JP7427585B2 - polyvalent feline vaccine - Google Patents

Description

CROSS-REFERENCES TO RELATED APPLICATIONS This application is filed under U.S. Provisional Patent Application No. 62/599,401, filed December 15, 2017, filed December 8, 2017, the entire contents of which are incorporated herein by reference. U.S. Provisional Patent Application No. 62/596508, filed on November 6, 2017, and U.S. Provisional Patent Application No. 62/581955, filed on November 6, 2017. Claims priority under 35 U.S.C. 119(e) of No.

The present invention relates to a novel multivalent feline vaccine. Also provided are methods of making and using multivalent vaccines alone or in combination with other protective agents.

Feline respiratory diseases include sinusitis, conjunctivitis, lacrimation, salivation, and oral ulcers. The two most common pathogens associated with upper respiratory tract disease in cats are feline calicivirus (FCV) and feline viral rhinotracheitis virus (FVR), also known as feline herpesvirus type 1 virus (FHV-1). ). These two feline viruses are thought to be responsible for approximately 80% of all feline respiratory diseases worldwide. The bacterium Chlamydophila felis is a third pathogen that can also play a role in feline respiratory disease. Therefore, vaccines against these three pathogens are currently commercially available.

FVR is an alphaherpesvirus related to canine herpesvirus 1 and is perhaps the most important of the feline respiratory pathogens. FVR is a large enveloped DNA virus that is highly contagious and can cause severe disease in kittens and cats. Therefore, most cats are exposed to FVR throughout their lives. The commercially available Nobivac® Feline-1 vaccine contains a modified live feline viral rhinotracheitis virus.

The most common feature and clinical sign of FCV infection is the development of vesicles (ulcers) on the tongue and oral mucosa, which begin as small individual ulcers but can spread and affect large parts of the tongue. . Fever is also often seen in infected cats. Certain strains of FCV also cause a disease in cats known as limping syndrome, which is characterized by fever, joint and muscle pain (limping), and occasional tongue/oral ulcers. Additionally, some strains of FCV have been associated with chronic stomatitis in infected cats. Cats infected with FCV can become persistently infected and can shed infectious virus over long periods of time.

FCV isolates are antigenically highly variable, and antibodies from cats vaccinated with older vaccine strains of FCV, such as FCV F9, do not effectively neutralize all current field isolates. Additionally, new FCV strains have been identified that are associated with systemic disease and high mortality [see US Pat. No. 7,449,323]. These “virulent systemic” (VS-FCV) isolates are responsible for local outbreaks, and current vaccines do not appear to protect cats from the disease caused by these strains. It won't happen.

FCV contains a single-stranded, positive-strand RNA genome consisting of three open reading frames (ORFs). The genome is polyadenylated at the 3' end and joined at the 5' end by virus-encoded proteins. The first open reading frame encodes the viral protease and RNA-dependent RNA polymerase expressed in a single polypeptide. This polypeptide is then post-translationally cleaved by viral proteases. The second open reading frame encodes the major capsid protein (ie, FCV capsid protein), which has six regions designated as AF [Scott et al. , 60 Am. J. Vet. Res. :652-658 (1999)]. Region A is cleaved to produce mature capsid protein. Regions B, D, and F of ORF2 are relatively conserved among FCV isolates, whereas regions C and E are variable, and region E of ORF2 contains the major B-cell epitope [Radford et al. , 38(2) Vet Res. :319-335 (2007)]. ORF3 encodes a minor structural protein [Sosnovtsev and Green, 277 Virology: 193-203 (2000)].

Chlamydophila felis is a bacterium endemic to domestic cats worldwide. Chlamydophila felis (C. felis) can cause conjunctival inflammation, rhinitis and respiratory problems in infected cats. Chlamydophila felis (C. felis) has a relatively small genome encoding only about 1000 proteins. This bacterium also contains a plasmid containing 75,000 base pairs. The commercially available Nobivac® Feline-1 vaccine contains modified live Chlamydophila felis.

In addition to the three pathogens associated with feline respiratory disease listed above, cats are usually susceptible to three other viruses: feline leukemia virus (FeLV), feline panleukopenia (FPV or FPLV), and rabies virus. will also be vaccinated. Feline leukemia virus is a retrovirus that infects domestic cats and causes significant morbidity and mortality worldwide. FeLV is primarily transmitted through saliva, but it has also been reported to spread through contact with body fluids [Pacitti et al. , Vet Rec 118:381-384 (1986) doi:10.1136/vr. 118.14.381; Levy et al. , J Feline Med Surg 10:300-316 (2008) doi:10.1016/j. jfms. 2008.03.002]. Clinical signs observed in cats during FeLV infection include cell proliferative disorders (lymphoid or myeloid tumors), cell suppressive disorders (immunosuppression, anemia, infectious diseases associated with myelosuppression), and inflammatory disorders. , neuropathy, miscarriage, and enterocolitis [Hoover et al. , J Am Vet Med Assoc 199:1287-1297 (1991); Levy and Crawford, Textbook of Veterinary Internal Medicine, 6th ed (Ettinger SJ, Feld (Man EC., eds.) WB Saunders, Philadelphia, PA. (2005)]. The prevalence of antigenemia varies from 1-5% in healthy cats to 15-30% in diseased cats [Hosie et al. Veterinary Records, 128:293-297 (1989); Braley, Feline Practice 22:25-29 (1994); Malik et al. , Australian Veterinary Journal 75:323-327 (1997); Arjona et al. , Journal of Clinical Microbiology 38:3448-3449 (2000)]. FeLV often establishes a persistent infection with concomitant persistent viremia, often leading to death of the host cat.

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

Feline panleukopenia (FPV or FPLV) is a highly contagious viral disease of cats that is often fatal. The name panleukopenia comes from the low number of white blood cells (white blood cells) in affected animals. FPLV infects and destroys actively dividing cells in lymphoid tissue, bone marrow, intestinal epithelium, and, in very young kittens, the retina and cerebellum. The virus can also spread transplacentally in pregnant cats, causing embryonic resorption, fetal mummification, stillbirth, or miscarriage. Infected cats shed the virus in their urine, feces, and nasal secretions, and susceptible cats become infected when they come into contact with these secretions or with fleas from infected cats. Clinical signs due to FPLV infection have also been classified as feline distemper or feline parvo.

Feline panleukopenia virus is a member of the genus Parvovirus in the family Parvoviridae. Therefore, FPLV is closely related to mink enteritis virus and canine parvovirus type 2 (CPV-2). FPLV has a single-stranded DNA genome that has been sequenced [Liu et al. , Genome Announce. Mar-Apr;3(2)(2015):e01556-14]. The commercially available Nobivac® Feline-1 vaccine contains a modified live feline panleukopenia virus.

Rabies is a preventable zoonotic disease that causes inflammation of the brain in humans and other mammals. Clinical rabies is an acute progressive encephalitis typically classified as rabid rabies or paralytic rabies. Violent rabies is characterized by restlessness, irritability and aggression. Paralytic rabies is characterized by excessive salivation, deep labored breathing, paralysis, and eventually coma. The causative agent of rabies is the rabies virus, which can infect most mammals, including cats, and maintains a reservoir of wild and susceptible domestic animals.

Rabies virus is an enveloped RNA virus that encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and an RNA-dependent RNA polymerase [Dietzschold et al. ．． , Crit Rev Immunol 10:427-439 (1991)]. Glycoprotein (G) is considered a protective antigen that induces virus-neutralizing antibodies [Cox et al. , Infect Immun 16:754-759 (1977)]. Several rabies vaccines have been produced to combat this disease. Whole virus killed rabies virus vaccine derived from inactivated cell culture is the most commonly used vaccine in the United States. These whole virus killed rabies virus vaccines require high levels of antigen and therefore require adjuvants. 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 successfully used in oral vaccine baits for immunization of wild animals [Mahl et al. , Vet Res 45(1):77 (2014)]. Additionally, a recombinant vaccine expressing glycoprotein (G) is currently marketed in the United States for use in cats. Nucleic acid vaccines are also being used in laboratory studies, but none are currently licensed in the United States.

Several vector strategies have been used in vaccines for many years in an effort to protect against certain pathogens. One such vector strategy involves the use of alphavirus-derived 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)], these are Venezuelan equine encephalitis virus (VEE) [Pushko et al. , Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al. , JOURNAL OF VIROLOGY 67: 6439-6466 (1993) and Semiriki Forest Virus (SFV) Developed from several different alpha viruses including] . RP vaccines deliver growth-defective alphavirus RNA replicons into host cells, resulting in expression of the desired antigenic transgene(s) in vivo [Pushko et al. , Virology 239(2):389-401 (1997)]. RP has an attractive safety and efficacy profile when compared to some conventional vaccine formulations [Vander Veen, et al. Anim Health Res Rev. 13(1):1-9. (2012)]. The RP platform has been used to encode pathogenic antigens and is the basis for several USDA-approved vaccines for pigs and poultry.

In particular, alphavirus RNA replicon particles, particularly Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles, catalyze a systemic antiviral state and protect against deadly viral attack [Konopka et al. , J. Virol. , 83 (29): 12432-12442 (2009)], particularly inducing rapid protection against foot-and-mouth disease virus [Segundo et al. , J. Virol. , 87(10):5447-5460 (2013)]. Therefore, alphavirus RNA replicon particles should not be combined with modified live viruses in vaccines, as they would be expected to be detrimental to the immune response, since they enhance the innate immune response against live viruses. Seem.

Citation of any reference herein shall not be construed as an admission that such reference is available as "prior art" to this application.

US Patent No. 7,449,323

Scott et al. , 60 Am. J. Vet. Res. :652-658 (1999) Radford et al. , 38(2) Vet Res. :319-335 (2007) Sosnovtsev and Green, 277 Virology: 193-203 (2000) Pacitti et al. , Vet Rec 118:381-384 (1986) doi:10.1136/vr. 118.14.381 Levy et al. , J Feline Med Surg 10:300-316 (2008) doi:10.1016/j. jfms. 2008.03.002 Hoover et al. , J Am Vet Med Assoc 199:1287-1297 (1991) Levy And Crawford, TextBook OF Veterinary Internal Medicine, 6th ED (Ettinger SJ, FELDMAN EC. HIA, Pa. (2005) Hosie et al. Veterinary Records, 128:293-297 (1989) Braley, Feline Practice 22:25-29 (1994) Malik et al. , Australian Veterinary Journal 75:323-327 (1997) Arjona et al. , Journal of Clinical Microbiology 38:3448-3449 (2000) Thomsen et al. , Journal of General Virology 73:1819-1824 (1992) DeNoronha, et al. , Virology 85:617-621 (1978) Nunberg et al. , PNAS 81:3675-3679 (1983) Mathes et al. , Nature 274:687-689 (1978) Flynn et al. , J. Virol. 76(5):2306-2315 (2002) Liu et al. , Genome Announce. Mar-Apr;3(2)(2015):e01556-14 Dietzschold et al. , Crit Rev Immunol 10:427-439 (1991) Cox et al. , Infect Immun 16:754-759 (1977) Mahl et al. , Vet Res 45(1):77 (2014) 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) Pushko et al. , Virology 239:389-401 (1997) Bredenbeek et al. , Journal of Virology 67:6439-6446 (1993) Liljestrom and Garoff, Biotechnology (NY) 9:1356-1361 (1991) Pushko et al. , Virology 239(2):389-401 (1997) Vander Veen, et al. Anim Health Res Rev. 13(1):1-9. (2012) Konopka et al. , J. Virol. , 83 (29): 12432-12442 (2009) Segundo et al. , J. Virol. , 87(10):5447-5460 (2013)

Accordingly, the present invention provides immunogenic compositions comprising alphavirus RNA replicon particles encoding one or more antigens from one or more feline pathogens, together with one or more modified live feline pathogens. include. All of the immunogenic compositions of the invention can also be used in multivalent vaccines. In a particular embodiment of this type, the subject being vaccinated is a cat. In a more specific embodiment, the subject being vaccinated is a domestic cat. Also provided are methods of making and using the immunogenic compositions and vaccines of the invention.

In certain embodiments, an immunogenic composition comprises alphavirus RNA replicon particles encoding one or more feline calicivirus (FCV) antigens and a modified live feline pathogen. In other embodiments, the immunogenic composition comprises alphavirus RNA replicon particles encoding one or more feline leukemia virus (FeLV) antigens and a modified live feline pathogen. In yet other embodiments, the immunogenic composition comprises alphavirus RNA replicon particles encoding one or more rabies virus antigens and a modified live feline pathogen. In certain embodiments, the modified live feline pathogen is feline viral rhinotracheitis virus (FVR). In other embodiments, the modified live feline pathogen is feline panleukopenia virus (FPLV). In yet other embodiments, the modified live feline pathogen is modified live Chlamydophila felis. In yet other embodiments, the modified live feline pathogen is a modified live F9-like feline calicivirus (FCV F9-like). In yet other embodiments, the modified live feline pathogen is modified live Bordetella bronchiseptica. The invention further provides immunogenic compositions comprising any combination of these alphavirus RNA replicon particles and a modified live feline pathogen. In specific embodiments of this type, the immunogenic composition comprises alphavirus RNA replicon particles encoding FCV antigens, alphavirus RNA replicon particles encoding FeLV antigens, modified live FVRs, modified live FPLVs. , and modified live Chlamydophila felis.

In certain embodiments of this type, the immunogenic composition comprises alphavirus RNA replicon particles encoding FCV capsid proteins. In more specific embodiments, the FCV capsid protein is FCV F9-like capsid protein. In other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of FCV F9-like capsid protein. In yet other embodiments, the FCV capsid protein is virulent systemic FCV (VS-FCV) capsid protein. In yet other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of the VS-FCV capsid protein. In yet other embodiments, the alphavirus RNA replicon particle encodes both an FCV F9-like capsid protein or an antigenic fragment thereof and a VS-FCV capsid protein or an antigenic fragment thereof.

In other embodiments, the immunogenic composition comprises an alphavirus RNA replicon particle encoding a VS-FCV capsid protein or an antigenic fragment thereof, wherein the VS-FCV capsid protein has an amino acid sequence of 95 Contains amino acid sequences that contain % or more identity. In a more specific embodiment, the VS-FCV capsid protein comprises the amino acid sequence of SEQ ID NO:2. In an even more specific embodiment, the VS-FCV capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 12. In a related embodiment, an alphavirus RNA replicon particle of the invention encodes an FCV F9-like capsid protein or an antigenic fragment thereof. In a specific embodiment of this type, the FCV F9-like capsid protein comprises an amino acid sequence that includes 95% or more identity with the amino acid sequence of SEQ ID NO:4. In a more specific embodiment, the FCV F9-like capsid protein comprises the amino acid sequence of SEQ ID NO:4. In an even more specific embodiment of this type, the FCV F9-like capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 13.

In a related embodiment, the immunogenic composition comprises alphavirus RNA replicon particles encoding one or more feline leukemia virus (FeLV) antigens. In certain embodiments, the FeLV antigen is FeLV glycoprotein (eg, gp85). In other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of FeLV gp85. In an even more specific embodiment of this type, the antigenic fragment of the FeLV glycoprotein is FeLV gp70. In a related embodiment, the antigenic fragment of FeLV glycoprotein is FeLV gp45. In a more specific embodiment of this type, FeLV gp85 comprises an amino acid sequence comprising 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 amino acid sequence of SEQ ID NO:6. In an even more specific embodiment of this type, FeLV gp85 is encoded by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 14. In a related embodiment, FeLV gp70 comprises an amino acid sequence that includes 95% or more identity with the amino acid sequence of SEQ ID NO:8. In a more specific embodiment of this type, FeLV gp70 comprises the amino acid sequence of SEQ ID NO:8. In an even more specific embodiment of this type, FeLV gp70 is encoded by the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 15.

In yet other embodiments, the immunogenic composition comprises alphavirus RNA replicon particles encoding rabies virus glycoprotein (G). In yet other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of rabies virus G. In a more specific embodiment of this type, rabies virus G comprises an amino acid sequence comprising 95% or more identity with the amino acid sequence of SEQ ID NO:10. In an even more specific embodiment of this type, rabies virus G comprises the amino acid sequence of SEQ ID NO:10. In an even more specific embodiment of this type, rabies virus G is encoded by the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 16.

In certain embodiments, the alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle. In a more specific embodiment of this type, the VEE alphavirus RNA replicon particle is a TC-83 VEE alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Sindbis (SIN) alphavirus RNA replicon particle. In yet other embodiments, the alphavirus RNA replicon particle is a Semliki Forest Virus (SFV) alphavirus RNA replicon particle. In an alternative embodiment, the naked DNA vector encodes one or more antigens derived from one or more feline pathogens. In certain embodiments of this type, the naked DNA vector encodes the FCV capsid protein or antigenic fragment thereof. In a specific embodiment of this type, the naked DNA vector encodes the VS-FCV capsid protein or antigenic fragment thereof. In other embodiments of this type, the naked DNA vector encodes FeLV gp85 or an antigenic fragment thereof.

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 glycoproteins or antigenic fragments thereof. fragments and/or alphavirus RNA replicon particles encoding one or more rabies virus G proteins or antigenic fragments thereof. In certain embodiments of this type, the alphavirus RNA replicon particle encodes both the VS-FCV capsid protein or antigenic fragment thereof and the FCV F9-like capsid protein or antigenic fragment thereof. In a related embodiment, the alphavirus RNA replicon particle encodes both VS-FCV capsid protein or antigenic fragment thereof and FeLV gp85 or antigenic fragment thereof. In other embodiments, the alphavirus RNA replicon particle encodes both FCV F9-like capsid protein or an antigenic fragment thereof and FeLV gp85 or an antigenic fragment thereof. In another embodiment, the alphavirus RNA replicon particle encodes both the VS-FCV capsid protein or antigenic fragment thereof and the rabies virus G protein or antigenic fragment thereof. In yet other embodiments, the alphavirus RNA replicon particle encodes both the FCV F9-like capsid protein or antigenic fragment thereof and the rabies virus G protein or antigenic fragment thereof. In yet other embodiments, the alphavirus RNA replicon particles encode VS-FCV capsid protein or antigenic fragment thereof, FeLV gp85 or antigenic fragment thereof, and rabies virus G protein or antigenic fragment thereof. In another embodiment, the alphavirus RNA replicon particles encode VS-FCV capsid protein or antigenic fragment thereof, FCV F9-like capsid protein or antigenic fragment thereof, and FeLV gp85 or antigenic fragment thereof. In yet other embodiments, the alphavirus RNA replicon particles include VS-FCV capsid protein or antigenic fragment thereof, FeLV gp85 or antigenic fragment thereof, rabies virus G protein or antigenic fragment thereof, and FCV F9-like capsid protein or antigenic fragment thereof. encodes its antigenic fragment. In yet other embodiments, the alphavirus RNA replicon particle encodes FeLV gp85 or an antigenic fragment thereof and rabies virus G protein or an antigenic fragment thereof. In yet other embodiments, the alphavirus RNA replicon particle encodes FeLV gp85 or an antigenic fragment thereof, FCV F9-like capsid protein or an antigenic fragment thereof, and rabies virus G protein or an antigenic fragment thereof.

Accordingly, the invention provides at least one modified live feline pathogen and any one or more alphavirus RNA replicon particles of the invention encoding multiple feline pathogen antigens and/or encoding a single feline pathogen antigen. and any one or more alphavirus RNA replicon particles of the invention. In certain embodiments, the modified live feline pathogen is a modified live FVR. In other embodiments, the modified live feline pathogen is modified live FPLV. In yet other embodiments, the modified live feline pathogen is modified live Chlamydophila felis. In yet other embodiments, the modified live feline pathogen is modified live FCV F9-like. In yet other embodiments, the modified live feline pathogen is modified live Bordetella bronchiseptica. In certain embodiments, two or more modified live feline pathogens are included in the immunogenic composition. 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 modified live FVR, modified live Chlamydophila, and the modified live feline pathogen is FPLV. In yet other embodiments, four or more modified live feline pathogens are included in the immunogenic composition. In specific embodiments of this type, the immunogenic composition comprises modified live FVR, modified live Chlamydophila, modified live FPLV, and modified live F9-like FCV. In certain embodiments, all of the alphavirus RNA replicon particles in the immunogenic composition are Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles. In an even more specific embodiment, all of the VEE alphavirus RNA replicon particles in the immunogenic composition are TC-83 VEE alphavirus RNA replicon particles.

In further embodiments, alphavirus RNA replicon particles may encode protein antigens (or antigenic fragments thereof) derived from other feline pathogens. In certain embodiments of this type, the protein antigen is derived from feline pneumovirus (FPN). In yet other embodiments, the protein antigen is derived from feline parvovirus (FPV). In yet other embodiments, the protein antigen is derived from feline infectious peritonitis virus (FIPV). In yet other embodiments, 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 feline influenza virus. In yet other embodiments, the protein antigen is derived from feline coronavirus (FCoV).

The present invention further provides immunogenic compositions and/or immunogenic compositions comprising alphavirus RNA replicon particles of the present invention, together with one or more modified live (e.g., attenuated) feline pathogens of the present invention, and/or killed feline pathogens. Provide vaccines (multivalent vaccines). In certain embodiments, the immunogenic composition comprises killed Chlamydophila felis, and/or killed FVR, and/or killed F9-like FCV, and/or killed VS-FCV, and/or killed FeLV, and/or killed FPLV. In certain embodiments, the vaccine comprises an immunologically effective amount of one or more of these immunogenic compositions.

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

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

Vaccines and multivalent vaccines of the invention may be administered as primer and/or booster vaccines. In a specific embodiment, the vaccines of the invention are administered as a single vaccine (one dose) without the need for subsequent administration. In certain embodiments, for administration of both primer and booster vaccines, the primer and booster vaccines may be administered by the same route. In certain embodiments of this type, both the primer vaccine and the booster vaccine are administered by subcutaneous injection. In an alternative embodiment, in the case of administration of both a primer and a booster vaccine, the administration of the primer vaccine may be carried out by one route and the booster vaccine by another route. In certain embodiments of this type, the primer vaccine may be administered by subcutaneous injection and the booster vaccine may be administered orally.

The present invention further provides a method of immunizing a cat against FCV, the method comprising injecting the cat with an immunologically effective amount of a vaccine of the invention as described above. In certain embodiments, the vaccine can include, for example, from about 1×10 ⁴ to about 1×10 ¹⁰ RPs or more. In more specific embodiments, the vaccine may comprise about 1×10 ⁵ to about 1×10 ⁹ RPs. In an even more particular embodiment, the vaccine may comprise about 1×10 ⁶ to about 1×10 ⁸ RPs. In certain embodiments, the cat is a domestic cat.

In certain embodiments, vaccines of the invention are administered in doses of 0.05 mL to 3 mL. In more specific embodiments, the dose administered is between 0.1 mL and 2 mL. In still more particular embodiments, the dose administered is between 0.2 mL and 1.5 mL. In an even more particular embodiment, the dose administered is 0.3-1.0 mL. In still more specific embodiments, the dose administered is between 0.4 mL and 0.8 mL.

These and other aspects of the invention will be better understood by reference to the detailed description below.

The present invention provides a safe and effective multivalent vaccine. In certain embodiments, the vaccine is non-adjuvanted. In this aspect of the invention, the vaccine does not induce feline injection site sarcoma, but still induces infection with feline calicivirus (FCV) and/or feline leukemia virus (FeLV), and feline viral rhinotracheitis virus (FVR), and Helps protect vaccinated cats from diseases caused by infection with feline panleukopenia virus (FPLV) and/or live Chlamydophila felis.

Despite the known enhancement of the innate immune system by alphavirus RNA replicon particles, the multivalent vaccines of the present invention, which include both alphavirus RNA replicon particles and modified live virus, do not require that the alphavirus RNA replicon particles be modified. It is unexpectedly safe and effective without significantly interfering with the immunological effects of live virus.

Indeed, alphavirus RNA replicon particles, particularly Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles, were previously shown to catalyze a systemic antiviral state and protect against deadly viral attack [Konopka et al. , J. Virol. , 83(29):12432-12442 (2009)]. Furthermore, VEE alphavirus RNA replicon particles have been reported to induce rapid protection against foot-and-mouth disease virus [Segundo et al. , J. Virol. , 87(10):5447-5460 (2013)]. Therefore, a vaccine containing both alphavirus RNA replicon particles and modified live virus would be expected to result in substantial inhibition of the immunological effects of the modified live virus. However, it was surprisingly found that the enhancement of the innate immune response by the presence of alphavirus RNA replicon particles was not detrimental to the immune response induced by the concomitant engineered live virus.

Thus, in certain aspects, the invention provides alphavirus RNA replicon particles encoding FCV capsid protein or antigenic fragments thereof and/or FeLV gp85 or antigenic fragments thereof, modified live feline FVR, and/or modified and/or modified live FPLV and/or modified live Chlamydophila felis. In yet another aspect of the invention, the vaccine comprises a naked DNA vector encoding FCV capsid protein or an antigenic fragment thereof and/or FeLV gp85 or an antigenic fragment thereof, a modified live feline FVR, and/or a modified live feline FVR. with modified live feline FPLV, and/or modified live Chlamydophila felis.

The vaccines of the invention can be administered to cats in the absence of an adjuvant and still effectively help protect vaccinated cats against FCV.

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

The use of the singular term for convenience in the description is not intended to be so limiting in any way. Thus, for example, reference to a composition comprising "a polypeptide" includes reference to one or more such polypeptides. Furthermore, reference to an "alphavirus RNA replicon particle" includes reference to a plurality of such alphavirus RNA replicon particles, unless otherwise indicated.

As used herein, the term "approximately" is used interchangeably with the term "about" to indicate that a value is within 50 percent of the indicated value, i.e., per milliliter. A composition containing "approximately" 1 x 10 ⁸ alphavirus RNA replicon particles contains 0.5 x 10 ⁸ to 1.5 x 10 ⁸ alphavirus RNA replicon particles per milliliter.

As used herein, the term "feline" refers to any member of the Felidae family. Domestic cats, purebred and/or mixed-breed companion cats, and wild or feral cats are all cats.

As used herein, the term "replicon" refers to one or more elements (e.g., structural proteins, refers to a modified RNA viral genome that lacks the coding sequence). In the appropriate cellular context, a replicon can amplify itself and produce one or more subgenomic RNA species.

As used herein, the term "alphavirus RNA replicon particle," abbreviated as "RP," refers to structural proteins, such as those described by Pushko et al. [Virology 239(2):389-401 (1997)]. As shown, it is an alphavirus-derived RNA replicon packaged into a capsid and glycoprotein also derived from alphaviruses. Because the replicon does not encode structural components of alphaviruses (eg, capsid and glycoproteins), RP cannot propagate in cell culture or animal hosts (without helper plasmids or similar components).

The terms "FCV F9-like" and "F9-like FCV" are used interchangeably and with the term "classical FCV," and as used herein, the FCV F9 strain is considered a typical representative. FCV can be characterized as an old, former universal vaccine strain of FCV. In direct contrast, FCV, referred to as virulent systemic "VS-FCV" or "(VS)FCV" used interchangeably herein, is unusually virulent and cannot be neutralized by antibodies of FCV F9-like strains. A new class of FCVs that cannot be combined [US Pat. No. 7,449,323; Radford et al. , 38(2) Vet res. 319-335 (2007)].

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. When used herein, it means that the unmodified and/or truncated amino acid sequence of a given protein antigen is encoded by the pathogen or strain of the pathogen. The coding sequences within the nucleic acid constructs of the invention for a protein antigen derived from a pathogen are expressed in the amino acid sequence of the protein antigen expressed relative to the corresponding sequence of that protein antigen in the pathogen or strain of the pathogen (including naturally attenuated strains). It may have been genetically engineered to result in sequence modifications and/or truncation.

As used herein, the terms "protect," or "provide protection," or "inducing protective immunity," "help prevent disease," and "help protect against" infection does not require complete protection from symptoms. For example, "helping to defend" is one of the mechanisms by which, after an attack, the defense causes at least the symptoms of the underlying infection to be reduced and/or the underlying cellular, physiological or biochemical causes or symptoms. or can mean sufficient such that a plurality is reduced and/or eliminated. "Reducing" as used in this context means relating to the state of the infection, including the physiological state of the infection as well as the molecular state of the infection.

As used herein, a "vaccine" is a vaccine that, when administered to an animal, is sufficiently potent to minimally help protect against disease resulting from infection with wild-type microorganisms, i.e., aids in the prevention of disease, and/or or one or more antigens in combination with a pharmaceutically acceptable carrier, such as a liquid, typically containing water, that induces an immune response strong enough to prevent, ameliorate or cure the disease. Compositions suitable for application to animals, such as cats (in certain embodiments, including humans, but in other embodiments, specifically non-humans).

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

The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, adjuvants are used to enhance the immune response against one or more vaccine antigens/isolates. Thus, an "adjuvant" non-specifically increases the immune response against a particular antigen and thus generates a sufficient immune response against the amount of antigen required for any given vaccine and/or against the antigen of interest. A drug that reduces the frequency of injections needed to In this context, adjuvants are used to enhance the immune response against one or more vaccine antigens/isolates. For example, the American Feline Veterinarian Association's Feline Vaccination Guidelines suggest the use of a non-adjuvanted FeLV vaccine [AAFP Feline Advisory Panel, 15:785-808 (2013)].

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

As used herein, the term "pharmaceutically acceptable" is used adjectively to mean that the modified noun is suitable for use in medicine. When this is used, for example, to describe an excipient in a pharmaceutical vaccine, this means that the excipient is compatible with the other ingredients of the composition and is disadvantageous to the intended recipient animal, e.g. a cat. characterized as not harmful.

"Parenteral administration" includes subcutaneous, submucosal, intravenous, intramuscular, intradermal and infusion.

As used herein, the term "antigenic fragment" with respect to a particular protein (e.g., a protein antigen) refers to a fragment that is antigenic, i.e., an immune system fragment such as an immunoglobulin (antibody) or a T-cell antigen receptor. is a fragment of that protein that can specifically interact with antigen-recognizing molecules. For example, an antigenic fragment of FCV capsid protein is a fragment of capsid protein that is antigenic. Preferably, antigenic fragments of the invention are immunodominant for antibody and/or T cell receptor recognition. In certain embodiments, an antigenic fragment for a given protein antigen is a fragment of that protein that retains at least 25% of the antigenicity of the full-length protein. In preferred embodiments, the antigenic fragment retains at least 50% of the antigenicity of the full-length protein. In more preferred embodiments, the antigenic fragment retains at least 75% of the antigenicity of the full-length protein. Antigenic fragments can be as small as 20 amino acids, or they can be large fragments in which only one amino acid is missing from the full-length protein. In certain embodiments, antigenic fragments contain 25-150 amino acid residues. In other embodiments, the antigenic fragment contains 50-250 amino acid residues. For example, in the case of FeLV, the FeLV gp45 glycoprotein and the FeLV gp70 glycoprotein are antigenic fragments of the FeLV gp85 glycoprotein, while in the case of FCV, one antigenic fragment of the FCV capsid protein comprises region E of ORF2.

As used herein, one amino acid sequence is 100% "identical" or has 100% "identity" with a second amino acid sequence if the amino acid residues of both sequences are the same. Thus, an amino acid sequence is 50% "identical" to a second amino acid sequence if 50% of the amino acid residues of the two amino acid sequences are identical. Sequence comparisons are performed over contiguous blocks of amino acid residues within a given protein, eg, the protein or polypeptide being compared. Certain embodiments contemplate selected deletions or insertions that may alter the correspondence between two amino acid sequences.

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

As used herein, the term "inactivated" microorganism is used interchangeably with the term "killed" microorganism. For purposes of the present invention, an "inactivated" microorganism is an organism that is capable of eliciting an immune response in an animal, but is incapable of infecting the animal. Antigens of the invention (eg, inactivated feline calicivirus) can be inactivated by an agent selected from the group consisting of binary ethyleneimine, formalin, β-propiolactone, thimerosal, or heat. In certain embodiments, the inactivated feline calicivirus isolate combined with the RP of the invention is inactivated by binary ethyleneimine.

Alphavirus RNA replicon particles of the invention can be lyophilized and rehydrated with sterile water diluent. On the other hand, if the alphavirus RNA replicon particles are stored separately but are intended to be mixed with other vaccine components prior to administration, the alphavirus RNA replicon particles may be combined with a stabilizing solution of those components, e.g. Can be stored in sucrose solution.

The vaccines of the invention can be readily administered by any standard route including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal and/or intraperitoneal vaccination. Those skilled in the art will appreciate that vaccine compositions are preferably formulated appropriately for each type of recipient animal and route of administration.

Accordingly, the present invention also provides methods of immunizing cats against feline pathogens. One such method involves injecting a cat with an immunologically effective amount of a vaccine of the invention such that the cat produces appropriate anti-pathogen antibodies.

Multivalent Vaccines Accordingly, the present invention provides multivalent vaccines comprising at least one modified live feline pathogen and one or more alphavirus RNA replicon particles. For example, coding sequences for protein antigens or antigenic fragments thereof, or combinations of such coding sequences for protein antigens useful in feline vaccines, are added to alphavirus RNA replicon particles (RPs) or in e.g. multivalent vaccines. can be combined into the same RP encoding the FCV capsid protein and/or the FeLV glycoprotein (gp85).

In a specific embodiment, the vaccine comprises at least one modified live feline pathogen and an alphavirus encoding an FCV F9-like capsid protein or an antigenic fragment thereof, and/or a VS-FCV capsid protein or an antigenic fragment thereof. An RNA replicon particle along with another alphavirus RNA replicon particle encoding FeLV gp85 or an antigenic fragment thereof. In other embodiments, the vaccine comprises at least one modified live feline pathogen, one alphavirus RNA replicon particle encoding a VS-FCV capsid protein or antigenic fragment thereof, and FeLV gp85 or an antigenic fragment thereof. another alphavirus RNA replicon particle encoding and a third alphavirus RNA replicon particle further encoding FCV F9-like capsid protein or an antigenic fragment thereof. In yet other embodiments, the vaccine comprises at least one modified live feline pathogen, FCV F9-like capsid protein or antigenic fragment thereof, VS-FCV capsid protein or antigenic fragment thereof, and FeLV gp85 or antigenic fragment thereof. alphavirus RNA replicon particles encoding fragments.

Examples of pathogens from which one or more such protein antigens may be derived include feline viral rhinotracheitis virus (FVR), feline leukemia virus (FeLV), feline panleukopenia virus (FPL), feline herpes virus (FHV), other FCV 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 Chlamydophila psittaci), Bordetella bronchiseptica, and B. Bartonella species (e.g. Bartonella henselae) B. henselae)). In certain embodiments, the coding sequences for capsid proteins or similar proteins from one or more of these feline or canine pathogens can be inserted into the same RP as the FCV antigen. Alternatively, or in combination, a coding sequence for a capsid protein or similar protein from one or more of these feline or canine pathogens can be inserted into one or more other RPs and used in a vaccine. Can be combined with RP encoding FCV F9-like capsid protein or antigenic fragment thereof and/or VS-FCV capsid protein or antigenic fragment thereof.

Accordingly, the invention provides one or more alphavirus RNA replicon particles (RPs) of the invention [e.g., VS-FCV capsid protein or antigenic fragment thereof] in one or more modified (attenuated) Viral isolates, such as live attenuated old vaccine strains of FCV, 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, and/or live attenuated feline infectious peritonitis virus, and/or live attenuated feline immunodeficiency virus, and/or live attenuated Borna disease virus, and/or live attenuated rabies virus, and/or live attenuated Vaccines comprising feline influenza virus, and/or live attenuated canine influenza virus, and/or live attenuated avian influenza, and/or live attenuated canine pneumovirus, and/or live attenuated feline pneumovirus are provided. Furthermore, live attenuated Chlamydophila felis, and/or live attenuated Bordetella bronchiseptica, and/or live attenuated Bartonella species (e.g., B. hensel ae) ) can also be included in such multivalent vaccines.

Additionally, a vaccine of the invention comprising one or more alphavirus RNA replicon particles of the invention [e.g., encoding a VS-FCV capsid protein or an antigenic fragment thereof] comprises one or more modified live viruses. Along with the isolate, one or more killed viral 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 contagious peritonitis virus, and/or killed feline immunodeficiency virus, and/or killed Borna disease virus, and/or killed rabies virus, and/or killed feline influenza virus, and/or killed canine influenza virus, and/or killed avian influenza virus. , and/or killed canine pneumovirus, and/or killed feline pneumovirus. Additionally, bacterins of Chlamydophila felis, and/or Bordetella bronchiseptica, and/or live attenuated Bartonella species (e.g., B. henselae) Like this too can be included in a multivalent vaccine.

It is also to be understood that the invention is not limited to the particular configurations, process steps, and materials disclosed herein, as such may vary somewhat. Additionally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention is limited only by the appended claims and their equivalents. I also want you to understand that this is not something you do.

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The following examples serve to provide a further understanding of the invention, but are in no way meant to limit the scope of the invention.

[Example]
[Example 1]
Introduction of FCV Capsid Protein Coding Sequence into Alphavirus RNA Replicon Particles RNA viruses have been used as vector vehicles to introduce genetically engineered vaccine antigens into the genome. However, their use to date has been primarily limited to incorporating viral antigens into RNA viruses and then introducing the virus into the recipient host. As a result, protective antibodies against the incorporated viral antigens are induced. Alphavirus RNA replicon particles have been used to encode pathogenic antigens. Such an alphavirus replicon platform is based on the Venezuelan equine encephalitis virus (VEE) [Pushko et al. , Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al. , Journal of Virology 67:6439-6446 (1993)] and Semliki Forest Virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9:1356-1361 (1991), the entire contents of which are hereby incorporated herein by reference. ) ] was developed from several different alphaviruses, including Additionally, alphavirus RNA replicon particles are the basis of several USDA-approved vaccines for pigs and poultry. These include swine epidemic diarrhea vaccine, RNA particles (product code 19U5.P1), swine influenza vaccine, RNA (product code 19A5.D0), avian influenza vaccine, RNA (product code 19O5.D0), and prescription products, Contains RNA particles (product code 9PP0.00).

Alphavirus RNA replicon particle construction RP-FCV:
The amino acid sequence of the FCV capsid protein was used to generate a codon-optimized (feline codon usage) nucleotide sequence in silico. Optimized sequences were prepared as synthetic DNA by a commercial vendor (ATUM, Newark, Calif.). Therefore, synthetic genes were designed based on the amino acid sequences of VS-FCV capsid protein and FCV F9-like capsid protein, respectively. Constructs encoding the amino acid sequence of the VS-FCV capsid protein [SEQ ID NO: 2] or the FCV F9-like capsid protein [SEQ ID NO: 4] were codon-optimized for cats and the flanking sequences were suitable for cloning into alphavirus replicon plasmids. was.

VEE replicon vectors designed to express FCV capsid proteins were constructed as previously described [see U.S. Pat. No. 9,441,247, the contents of which are incorporated herein by reference], with the following modifications. It was constructed. The TC-83 derived replicon vector "pVEK" [disclosed and described in US Pat. No. 9,441,247] was digested with restriction enzymes AscI and PacI. DNA comprising the codon-optimized open reading frame nucleotide sequence of the FCV capsid gene and having a 5' flanking sequence (5'-GGCGCGCCGCACC-3') [SEQ ID NO: 11] and a 3' flanking sequence (5'-TTAATTAA-3') The plasmid was similarly digested with restriction enzymes AscI and PacI. The synthetic gene cassette was then ligated into the digested pVEK vector.

Generation of TC-83 RNA replicon particles (RP) was performed according to previously described methods [U.S. Pat. No. 9,441,247 and U.S. Pat. No. 8,460,913, the contents of which are incorporated herein by reference]. . Briefly, pVHV replicon vector DNA and helper DNA plasmids were linearized with NotI restriction enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and cap analog (Promega, Madison, WI). Importantly, the helper RNA used in the production was as previously described [Kamrud et al. , J Gen Virol. 91(Pt 7):1723-1727 (2010)], lacking the VEE subgenomic promoter sequence. Purified RNA of replicon and helper components were combined, mixed with a suspension of Vero cells, electroporated in 4 mm cuvettes, and returned to OptiPro® SFM cell culture medium (Thermo Fisher, Waltham MA). After overnight incubation, alphavirus RNA replicon particles were filtered in phosphate-buffered saline containing 5% sucrose (w/v) by passing the suspension through a ZetaPlus BioCap depth filter (3M, Maplewood, MN). It was purified from the cells and medium by washing and finally eluting the retained RP with 400mM NaCl buffer. The eluted RP was formulated in a final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and aliquoted for storage. Functional RP titers were determined by immunofluorescence assay of infected Vero cell monolayers.

RP-FeLV:
The amino acid sequence of FeLV gp85 was used to generate a codon-optimized (feline codon usage) nucleotide sequence in silico. Optimized sequences were prepared as synthetic DNA by a commercial vendor (ATUM, Newark, CA). Therefore, 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 cat, and flanking sequences were appropriate for cloning into the alphavirus replicon plasmid.

The VEE replicon vector designed to express FeLV gp85 was as previously described [see US Pat. No. 9,441,247, the entire contents of which are incorporated herein by reference] with the following modifications: It was constructed. The TC-83 derived replicon vector "pVEK" [disclosed and described in US Pat. No. 9,441,247] was digested with restriction enzymes AscI and PacI. DNA comprising a codon-optimized open reading frame nucleotide sequence of the FeLV gp85 gene and having a 5' flanking sequence (5'-GGCGCGCCGCACC-3') [SEQ ID NO: 11] and a 3' flanking sequence (5'-TTAATTAA-3') The plasmid 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 renamed "pVHV-FeLV gp85." The "pVHV" vector nomenclature was chosen to refer to the pVEK-derived replicon vector containing the transgene cassette cloned through the AscI and PacI sites of the multiple cloning site of pVEK.

Generation of TC-83 RNA replicon particles (RP) was performed according to previously described methods [U.S. Pat. Ta. 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 RNA used in the production was as previously described [Kamrud et al. , J Gen Virol. 91(Pt 7):1723-1727 (2010)], lacking the VEE subgenomic promoter sequence. Purified RNA of replicon and helper components were combined, mixed with a suspension of Vero cells, electroporated in 4 mm cuvettes, and returned to OptiPro® SFM cell culture medium (Thermo Fisher, Waltham MA). After overnight incubation, alphavirus RNA replicon particles were filtered in phosphate-buffered saline containing 5% sucrose (w/v) by passing the suspension through a ZetaPlus BioCap depth filter (3M, Maplewood, MN). It was purified from the cells and medium by washing and finally eluting the retained RP with 400mM NaCl buffer. The eluted RP was formulated in a final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and aliquoted for storage. Functional RP titers were determined by immunofluorescence assay of infected Vero cell monolayers.

RP-RV
A vaccine was prepared containing alphavirus RNA replicon particles encoding rabies virus glycoprotein (G) from rabies virus (RV) packaged with the capsid protein and glycoprotein of the nonvirulent TC-83 strain of Venezuelan equine encephalitis virus. . The nucleotide sequence of the rabies virus G protein was codon-optimized for humans. The resulting sequence, despite having 100% amino acid identity, has only about 85% nucleotide identity to the live rabies virus glycoprotein (G) sequence. The vaccine is used as a single dose administered to mammalian subjects, e.g., subcutaneously to cats and dogs over 12 weeks of age, or in multiple doses, including an initial dose followed by one or more booster doses. can do.

The amino acid sequence of rabies glycoprotein (G) was used to generate a codon-optimized (human codon usage) nucleotide sequence in silico. Optimized sequences were prepared as synthetic DNA by a commercial vendor (ATUM, Newark, CA). Therefore, a synthetic gene [SEQ ID NO: 9] was designed based on the amino acid sequence of rabies glycoprotein. The construct (RABV-G) was wild type amino acid sequence [SEQ ID NO: 10], codon optimized for human, and flanking sequences were appropriate for cloning into the alphavirus replicon plasmid.

VEE replicon vectors designed to express rabies G were constructed as previously described [see U.S. Pat. No. 9,441,247, the contents of which are incorporated herein by reference] with the following modifications: did. The TC-83 derived replicon vector "pVEK" [disclosed and described in US Pat. No. 9,441,247] was digested with restriction enzymes AscI and PacI. DNA comprising a codon-optimized open reading frame nucleotide sequence of the rabies G gene and having a 5' flanking sequence (5'-GGCGCGCCGCACC-3') [SEQ ID NO: 11] and a 3' flanking sequence (5'-TTAATTAA-3') The plasmid 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 renamed "pVHV-RABV-G". The "pVHV" vector nomenclature was chosen to refer to the pVEK-derived replicon vector containing the transgene cassette cloned through the AscI and PacI sites of the multiple cloning site of pVEK.

Generation of TC-83 RNA replicon particles (RP) was performed according to previously described methods [U.S. Pat. Ta. 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 RNA used in the production was as previously described [Kamrud et al. , J Gen Virol. 91(Pt 7):1723-1727 (2010)], lacking the VEE subgenomic promoter sequence. Purified RNA of replicon and helper components were combined, mixed with a suspension of Vero cells, electroporated in a 4 mm cuvette, and returned to OptiPro® SFM cell culture medium (Thermo Fisher, Waltham, MA). After overnight incubation, the alphavirus RNA replicon particles were filtered by passing the suspension through a ZetaPlus BioCap® depth filter (3M, Maplewood, MN) in a phosphate buffer containing 5% sucrose (w/v). Cells and media were purified by washing with saline and finally eluting retained RP with 400mM NaCl buffer. The eluted RP was formulated in a final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and aliquoted for storage. Functional RP titers were determined by immunofluorescence assay of infected Vero cell monolayers.

[Example 2]
Evaluation of the safety of a combination vaccine containing two RP constructs and three modified live fractions in cats Various formulations and reconstitution methods of lyophilized pentavalent feline combination vaccines were evaluated for their safety in cats. We evaluated the following. The preferred presentation of a pentavalent vaccine is a 0.5 mL dose, and the optimal formulation and filling method is a 0.5 mL fill. The stabilizer contains 1.1% NZ-amine (casein enzyme hydrolyzate), 1.1% gelatin and 7.5% sucrose; percentages provided represent final concentrations. If more than 0.5 mL is required due to volume and final potency constraints for adding the 5 fractions, formulate the product to fill to 1.0 mL and add 0.5 mL of diluent. Can be reconfigured. This doubles the concentration of the stabilizer component of the administered dose. The safety of such concentrated dose stabilizers containing these antigens has not been previously tested in cats.

If the 1.0 mL fill/0.5 mL rehydrated format was not safe for cats, a third option of a 1.0 mL fill volume rehydrated with 1.0 mL diluent was also tested. The vaccine was blended and then lyophilized. The vaccine consists of alphavirus RNA replicon particles encoding feline leukemia virus glycoprotein (RP-FeLV) and feline calicivirus in a stabilizer containing 1.1% gelatin, 1.1% NZ amine and 7.5% sucrose. Alphavirus RNA replicon particles encoding the capsid protein (RP-FCV) were used as a component of the commercially available vaccine, Nobivac® Feline-1, i.e., modified live (MLV) feline panleukopenia virus (FPL). , along with a modified live feline viral rhinotracheitis virus (FVR) and a modified live Chlamydophila felis. Various formulations of pentavalent feline combination vaccine were prepared and reconstituted as described in Table 1 below.

Vaccines were formulated to contain the same dose of each antigen (0.5 mL cake vaccines were formulated with twice the concentration of each antigen), and stabilizers were used at a constant concentration in all formulations (0.5 mL The 1.0 mL cake vaccine rehydrated with diluent contained twice the concentration of stabilizer upon rehydration).

Experimental feline subjects were vaccinated at 7-8 weeks of age (typically with the minimum vaccination age for feline core vaccines) and again 21 days later with the indicated volumes of each test vaccine. Cats were tested for reactions to the test vaccine that may exhibit pain or discomfort, such as barking, stinging, itching, biting, sudden movements upon injection of the vaccine, or unusual reactions (see Tables 2 and 3 below). The animals were observed for 15 minutes immediately after each vaccination. Clinical evaluations were performed by a veterinarian 4-6 hours after vaccination and daily for 3 days after vaccination.

Clinical evaluation included palpation of the injection site and observation for any local reactions, including pain to touch, swelling, redness, or abscess. Cats were also observed for any systemic reactions such as depression, lethargy, lameness, vomiting, tremors, agitation and diarrhea. Body temperature was also measured and recorded 4-6 hours after vaccination and daily for 2 days after each vaccination. Palpation of the injection site for local reactions was further performed three times a week from 7 to 21 days after each vaccination.

All vaccine formulations and rehydration protocols were found to be safe. No local or systemic reactions were observed in any of the cats. All cats at each measurement period except for one cat in treatment group 3 (0.5 mL cake/0.5 mL diluent) who had a temperature of 103.6 °C 5 hours after the second vaccination. Body temperature was normal. Therefore, all three preparations of pentavalent vaccine were found to be tolerated.

The 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 will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims.

Furthermore, it is to be understood that all base or amino acid sizes and all molecular weight or molecular mass values given for nucleic acids or polypeptides are approximations and are provided for illustrative purposes.

Claims (11)

a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle encoding a feline calicivirus (FCV) capsid protein or an antigenic fragment thereof;
From the group consisting of modified live feline viral rhinotracheitis virus (FVR), modified live feline panleukopenia virus (FPLV), modified live Chlamydophila felis, and any combination thereof. a selected modified live feline pathogen;
An immunogenic composition, wherein the FCV capsid protein is an FCV F9-like capsid protein. 2. The immunogenic composition of claim 1, further comprising an additional VEE alphavirus RNA replicon particle encoding a virulent systemic FCV (VS-FCV) capsid protein or an antigenic fragment thereof. 3. The method of claim 1 or 2 comprising one or more additional VEE alphavirus RNA replicon particles encoding an antigen selected from the group consisting of FeLV glycoprotein (gp85), antigenic fragments of gp85, and combinations thereof. The immunogenic composition described. 2. The VEE alphavirus RNA replicon particle further encodes an antigen selected from the group consisting of feline leukemia virus (FeLV) glycoprotein (gp85), antigenic fragments of said gp85, and combinations thereof. or the immunogenic composition according to 3. 3. The immunogenic composition of claim 2, wherein the VS-FCV capsid protein comprises an amino acid sequence that includes at least 95% identity with the amino acid sequence of SEQ ID NO: 2 . 5. The immunogenic composition of claim 1, 2, 3 or 4, wherein the FCV F9-like capsid protein comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO:4. 5. The immunogenic composition of claim 3 or 4, wherein the FeLV glycoprotein (gp85) comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO:6. A vaccine to help prevent diseases caused by FCV, comprising the immunogenic composition according to claim 1, 2, 3, 4, 5 or 6 and a pharmaceutically acceptable carrier. VEE alphavirus RNA replicon particles encoding virulent systemic feline calicivirus VS-FCV capsid protein and FCV F9-like capsid protein;
a VEE alphavirus RNA replicon particle encoding a feline leukemia virus (FeLV) glycoprotein (gp85) or an antigenic fragment of the gp85;
a modified live feline viral rhinotracheitis virus (FVR);
a modified live feline panleukopenia virus (FPLV);
A vaccine to help prevent disease caused by FCV, comprising modified live Chlamydophila felis. 10. The vaccine according to claim 8 or 9, which is a non-adjuvanted vaccine. 11. A method of immunizing a cat against pathogenic FCV, the method comprising the step of administering to said cat an immunologically effective amount of the vaccine of claim 8, 9 or 10.