Rabies virus vaccine

Abstract

To provide rabies vaccines to replace whole virus inactivated rabies virus vaccine derived from inactivated cell culture without requiring high levels of antigen and an adjuvant.SOLUTION: Provided is an immunogenic composition containing a vector encoding one or more rabies virus antigens and an alphavirus RNA replicon particle encoding one or more antigens derived from rabies virus.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under 35 U.S.C.§119(e) to Provisional Application U.S.Serial No.62 / 581,955, filed on November 6, 2017 the entire content of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a novel vaccine for rabies virus. Methods of manufacturing the vaccine and using it alone or in combination with other protective agents are also provided.

Background Art

[0003] Rabies is a preventable zoonotic infection that leads to inflammation of the brain in humans and other mammals Clinical rabies is an acute progressive encephalitis and is typically classified as furious rabies or paralytic rabies Furious rabies is characterized by restlessness, irritability, and aggression. Paralytic rabies is characterized by excessive salivation, deep labored breathing, paralysis, and ultimately coma.

[0004] The causative agent of rabies is the rabies virus, which can infect most mammals and maintains disease reservoirs in wild and domestic animals, including highly susceptible livestock. Although different species act as major reservoirs of rabies virus in various geographical regions, including wild dogs, raccoons, skunks, foxes, bats, and mongooses [Robinson et al., Semin Vet Med Surg (small Anim)6:203 - 2 11(1991)], the rabies virus is present in most regions of the world. The rabies virus Rabies is most commonly transmitted through bites from infected animals. Once the rabies virus infects the central nervous system, the clinical symptoms of rabies appear.

[0005] The rabies virus is an enveloped RNA virus and encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase [Dietzschold et al., Crit Rev Immunol. 10:427-439 (1991)]. The glycoprotein (G) is considered a protective antigen that induces virus-neutralizing antibodies [Cox et al., Infect Immun. 16:754-759 (1977)]. To combat this disease, several types of rabies vaccines have been produced. Whole virus inactivated rabies virus vaccines derived from inactivated cell cultures are the most commonly used vaccines in the United States. These whole virus killed rabies virus vaccines require high levels of antigen and thus an adjuvant. Unfortunately, this use of adjuvant is associated with injection site reactivity, hypersensitivity, and even the recognized risk of injection site sarcomas in cats. Recently, modified live vaccines have been successfully used with oral vaccine baits for the immunization of wildlife [Mahl et al., Vet Res. 45(1):77 2014)]. In addition, recombinant vaccines expressing the glycoprotein (G) are currently commercially available in the United States for use in cats. Nucleic acid vaccines are also used in laboratory research but are not currently approved in the United States. Numerous vector strategies have been used over the years to protect against specific animal pathogens.

[0006] For many years, a number of vector strategies have been used to protect against specific animal ​have been used in. One such vector strategy involves the use of alphavirus-derived replicon RNA particles (RPs) [Vander Veen et al., Anim Health Res Rev. 13(1):1-9, (2012) doi:10.1017 / S 1466252312000011; Kamrud et al., J Gen Virol. 91 (Pt7):1723-1727, (2010)], which are derived from several different alphaviruses including Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virology. 239:389-401 (1997 )], Sindbis (SIN) [Bredenbeek et al., Journal of Vir ology 67:6439-644, (1993)], and Semliki Forest virus (SFV) [Liljestrom and Garoff, Biotechnology( NY). 9:1356-1361, (1991)]. RP vaccines deliver a replication-defective alphavirus RNA replicon to host cells, resulting in the expression of the desired antigen transgene in vivo [Pushko et al., Virology 239(2):389-401 (1997)]. RPs have an attractive safety and efficacy profile when compared to some traditional 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 forms the basis for several US-approved vaccines for pigs and poultry.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0008] Despite the widespread availability of whole - virus killed rabies vaccines and the introduction of new vaccines, rabies continues to pose a threat to both livestock and humans. Therefore, new rabies vaccines that can help protect mammals, including cats, dogs, horses, ferrets, sheep, and cows, from this debilitating disease have been needed for many years. from this debilitating disease new rabies vaccines that can help protect mammals, including cats, dogs, horses, ferrets, sheep, and cows, from this debilitating disease have been needed for many years. from this debilitating disease

[0009] The citation of reference matters in this specification should not be construed as an admission that such reference matters are available as "prior art" for this application. for this application

[0010] Summary of the Invention Accordingly, the present invention provides a vector encoding one or more rabies virus antigens. Such vectors can be used in immunogenic compositions containing these vectors. The immunogenic compositions of the present invention can be used as vaccines. In one aspect of the present invention, the vaccine protects the vaccinated subject (e.g., a mammal) from rabies virus. In one embodiment of this type, the vaccinated subject is a dog. In another embodiment, the vaccinated subject is a cat. In a more specific embodiment of this type, the vaccinated subject is a pet cat. In yet another embodiment, the vaccinated subject is a pet cat. In yet another embodiment, the vaccinated subject is a pet cat. In yet another embodiment, , the mammal is equine (e.g., horse). The present invention further provides a combination vaccine for inducing protective immunity against rabies and other diseases, such as infectious diseases of other dogs, horses and / or cats. The present invention also provides methods for manufacturing and using the immunogenic compositions and vaccines. In certain embodiments, the vector is an alphavirus RNA replicon particle encoding one or more antigens derived from rabies virus. In further specific embodiments , the alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle. In more specific embodiments

[0011] , the VEE alphavirus RNA replicon particle is a TC-83 VEE alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Sindbis (SIN) alphavirus RNA replicon particle. In still other embodiments , the alphavirus RNA replicon particle is a Semliki Forest virus (SFV) alphavirus RNA replicon particle. In another embodiment, the naked DNA vector contains a nucleic acid construct encoding the rabies virus glycoprotein (G) antigen. The present invention includes all of the nucleic acid constructs of the present invention, including RNA plasmids, RNA replicons, as well as all of the alphavirus RNA replicon particles of the present invention, naked DNA vectors, and nucleic acid constructs (e.g., RNA plasmids, RNA replicons) of the alphavirus RNA replicon particles and / or naked DNA vectors of the present invention, and immunogenic compositions and / or vaccines containing the same. In certain embodiments, the vector is an alphavirus RNA replicon particle encoding one or more antigens derived from rabies virus. In further specific embodiments , the alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle. In more specific embodiments , the VEE alphavirus RNA replicon particle is a TC-83 VEE alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Sindbis (SIN) alphavirus RNA replicon particle. In still other embodiments , the alphavirus RNA replicon particle is a Semliki Forest virus (SFV) alphavirus RNA replicon particle. In another embodiment, the naked DNA vector contains a nucleic acid construct encoding the rabies virus glycoprotein (G) antigen. The present invention includes all of the nucleic acid constructs of the present invention, including RNA plasmids, RNA replicons, as well as all of the alphavirus RNA replicon particles of the present invention, naked DNA vectors, and nucleic acid constructs (e.g., RNA plasmids, RNA replicons) of the alphavirus RNA replicon particles and / or naked DNA vectors of the present invention, and immunogenic compositions and / or vaccines containing the same. The present invention includes all of the nucleic acid constructs of the present invention, including RNA plasmids, RNA replicons, as well as all of the alphavirus RNA replicon particles of the present invention, naked DNA vectors, and nucleic acid constructs (e.g., RNA plasmids, RNA replicons) of the alphavirus RNA replicon particles and / or naked DNA vectors of the present invention, and immunogenic compositions and / or vaccines containing the same. The present invention includes immunogenic compositions and / or vaccines containing nucleic acid constructs (e.g., RNA plasmids, RNA replicons) of the alphavirus RNA replicon particles and / or naked DNA vectors of the present invention. In certain embodiments, the vector is an alphavirus RNA replicon particle encoding one or more antigens derived from rabies virus. In further specific embodiments , the alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle. In more specific embodiments

[0012] In one aspect, the alphavirus RNA replicon particle encodes one rabies virus G antigen. In related aspects, the alphavirus RNA replicon particle encodes one or more rabies virus G antigens or antigenic fragments thereof. In yet other aspects, the alphavirus RNA replicon particle encodes 2 to 4 rabies virus G antigens or antigenic fragments thereof. In a specific embodiment, the alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEE) alphavirus RN A replicon particle.

[0013] The present invention also provides an immunogenic composition comprising an alphavirus RNA repl icon particle encoding one rabies virus G antigen. In related aspects, the immunogenic composition comprises an alphavirus RNA replicon particle encoding one or more rabies virus G antigens or antigenic fragments thereof. In a specific aspect of this type, the immunogenic composition comprises an alphavirus RNA replicon particle encoding 2 to 4 rabies virus G antigens or antigenic fragments thereof. In a more specific aspect, the immunogenic composition comprises an alphavirus RNA replicon particle that is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle. In yet other aspects, the immunogenic composition comprises two or more sets of alphavirus RNA replicon particles. In a specific embodiment of this type, the first set of alphavirus RNA repl icon particles encodes a rabies virus G antigen or antigenic fragment thereof, and the second set

[0014] of alphavirus RNA replicon particles encodes a feline calicivirus (FCV) antigen or In a specific embodiment of this type, the first set of alphavirus RNA replicon particles encodes a rabies virus G antigen or antigenic fragment thereof, and the second set of alphavirus RNA replicon particles encodes a rabies virus G antigen or antigenic fragment thereof, and the second set of alphavirus RNA replicon particles encodes a feline calicivirus (FCV) antigen or It encodes the antigenic fragment. In certain embodiments of this type, the FCV antigen is derived from a virulent systemic feline calicivirus. In other embodiments, the FCV antigen is derived from a classical (F9-like) feline calicivirus. In yet other embodiments, the second set of alphavirus RNA replicon particles encodes two FCV antigens, one of which is derived from a virulent systemic feline calicivirus and the other is derived from a classical (F9-like) feline calicivirus. Accordingly, in one aspect, the nucleic acid construct of the invention encodes one or more rabies virus G antigens or antigenic fragments thereof. In certain embodiments of this type, the nucleic acid construct encodes 2 to 4 rabies virus G antigens or antigenic fragments thereof. In related

[0015] embodiments, the alphavirus RNA replicon particles contain a nucleic acid construct encoding one or more rabies virus G antigens or antigenic fragments thereof. In yet other embodiments, the alphavirus RNA replicon particles contain a nucleic acid construct encoding 2 to 4 rabies virus G antigens or antigenic fragments thereof. In certain embodiments, the immunogenic composition contains alphavirus RNA replicon particles containing a nucleic acid construct encoding one or more rabies virus G antigens or antigenic fragments thereof. In related embodiments, the immunogenic composition contains alphavirus RNA replicon particles encoding 2 to 4 rabies virus G

[0016] antigens or antigenic fragments thereof. In certain embodiments of this type, the alphavirus RNA replicon particles are rabies virus G antigen or antigenic fragments thereof encoding nucleic acid constructs. In related embodiments, the immunogenic composition contains alphavirus RNA replicon particles encoding 2 to 4 rabies virus G antigens or antigenic fragments thereof. In certain embodiments of this type, the alphavirus RNA replicon particles are rabies virus G antigen or antigenic fragments thereof encoding nucleic acid constructs. In related embodiments, the immunogenic composition contains alphavirus RNA replicon particles encoding 2 to 4 rabies virus G antigens or antigenic fragments thereof. In certain embodiments of this type, the alphavirus RNA replicon particles are rabies virus G antigen or antigenic fragments thereof encoding nucleic acid constructs. In related embodiments, the immunogenic composition contains alphavirus RNA replicon particles encoding 2 to 4 rabies virus G antigens or antigenic fragments thereof. In certain embodiments of this type, the alphavirus RNA replicon particles are rabies virus G antigen or antigenic fragments thereof encoding nucleic acid constructs. In related embodiments, the immunogenic composition contains alphavirus RNA replicon particles encoding 2 to 4 rabies virus G antigens or antigenic fragments thereof. In certain embodiments of this type, the alphavirus RNA replicon particles are rabies It encodes a pathogenic virus G or an antigenic fragment thereof. In a more specific embodiment, the immune genic composition comprises Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles and certain alphavirus RNA replicon particles. In other embodiments, the immunogenic composition comprises two or more sets of alphavirus RNA replicon particles. In a specific implementation of this type, the first set of alphavirus RNA replicon particles contains a first nucleic acid construct , while the other set of alphavirus RNA replicon particles contains a second nucleic acid construct .

[0017] In yet another embodiment, the immunogenic composition comprises a first set of al phavirus RNA replicon particles containing a first nucleic acid construct, another set of alphavirus RNA replicon particles containing a second nucleic acid construct, and a third set of alphavirus RN A replicon particles containing a third nucleic acid construct. In a specific embodiment of this type, the first nucleic acid construct encodes a rabies virus G antigen or an antigenic fragment thereof, the second nucleic acid construct encodes a feline calicivirus (FCV) antigen derived from a virulent systematic feline calicivirus or an antigenic fragment thereof, and the third nucleic acid construct encodes a feline calicivirus (FCV) antigen derived from a classical (F9-like) feline calicivirus or an antigenic fragment thereof. In a specific embodiment, the feline calicivirus (FCV) antigen is an FCV capsid protein.

[0018] In yet another embodiment, the immunogenic composition comprises a first set of al phavirus RNA replicon particles containing a first nucleic acid construct, another set of alphavirus An alphavirus RNA replicon particle, a third set of alphavirus RNA replicon particles comprising a third nucleic acid construct, a fourth set of alphavirus RNA replicon particles comprising a fourth nucleic acid construct, and a fifth set of alphavirus RNA replicon particles comprising a fifth nucleic acid construct. In such embodiments, the nucleotide sequences of the first nucleic acid construct, the second nucleic acid construct, the third nucleic acid construct, the fourth nucleic acid construct and the fifth nucleic acid construct are all different.

[0019] Accordingly, the immunogenic composition of the present invention can contain alphavirus RNA replicon particles comprising a nucleic acid construct encoding at least one non-rabies virus antigen for inducing protective immunity against non-rabies virus pathogens. In a specific example of this type, the non-rabies virus antigen is a protein antigen derived from feline herpesvirus (FHV). In other embodiments, the non-rabies virus antigen is a protein antigen derived from feline calicivirus (FCV). In still other embodiments, the non-rabies virus antigen is a protein antigen derived from feline pneumovirus (FPN). In still other embodiments, the non-rabies virus antigen is a protein antigen derived from feline panleukopenia virus (FPV). In still other embodiments, the immunogenic composition comprises a first set of alphavirus RNA replicon particles comprising a first nucleic acid construct, another set of alphavirus RNA replicon particles comprising a second nucleic acid construct, a third set of alphavirus RNA replicon particles comprising a third nucleic acid construct, and a fourth set of alphavirus RNA replicon particles comprising a fourth nucleic acid construct. In still other embodiments, the non-rabies virus antigen is a protein antigen derived from feline calicivirus (FCV). In still other embodiments, the non-rabies virus antigen is a protein antigen derived from feline pneumovirus (FPN). In still other embodiments, the non-rabies virus antigen is a protein antigen derived from feline pneumovirus (FPN). In still other embodiments, the non-rabies virus antigen is a protein antigen derived from feline panleukopenia virus (FPV). In still other embodiments, the non-rabies virus antigen is a protein antigen derived from feline panleukopenia virus (FPV). In still other embodiments, the non-rabies virus antigen is a protein antigen derived from feline panleukopenia virus (FPV).

[0020] In still other embodiments, the immunogenic composition comprises a first set of alphavirus RNA replicon particles comprising a first nucleic acid construct, another set of alphavirus RNA replicon particles comprising a second nucleic acid construct, a third set of alphavirus RNA replicon particles comprising a third nucleic acid construct, and a fourth set of alphavirus RNA replicon particles comprising a fourth nucleic acid construct. Another set of alphavirus RNA replicon particles comprising a second nucleic acid construct, a third set of alphavirus RNA replicon particles comprising a third nucleic acid construct, and a fourth set of alphavirus RNA replicon particles comprising a fourth nucleic acid construct. ​​It contains particles. In certain embodiments of this type, the first nucleic acid construct encodes rabies virus G antigen or an antigenic fragment thereof, and the second nucleic acid construct encodes a virulent strain feline calicivirus or a feline calicivirus ( FCV) antigen derived from an antigenic fragment thereof, and the third nucleic acid construct encodes a classical (F9-like) feline calicivirus or a feline calicivirus (FCV) antigen derived from an antigenic fragment thereof , and the fourth nucleic acid construct encodes a FeLV antigen or an antigenic fragment thereof .

[0021] In yet other embodiments, the immunogenic composition comprises a first set of alphavirus RNA replicons containing the first nucleic acid construct, another set of alphavirus RNA replicons containing the second nucleic acid construct, a third set of alphavirus RNA repl icons containing the third nucleic acid construct, a fourth set of alphavirus RNA replicons containing the fourth nucleic acid construct, and a fifth set of alphavirus RNA replicons containing the fifth nucleic acid construct. In such embodiments, the nucleotide sequences of the first nucleic acid construct, the second nucleic acid construct, the third nucleic acid construct, the fourth nucleic acid construct and the fifth nucleic acid construct are all different.

[0022] Thus, the immunogenic composition of the present invention can contain alphavirus RNA replicons containing a nucleic acid construct encoding at least one non-rabies virus antigen for inducing protective immunity against non-rabies virus pathogens. In certain embodiments of this type, the non-rabies virus antigen is a protein antigen derived from feline herpesvirus (FHV). In other embodiments, the non-rabies virus antigen is feline calicivirus (F ​ a protein antigen derived from CV). Further in other embodiments, the non-rabies virus antigen is a protein antigen derived from feline pneumovirus (FPN). Further in other embodiments, the non-rabies virus antigen is derived from feline panleukopenia virus (FPV) and is a protein antigen.

[0023] The present invention further provides a combined immunogenic composition and / or vaccine comprising an alphavirus RNA replicon particle encoding an antigen derived from rabies virus or an antigenic fragment thereof, together with one or more modified live (e.g., attenuated) or inactivated mammalian pathogens.

[0024] In certain embodiments of the present invention, the rabies virus antigen is rabies virus G. In certain embodiments of this type, rabies virus G comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 2. In more specific embodiments of this type, rabies virus G comprises the amino acid sequence of SEQ ID NO: 2. In even more specific embodiments of this type, rabies virus G is encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4.

[0025] The present invention further includes vaccines and multivalent vaccines comprising the immunogenic composition of the present invention. In certain embodiments, the vaccine is a non-adjuvanted vaccine. The vaccines of the present invention can assist in the prevention of diseases associated with rabies virus. In certain embodiments, antibodies are induced in a mammalian subject when the mammalian subject is immunized with the vaccine. In certain embodiments, the mammalian subject is a dog. In other embodiments, the mammalian subject is ​​​​It is a cat. In yet other embodiments, the mammal is a horse. In yet other aspects the mammal is a mustelid. In certain embodiments of this type, the mustelid is a ferret. In yet other embodiments, the mammal is a bovine. In certain embodiments of this type, the bovine is a cow. In other embodiments of this type, the bovine is a sheep.

[0026] The present invention also provides a method of immunizing a mammal against rabies virus, which comprises administering to the mammal an immunologically effective amount of the vaccine of the present invention. In certain embodiments the vaccine is administered by intramuscular injection. In another embodiment, the vaccine is administered by subcutaneous injection. In other embodiments, the vaccine is administered by intravenous injection. In yet other embodiments, the vaccine is administered by intradermal injection. In yet other embodiments, the vaccine is administered orally. In yet other embodiments, the vaccine is administered intranasally. In certain embodiments, the mammal is a cat. In other

[0027] The vaccines of the present invention (including multivalent vaccines) can be administered as a primer vaccine and / or a booster vaccine. In certain embodiments, the vaccine of the present invention is administered as a one-shot vaccine (a single administration) without the need for subsequent administrations. In certain embodiments, when both a primer vaccine and a booster vaccine are administered, In other embodiments of this type, both the primer vaccine and the booster vaccine are administered by subcutaneous injection. In alternative embodiments, the administration of the primer vaccine can be by one route and the booster vaccine by another route. In certain embodiments of this type, the primer vaccine can be administered by subcutaneous injection and the booster vaccine can be administered orally.

[0028] The present invention further provides a method of immunizing a mammal against rabies virus, comprising injecting an immunologically effective amount of the vaccine of the present invention into the mammal. In certain embodiments, the vaccine can comprise from about 1×10 to about 1×10 5 or more RP. In more specific embodiments, the vaccine can comprise from about 1×10 10 to about 1×10 of RP. In even more specific embodiments, the vaccine can comprise from about 1×10 6 to about 1×10 9 of RP. In certain embodiments, the vaccine of the present invention is administered in a dose of from 0.03 mL to 5 mL. 7 In specific embodiments, the vaccine of the present invention is administered in a dose of from 0.05 mL to 3 mL. 8 In more specific aspects, the dose administered is from 0.1 mL to 2 mL. In even

[0029] more specific aspects, the dose administered is from 0.2 mL to 1.5 mL. In yet more specific aspects, the dose administered is from 0.3 to 1.0 mL. In even more specific aspects, the dose administered is from 0.4 mL to 0.8 mL. In yet more specific aspects, the dose administered is from 0.4 mL to 0.8 mL. In yet more specific embodiments, the dose administered is from 0.3 to 1.0 mL. In yet more specific embodiments, the dose administered is from 0.4 mL to 0.8 mL.

[0030] These and other aspects of the invention will be better understood by reference to the following detailed description. will be understood.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides a vaccine composition comprising an antigen derived from one or more strains of rabies virus in an immunologically effective amount, which aids in inducing protective immunity in vaccinated recipient animals. In one aspect of the invention, the vaccine comprises the capsid protein and glycoprotein of Venezuelan equine encephalitis virus (VEE), and an alphavirus RNA replicon particle (RP) encoding rabies glycoprotein (G) or an antigenic fragment thereof. In an even more specific embodiment, the vaccine comprises the capsid protein and glycoprotein of the non-pathogenic TC-83 strain of VEE, and an alphavirus RNA replicon particle (RP) encoding rabies glycoprotein (G) or an antigenic fragment thereof. In another aspect of the invention, the vaccine comprises a naked DNA vector encoding rabies glycoprotein (G). A vaccine comprising an alphavirus RNA replicon particle encoding rabies glycoprotein (G) can be administered in the absence of an adjuvant and is still effective in inducing protective immunity in mammals vaccinated against rabies virus. will be understood. In one aspect of the invention, the vaccine comprises the capsid protein and glycoprotein of Venezuelan equine encephalitis virus (VEE), and an alphavirus RNA replicon particle (RP) encoding rabies glycoprotein (G) or an antigenic fragment thereof. In one aspect of the invention, the vaccine comprises the capsid protein and glycoprotein of Venezuelan equine encephalitis virus (VEE), and an alphavirus RNA replicon particle (RP) encoding rabies glycoprotein (G) or an antigenic fragment thereof. In an even more specific embodiment, the vaccine comprises the capsid protein and glycoprotein of the non-pathogenic TC-83 strain of VEE, and an alphavirus RNA replicon particle (RP) encoding rabies glycoprotein (G) or an antigenic fragment thereof. In an even more specific embodiment, the vaccine comprises the capsid protein and glycoprotein of the non-pathogenic TC-83 strain of VEE, and an alphavirus RNA replicon particle (RP) encoding rabies glycoprotein (G) or an antigenic fragment thereof. In an even more specific embodiment, the vaccine comprises the capsid protein and glycoprotein of the non-pathogenic TC-83 strain of VEE, and an alphavirus RNA replicon particle (RP) encoding rabies glycoprotein (G) or an antigenic fragment thereof. In an even more specific embodiment, the vaccine comprises the capsid protein and glycoprotein of the non-pathogenic TC-83 strain of VEE, and an alphavirus RNA replicon particle (RP) encoding rabies glycoprotein (G) or an antigenic fragment thereof. In another aspect of the invention, the vaccine comprises a naked DNA vector encoding rabies glycoprotein (G). In another aspect of the invention, the vaccine comprises a naked DNA vector encoding rabies glycoprotein (G). A vaccine comprising an alphavirus RNA replicon particle encoding rabies glycoprotein (G) can be administered in the absence of an adjuvant and is still effective in inducing protective immunity in mammals vaccinated against rabies virus. A vaccine comprising an alphavirus RNA replicon particle encoding rabies glycoprotein (G) can be administered in the absence of an adjuvant and is still effective in inducing protective immunity in mammals vaccinated against rabies virus. is useful.

[0032] Accordingly, one aspect of the invention provides an improved, safe, non-adjuvanted rabies virus vaccine. In a related aspect, the vaccine of the invention does not induce injection site sarcomas and yet still protects against the debilitating pathologies caused by rabies virus infection. Accordingly, one aspect of the invention provides an improved, safe, non-adjuvanted rabies virus vaccine. In a related aspect, the vaccine of the invention does not induce injection site sarcomas and yet still protects against the debilitating pathologies caused by rabies virus infection. Accordingly, one aspect of the invention provides an improved, safe, non-adjuvanted rabies virus vaccine. In a related aspect, the vaccine of the invention does not induce injection site sarcomas and yet still protects against the debilitating pathologies caused by rabies virus infection. Provides protection to inoculated mammals, which has efficacy equal to or higher than that of the corresponding adjuvant vaccine. It has strength.

[0033] To more fully understand the present invention, the following definitions are provided.

[0034] The use of singular terms for convenience of explanation is never intended to be so limiting. Thus, for example, a reference to a composition containing "polypeptide" includes a reference to one or more of such polypeptides. Further, a reference to "alpha virus RNA A replicon particle" includes, unless otherwise indicated, a reference to a plurality of such alpha virus R NA replicon particles.

[0035] As used herein, the term "about" is used interchangeably with the term "approximately" and indicates that the value is within 50 percent of the indicated value, i.e., a composition containing "about" 1x10 8 al pha virus RNA replicon particles per milliliter contains from 5x10 to 1.5x10 7~ alpha virus RNA replicon particles per milliliter. 8

[0036] As used herein, the term "cat" refers to any member of the Felidae family. Domestic cats, purebred and / or hybrid cats, as well as wild or feral cats are all cats.

[0037] As used herein, the term "dog" includes, unless otherwise indicated, all domestic dogs, Canis lupus family or Canidae family.

[0038] As used herein, "ferret" is a mammal belonging to the family Mustelidae and is one of the mammals

[0039] As used herein, "bovine family" refers to a mammalian family of ruminant mammals of the even-toed ungulates, including chamois, sheep (ovis), goats, musk oxen, and cattle, such as bison, African buffalo, buffalo, and cattle (cot tle).

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

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

[0042] The term "non-rabies virus" modifies terms such as pathogen and / or antigen (or immunogen) ​​​​Used for decoration, each pathogen and / or antigen (or immunogen) is neither a rabies virus nor a rabies virus antigen (or immunogen), and the non-rabies virus protein antigen (or immunogen) means not derived from the rabies virus. The terms "originate from", "originates from" and "originating from" are used interchangeably with respect to a given protein antigen and the pathogen or

[0043] its strain that naturally encodes it, and when used herein, the unmodified and / or cleaved amino acid sequence of the given protein antigen means being encoded by that pathogen or strain of that pathogen. The coding sequence in the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may be genetically engineered to result in modification and / or cleavage of the amino acid sequence of the expressed protein antigen as compared to the corresponding sequence of the protein antigen in the pathogen or strain of that pathogen (including naturally attenuated strains). The terms "protection" or "provide protection" or "induce protective immunity" or "assist in the prevention of disease" and "assist in protection" as used herein do not require complete protection from any signs of infection. For example, "assist in protection" may mean that after challenge, the signs 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 symptoms are reduced and / or removed such that the

[0044] protection is sufficient. As used herein, "reduced" refers only to the physiological state of the infection and / or one or more of the underlying causes or mechanisms that cause symptoms are reduced and / or removed such that the protection is sufficient. As used herein, "reduced" refers only to the physiological state of the infection and / or one or more of the underlying causes or mechanisms that cause symptoms are reduced and / or removed such that the protection is sufficient. As used herein, "reduced" refers only to the physiological state of the infection and / or one or more of the underlying causes or mechanisms that cause symptoms are reduced and / or removed such that the Rather, it is understood to mean something related to the symptoms of infection, including the molecular state of the infection.

[0045] As used herein, "vaccine" refers to one or more antigens typically combined with a pharmaceutically acceptable carrier, such as a liquid containing water, suitable for administration to an animal, e.g., a dog (in certain embodiments includes humans, but in other embodiments does not specifically include humans), which, when administered to the animal, induces an immune response of sufficient strength to minimally assist in protecting against diseases resulting from infection with wild-type microorganisms, i.e., to assist in preventing the disease and / or to prevent, ameliorate or treat the disease. When used herein, a multivalent vaccine is a vaccine containing two or more different antigens. In certain embodiments of this type, the multivalent vaccine stimulates the recipient's immune system against two or more different pathogens. The terms "adjuvant" and "immunostimulant" are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance the immune response to one or more vaccine antigens / isolate. Thus, an "adjuvant" is an agent that nonspecifically increases the immune response to a particular 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 antigen of interest. In this context, an adjuvant is used to enhance the immune response to one or more vaccine antigens / isolate.

[0046]

[0047]

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

[0049] As used herein, the term "pharmaceutically acceptable" is used adjectivally to mean that the modified noun is suitable for use in a pharmaceutical product. For example, when used to describe an additive for a pharmaceutical vaccine, it characterizes that the excipient is compatible with the other components of the composition and is not detrimentally harmful to the intended recipient animal, e.g., a dog.

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

[0051] As used herein, the term "antigenic fragment" with respect to a particular protein (e.g., a protein antigen) refers to a fragment of that protein that is antigenic, i.e., capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or a T cell antigen receptor. Preferably, the antigenic fragments of the present invention are immunodominant for antibody and / or T cell receptor recognition. In certain embodiments, an antigenic fragment with respect to a given protein antigen is a fragment of that protein that retains at least 25% of the antigenicity of the full-length protein. In preferred embodiments, the antigenic fragment retains at least 50% of the antigenicity of the full-length protein. In more preferred embodiments, the antigenic fragment retains at least 75% of the antigenicity of the full-length protein. The antigenic fragment can be as small as a fragment of about 20 amino acids or , or conversely, a large fragment lacking only one amino acid from the full-length protein may also be present. In certain embodiments, the antigenic fragment comprises 25-150 amino acids residues. In other embodiments, the antigenic fragment comprises 50-250 amino acid residues .

[0052] As used herein, when the amino acid residues of two sequences are identical, one amino acid sequence is 100% "identical" to the second amino acid sequence, or has 100% " identity". Thus, when 50% of the amino acid residues of two amino acid sequences are identical, the amino acid sequence is 50% "identical" to the second amino acid sequence. Sequence comparison is performed over a continuous block of amino acid residues that make up a given protein, e.g., a protein or a portion of the polypeptide being compared. In certain embodiments, selected deletions or insertions that can otherwise alter the correspondence between two amino acid sequences are considered .

[0053] As used herein, nucleotide and amino acid sequence percent identity can be determined using C, MacVector (MacVector, Inc. Cary, NC 27519), Ve ctor NTI (Informax, Inc. MD), Oxford Molecul ar Group PLC (1996) and the Clustal W algorithm, with alignment default parameters and default parameters for identity. Using these commercially available programs, sequence similarity can also be determined using the same or similar default para meters. Alternatively, with default filter criteria Under the conditions, for example, using default parameters, the advanced blast search can be used with the pile-up program of GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin). Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin In the context of the present invention, an "inactivated" microorganism is a microorganism that can induce an immune response in an animal but cannot infect the animal. For example, inactivated rabies virus can be inactivated by a substance selected from the group consisting of binary ethyleneimine, formalin, β-propiolactone, thimerosal or heat.

[0054] For the purposes of the present invention, an "inactivated" microorganism is a microorganism that can induce an immune response in an animal but cannot infect the animal. For example, inactivated rabies virus can be inactivated by a substance selected from the group consisting of binary ethyleneimine, formalin, β-propiolactone, thimerosal or heat. virus can be inactivated by a substance selected from the group consisting of binary ethyleneimine, formalin, β-propiolactone, thimerosal or heat. virus can be inactivated by a substance selected from the group consisting of binary ethyleneimine, formalin, β-propiolactone, thimerosal or heat. virus can be inactivated by a substance selected from the group consisting of binary ethyleneimine, formalin, β-propiolactone, thimerosal or heat.

[0055] The alpha virus RNA replicon particles of the present invention can be lyophilized and rehydrated with a sterile water diluent. On the other hand, if the alpha virus RNA replicon particles are stored separately but are intended to be mixed with other vaccine components before administration, the alpha virus RNA replicon particles can be stored in a stabilizing solution of their composition, such as a high sucrose solution. The alpha virus RNA replicon particles of the present invention can be lyophilized and rehydrated with a sterile water diluent. On the other hand, if the alpha virus RNA replicon particles are stored separately but are intended to be mixed with other vaccine components before administration, the alpha virus RNA replicon particles can be stored in a stabilizing solution of their composition, such as a high sucrose solution. The alpha virus RNA replicon particles of the present invention can be lyophilized and rehydrated with a sterile water diluent. On the other hand, if the alpha virus RNA replicon particles are stored separately but are intended to be mixed with other vaccine components before administration, the alpha virus RNA replicon particles can be stored in a stabilizing solution of their composition, such as a high sucrose solution. The alpha virus RNA replicon particles of the present invention can be lyophilized and rehydrated with a sterile water diluent. On the other hand, if the alpha virus RNA replicon particles are stored separately but are intended to be mixed with other vaccine components before administration, the alpha virus RNA replicon particles can be stored in a stabilizing solution of their composition, such as a high sucrose solution. The alpha virus RNA replicon particles of the present invention can be lyophilized and rehydrated with a sterile water diluent. On the other hand, if the alpha virus RNA replicon particles are stored separately but are intended to be mixed with other vaccine components before administration, the alpha virus RNA replicon particles can be stored in a stabilizing solution of their composition, such as a high sucrose solution.

[0056] The vaccine of the present invention can be easily administered by any standard route including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal, and / or intraperitoneal vaccination. Those skilled in the art will understand that the vaccine composition is preferably appropriately formulated for each type of recipient animal and route of administration. Accordingly, the present invention also provides a method of immunizing a mammal against rabies and / or other mammalian pathogens. One such method is to administer to the mammal The vaccine of the present invention can be easily administered by any standard route including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal, and / or intraperitoneal vaccination. Those skilled in the art will understand that the vaccine composition is preferably appropriately formulated for each type of recipient animal and route of administration. Accordingly, the present invention also provides a method of immunizing a mammal against rabies and / or other mammalian pathogens. One such method is to administer to the mammal The vaccine of the present invention can be easily administered by any standard route including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal, and / or intraperitoneal vaccination. Those skilled in the art will understand that the vaccine composition is preferably appropriately formulated for each type of recipient animal and route of administration. Accordingly, the present invention also provides a method of immunizing a mammal against rabies and / or other mammalian pathogens. One such method is to administer to the mammal The vaccine of the present invention can be easily administered by any standard route including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal, and / or intraperitoneal vaccination. Those skilled in the art will understand that the vaccine composition is preferably appropriately formulated for each type of recipient animal and route of administration. Accordingly, the present invention also provides a method of immunizing a mammal against rabies and / or other mammalian pathogens. One such method is to administer to the mammal The vaccine of the present invention can be easily administered by any standard route including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal, and / or intraperitoneal vaccination. Those skilled in the art will understand that the vaccine composition is preferably appropriately formulated for each type of recipient animal and route of administration. Accordingly, the present invention also provides a method of immunizing a mammal against rabies and / or other mammalian pathogens. One such method is to administer to the mammal immunologically effective amounts to cause the subject to produce a suitable rabies virus glycoprotein (G) antibody comprising injecting a mammal with the vaccine of the invention

[0057] multivalent vaccine The invention also provides a multivalent vaccine. Any antigen useful in mammalian vaccines or a combination of such antigens can be added to the vaccine to a replication-defective alphavirus RNA replicon particle (RP) encoding a mammalian antigen of rabies virus [e.g., rabies glycoprotein (G)]. Thus, such multivalent vaccines are included in the invention

[0058] [Table 1]

[0059] sequence The rabies glycoprotein (G) gene was codon-optimized for humans. The resulting gene has only about 85% nucleotide identity to the wild-type rabies virus glycoprotein (G) sequence, despite having 100% amino acid identity

[0060] TIFF0007714009000002.tif83165

[0061] TIFF0007714009000003.tif48166

[0062] TIFF0007714009000004.tif86168 [Examples]

[0063] The following examples are useful for providing a further understanding of the invention, but do not limit the effective scope of the invention

[0064] ​​​​​​ Example 1 Integration of the coding sequence of rabies virus glycoprotein into alphavirus RNA replicon particles Incorporation Introduction RNA viruses can be used as vectors for introducing genetically engineered vaccine antigens into the genome - vehicles. However, their use to date has been mainly limited to incorporating viral antigens into RNA viruses and then introducing the viruses into recipient hosts As a result, protective antibodies against the incorporated viral antigens are induced Alphavirus RNA replicon particles have been used to encode pathogenic antigens such alphavirus replicon platforms include Venezuelan equine encephalitis virus [Pushko et al., Virology, 239: 389-401 (1997 )], Sindbis (SIN) [Bredenbeek et al., Journal of Vir ology, 67: 6439-6446, (1993), the entire content of which is incorporated herein ), and Semliki Forest virus (SFV) [Liljestrom and G arovff, Biotechnology (NY), 9: 1356-1361 (1991 ), the entire content of which is incorporated herein], and have been developed from several different alphaviruses including In addition, alphavirus RNA replicon particles are the basis for several US Department of Agriculture-approved vaccines against pigs and poultry These include porcine epidemic diarrhea vaccine, RNA particles (product code 19U5.P1), porcine influenza vaccine, RNA (product code 19A5.D0), avian influenza vaccine, RNA (product code 19O 5.D0), and a formulated product, RNA particles (product code 9PP0.00) (product code 19O 5.D0), and a formulated product, RNA particles (product code 9PP0.00)

[0065] Construction of alphavirus RNA replicon Capsid protein and glycoprotein of the attenuated TC-83 strain of Venezuelan equine encephalitis virus packaging a rabies virus glycoprotein (G) derived from rabies virus. A vaccine containing alphavirus RNA replicon particles was prepared. The nucleotide sequence of the rabies virus G protein was codon-optimized for humans. Despite having 100% amino acid identity, the resulting sequence has only about 85% nucleotide identity to the sequence of the live rabies virus glycoprotein (G). The vaccine can be used as a single dose administered subcutaneously to mammalian subjects, e.g., cats and dogs 12 weeks of age or older, or alternatively, as a multiple dose regimen comprising one or more booster doses following a primary dose. Using the amino acid sequence of the rabies virus glycoprotein (G), a codon-optimized (human codon usage) nucleotide sequence was generated in silico. The optimized sequence was prepared as synthetic DNA by a commercial vendor (ATUM, Newark, California, USA). Thus, synthetic gene [SEQ ID NO:1] was designed based on the amino acid sequence of the rabies virus glycoprotein. The construct (RABV-G) was a human codon-optimized wild-type amino acid sequence [SEQ ID NO:2] with flanking sequences suitable for cloning into an alphavirus replicon plasmid. The VEE replicon vector designed to express rabies virus G has the following modifications to the original VEE

[0066] vector and is described below

[0067] The VEE replicon vector designed to express rabies virus G has the following modifications ​​​​Modify and construct as described above [see U.S. Patent No. 9,441,247 B2; the contents of which are incorporated herein by reference]. Digest the TC-83-derived replicon vector "p VEK" [disclosed and described in U.S. Patent No. 9,441,247 B2] with the restriction enzymes AscI and PacI. Digest a DNA plasmid containing the codon-optimized open reading frame nucleotide sequence of the rabies G gene with the 5' flanking sequence (5'-GGCGCGCCGC ACC-3') [SEQ ID NO: 3] and the 3' flanking sequence (5'-TTAATTAA-3 ') with the restriction enzymes AscI and PacI in the same manner. Then, ligate the synthetic gene cassette to the digested pVEK vector, and rename the resulting clone "pVHV-RABV-G". The "pVHV" vector nomenclature was selected to refer to a pVEK-derived replicon vector containing the transgene cassette cloned via the AscI and PacI sites in the multiple cloning site of p VEK.

[0068] The production of TC-83 RNA replicon particles (RPs) was performed according to the previously described methods [U.S. Patent Nos. 9,441,247 B2 and 8,460,913 B2; the contents of which are incorporated herein by reference]. Briefly, MegaScr ipt T7 RNA polymerase and a cap analog (Promega, Madison, Wisconsin (USA)) were used to linearize the pVHV replicon vector DNA and the helper DNA plasmid with the NotI restriction enzyme before in vitro transcription. Importantly, the helper RNA used for production was the VEE subgenomic promoter as described above Lacking an array [Kamrud et al., J Gen Virol. 91 (Pt 7): 17 23-1727 (2010)]. Purified RNAs for the replicon and helper components were combined with a suspension of Vero cells, mixed, and electroporated in a 4 mm cuvette and then returned to OptiPro® SFM cell culture medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA). After an overnight incubation the alphavirus RNA replicon particles were passed through a ZetaPlus BioCap® depth filter (3M, Maplewood, Minnesota, USA), washed with phosphate-buffered saline containing 5% sucrose (w / v), and finally eluted with 400 mM NaCl buffer to retain the RP, and purified from the cells and medium by eluting the RP. The eluted RP was finally formulated into 5% sucrose (w / v), passed through a 0.22 micron membrane filter, and aliquoted for storage. The titer of functional R P was measured by an immunofluorescence assay on infected Vero cell monolayers.

[0069] Example 2 Administration of a vaccine containing alphavirus RNA replicon particles encoding viral glycoprotein to dogs An initial study was conducted to evaluate the safety and serological response in dogs after RP-rabies virus G vaccination. The RP rabies virus G vaccine for the study was formulated in 5% sucrose and 1% canine serum as stabilizers, and the liquid vaccine was frozen for storage. Five groups of five dogs each were vaccinated as follows.

[0070]

Table 2

[0071] Dogs aged 12-13 weeks were vaccinated with 1.0 mL of each vaccine (see Table 1 above). Dogs in group 4 were given zoetis injections subcutaneously in the right scapular region as described. The currently licensed commercial rabies vaccine, DEFENSOR, is marketed by Dogs in group 5 received a non-related RP construct, canine non-rabies virus-infected After vaccination, dogs received the vaccinated The injection site was palpated 4 to 8 hours after administration and daily for 7 days, and clinical evaluation was performed. Adverse reactions to the vaccine were observed. No local or systemic side effects were observed for any of the vaccines. No serious adverse reactions were observed. Dogs were monitored daily for 14 days prior to vaccination and for the first 3 months of the study. Blood samples were taken for serum at monthly intervals. Rapid fluorescent focus inhibition test (RFFIT) confirmed Serum antibody titers against canine disease viruses were tested.

[0072] Antirabies serological results are shown in Table 2 below. Titers are expressed in International Units / mL (IU / mL). and 0.5 IU / mL is considered the protective titer.

[0073] [Table 3]

[0074] The trial was originally intended to end three months after vaccination, but Remarkably, rabies virus serum titers (i) remained at protective levels over this period; and (ii) was superior to currently licensed commercial rabies vaccines, The study was extended, retaining five dogs from Group 1, three from Group 2, and two from Group 4. The remaining dogs were bled for serum at approximately monthly intervals for one year of post-vaccination testing. It was done.

[0075] The anti-rabies virus serological results for selected dogs over the first year are shown in Table 3 below. Potency is expressed in international units / mL (IU / mL), with 0.5 IU / mL considered a protective titer:

[0076] [Table 4]

[0077] This first study was followed up with a second study (currently ongoing) that yielded similar results. At least six months have passed.

[0078] Example 3 A virus containing alphavirus RNA replicon particles encoding viral glycoproteins Administration of Cutin to Cats To evaluate the safety and serological response in cats after RP-rabies virus G vaccination. For this study, the RP rabies virus G vaccine was prepared in an experimental liquid form. Formulated in a stabilizer (see, e.g., U.S. Pat. No. 9,314,519 B2) and stored at 2-7°C. Stored refrigerated. Four groups of cats were vaccinated, as summarized in Table 4 below.

[0079] TIFF0007714009000008.tif77167

[0080] Cats aged 15-16 weeks were vaccinated with 1.0 mL of each vaccine (see Table 4 above). The animals were seeded and administered subcutaneously in the right scapular region. As shown, cats in Group 4 received Zoetis or received the currently approved commercially available rabies vaccine DEFENSOR (registered trademark )3, which contains chemically inactivated rabies virus together with aluminum hydroxide adjuvant.

[0081] After vaccination, cats were clinically evaluated and the injection sites were palpated 4 - 8 hours after vaccination and daily for 7 days after vaccination to observe the side effects of the vaccine. Cats were observed for 10 - 15 minutes immediately after vaccination for systemic reactions. Some immediate systemic reactions were observed in the cats of Group 1, Group 2, and Group 3, indicating that the cats experienced a stinging or pain reaction during injection. These reactions did not last more than 5 minutes. These injection reactions were due to the composition of the experimental liquid stable formulation. No local side effects were observed after vaccination. Cats had their sera collected at 1 - month intervals after vaccination on the day before vaccination and for the first 3 months of the test. The serum antibody titers against rabies virus were tested by rapid fluorescent focus inhibition test (RFFIT). The anti - rabies virus serological results are shown in Table 5 below. The titer is expressed in international units / mL (IU / mL), and a titer of 0.5 IU / mL is considered the protective titer.

[0082] TIFF0007714009000009.tif240167 TIFF0007714009000010.tif30166

[0083] The RP - rabies virus vaccine induces a high serum anti - rabies titer in cats with a single dose. In particular, the RFFIT titer in cats was higher than that observed in dogs vaccinated with the same dose of the vaccine. A titer of 0.5 IU / mL in the RFFIT test is considered the protective titer. ​​​​​​​​​​​​Cats having serological antibody titers below this level are often protected from virulent challenge in long-term immunological studies. All three groups vaccinated with various doses of the RP rabies virus vaccine have higher geometric mean anti-rabies virus RFFIT titers than the groups vaccinated with currently approved commercial rabies products. The immunization period is indicated to be 3 years.

[0084] This first study was followed by a second study (currently in progress) that obtained similar results, and at least six months have elapsed.

[0085] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the

[0086] appended claims. All base sizes or amino acid sizes and all molecular weights or molecular weight values given for nucleic acids or polypeptides are approximate and are provided for

Claims

1. A vaccine for assisting in the prevention of diseases caused by rabies virus, comprising Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles containing a nucleotide sequence encoding a rabies virus antigen or an antigenic fragment thereof, and a pharmaceutically acceptable carrier, wherein the nucleotide sequence encoding the rabies virus antigen comprises 85% sequence identity with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, the vaccine.

2. The vaccine according to claim 1, comprising one or more additional alphavirus RNA replicon particles encoding a second rabies virus antigen derived from a strain of rabies virus different from that from which the rabies virus antigen is derived.

3. The vaccine according to claim 2, wherein the second rabies virus antigen is a glycoprotein (G) or an antigenic fragment thereof.

4. The vaccine according to claim 2 or 3, wherein the one or more additional alphavirus RNA replicon particles are VEE alphavirus RNA replicon particles.

5. The vaccine according to claim 3, wherein the glycoprotein (G) or an antigenic fragment thereof comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

2.

6. The vaccine according to any one of claims 1 to 5, wherein antibodies are induced in a non-human mammal when the non-human mammal is immunized with the vaccine.

7. The vaccine according to claim 6, wherein the non-human mammal is selected from the group consisting of dogs, cats, horses, ferrets, sheep and cows.

8. The vaccine according to claim 1, further comprising at least one non-rabies virus antigen for inducing protective immunity against non-rabies virus pathogens.

9. The vaccine according to any one of claims 1 to 8, further comprising at least one recombinant vector and / or alphavirus RNA replicon particles containing a nucleotide sequence encoding at least one protein antigen or an antigenic fragment thereof derived from a non-rabies virus pathogen.

10. The vaccine according to any one of claims 1 to 9, which is a non-adjuvanted vaccine.

11. A method of immunizing a mammal against rabies virus, comprising administering an immunologically effective single dose of the vaccine according to any one of claims 1 to 10 to a non-human mammal.

12. The method according to claim 11, wherein the non-human mammal is selected from the group consisting of dogs, cats and horses.

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