FCV, and / or killed FeLV, and / or killed FPLV In certain embodiments, the vaccine may further comprise an immunologically effective amount of one or more These immunogenic compositions include:

[0031] The present invention further includes vaccines and multivalent vaccines comprising the immunogenic compositions of the present invention. In certain embodiments, the multivalent vaccine is a non-adjuvanted vaccine. If the vaccine is FCV, and / or FeLV, and / or FVR, and / or or FPLV, and / or rabies virus, and / or Chlamydophila ferri Helps prevent disease caused by Chlamydophila felis. In an embodiment, antibodies are induced in the feline subject when the cat is immunized with the vaccine. Additionally, all of the alphavirus RNA replicon particles and naked DNA vectors of the present invention Includes:

[0032] The present invention also provides a method of immunizing a cat against a feline pathogen, such as FCV, comprising administering to the cat a and a method comprising administering to a cat a therapeutically effective amount of a vaccine or multivalent vaccine of the present invention. In certain embodiments, the vaccine is administered via intramuscular injection. In some embodiments, the vaccine is administered via subcutaneous injection. In yet another embodiment, the vaccine is administered via intradermal injection. In yet other embodiments, the vaccine is administered via intradermal injection. In embodiments, the vaccine is administered via oral administration. In a specific embodiment, the cat is a domestic cat.

[0033] The vaccines and multivalent vaccines of the present invention may be used as a primer vaccine and / or a booster vaccine. In a specific embodiment, the vaccine of the present invention can be administered as a subsequent In certain embodiments, the vaccine is administered as a single vaccine (one dose), without the need for repeated administration. In the case of administration of both primer and booster vaccines, the primer and booster vaccines The primary and booster vaccines may be administered by the same route. In this condition, both the primer and booster vaccines are administered by subcutaneous injection. In an alternative embodiment, for administration of both a primer vaccine and a booster vaccine: Primer vaccines are administered by one route and booster vaccines by another route. In certain embodiments of this type, the primer vaccine may be administered subcutaneously. The vaccine may be administered by injection and a booster vaccine may be administered orally.

[0034] The present invention further provides a method of immunizing a cat against FCV, comprising administering to the cat an immunologically effective amount of the above-described In certain embodiments, the method comprises injecting a cat with a vaccine of the present invention. is about 1 x 10 4 ~Approx. 1×10 10 Contains RP or more In a more particular embodiment, the vaccine is obtained at about 1 x 10 5 ~Approx. 1×10 9 RP In an even more particular embodiment, the vaccine may comprise about 1 x 10 6 ~Approx. 1×10 8 pieces In certain embodiments, the cat is a domestic cat.

[0035] In certain embodiments, the vaccines of the invention are administered in doses of 0.05 mL to 3 mL. In a more particular embodiment, the administered dose is between 0.1 mL and 2 mL. In certain embodiments, the administered dose is between 0.2 mL and 1.5 mL. In embodiments, the administered dose is 0.3 to 1.0 mL. In this case, the administered dose is 0.4 mL to 0.8 mL.

[0036] These and other aspects of the present invention are better understood by reference to the following detailed description. Let it be understood. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention provides a safe and effective multivalent vaccine. In certain embodiments, the vaccine comprises a non- In this aspect of the invention, the vaccine does not induce feline injection site sarcomas. However, feline calicivirus (FCV) and / or feline leukemia virus (FeL) V), and feline viral rhinotracheitis virus (FVR), and / or Feline panleukopenia virus (FPLV), and / or live Chlamydophila felis Is it a disease caused by infection with Chlamydophila felis? Helps protect cats from vaccinates.

[0038] Despite the known enhancement of the innate immune system by alphavirus RNA replicon particles The present invention, including both alphavirus RNA replicon particles and modified live viruses, Multivalent vaccines are immunizations containing live viruses engineered with alphavirus RNA replicon particles. The study is unexpectedly safe and effective without significantly interfering with clinical efficacy.

[0039] Indeed, alphavirus RNA replicon particles, particularly Venezuelan equine encephalitis (VEE) The lufavirus RNA replicon particle catalyzes a systemic antiviral state, preventing the deadly virus It has previously been shown to protect against virulence attacks [Konopka et al., J. Vir ol.,83(29):12432-12442(2009)]. Furthermore, VEE Alf Antiviral RNA replicon particles induce rapid protection against foot-and-mouth disease virus It has been reported [Segundo et al., J. Virol., 87(10): 5447-5460(2013)]. Therefore, alphavirus RNA replicon particles Vaccines containing both the modified live virus and the modified live virus have the immunological efficacy of the modified live virus. Surprisingly, however, the Enhancement of the innate immune response by the presence of viral RNA replicon particles is accompanied by altered immune responses. The results showed that the immune response induced by the live virus was not adversely affected.

[0040] Thus, in a particular embodiment, the present invention provides an FCV capsid protein or an antigen thereof. alpha encoding FeLV gp85 or an antigenic fragment thereof The viral RNA replicon particles are then transfected with modified live feline FVR and / or modified Live FPLV, and / or modified live Chlamydophila felis (Chlamydo In yet another aspect of the present invention, a vaccine is provided comprising the virus in combination with a strain of the virus known as genus phila felis. , the vaccine comprises an FCV capsid protein or an antigenic fragment thereof and / or FeL A naked DNA vector encoding V gp85 or an antigenic fragment thereof is introduced into a modified live Feline FVR, and / or modified live feline FPLV, and / or modified live cat Includes Chlamydophila felis.

[0041] The vaccine of the present invention can be administered to cats in the absence of adjuvant and still This can effectively help protect vaccinated cats against FCV.

[0042] In order to more fully understand the present invention, the following definitions are provided.

[0043] The use of singular terms in the description for convenience is in no way intended to be limiting. Thus, for example, "a polypeptide" is not included. Reference to a composition comprising includes reference to one or more such polypeptides. References to "alphavirus RNA replicon particles" are plural unless otherwise indicated. This includes reference to such alphavirus RNA replicon particles.

[0044] As used herein, the term "approximately" is used interchangeably with the term "about." is used to indicate that the value is within 50 percent of the indicated value, i.e., 1 mm Approximately 1 x 10 per liter 8 Contains alphavirus RNA replicon particles The composition is 0.5 x 10 per milliliter 8 ~1.5×10 8 Alphawii Contains RNA replicon particles.

[0045] As used herein, the term "cat" refers to any member of the family Felidae. refers to members of the All feral cats are cats.

[0046] As used herein, the term "replicon" refers to a vector that, when present, is a vector that is capable of expressing a replicon in a cell culture medium. One or more factors that allow successful propagation of the parent virus in a human or animal host ( For example, it refers to a modified RNA virus genome that lacks coding sequences for structural proteins. In the appropriate cellular context, the replicon amplifies itself and produces one or more subgenomic RNA species. can be produced.

[0047] As used herein, "alphavirus RNA replicon," abbreviated as "RP," The term "particle" refers to structural proteins, e.g., Pushko et al. [Virology 2 39(2):389-401(1997)], as well as alpha Alphaviruses packaged in viral capsids and glycoproteins The replicon is a structural component of the alphavirus (e.g., the alphavirus). Since the RP does not encode the nucleotides (nucleotides and glycoproteins), it is not necessary to use a helper plasmid or similar cannot grow in cell culture or animal hosts (without similar components).

[0048] The terms "FCV F9-like" and "F9-like FCV" are used interchangeably with each other and with "classical FCV" The term "FCV" is used interchangeably with the term "V," and as used herein, the FCV F9 strain is representative. It can be characterized as an old, pre-universal vaccine strain of FCV that is considered a representative In direct contrast, highly virulent systemic "VS-FCV" or interchangeably referred to herein as The FCV used, called "(VS)FCV," is unusually virulent and FCV F9-like. This is a new class of FCV that cannot be neutralized by antibodies from the same strain [U.S. Patent No. 7,449,323 No. specification; Radford et al.,38(2)Vet res.319-335 (2007)].

[0049] "originate from", "originates" The terms "from" and "originating from" , for a given protein antigen and the pathogen or strain of that pathogen that naturally encodes it. and as used herein refer to the unmodified form of a given protein antigen. and / or the truncated amino acid sequence is encoded by the pathogen or a strain of the pathogen. The nucleic acid construct of the present invention for a protein antigen derived from a pathogen The coding sequence in the vector is the same as that of the pathogen or strain of the pathogen (including naturally attenuated strains). Alteration of the amino acid sequence of the expressed protein antigen relative to the corresponding sequence of the protein antigen and may have been genetically engineered to produce cleavage and / or amputation.

[0050] As used herein, "protect" or "provide protection" or "protection" means The terms "inducing immunity," "helping prevent disease," and "helping protect" are used to describe the effects of sensitization. It does not require complete protection from the onset of infection. For example, "help with defense" means that defense is At least a reduction in the symptoms of the underlying infection and / or a reduction in the underlying cellular, physiological One or more of the biological or biochemical causes or mechanisms that cause the symptoms are reduced and It can mean sufficient to exclude and / or to be excluded. The "reduced" refers to the state of infection, including not only the physiological state of infection but also the molecular state of infection. It means that the state is related to the

[0051] As used herein, a "vaccine" refers to a substance that, when administered to an animal, prevents the development of a disease caused by a wild-type microorganism. Strong enough to minimally help protect against disease resulting from infection, i.e., prevent disease. and / or induce an immune response strong enough to prevent, ameliorate, or cure disease. Inducing the or multiple antigens, including animals, such as cats (and in certain embodiments, humans, but also other In embodiments, the composition is suitable for application to a subject (specifically not a human).

[0052] As used herein, a multivalent vaccine is a vaccine that contains two or more different antigens. In certain embodiments of this type, the multivalent vaccine is directed against two or more different pathogens. stimulates the recipient's immune system against the virus.

[0053] The terms "adjuvant" and "immunostimulant" are used interchangeably herein, It is defined as one or more substances that cause stimulation of the immune system. Adjuvants are used to enhance the immune response to one or more vaccine antigens / isolates. Therefore, an "adjuvant" is used to direct the immune response to a specific antigen in a non-specific manner. thus significantly increasing the amount of antigen required for any given vaccine, and / or Agents that reduce the frequency of injections required to generate an adequate immune response to an antigen of interest In this context, adjuvants are used to enhance the immune response of one or more vaccine antigens / isolates. For example, the American Association of Feline Veterinarians' Cat Vaccine The vaccination guidelines suggest the use of non-adjuvanted FeLV vaccines [AAF P Feline Advisory Panel, 15:785-808 (201 3)].

[0054] As used herein, a "non-adjuvanted vaccine" is a vaccine that does not contain an adjuvant. It is a non-immune or multivalent vaccine.

[0055] As used herein, the term "pharmaceutically acceptable" means that the modified noun Used adjectively to mean suitable for use in medicines. For example, when used to describe an excipient in a pharmaceutical vaccine, this refers to the compatible with the other ingredients of the composition and not adversely or harmful to the intended recipient animal, e.g., cats. characterize it as not.

[0056] "Parenteral administration" includes subcutaneous injection, submucosal injection, intravenous injection, intramuscular injection, intradermal injection, and Including injections.

[0057] As used herein, "antibody" refers to a specific protein (e.g., a protein antigen). The term "antigenic fragment" refers to a fragment that is antigenic, i.e., does not react with an immunoglobulin (antibody) or T Its ability to specifically interact with antigen-recognizing molecules of the immune system, such as cell antigen receptors, For example, an antigenic fragment of the FCV capsid protein is an antigenic fragment of the FCV capsid protein. Preferably, the antigenic fragment of the present invention is an antibody and / or a fragment of the capsid protein. or immunodominant for T cell receptor recognition. An antigenic fragment of a protein antigen retains at least 25% of the antigenicity of the full-length protein. In a preferred embodiment, the antigenic fragment is a fragment of the full-length protein. In a more preferred embodiment, the antigenic fragment retains at least 50% of the antigenicity of the complete Retains at least 75% of the antigenicity of the full-length protein. Antigenic fragments are 20 amino acids long. They can be as small as an amino acid, or as small as a single amino acid from a full-length protein. In certain embodiments, the antigenic fragment may be a larger fragment in which only the amino acid is missing. In another embodiment, the antigenic fragment comprises 25 to 150 amino acid residues. It contains 50 amino acid residues. For example, in the case of FeLV, the FeLV gp45 glycoprotein The FeLV gp70 glycoprotein is an antigenic fragment of the FeLV gp85 glycoprotein. In the case of FCV, one antigenic fragment of the FCV capsid protein is located in ORF2. Includes area E.

[0058] As used herein, if the amino acid residues in both sequences are identical, one amino acid A sequence of amino acids is 100% "identical" to or has 100% "identity" with a second amino acid sequence. Therefore, if 50% of the amino acid residues in two amino acid sequences are identical, An amino acid sequence is 50% "identical" to a second amino acid sequence. Sequence comparison is amino acid residues contained in a portion of a protein, e.g., a protein or polypeptide to be compared In certain embodiments, the correspondence between the two amino acid sequences is Selected deletions or insertions that may alter the sequence are considered.

[0059] As used herein, % nucleotide and amino acid sequence identity is used to determine alignment. C, along with the default parameters for the input and the default parameters for the identity MacVector(MacVector, Inc.Cary, NC 27519) , Vector NTI (Informax, Inc. MD), Oxford Mol The vascular Group PLC (1996) and Clustal W algorithms These commercially available programs can be used to determine the same or similar Sequence similarity can also be determined using the default parameters of , using default parameters. oup, Program Manual for the GCG Package, Version 7, Madison, Wis. Using the pileup program, Ad under default filter conditions A vanished Blast search can be used.

[0060] As used herein, the term "inactivated" microorganisms refers to "dead" microorganisms. For purposes of the present invention, an "inactivated" microorganism is one that has been immunized in an animal. The antibodies of the present invention are organisms that can induce a response but are unable to infect animals. The original (e.g., inactivated feline calicivirus) is treated with binary ethyleneimine (BIN). ethyleneimine), formalin, β-propiolactone, thimerosal, or heat. In certain embodiments, The inactivated feline calicivirus isolate combined with the RP of the present invention is It is inactivated by phenylalanine.

[0061] The alphavirus RNA replicon particles of the present invention can be lyophilized and reconstituted in a sterile water diluent. On the other hand, alphavirus RNA replicon particles are individually preserved, but , alphavirus RNase A, if intended to be mixed with other vaccine components before administration A. Storing replicon particles in a stabilizing solution of their components, e.g., a high sucrose solution. This can be done.

[0062] The vaccines of the present invention may be administered intravenously, intramuscularly, subcutaneously, orally, intranasally, intradermally and / or intraperitoneally. It can be readily administered by any standard route, including intravenous vaccination. If so, the vaccine composition is preferably adapted to each type of recipient animal and route of administration. It will be understood that the formulation will be appropriately tailored to the individual.

[0063] Thus, the present invention also provides a method for immunizing a cat against a feline pathogen. One method involves administering an immunologically effective amount of the present invention to the cat so that the cat produces the appropriate anti-pathogen antibodies. The method includes injecting the vaccine into a cat.

[0064] Multivalent vaccines Thus, the present invention provides a method for producing a feline pathogen comprising administering to a feline animal a live feline pathogen and one or more and a multivalent vaccine comprising the alphavirus RNA replicon particles. coding sequences for protein antigens or antigenic fragments thereof, or proteins useful in feline vaccines Such combinations of coding sequences for antigens are then incorporated into alphavirus RNA replicon particles. (RP), or, for example, FCV capsid proteins and and / or combined with the same RP as that encoding the FeLV glycoprotein (gp85) It is possible.

[0065] In a specific embodiment, the vaccine comprises at least one modified live feline pathogen and F CV F9-like capsid protein or an antigenic fragment thereof, and / or VS-FCV Alphavirus RNA replicas encoding capsid proteins or antigenic fragments thereof The conjugate particle was then subjected to a transfection of another alphavirus encoding FeLV gp85 or an antigenic fragment thereof. In another embodiment, the vaccine comprises at least one and the VS-FCV capsid protein or its antigenic fragment. One alphavirus RNA replicon particle encoding FeLV gp85 or Another alphavirus RNA replicon particle encoding the antigenic fragment and further FC A third alphavirus encoding a VF9-like capsid protein or an antigenic fragment thereof In yet another embodiment, the vaccine comprises at least one Two modified live feline pathogens and the FCV F9-like capsid protein or its antigenic fragments fragment, VS-FCV capsid protein or its antigenic fragment, and FeLV gp85 or an alphavirus RNA replicon particle encoding an antigenic fragment thereof.

[0066] Examples of pathogens from which one or more such protein antigens may be derived include cat cows. Viral rhinotracheitis virus (FVR), feline leukemia virus (FeLV), feline panleukemia virus (FeLV) Feline pneumonia virus (FPL), feline herpesvirus (FHV), other FCV strains, cat pal Feline infectious peritonitis virus (FPV), feline immunodeficiency 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 Chlamydophila psittaci, Bordetella bronchiseptica , and Bartonella species (e.g., Bartonella henselae (B In certain embodiments, these feline or canine diseases include a coding sequence for a capsid protein or a similar protein from one or more of the biologics, Alternatively, or in combination with, the FCV antigen can be inserted into the same RP. Capsid proteins or similar proteins from one or more of these feline or canine pathogens The coding sequence for the protein can be inserted into one or more other RPs, which can then be used to FCV F9-like capsid protein or an antigenic fragment thereof and / or VS - in combination with an RP encoding an FCV capsid protein or an antigenic fragment thereof This can be done.

[0067] Thus, the present invention provides one or more alphavirus RNA replicons of the invention. particle (RP) [e.g., VS-FCV capsid protein or an antigenic fragment thereof]. or multiple modified live (attenuated) virus isolates, e.g., live attenuated FCV vaccines. feline strains, such as live attenuated FCV F9, and / or live attenuated feline herpesvirus, and / or live attenuated feline parvovirus, and / or live attenuated feline leukemia virus feline infectious peritonitis virus, and / or live attenuated feline immunoglobulin A, and / or live attenuated feline immunoglobulin B Viruses containing HIV and / or live attenuated Borna disease viruses and / or live attenuated Borna disease viruses Canine influenza virus, and / or live attenuated feline influenza virus, and / or live attenuated canine influenza virus and / or live attenuated avian influenza, and and / or live attenuated canine pneumovirus and / or live attenuated feline pneumovirus Further, a vaccine comprising live attenuated Chlamydophila felis (Chla mydophila felis), and / or live attenuated Bordetella bronchiseptica Chika (Bordetella bronchiseptica), and / or live attenuated Bartonella species (e.g., B. henselae) nselae) can also be included in such a multivalent vaccine.

[0068] Additionally, one or more alphavirus RNA replicon particles of the invention [e.g., The vaccine of the present invention comprises a vaccine encoding a VS-FCV capsid protein or an antigenic fragment thereof. The cutin is mixed with one or more killed virus isolates along with one or more modified live virus isolates. Viral isolates, such as killed FCV strains and / or killed feline herpesvirus, and and / or killed feline parvovirus, and / or killed feline leukemia virus, and / or or killed feline infectious peritonitis virus, and / or killed feline immunodeficiency virus, and and / or killed Borna disease virus, and / or killed rabies virus, and / or Killed feline influenza virus and / or killed canine influenza virus, and and / or killed avian influenza virus and / or killed canine pneumovirus and / or killed feline pneumovirus. Chlamydophila felis, and / or Boll Bordetella bronchiseptica, and / or live attenuated Bartonella species (e.g., Bartonella Bacterins from B. henselae may also be included in such multivalent vaccines. It is possible.

[0069] Also, the specific configurations, process steps, and materials disclosed herein may be somewhat The present invention is not limited to such configurations, process steps, and materials, as these may vary. It should also be understood that the scope of the present invention is limited by the appended claims and their equivalents. The terminology used herein is intended to describe specific embodiments only as limited by the terms and conditions set forth herein. It should also be understood that these are for illustrative purposes only and are not intended to be limiting. [Table 1]

[0070] array TIFF2025170230000002.tif57159

[0071] TIFF2025170230000003.tif65161

[0072] TIFF2025170230000004.tif48163

[0073] TIFF2025170230000005.tif116163

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[0078] TIFF2025170230000010.tif119159

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[0080] TIFF2025170230000012.tif119166

[0081] TIFF2025170230000013.tif84169

[0082] TIFF2025170230000014.tif33169

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[0085] TIFF2025170230000017.tif45165

[0086] TIFF2025170230000018.tif78169

[0087] The following examples serve to provide a further understanding of the present invention, but do not limit the scope of the invention. is in no way meant to limit

[0088] [Example] [Example 1] Incorporation of the coding sequence for the FCV capsid protein into alphavirus RNA replicon particles. Mikomi introduction RNA viruses are vectors for introducing genetically engineered vaccine antigens into the genome. However, their use to date has been primarily as an antiviral agent. by incorporating the gene into an 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 are used to encode pathogenic antigens Such alphavirus replicon platforms have been used to identify Venezuelan equine encephalitis viruses. Virus (VEE) [Pushko et al., Virology 239:389- 401 (1997)], Sindbis (SIN) [the entire contents of which are hereby incorporated herein by reference]. Bredenbeek et al., Journal of Virol ogy 67:6439-6446(1993)] and Semliki Forest virus (SFV ) [Liljestrom and Garoff,Biotechnology (NY)9:1356-1361(19 91)]. Virus RNA replicon particles are used in several USDA-licensed vaccines for swine and poultry. These include the porcine epidemic diarrhea vaccine, RNA particles (product code 19 U5.P1), Swine Influenza Vaccine, RNA (Product Code 19A5.D0), Avian Influenza Vaccine, RNA (Product Code 19O5.D0), and Prescription Products, RN Contains A particles (product code 9PP0.00).

[0089] Alphavirus RNA replicon particle construction RP-FCV: The amino acid sequence of the FCV capsid protein was used to perform in silico codon optimization (Neurogene). The optimized sequences were generated using a commercial vendor (ATUM, The synthetic gene was prepared as synthetic DNA by the NIH Group (Newark, CA). , VS-FCV capsid protein and FCV F9-like capsid protein, respectively The amino acid sequence of the VS-FCV capsid protein [sequence sequence number 2], or a construct encoding the FCV F9-like capsid protein [SEQ ID NO: 4] The construct was codon-optimized for cats and the flanking sequences were derived from an alphavirus replicon plasmid. The clone was suitable for cloning into the

[0090] VEE replicon vectors designed to express FCV capsid proteins are listed below. As previously described, with the following modifications [the contents of which are incorporated herein by reference]: (See U.S. Patent No. 9,441,247) A TC-83-derived replicon vector was constructed. - "pVEK" [disclosed and described in U.S. Pat. No. 9,441,247], The fragment was digested with restriction enzymes AscI and PacI. It contains the open reading frame nucleotide sequence and the 5' flanking sequence (5'-GGCGCG CCGCACC-3') [SEQ ID NO: 11] and the 3' flanking sequence (5'-TTAATTAA The DNA plasmid carrying the nucleotide sequence (-3') was similarly digested with the restriction enzymes AscI and PacI. The synthetic gene cassette was then ligated into the digested pVEK vector.

[0091] TC-83 RNA replicon particles (RP) were prepared as previously described [ No. 9,441,247 and U.S. Pat. No. 6,441,247, which are incorporated herein by reference. Briefly, the pVHV replicon vector was The target DNA and helper DNA plasmids were transfected with MegaScript T7 RNA polymerase. Immunoprecipitation using ribosomal enzyme and cap analogue (Promega, Madison, WI) Prior to in vitro transcription, the fragment was linearized with NotI restriction enzyme. Luper RNA was prepared as previously described [Kamrud et al., J Gen V irol.91(Pt 7):1723-1727(2010)], VEE subgenome project The purified RNA of the replicon and helper components was combined and used as a vector. The cells were mixed with a suspension of ro ​​cells and electroporated in a 4 mm cuvette using OptiPro (registered trademark). (Target) SFM cell culture medium (Thermo Fisher, Waltham MA) After overnight incubation, the alphavirus RNA replicon particles were isolated from the suspension. ZetaPlus BioCap depth filters (3M, Maplewood, MN) The tube was then passed through a tube, washed with phosphate-buffered saline containing 5% sucrose (w / v), and finally from cells and medium by eluting the retained RP with 400 mM NaCl buffer. The eluted RP was formulated in a final 5% sucrose (w / v) solution and diluted to 0.22 μg. The cells were passed through a lon membrane filter and aliquoted for storage. The titer of functional RP was determined by the 100% RT-PCR of 100 μg of the infected cells. The immunofluorescence assay of Vero cell monolayers was performed.

[0092] RP-FeLV: The amino acid sequence of FeLV gp85 was used to perform in silico codon optimization (Fecocode). Nucleotide sequences were generated using a commercial vendor (ATUM, New The amino acid sequence of gp85 was prepared as a synthetic DNA fragment by the company Merced (McGraw-Hill, CA). A synthetic gene was designed based on the sequence. The construct (gp85_wt) contains the wild-type amino acid sequence [SEQ ID NO: 2], codon-optimized for cats, with flanking sequences containing alphavirus reptiles. It was suitable for cloning into a recombinant plasmid.

[0093] The VEE replicon vector designed to express FeLV gp85 was modified as follows: Correction was made as previously described [the entire contents of which are incorporated herein by reference, [See U.S. Patent No. 9,441,247]. TC-83-derived replicon vector "pVEK" [disclosed and described in U.S. Pat. No. 9,441,247] is a controlled The DNA was digested with the restriction enzymes AscI and PacI. The open reading frame nucleotide sequence includes the 5' flanking sequence (5'-GGCGC GCCGCACC-3') [SEQ ID NO: 11] and the 3' flanking sequence (5'-TTAATTA A DNA plasmid carrying the nucleotide sequence (A-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 The clone was renamed "pVHV-FeLV gp85." - The nomenclature is via the AscI and PacI sites in the multiple cloning site of pVEK. Refers to a pVEK-derived replicon vector containing a transgene cassette cloned into I chose to do so.

[0094] TC-83 RNA replicon particles (RP) were prepared as previously described [ No. 9,441,247 and U.S. Pat. No. 6,441,247, which are incorporated herein by reference in their entireties. Briefly, the pVHV replicon Vector DNA and helper DNA plasmids were transfected with MegaScript T7 RNase A polymerase and cap analog (Promega, Madison, WI) were used. The DNA fragments were linearized with NotI restriction enzyme before in vitro transcription. The helper RNA was prepared as previously described [Kamrud et al., J Gen Virol.91(Pt 7):1723-1727(2010)], VEE Subgeno The purified RNA of the replicon and helper components is combined and Mix with a suspension of Vero cells, electroporate in a 4 mm cuvette, and inject into OptiPro ( SFM cell culture medium (Thermo Fisher, Waltham MA) After overnight incubation, the alphavirus RNA replicon particles were transferred to the suspension. The solution was filtered through a ZetaPlus BioCap depth filter (3M, Maplewood, M N) and washed with phosphate buffered saline containing 5% sucrose (w / v), Finally, cells and medium were separated by eluting the retained RP with 400 mM NaCl buffer. The eluted RP was formulated in a final 5% sucrose (w / v) and diluted to 0.22 The resulting solution was passed through a micron membrane filter and aliquoted for storage. The titer of functional RP was determined as follows: was determined by immunofluorescence assay of infected Vero cell monolayers.

[0095] RP-RV Capsid proteins and glycoproteins of the non-virulent TC-83 strain of Venezuelan equine encephalitis virus Rabies virus glycoprotein from protein-packaged rabies virus (RV) A vaccine containing alphavirus RNA replicon particles encoding the protein (G) was prepared. The nucleotide sequence of the rabies virus G protein was codon-optimized for humans. The resulting sequence has 100% amino acid identity, but is not identical to the live rabies virus. It shares only about 85% nucleotide identity with the rhus glycoprotein (G) sequence. The vaccine may be administered as a single dose to a mammalian subject, for example, a cat or cats 12 weeks of age or older. and dogs subcutaneously, or as a primary dose followed by one or more booster doses. It can be used in multiple doses including:

[0096] The amino acid sequence of the rabies glycoprotein (G) was used to perform in silico codon optimization (Hu The optimized sequences were generated using a commercial vendor (ATUM, The rabies protein was prepared as synthetic DNA by the National Institute of Allergy and Immunology (NIA), Newark, CA. A synthetic gene (SEQ ID NO: 9) was designed based on the amino acid sequence of the protein. VG) is the wild-type amino acid sequence [SEQ ID NO: 10], codon-optimized for human, The flanking sequences were suitable for cloning into an alphavirus replicon plasmid.

[0097] The VEE replicon vector designed to express rabies G was constructed with the following modifications: As previously described [U.S. Pat. No. 94,494,494, the contents of which are incorporated herein by reference]. TC-83-derived replicon vector "pVEK" [see specification of No. 41247] was constructed. No. 9,441,247] with the restriction enzyme AscI and PacI. The codon-optimized open reading frame of the rabies G gene was nucleotide sequence, and the 5' flanking sequence (5'-GGCGCGCCGCACC-3') DNA having [SEQ ID NO: 11] and the 3' adjacent sequence (5'-TTAATTAA-3') The plasmid was similarly digested with the restriction enzymes AscI and PacI. The cassette was ligated into the digested pVEK vector and the resulting clone was named "pV The "pVHV" vector nomenclature was changed to "pVEK multi- Transgenes cloned via the AscI and PacI sites of the cloning site It was chosen to refer to the pVEK-derived replicon vector containing the cassette.

[0098] TC-83 RNA replicon particles (RP) were prepared as previously described [ No. 9,441,247 and U.S. Pat. No. 6,441,247, which are incorporated herein by reference in their entireties. Briefly, the pVHV replicon Vector DNA and helper DNA plasmids were transfected with MegaScript T7 RNase A polymerase and cap analog (Promega, Madison, WI) were used. The DNA fragments were linearized with NotI restriction enzyme before in vitro transcription. The helper RNA was prepared as previously described [Kamrud et al., J Gen Virol.91(Pt 7):1723-1727(2010)], VEE Subgeno The purified RNA of the replicon and helper components is combined and Mix with a suspension of Vero cells, electroporate in a 4 mm cuvette, and inject into OptiPro ( SFM cell culture medium (Thermo Fisher, Waltham, MA) After overnight incubation, the alphavirus RNA replicon particles were transferred to the suspension. The solution was filtered through a ZetaPlus BioCap® depth filter (3M, Maple wood, MN) and phosphate-buffered saline containing 5% sucrose (w / v). The cells were washed with water and finally eluted with 400 mM NaCl buffer. The eluted RP was purified from cells and culture medium. The eluted RP was formulated in a final 5% sucrose (w / v). The solution was filtered, passed through a 0.22 micron membrane filter, and aliquoted for storage. The titer of P was determined by immunofluorescence assay of infected Vero cell monolayers.

[0099] [Example 2] Safety of a combination vaccine containing two RP constructs and three modified live fractions in cats Rating Various formulations and reconstitution methods of lyophilized pentavalent feline combination vaccines were investigated in cats. Their safety was evaluated. The recommended dose of the pentavalent vaccine is 0.5 mL. The optimal formulation and filling method is 0.5 mL filling. The stabilizer is 1.1% NZ-amine. (enzymatic casein hydrolysate), 1.1% gelatin and 7.5% sucrose. The percentages provided represent the final concentrations. The volumes for adding the five fractions and the final potency are given in Table 1. If constraints require a volume greater than 0.5 mL, the product should be formulated to reduce the volume to 1.0 mL. The drug can be filled and reconstituted with 0.5 mL of diluent. This stabilizes the dose. The concentration of the ingredients is doubled. The safety of such concentrated doses of stabilizers containing these antigens is unclear. It has not been previously tested in cats.

[0100] If the 1.0 mL fill / 0.5 mL rehydrate format is not safe for cats, use the 1.0 mL A third option was also tested, a 1.0 mL fill volume rehydrated with diluent. The vaccine was prepared by centrifugation and then freeze-dried. Feline leukemia virus glycoprotein (RP-F) in a stabilizer containing 7.5% sucrose Alphavirus RNA replicon particles encoding eLVs and feline calicivirus Alphavirus RNA replicon particle encoding capsid protein (RP-FCV) The pups were given a component of the commercially available vaccine, Nobivac® Feline-1, i.e. , modified live (MLV) feline panleukopenia virus (FPL), modified live feline Viral rhinotracheitis virus (FVR) and modified live Chlamydophila felis (C hlamydophila felis) as described in Table 1 below. Various formulations of pentavalent feline combination vaccines were prepared and reconstituted as described above. [Table 2]

[0101] The vaccines were formulated to contain the same dose of each antigen (0.5 mL cake vaccine was doubled). The stabilizer was used at a constant concentration in all formulations (0.5 ml). The 1.0 mL cake vaccine rehydrated with L diluent contains twice the concentration of stabilizer upon rehydration. contained).

[0102] Experimental feline subjects were 7-8 weeks of age (typically the minimum vaccination age for feline core vaccines). and then vaccinated again 21 days later with the indicated volume of each test vaccine. Cats should be monitored for signs of vocalization, stinging, scratching, biting, sudden movements during vaccination, or Any test vaccine that may cause pain or discomfort, such as abnormal reactions (see Tables 2 and 3 below) The vaccines were observed for 15 minutes immediately after each vaccination for reactions to the vaccine. The test was performed by a veterinarian 4-6 hours after vaccination and daily for 3 days after vaccination.

[0103] Clinical evaluation included palpation of the injection site and any localized tenderness, swelling, redness, or abscess. Observations included observation of reactions to the drug. Cats were monitored for depression, lethargy, lameness, vomiting, tremors, agitation, and The subjects were also observed for any systemic reactions, such as diarrhea, fever, and fever. Injection site responses for local reactions were recorded 24 hours after vaccination and daily for 2 days after each vaccination. Palpation was further performed 3 times per week from 7 to 21 days after each vaccination.

[0104] All vaccine formulations and rehydration protocols were found to be safe. No local or systemic reactions were observed. One cat in treatment group 3 (0.5 mL cake / 0.5 mL diluent) showed a temperature of 103.6°C. All cats had normal body temperatures at each measurement period except for the pentavalent vaccine. All three preparations were found to be acceptable. [Table 3]

[0105] [Table 4]

[0106] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein may be made in light of the foregoing description. Such modifications will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims. This is the intention.

[0107] Additionally, any base size or amino acid given for a nucleic acid or polypeptide Sizes and all molecular weight or molecular mass values ​​are approximate and are provided for illustrative purposes. I want you to understand that.

Claims

1. An immunogenic composition comprising an alphavirus RNA replicon particle encoding one or more feline leukemia virus (FeLV) glycoproteins and one or more modified live feline pathogens selected from the group consisting of a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), a modified live Chlamydophila felis, and any combination thereof.

2. The immunogenic composition of claim 1, wherein the immunogenic composition comprises at least two modified live pathogens selected from the group consisting of modified live FCV, modified live FVR, modified live FPLV and modified live Chlamydophila felis.

3. The immunogenic composition of claim 1, wherein the immunogenic composition comprises at least three modified live pathogens selected from the group consisting of modified live FCV, modified live FVR, modified live FPLV and modified live Chlamydophila felis.

4. An immunogenic composition comprising an alphavirus RNA replicon particle encoding one or more FeLV glycoproteins and a modified live feline pathogen, including a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), or a modified live Chlamydophila felis.

5. An immunogenic composition comprising an alphavirus RNA replicon particle encoding one or more FeLV glycoproteins and a modified live feline pathogen, including a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), and a modified live Chlamydophila felis.

6. The immunogenic composition of any one of claims 1 to 5, wherein the one or more FeLV glycoproteins are selected from the group consisting of FeLV glycoprotein gp85, FeLV glycoprotein gp70, FeLV glycoprotein gp45, and any combination thereof.

7. The immunogenic composition of any one of claims 1 to 6, wherein the alphavirus RNA replicon particles are Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles.

8. The immunogenic composition of claim 7, wherein the FeLV glycoprotein is selected from the group consisting of FeLV glycoprotein gp85 having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6, FeLV glycoprotein gp70 having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8, and combinations thereof.

9. An immunogenic composition comprising a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), a modified live Chlamydophila felis, and a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle encoding FeLV glycoprotein gp85 comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

10. The immunogenic composition of any one of claims 7 to 9, wherein the VEE alphavirus RNA replicon particles further encode one or more non-FeLV antigens selected from the group consisting of FCV F9-like capsid protein, virulent systemic FCV (VS-FCV) capsid protein, rabies virus G protein, and any combination thereof.

11. The immunogenic composition of any one of claims 7 to 9, further comprising additional VEE alphavirus RNA replicon particles encoding one or more non-FeLV antigens selected from the group consisting of FCV F9-like capsid protein, VS-FCV capsid protein, rabies virus G protein, and any combination thereof.

12. The immunogenic composition of claim 10 or 11, wherein the one or more non-FeLV antigens are selected from the group consisting of an FCV F9-like capsid protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 4, a VS-FCV capsid protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2, a rabies virus G protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 10, and any combination thereof.

13. The immunogenic composition of any one of claims 7 to 10, further comprising an additional VEE alphavirus RNA replicon particle encoding FeLV glycoprotein gp70 comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

8.

14. A vaccine to help prevent disease caused by feline leukemia virus (FeLV), comprising the immunogenic composition of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. A vaccine for helping to prevent disease caused by feline leukemia virus (FeLV), comprising VEE alphavirus RNA replicon particles encoding one or more FeLV glycoproteins, and modified live feline pathogens, including modified live feline viral rhinotracheitis virus (FVR), modified live feline panleukopenia virus (FPLV), and modified live Chlamydophila felis, and a pharmaceutically acceptable carrier.

16. 1. A vaccine for helping to prevent disease caused by feline leukemia virus (FeLV), comprising VEE alphavirus RNA replicon particles encoding one or more FeLV glycoproteins, and a modified live feline pathogen, including a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), and a modified live Chlamydophila felis, and a pharmaceutically acceptable carrier.

17. 1. A vaccine to help prevent disease caused by feline leukemia virus (FeLV), comprising a modified live feline calicivirus (FCV), a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), a modified live Chlamydophila felis, and a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle encoding FeLV glycoprotein gp85 comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

18. 18. The vaccine of any one of claims 15 to 17, wherein the VEE alphavirus RNA replicon particles further encode one or more non-FeLV antigens selected from the group consisting of FCV F9-like capsid protein, virulent systemic FCV (VS-FCV) capsid protein, rabies virus G protein, and any combination thereof.

19. 18. The vaccine of any one of claims 15 to 17, further comprising additional VEE alphavirus RNA replicon particles encoding one or more non-FeLV antigens selected from the group consisting of FCV F9-like capsid protein, VS-FCV capsid protein, rabies virus G protein, and any combination thereof.

20. 20. The vaccine of claim 18 or 19, wherein the one or more non-FeLV antigens are selected from the group consisting of an FCV F9-like capsid protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 4, a VS-FCV capsid protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 2, a rabies virus G protein comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 10, and any combination thereof.

21. The vaccine of any one of claims 15 to 18, further comprising an additional VEE alphavirus RNA replicon particle encoding FeLV glycoprotein gp70 comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

8.

22. The vaccine composition according to any one of claims 14 to 21, which is a non-adjuvanted vaccine.

23. A method of immunizing a cat against pathogenic FeLV, comprising administering to said cat an immunologically effective amount of a vaccine according to any one of claims 14 to 22.

24. 16. A method of immunizing a cat against pathogenic FeLV, comprising administering to said cat an immunologically effective amount of the vaccine of claim 15.

25. 17. A method of immunizing a cat against pathogenic FeLV, comprising administering to said cat an immunologically effective amount of the vaccine of claim 16.

26. 20. A method of immunizing a cat against pathogenic FeLV, comprising administering to said cat an immunologically effective amount of the vaccine of claim 17.