JP7742695B2 - Polyvalent feline vaccine - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on December 15, 2017, the entire contents of which are incorporated herein by reference.
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/599,401, filed on 2016-02-16, U.S. Provisional Patent Application No. 62/596,508, filed on December 8, 2017, U.S. Provisional Patent Application No. 62/582,050, filed on November 6, 2017, and U.S. Provisional Patent Application No. 62/581,955, filed on November 6, 2017.

The present invention relates to a novel multivalent vaccine for cats. Methods for making and using the multivalent vaccine alone or in combination with other protective agents are also provided.

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

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

The most common characteristic and clinical sign of FCV infection is vesicles (ulcers) on the tongue and oral mucosa.
The development of FCV, which begins as small, isolated ulcers but can spread and affect large portions of the tongue, is also frequently 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 shed infectious virus for extended 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. Furthermore, new FCV strains associated with systemic disease and high mortality have been identified.
See U.S. Patent No. 7,449,323.
"Vaccine systemic" (VS-FCV) isolates are responsible for local outbreaks, and current vaccines do not appear to protect cats from disease caused by these strains.

FCV contains a single-stranded, positive-sense RNA genome consisting of three open reading frames (ORFs). The genome is polyadenylated at the 3' end and bounded at the 5' end by virally encoded proteins. The first ORF encodes the viral protease and RNA-dependent RNA polymerase, expressed as a single polypeptide. This polypeptide is then post-translationally cleaved by the viral protease. The second ORF encodes the major capsid protein (i.e., FC).
V capsid protein), which has six regions designated as A-F [
Scott et al. , 60 Am. J. Vet. Res. :652-658(19
99)]. Region A is cleaved to produce the 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.
al. , 38(2) Vet Res. :319-335 (2007)]. ORF3
encodes a minor structural protein [Sosnovtsev and Green
n, 277 Virology: 193-203 (2000)].

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

In addition to the three pathogens associated with feline respiratory disease mentioned above, cats commonly infect three other viruses: feline leukemia virus (FeLV), feline panleukopenia virus (FPV or FPLV), and
, and rabies virus. Feline leukemia virus is a retrovirus that infects domestic cats and causes significant morbidity and mortality worldwide. FeLV is primarily transmitted via 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
Clinical signs observed in cats during FeLV infection include cytoproliferative disorders (lymphoid or myeloid tumors), cytostatic disorders (immunosuppression, anemia, infectious diseases associated with bone marrow suppression), inflammatory disorders, neurological disorders, abortion,
and enteritis [Hoover et al., J Am Vet Med As
soc 199:1287-1297 (1991); Levy and Crawford
d.Textbook of Veterinary Internal Medicine
ne, 6th ed (Ettinger SJ, Feldman EC., eds
．． ) WB 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 frequently establishes persistent infection with associated persistent viremia, often leading to death of the host cat.

The single-stranded RNA genome of FeLV encodes only three genes: (i) the ENV gene, which encodes the envelope glycoprotein, (ii) the GAG gene, which encodes the major structural elements of the virus, and (iii) the POL gene, which encodes the RNA polymerase [T
Homsen et al. , Journal of General Virolog
y 73:1819-1824 (1992)]. The FeLV envelope (ENV) gene encodes the gp85 precursor protein, which is proteolytically processed by one or more cellular enzymes to produce the major envelope glycoprotein gp70 and the associated transmembrane protein p15E [DeNoronha, et al., Virology 8
5:617-621 (1978); Nunberg et al. , PNAS 81:3
675-3679 (1983)]. The transmembrane protein p15E contains a sequence conserved among gammaretroviruses that has immunosuppressive properties [Mathes et al.,
Nature 274:687-689 (1978)]. Recently, the European Medicines Agency's Committee for Veterinary Medicinal Products (CVMP) has approved the use of Escherichia coli as an active ingredient.
) recombinant p53 derived from the gp70 surface glycoprotein of FeLV subgroup A expressed in
45 A positive opinion was adopted for vaccines containing FeLV envelope antigens.
The LV envelope glycoprotein is the target of FeLV-specific cytotoxic T cell responses as well as neutralizing antibodies and is therefore 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 white blood cell count (
The disease is caused by a lack of white blood cells (FPLV). 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 be spread transplacentally in pregnant cats, causing embryonic resorption, fetal mummification, stillbirth, or abortion. Infected cats shed the virus in urine, feces, and nasal secretions, and susceptible cats become infected when they come into contact with these secretions or infected cat fleas. The clinical signs of FPLV infection are also classified as feline distemper or feline parvo.

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

Rabies is a preventable zoonotic disease that results in inflammation of the brain in humans and other mammals. Clinical rabies is an acute, progressive encephalitis typically classified as violent 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 eventual coma. The causative agent of rabies is the rabies virus, which can infect most mammals, including cats, and maintains a reservoir in wild and susceptible domestic animals.

The rabies virus consists of five structural proteins: nucleoprotein (N), phosphoprotein (P),
), a matrix protein (M), a glycoprotein (G), and an RNA-dependent RNA polymerase.
tal. , 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 types of rabies vaccines have been produced to combat this disease. Inactivated cell culture-derived whole virus killed rabies virus vaccines are the most commonly used vaccines in the United States. These whole virus killed rabies virus vaccines require high levels of antigen, thus necessitating the use of adjuvants. Unfortunately, the use of adjuvants has been associated with injection site reactivity, hypersensitivity, and even a perceived risk of injection site sarcoma in cats. Recently, modified live vaccines have been successfully used in oral vaccine baits for the immunization of wild animals [Mahl et al., Vet Res 45(1):77(2)].
014)]. 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 over the years in vaccines 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)], which are Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virol
logy 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)]. RP vaccines deliver a growth-deficient alphavirus RNA replicon to 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 with some conventional vaccine formulations [Vander Veen, et al. Anim Health Res Rev. 1
3(1):1-9. (2012)]. The RP platform has been used to encode pathogenic antigens and is the basis for several USDA-licensed vaccines for swine 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 lethal viral attack [Konopka et al., J. Virol., 83 (2
9): 12432-12442 (2009)], and induces rapid protection against foot-and-mouth disease virus in particular [Segundo et al., J. Virol., 87(10):544
7-5460 (2013)]. Therefore, alphavirus RN
Since the A replicon particles enhance the innate immune response to the live virus, it appears that alphavirus RNA replicon particles should not be combined with modified live virus in a vaccine, as this would be expected to be detrimental to the immune response.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

U.S. 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., eds.) WB Saunders, Philadelphia, 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)

Thus, the present invention includes 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. All of the immunogenic compositions of the present invention can also be used in multivalent vaccines. In particular embodiments of this type, the subject to be vaccinated is a cat. In even more particular embodiments, the subject to be vaccinated is a domestic cat. Methods of making and using the immunogenic compositions and vaccines of the present invention are also provided.

In certain embodiments, the immunogenic composition comprises one or more feline caliciviruses (FCs).
In other embodiments, the immunogenic composition comprises an alphavirus RNA replicon particle encoding one or more feline leukemia virus (FeLV) antigens and a modified live feline pathogen. In yet other embodiments, the immunogenic composition comprises an alphavirus RNA replicon particle 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
In yet another embodiment, the modified live feline pathogen is a modified live Chlamydophila felis.
In yet another embodiment, the modified live feline pathogen is a modified live F9-like feline calicivirus (FCV F9-like). In yet another embodiment, the modified live feline pathogen is a modified live Bordetella bronchiseptica (Bordetella
The present invention further provides immunogenic compositions comprising any combination of these alphavirus RNA replicon particles and modified live feline pathogens. In a specific embodiment of this type, the immunogenic composition comprises alphavirus RNA replicon particles encoding FCV antigens, alphavirus RNA replicon particles encoding FeLV antigens, modified live FVR, modified live FPLV, and modified live Chlamydophila felis.
Includes:

In certain embodiments of this type, the immunogenic composition comprises an alphavirus RNA replicon particle encoding an FCV capsid protein.
The CV capsid protein is an FCV F9-like capsid protein. In other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of an FCV F9-like capsid protein. In yet other embodiments, the FCV capsid protein is a virulent systemic FCV (VS-FCV) capsid protein. In yet other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of a VS-FCV capsid protein. In yet other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of an FCV
It encodes both an 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 comprises an amino acid sequence comprising 95% or greater identity to the amino acid sequence of SEQ ID NO: 2. In a more specific embodiment, the VS-FCV capsid protein comprises an amino acid sequence comprising 95% or greater identity to 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 comprising 95% or more identity to 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 one or more feline leukemia viruses (F
In certain embodiments, the alphavirus RNA replicon particle encodes an FeLV (e.g., FeLV) antigen. In certain embodiments, the FeLV antigen is an FeLV glycoprotein (e.g., gp85). In other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of FeLV gp85. In even more specific embodiments of this type, the antigenic fragment of the FeLV glycoprotein is FeLV gp70. In related embodiments, the antigenic fragment of the FeLV glycoprotein is FeLV gp45. In more specific embodiments of this type, the antigenic fragment of the FeLV glycoprotein is FeLV gp45.
LV gp85 comprises an amino acid sequence that comprises 95% or more identity to the amino acid sequence of SEQ ID NO: 6. In a more specific embodiment of this type, FeLV gp85 comprises an amino acid sequence that comprises 95% or more identity to the amino acid sequence of SEQ ID NO: 6.
In an even more specific embodiment of this type, the amino acid sequence comprises the amino acid sequence of FeLV gp
85 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 comprises 95% or more identity to 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 even more specific embodiments of this type, FeLV gp70 is encoded by the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 15.

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

In particular embodiments, the alphavirus RNA replicon particles are Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particles. In more specific embodiments of this type, the VEE alphavirus RNA replicon particles are TC-83 VEE alphavirus RNA replicon particles. In other embodiments, the alphavirus RNA
The replicon particle is a Sindbis (SIN) alphavirus RNA replicon particle. In yet another embodiment, 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 from one or more feline pathogens. In a particular embodiment of this type, the naked DNA vector encodes an FCV capsid protein or an antigenic fragment thereof. In a specific embodiment of this type, the naked DNA vector encodes a VS-FCV capsid protein or an antigenic fragment thereof. In another embodiment of this type, the naked DNA vector encodes FeLV gp85 or an antigenic fragment thereof.

In certain embodiments, the immunogenic composition can comprise at least one modified live feline pathogen and an alphavirus RNA replicon particle encoding one or more FCV capsid proteins or antigenic fragments thereof, one or more FeLV glycoproteins or antigenic fragments thereof, and/or one or more rabies virus G proteins or antigenic fragments thereof. In particular embodiments of this type, the alphavirus RNA replicon particle encodes a VS-FCV capsid protein or antigenic fragment thereof and an FCV
In a related embodiment, the alphavirus RNA replicon particle encodes both a VS-FCV capsid protein or antigenic fragment thereof and FeLV gp85 or an antigenic fragment thereof.
In other embodiments, the alphavirus RNA replicon particle encodes both an FCV F9-like capsid protein, or an antigenic fragment thereof, and an FeLV gp85, or an antigenic fragment thereof.
In yet another embodiment, the alphavirus RNA replicon particle encodes both an FCV F9-like capsid protein or an antigenic fragment thereof and a rabies virus G protein or an antigenic fragment thereof.
In yet another embodiment, the alphavirus RNA replicon particle encodes both a VS-FCV capsid protein or an antigenic fragment thereof, an FeLV gp85 or an antigenic fragment thereof, and a rabies virus G protein or an antigenic fragment thereof. In another embodiment, the alphavirus RNA replicon particle encodes a VS-FCV capsid protein or an antigenic fragment thereof, an FCV F9-like capsid protein or an antigenic fragment thereof, and an FeLV gp85 or an antigenic fragment thereof. In yet another embodiment, the alphavirus RNA replicon particle encodes a V
In yet another embodiment, the alphavirus RNA replicon particle encodes an S-FCV capsid protein or an antigenic fragment thereof, FeLV gp85 or an antigenic fragment thereof, rabies virus G protein or an antigenic fragment thereof, and an FCV F9-like capsid protein or an antigenic fragment thereof. In yet another embodiment, the alphavirus RNA replicon particle encodes an FeLV gp85 or an antigenic fragment thereof, and rabies virus G protein or an antigenic fragment thereof. In yet another embodiment, the alphavirus RNA replicon particle encodes an FeLV gp85 or an antigenic fragment thereof, an FCV F9-like capsid protein or an antigenic fragment thereof, and rabies virus G protein or an antigenic fragment thereof.
It encodes a protein or an antigenic fragment thereof.

Thus, the present invention provides immunogenic compositions comprising at least one modified live feline pathogen and any one or more alphavirus RNA replicon particles of the present invention encoding multiple feline pathogen antigens and/or any one or more alphavirus RNA replicon particles of the present invention encoding a single feline pathogen antigen. In certain embodiments, the modified live feline pathogen is a modified live FVR. In other embodiments, the modified live feline pathogen is a modified live FPLV. In yet other embodiments,
The modified live feline pathogen is modified live Chlamydophila felis.
In yet another embodiment, the modified live feline pathogen is a modified live FCV F9-like. In yet another embodiment, the modified live feline pathogen is a 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 a modified live FVR, a 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 a specific embodiment of this type, the immunogenic composition comprises a modified live FVR, a modified live Chlamydophila, a modified live FPLV, and a modified live F9-like FCV. In a specific embodiment, all of the alphavirus RNA replicon particles in the immunogenic composition are those encoding Venezuelan equine encephalitis (VEE) virus.
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 particle.

In further embodiments, the alphavirus RNA replicon particles may encode protein antigens (or antigenic fragments thereof) derived from other feline pathogens. In particular 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 a feline influenza virus.
In yet other embodiments, the protein antigen is derived from a feline coronavirus (FCoV).

The present invention further provides immunogenic compositions and/or vaccines (multivalent vaccines) comprising the alphavirus RNA replicon particles of the present invention, together with one or more modified live (e.g., attenuated) feline pathogens of the present invention, together with killed feline pathogens. In certain embodiments, the immunogenic composition comprises killed Chlamydophila felis.
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 present invention further includes vaccines and multivalent vaccines comprising the immunogenic compositions of the present invention.
In particular embodiments, the multivalent vaccine is a non-adjuvanted vaccine. In certain embodiments, the vaccine aids in the prevention of disease caused by FCV, and/or FeLV, and/or FVR, and/or FPLV, and/or rabies virus, and/or Chlamydophila felis. In related embodiments, antibodies are induced in feline subjects when cats are immunized with the vaccine. The present invention further includes all of the alphavirus RNA replicon particles and naked DNA vectors of the present invention.

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

The vaccines and multivalent vaccines of the present invention can be administered as primer vaccines and/or booster vaccines. In a specific embodiment, the vaccines of the present invention are administered as single-shot vaccines (single doses) without the need for subsequent administration. In certain embodiments, when both primer and booster vaccines are administered, the primer and booster vaccines can be administered by the same route. In certain embodiments of this type, both the primer and booster vaccines are administered by subcutaneous injection. In an alternative embodiment, when both primer and booster vaccines are administered,
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 for immunizing a cat against FCV, comprising the step of injecting the cat with an immunologically effective amount of the vaccine of the present invention described above. In certain embodiments, the vaccine may contain, for example, about 1 x ¹⁰ to about 1 x ¹⁰ RP or more. In more specific embodiments, the vaccine may contain about 1 x ¹⁰ to about 1 x ¹⁰ RP. In even more specific embodiments, the vaccine may contain about 1 x ¹⁰ to about 1 x ¹⁰ RP. In certain embodiments, the cat is a domestic cat.

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

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

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, yet still inhibits feline calicivirus (FCV) and/or feline leukemia virus (FeL).
V), and infection with feline viral rhinotracheitis virus (FVR), and/or 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 vaccine of the present invention, which contains both alphavirus RNA replicon particles and modified live viruses, is unexpectedly safe and effective, without significantly interfering with the immunological effects of the live viruses with which the alphavirus RNA replicon particles have been modified.

Indeed, alphavirus RNA replicon particles, particularly Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles, have previously been shown to catalyze a systemic antiviral state and protect against lethal viral challenge [Konopka et al., J. Vir.
ol., 83(29):12432-12442(2009)]. Furthermore, it has been reported that VEE alphavirus RNA replicon particles induce rapid protection against foot-and-mouth disease virus [Segundo et al., J. Virol., 87(10):
5447-5460 (2013)]. Therefore, one would have predicted that a vaccine containing both alphavirus RNA replicon particles and a modified live virus would result in substantial inhibition of the immunological effects of the modified live virus. Surprisingly, however, it was 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 modified live virus.

Thus, in a particular aspect, the present invention provides an alphavirus RNA replicon particle encoding an FCV capsid protein or an antigenic fragment thereof and/or FeLV gp85 or an antigenic fragment thereof, wherein the alphavirus RNA replicon particle is a modified live feline FVR, and/or a modified live FPLV, and/or a modified live Chlamydophila felis (
In yet another aspect of the present invention, the vaccine comprises an FCV capsid protein or an antigenic fragment thereof and/or an FeL.
The present invention also includes a naked DNA vector encoding V gp85 or an antigenic fragment thereof, together with a modified live feline FVR, and/or a modified live feline FPLV, and/or a modified live Chlamydophila felis.

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

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

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

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

As used herein, the term "cat" 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., replicon-specific vectors) that, when present, allow for the successful propagation of a parent virus in cell culture or an animal host.
For example, it refers to a modified RNA viral genome that lacks coding sequences for structural proteins.
In the appropriate cellular context, the 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 the alphavirus RNA replicon particle that is a particle that contains structural proteins, e.g., Pushko et al. [Virology 2
39(2):389-401 (1997)], RP is an alphavirus-derived RNA replicon packaged in a capsid and glycoproteins also derived from an alphavirus. Because the replicon does not encode the structural components of an alphavirus (e.g., capsid and glycoproteins), the RP cannot be propagated in cell culture or in an animal host (without a helper plasmid or similar components).

The terms "FCV F9-like" and "F9-like FCV" are used interchangeably with each other and with "classical FC
"VS-FCV" is used interchangeably with the term "VS-FCV" and, as used herein, is an FCV that can be characterized as an older, pre-universal vaccine strain of FCV, for which the FCV F9 strain is considered the archetypal representative. In direct contrast, an FCV termed highly virulent systemic "VS-FCV" or "(VS)FCV," used interchangeably herein, is a new class of FCV that is unusually virulent and cannot be neutralized by antibodies of FCV F9-like strains [U.S. Patent No. 7,449,323
Specification; Radford et al. , 38(2) Vet res. 319-335
(2007)].

"Originate from", "Originates
The terms "from" and "originating from" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it, and as used herein mean that the unmodified and/or truncated amino acid sequence of the given protein antigen is encoded by the pathogen or strain of pathogen. The coding sequence within a nucleic acid construct of the invention for a protein antigen derived from a pathogen may have been genetically engineered to result in the modification and/or truncation of the amino acid sequence of the expressed protein antigen relative to the corresponding sequence of that protein antigen in the pathogen or strain of pathogen (including naturally attenuated strains).

As used herein, the terms "protect," or "provide protection," or "induce protective immunity,""help prevent disease," and "help protect" do not require complete protection from the onset of infection. For example, "help protect" may mean that protection is achieved after an attack,
It can mean sufficient such that the symptoms of the underlying infection are at least reduced and/or one or more of the underlying cellular, physiological or biochemical causes or mechanisms that cause the symptoms are reduced and/or eliminated. As used in this context, "reducing" means relating to the state of the infection, including not only the physiological state of the infection, but also the molecular state of the infection.

As used herein, a "vaccine" is a composition suitable for administration to an animal, e.g., a cat (which in certain embodiments includes a human, but in other embodiments is not specifically a human), comprising one or more antigens combined with a pharmaceutically acceptable carrier, typically a liquid containing water, which, when administered to the animal, induces an immune response that is strong enough to at least help protect against disease resulting from infection with a wild-type microorganism, i.e., to help prevent disease, and/or is strong enough to prevent, ameliorate, or cure disease.

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

The terms "adjuvant" and "immunostimulant" are used interchangeably herein,
An adjuvant is defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance the immune response to one or more vaccine antigens/isolates. Thus, an "adjuvant" is an agent that nonspecifically increases the immune response to a specific antigen, thus reducing the amount of antigen required for any given vaccine and/or the frequency of injections required to generate a sufficient immune response to the target antigen. In this context, an adjuvant is used to enhance the immune response to one or more vaccine antigens/isolates. For example, the American Association of Feline Veterinarians' feline vaccination guidelines suggest the use of non-adjuvanted FeLV vaccines [AAF
P Feline Advisory Panel, 15:785-808 (201
3).

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

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

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

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 contain an immunoglobulin (antibody) or T
It is a fragment of that protein that can specifically interact with an antigen-recognizing molecule of the immune system, such as a cellular antigen receptor. For example, an antigenic fragment of an FCV capsid protein is a fragment of the capsid protein that is antigenic. Preferably, the antigenic fragment of the present invention is an antibody and/or a fragment of the FCV capsid protein that is antigenic.
or immunodominant for T cell receptor recognition. In certain embodiments, an antigenic fragment with respect to a given protein antigen is a fragment of that protein that retains at least 25% of the antigenicity of the full-length protein. In preferred embodiments, the antigenic fragment retains at least 50% of the antigenicity of the full-length protein. In 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 larger fragments that are missing only a single amino acid from the full-length protein. In certain embodiments, the antigenic fragment comprises 25-150 amino acid residues. In other embodiments, the antigenic fragment comprises 50-200 amino acid residues.
It contains 50 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 contains region E of ORF2.

As used herein, an 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 identical. 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 comparison is performed over contiguous blocks of amino acid residues contained in a given protein, e.g., a portion of a protein or polypeptide being compared. In certain embodiments, selected deletions or insertions that may alter the correspondence between the two amino acid sequences are taken into account.

As used herein, percent nucleotide and amino acid sequence identity is calculated using C, with default parameters for alignment and identity.
MacVector (MacVector, Inc. Cary, NC 27519)
, Vector NTI (Informax, Inc. MD), Oxford Mol
Sequence similarity can be determined using the Clustal Group PLC (1996) and Clustal W algorithms. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, for example, GCG (Genetics Computer Group) using default parameters can be used.
oup, Program Manual for GCG Package, Version 7, Madison, WI) pileup program was used to search for Ad under default filter conditions.
A vanced Blast search can be used.

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 unable to infect the animal. The antigens of the present invention (e.g., inactivated feline calicivirus) are derivatized using binary ethyleneimine (BEE) and/or BEE.
ethyleneimine), formalin, β-propiolactone, thimerosal,
or heat. In certain embodiments,
The inactivated feline calicivirus isolate combined with the RP of the present invention is inactivated by binary ethyleneimine.

The alphavirus RNA replicon particles of the present invention can be lyophilized and rehydrated with a 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 can be lyophilized and rehydrated with a sterile water diluent.
The A replicon particles can be stored in a stabilizing solution of their components, such as a high sucrose solution.

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

Thus, the present invention also provides methods for immunizing a cat against a feline pathogen, one such method comprising injecting the cat with an immunologically effective amount of a vaccine of the present invention so 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, the coding sequence for a protein antigen or antigenic fragment thereof, or a combination of such coding sequences for protein antigens useful in a feline vaccine, can be added to an alphavirus RNA replicon particle (RP) or combined in the same RP that encodes, for example, the FCV capsid protein and/or FeLV glycoprotein (gp85) in a multivalent vaccine.

In a specific embodiment, the vaccine comprises at least one modified live feline pathogen and F
CV F9-like capsid protein or antigenic fragment thereof, and/or VS-FCV
In another embodiment, the vaccine comprises at least one modified live feline pathogen, one alphavirus RNA replicon particle encoding a VS-FCV capsid protein or an antigenic fragment thereof, another alphavirus RNA replicon particle encoding FeLV gp85 or an antigenic fragment thereof, and another alphavirus RNA replicon particle encoding FeLV gp85 or an antigenic fragment thereof.
and a third alphavirus RNA replicon particle encoding a VF9-like capsid protein or an antigenic fragment thereof.
and two modified live feline pathogens, an FCV F9-like capsid protein or an antigenic fragment thereof, a VS-FCV capsid protein or an antigenic fragment thereof, and FeLV gp85.
or an alphavirus RNA replicon particle encoding an antigenic fragment thereof.

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 herpesvirus (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), and the like.
Chlamydophila psittaci, Bordetella bronchiseptica
, and Bartonella species (e.g., Bartonella henselae (B
In certain embodiments, the coding sequence for the capsid protein or similar protein from one or more of these feline or canine pathogens is selected from the group consisting of:
It can be inserted into the same RP as the FCV antigen. Alternatively, or in combination therewith,
The coding sequence for the capsid protein or similar protein from one or more of these feline or canine pathogens can be inserted into one or more other RPs, which can then be used in a vaccine to encode the FCV F9-like capsid protein or an antigenic fragment thereof and/or the VS.
- can be combined with a RP encoding the FCV capsid protein or an antigenic fragment thereof.

Thus, the present invention provides for the incorporation of one or more alphavirus RNA replicon particles (RP) of the present invention [e.g., VS-FCV capsid protein or antigenic fragment thereof] into one or more modified live (attenuated) virus isolates, such as a live attenuated ancient vaccine strain of FCV, e.g., live attenuated FCV F9, and/or a live attenuated feline herpesvirus,
and/or live attenuated feline parvovirus, and/or live attenuated feline leukemia virus, and/or live attenuated feline infectious peritonitis virus, and/or live attenuated feline immunodeficiency virus, and/or live attenuated Borna disease virus, and/or live attenuated rabies virus, and/or live attenuated feline influenza virus, and/or live attenuated canine influenza virus, and/or live attenuated avian influenza, and/or live attenuated canine pneumovirus, and/or live attenuated feline pneumovirus.
mydophila felis, and/or live attenuated Bordetella bronchiseptica, and/or live attenuated Bartonella species (e.g., Bartonella henselae, B. ...
nselae) can also be included in such a multivalent vaccine.

Additionally, one or more alphavirus RNA replicon particles of the invention [e.g.,
The vaccine of the present invention, comprising a VS-FCV capsid protein or an antigenic fragment thereof, may comprise one or more modified live virus isolates, together with one or more killed virus isolates, such as killed FCV strains, and/or killed feline herpesvirus, 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/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.
and/or live attenuated Bartonella species (eg, B. henselae) bacterins may also be included in such multivalent vaccines.

It is also to be understood that the invention is not limited to the particular configurations, process steps, and materials disclosed herein, as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

array

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

[Example]
[Example 1]
Incorporation of the 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 limited primarily to incorporating viral antigens into the RNA virus and then introducing the virus into a recipient host, resulting in the induction of protective antibodies against the incorporated viral antigens.
Alphavirus RNA replicon particles have been used to encode pathogenic antigens. Such alphavirus replicon platforms include those derived from Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virology 239:389-
401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of Virol, vol. 1, pp. 111-115, 1997, the entire contents of which are hereby incorporated by reference].
logy 67:6439-6446 (1993)] and Semliki Forest virus (SFV
) [Liljestrom and
Garoff, Biotechnology (NY) 9:1356-1361 (19
91). In addition, alphavirus RNA replicon particles are the basis of several USDA-licensed vaccines for swine and poultry. These include the Porcine Epidemic Diarrhea Vaccine, RNA Particle (Product Code 19
U5.P1), Swine Influenza Vaccine, RNA (Product Code 19A5.D0), Avian Influenza Vaccine, RNA (Product Code 19O5.D0), and Formulated Products, RN
Contains A particles (product code 9PP0.00).

Alphavirus RNA replicon particle construct RP-FCV:
The amino acid sequence of the FCV capsid protein was used to generate a codon-optimized (feline codon) nucleotide sequence in silico. The optimized sequence was obtained from a commercial vendor (ATUM,
The constructs were prepared as synthetic DNA by the National Institute of Health Sciences (Newark, CA). Synthetic genes were therefore designed based on the amino acid sequences of the VS-FCV capsid protein and the 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 an alphavirus replicon plasmid.

A VEE replicon vector designed to express FCV capsid proteins was 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: The TC-83 derived replicon vector "pVEK" [disclosed and described in U.S. Pat. No. 9,441,247] was transfected with:
The fragment was digested with the restriction enzymes AscI and PacI. It contains the codon-optimized open reading frame nucleotide sequence of the FCV capsid gene 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.

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

RP-FeLV:
The amino acid sequence of FeLV gp85 was used to generate a codon-optimized (feline codon) nucleotide sequence in silico. The optimized sequence was obtained from a commercial vendor (ATUM, New
The gp85 construct was prepared as synthetic DNA by Sigma-Aldrich (St. Louis, MO). Therefore, a synthetic gene was designed based on the amino acid sequence of gp85. The construct (gp85_wt) had the wild-type amino acid sequence [SEQ ID NO: 2], codon-optimized for feline use, and flanking sequences suitable for cloning into an alphavirus replicon plasmid.

A VEE replicon vector designed to express FeLV gp85 was prepared as previously described [the entire contents of which are incorporated herein by reference] with the following modifications.
[See U.S. Patent No. 9,441,247]. The TC-83 derived replicon vector "pVEK" [disclosed and described in U.S. Patent No. 9,441,247] was digested with the restriction enzymes AscI and PacI. A 5' flanking sequence (5'-GGCGC) was inserted into the TC-83 derived replicon vector pVEK.
GCCGCACC-3') [SEQ ID NO: 11] and the 3' flanking sequence (5'-TTAATTA
A DNA plasmid carrying the transgene cassette (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 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 via the AscI and PacI sites of the multiple cloning site of pVEK.

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

RP-RV
A vaccine was prepared containing alphavirus RNA replicon particles encoding the rabies virus glycoprotein (G) from rabies virus (RV) packaged with the capsid protein and glycoprotein of the non-virulent 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 can be used as a single dose administered to mammalian subjects, for example, subcutaneously to cats and dogs aged 12 weeks or older, or in multiple doses including a primary dose followed by one or more booster doses.

The amino acid sequence of the rabies glycoprotein (G) was used to generate a codon-optimized (human codon-using) nucleotide sequence in silico. The optimized sequence was obtained from a commercial vendor (ATUM,
The construct (RAB) was prepared as synthetic DNA by the Rabies Glycoprotein Research Center (Newark, CA). Therefore, a synthetic gene [SEQ ID NO: 9] was designed based on the amino acid sequence of the rabies glycoprotein.
V-G) 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.

The VEE replicon vector designed to express rabies G was constructed with the following modifications:
As previously described [U.S. Pat. No. 94,944, 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 nucleotide sequence of the codon-optimized open reading frame of the rabies G gene was included, along with the 5' flanking sequence (5'-GGCGCGCCGCACC-3').
DNA having [SEQ ID NO: 11] and the 3' flanking sequence (5'-TTAATTAA-3')
The plasmid was similarly digested with the restriction enzymes AscI and PacI. The synthetic gene cassette was then ligated into the digested pVEK vector, and the resulting clone was named "pVEK".
The "pVHV" vector nomenclature was chosen to refer to a pVEK-derived replicon vector containing a transgene cassette cloned via the AscI and PacI sites of the multiple cloning site of pVEK.

TC-83 RNA replicon particles (RP) were produced according to previously described methods [U.S. Pat. Nos. 9,441,247 and 8,460,913, the entire contents of which are incorporated herein by reference]. Briefly, pVHV replicon vector DNA and helper DNA plasmids were transfected with MegaScript T7 RNase A (MegaScript T7 RNase A).
The primers were linearized with NotI restriction enzyme before in vitro transcription using Promega A polymerase and cap analog (Promega, Madison, WI). Importantly, the helper RNA used in the construction was prepared as previously described [Kamrud et al., J Gen
Virol. 91(Pt 7):1723-1727(2010)], lacking the VEE subgenomic promoter sequence. Purified RNA of the replicon and helper components was combined and
The cells were mixed with a suspension of Vero cells, electroporated in a 4 mm cuvette, and placed back into OptiPro® SFM cell culture medium (Thermo Fisher, Waltham, MA). After overnight incubation, the alphavirus RNA replicon particles were isolated by loading the suspension onto a ZetaPlus BioCap® depth filter (3M, Maple).
The RP was purified from cells and media by passage through a 0.22 micron membrane filter (Wood, MN), washing with phosphate-buffered saline containing 5% sucrose (w/v), and finally eluting the retained RP with 400 mM NaCl buffer. The eluted RP was formulated to a final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and aliquoted for storage. Functional RP
P 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 a lyophilized pentavalent feline combination vaccine were evaluated for their safety in cats. The desired presentation of the 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 (enzymatic casein hydrolysate), 1.1% gelatin, and 7.5% sucrose, with the percentages provided representing final concentrations. If a volume greater than 0.5 mL is required due to volume and final potency constraints for adding the five fractions, the product can be formulated and filled to 1.0 mL and reconstituted with 0.5 mL of diluent. This doubles the concentration of stabilizer components in the administered dose. The safety of such concentrated doses of stabilizer containing these antigens is evaluated.
It has not been previously tested in cats.

If the 1.0 mL fill/0.5 mL rehydration format is not safe for cats,
A third option was also tested, with a 1.0 mL fill volume rehydrated with a diluent of 1.1% feline leukemia virus glycoprotein (RP-F) in a stabilizer containing 1.1% gelatin, 1.1% NZ amine, and 7.5% sucrose.
Alphavirus RNA replicon particles encoding feline calicivirus capsid protein (RP-FCV) and feline calicivirus capsid protein (RP-FCV) were co-injected with components of the commercially available vaccine, Nobivac® Feline-1, namely, modified live (MLV) feline panleukopenia virus (FPL), modified live feline viral rhinotracheitis virus (FVR), and modified live Chlamydophila felis (C
Various formulations of the pentavalent feline combination vaccine were prepared and reconstituted as described in Table 1 below.

The vaccines were formulated to contain the same dose of each antigen (0.5 mL cake vaccine was 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 L diluent contained twice the concentration of stabilizer upon rehydration).

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

Clinical evaluation included palpation of the injection site and observation for any local reactions, including tenderness to the touch, swelling, redness, or abscesses. Cats were also observed for any systemic reactions, such as depression, lethargy, lameness, vomiting, tremors, agitation, and diarrhea. Temperatures were also taken and were monitored between 4 and 6 days after vaccination.
Injection site palpation for local reactions was also performed three times weekly 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 had normal temperatures at each measurement period, except for one cat in treatment group 3 (0.5 mL cake/0.5 mL diluent) that had a temperature of 103.6°C 5 hours after the second vaccination. Therefore, all three preparations of the pentavalent vaccine were found to be well tolerated.

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

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

Claims (11)

1. An immunogenic composition comprising: a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle encoding a virulent systemic feline calicivirus (VS-FCV) capsid protein or an antigenic fragment thereof; and a modified live feline pathogen selected from the group consisting of a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), a modified live Chlamydophila felis, and any combination thereof . The immunogenic composition of claim 1, wherein the VEE alphavirus RNA replicon particles further encode one or more feline calicivirus (FCV) antigens and/or one or more feline leukemia virus (FeLV) antigens. The immunogenic composition of claim 2, wherein the FCV antigen is selected from the group consisting of an FCV F9-like capsid protein or an antigenic fragment thereof and a combination thereof, and/or the FeLV antigen is selected from the group consisting of an FeLV glycoprotein (gp85), an antigenic fragment of gp85 and a combination thereof. The immunogenic composition of any one of claims 1 to 3, comprising one or more additional VEE alphavirus RNA replicon particles encoding an FeLV antigen selected from the group consisting of FeLV glycoprotein (gp85), antigenic fragments of gp85, and combinations thereof. The immunogenic composition according to any one of claims 1 to 4, wherein the VS-FCV capsid protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2. The immunogenic composition of any one of claims 2 to 4, wherein the VEE alphavirus RNA replicon particle further encodes one or more feline calicivirus (FCV) antigens selected from the group consisting of an FCV F9-like capsid protein or an antigenic fragment thereof, and a combination thereof, and the FCV F9-like capsid protein comprises an amino acid sequence that comprises at least 95% identity to the amino acid sequence of SEQ ID NO:4. The immunogenic composition of any one of claims 2 to 4, wherein the VEE alphavirus RNA replicon particle further encodes one or more feline leukemia virus (FeLV) antigens selected from the group consisting of FeLV glycoprotein (gp85), antigenic fragments of gp85, and combinations thereof, and the FeLV glycoprotein (gp85) comprises an amino acid sequence comprising at least 95% identity to the amino acid sequence of SEQ ID NO:6. A vaccine for helping to prevent diseases caused by feline calicivirus (FCV) and/or feline leukemia virus (FeLV), comprising the immunogenic composition of any one of claims 1 to 7 and a pharmaceutically acceptable carrier. a VEE alphavirus RNA replicon particle encoding a virulent systemic feline calicivirus VS-FCV capsid protein or a VEE alphavirus RNA replicon particle encoding an FCV F9-like capsid protein;
a VEE alphavirus RNA replicon particle encoding feline leukemia virus (FeLV) glycoprotein (gp85) or an antigenic fragment of said gp85;
a modified live feline viral rhinotracheitis virus (FVR);
a modified live feline panleukopenia virus (FPLV);
A vaccine to help prevent disease caused by feline calicivirus (FCV), comprising a modified live Chlamydophila felis. The vaccine according to claim 8 or 9, which is a non-adjuvanted vaccine. A method for immunizing a cat against pathogenic feline calicivirus (FCV), comprising administering to the cat an immunologically effective amount of the vaccine described in any one of claims 8 to 10.