Rabies virus vaccine

Abstract

To provide a vaccine for rabies virus.SOLUTION: Provided is an immunogenic composition comprising an alphavirus RNA replicon particle encoding a rabies virus antigen.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on November 6, 2017, under 35 U.S.C. § 119(e). This application claims priority to provisional application U.S. Serial No. 62 / 581,955 filed in , the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to a novel vaccine for rabies virus. Also provided are methods for using the agent alone or in combination with other protective agents. [Background technology]

[0003] Rabies is a preventable zoonotic disease that leads to brain inflammation in humans and other mammals. Clinical rabies is an acute, progressive encephalitis, typically fulminant rabies or paralytic rabies. It is classified as a canine disease. Violent rabies is characterized by restlessness, irritability and aggression. Paralytic rabies is characterized by excessive salivation, deep labored breathing, paralysis and eventually coma.

[0004] The causative agent of rabies is the rabies virus, which is infective in most mammals. It can transmit disease and maintain a reservoir of disease in wild and domestic animals and susceptible domestic animals. various geographical regions, including dogs, raccoons, skunks, foxes, bats, and mongooses Within the rabies virus, different species act as the primary reservoirs [Robinson et al., Semin Vet Med Surg (small Anim)6:203-2 11 (1991)], the rabies virus is present in most parts of the world. Rabies is most commonly transmitted by being bitten by an infected animal. Once the system is infected, clinical signs of rabies appear.

[0005] Rabies virus is an enveloped RNA virus that contains nucleoprotein (N), phosphate Protein (P), matrix protein (M), glycoprotein (G) and RNA-dependent It encodes the five structural proteins of the ubiquitous RNA polymerase [Dietzscho ld et al., Crit Rev Immunol. 10:427-439 (1991)]. Protein (G) is thought to be a protective antigen that induces virus-neutralizing antibodies [Cox et al., In fect Immun.16:754-759(1977)]. There are several rabies vaccines available: inactivated cell culture-derived whole virus inactivated rabies Viral vaccines are the most commonly used vaccines in the United States. Killed rabies virus vaccines require high levels of antigen, so adjuvants are used. Unfortunately, this use of adjuvants can lead to injection site reactivity, hypersensitivity, and and even associated with a recognized risk of injection site sarcoma in cats. has been used successfully with oral vaccine baits for immunization of wild animals [Ma hl et al., Vet Res. 45(1):77 2014)]. In addition, glycoproteins (G ) is currently commercially available 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. There is no.

[0006] A number of vector strategies have been developed over the years to protect against specific animal pathogens. One such vector strategy is the use of alphavirus-derived replicon vectors. This includes the use of RNA particles (RP) [Vander Veen et al., Animal Health h 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 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 Semiliquiforest virus SFV [Liljestrom and Garoff, Biotechnology ( NY).9:1356-1361, (1991)], including several different alphawilds. The RP vaccine was developed from a virus carrying a replication-deficient alphavirus RNA replicon. delivery to host cells, resulting in expression of the desired antigen transgene in vivo [Pushko et al., Virology 239(2):389-401(1997)]. RP has several Possesses an attractive safety and efficacy profile when compared to traditional vaccine formulations [Vander Veen et al., Anim Health Res Rev.13(1): 1-9, (2012)]. The RP platform is used to encode pathogenic antigens. It is the basis for several US-licensed vaccines for swine and poultry. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 9,441,247B2 [Patent Document 2] U.S. Patent No. 8,460,913B2 [Patent Document 3] U.S. Patent No. 9,314,519B2 Summary of the Invention [Problem to be solved by the invention]

[0008] Despite the widespread availability of whole-virus killed rabies vaccines and the introduction of new vaccines, However, rabies continues to pose a threat to both livestock and humans. New technology to help protect mammals, including cats, dogs, horses, ferrets, sheep and cattle There has been a long-standing need for a rabies vaccine.

[0009] The citation of a reference herein does not imply that the reference is available as "Prior Art" to the present application. This should not be construed as an admission that it is possible.

[0010] Summary of the Invention Thus, the present invention provides vectors encoding one or more rabies virus antigens. Such vectors can be used in immunogenic compositions containing these vectors. The immunogenic composition of the present invention can be used in a vaccine. Thus, the vaccine protects the vaccinated subject (e.g., a mammal) from the rabies virus. In one embodiment of this type, the vaccinated subject is a dog. In an embodiment, the vaccinated subject is a cat. In one embodiment, the vaccinated subject is a domestic cat. The mammal is an equine (e.g., a horse). In addition, protection against rabies and other diseases, e.g., other canine, equine and / or feline infections. The immunogenic compositions and vaccines of the present invention are also provided. Methods for making and using the compounds are also provided.

[0011] In certain embodiments, the vector encodes one or more antigens derived from rabies virus. In a more particular embodiment, the alphavirus RNA replicon particle is a Alphavirus RNA replicon particles are the vectors responsible for Venezuelan equine encephalitis (VEE) alphaviruses. In a more specific embodiment, the virus RNA replicon particle is a VEE alpha virus. The viral RNA replicon particles are TC-83VEE alphavirus RNA replicon particles. In another embodiment, the alphavirus RNA replicon particle is a Sindhivirus. In yet another embodiment, the alphavirus RNA replicon particle is a single stranded (SIN) alphavirus RNA replicon particle. Alphavirus RNA replicon particles are derived from Semliki Forest virus (SFV) In another embodiment, the vector is a naked DNA vector. The target comprises a nucleic acid construct encoding the rabies virus glycoprotein (G) antigen. The present invention also relates to all of the nucleic acid constructs of the present invention, including RNA plasmids and RNA replicons, as well as the All of the alphavirus RNA replicon particles, naked DNA vectors, and the present invention Alphavirus RNA replicon particles and / or naked DNA vector nucleic acid constructs (e.g., RNA plasmids, RNA replicons) and / or vaccines Including Chin.

[0012] In some embodiments, the alphavirus RNA replicon particle comprises one rabies virus In a related embodiment, the alphavirus RNA replicon particle encodes the G antigen. , encoding one or more rabies virus G antigens or antigenic fragments thereof. In this embodiment, the alphavirus RNA replicon particle comprises 2 to 4 rabies viruses. In a specific embodiment, the alpha G antigen or an antigenic fragment thereof is encoded by the alpha G antigen. The viral RNA replicon particles are Venezuelan equine encephalitis (VEE) alphavirus RNA A replicon particle.

[0013] The present invention also provides an alphavirus RNA replicative vector encoding a single rabies virus G antigen. In a related embodiment, the immunogenic composition comprises a recombinant protein particle. alphavirus encoding one or more rabies virus G antigens or antigenic fragments thereof; In a particular embodiment of this type, the immunogenic composition comprises a viral RNA replicon particle. is an alpha fragment encoding 2 to 4 rabies virus G antigens or their antigenic fragments. In a more particular embodiment, the immunogenic composition comprises a viral RNA replicon particle. Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle, alphavirus Contains RNA replicon particles.

[0014] In other embodiments, the immunogenic composition comprises two or more sets of alphavirus RNA replicons. In certain embodiments of this type, the first set of alphavirus RNA transcripts comprises The plicon particle encodes the rabies virus G antigen or an antigenic fragment thereof, and Alphavirus RNA replicon particles of feline calicivirus (FCV) antigens or In some embodiments of this type, the FCV antigen encodes an antigenic fragment thereof. In another embodiment, the FCV antigen is derived from the classical feline calicivirus. In yet another embodiment, the second set of antigens is derived from a feline calicivirus (F9-like). The FCV RNA replicon particle encodes two FCV antigens, one of which is , derived from the virulent systemic feline calicivirus, and the other from the classical (F9-like) feline calicivirus. It is derived from the Lysivirus.

[0015] Thus, in one embodiment, the nucleic acid construct of the invention comprises one or more rabies virus G In a particular embodiment of this type, the nucleic acid construct encodes an antigen or an antigenic fragment thereof. The product encodes 2 to 4 rabies virus G antigens or antigenic fragments thereof. In certain embodiments, the alphavirus RNA replicon particles are In yet another embodiment, the nucleic acid construct encodes a human serotype G antigen or an antigenic fragment thereof. In this case, the alphavirus RNA replicon particle contains 2 to 4 rabies virus G antigens or includes nucleic acid constructs encoding antigenic fragments thereof.

[0016] In certain embodiments, the immunogenic composition comprises one or more rabies virus G antigens or Alphavirus RNA replicon containing a nucleic acid construct encoding an antigenic fragment of In a related embodiment, the immunogenic composition comprises two to four rabies virus G antigens. containing alphavirus RNA replicon particles encoding the antigen or antigenic fragment thereof. In certain embodiments of this type, the alphavirus RNA replicon particles are In a more particular embodiment, the immunization vector encodes the immunization vector G or an antigenic fragment thereof. The progenitor composition is Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particles. In another embodiment, the immunogenic composition comprises an alphavirus RNA replicon particle. contains two or more sets of alphavirus RNA replicon particles. In the form, the first set of alphavirus RNA replicon particles comprises a first nucleic acid construct while the other set of alphavirus RNA replicon particles contains a second nucleic acid construct. nothing.

[0017] In yet another embodiment, the immunogenic composition comprises a first set of antigens comprising a first nucleic acid construct. lupus virus RNA replicon particles, another set of alphaviruses containing a second nucleic acid construct. a third set of alphavirus RNA replicon particles and a third set of alphavirus RNA replicon particles comprising a third nucleic acid construct; In a particular embodiment of this type, the first nucleic acid construct comprises a replicon particle. the second nucleic acid construct encodes a rabies virus G antigen or an antigenic fragment thereof; A feline calicivirus derived from a potentially phylogenetic feline calicivirus or an antigenic fragment thereof The third nucleic acid construct encodes a classical (F9-like) FCV antigen. Feline calicivirus (FCV) derived from feline calicivirus or its antigenic fragment ) antigen. In a specific embodiment, the feline calicivirus (FCV) antigen encodes It is an FCV capsid protein.

[0018] In yet another embodiment, the immunogenic composition comprises a first set of antigens comprising a first nucleic acid construct. lupus virus RNA replicon particles, another set of alphaviruses containing a second nucleic acid construct. 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 an embodiment, the first nucleic acid construct, the second nucleic acid construct, the third nucleic acid construct, The nucleotide sequences of the fourth nucleic acid construct and the fifth nucleic acid construct are all different.

[0019] Thus, the immunogenic compositions of the present invention induce protective immunity against non-rabies viral pathogens. a nucleic acid construct encoding at least one non-rabies virus antigen for use in the manufacture of an alpha-rabies virus vaccine; In particular embodiments of this type, the vector may contain a viral RNA replicon particle. Non-rabies virus antigens are protein antigens derived from feline herpesvirus (FHV). In another embodiment, the non-rabies virus antigen is feline calicivirus (FC In yet another embodiment, the antigen is a protein antigen derived from a non-rabies virus. The antigen is a protein antigen derived from feline pneumovirus (FPN). In this embodiment, the non-rabies viral antigen is derived from feline parvovirus (FPV). It is a protein antigen.

[0020] In yet another embodiment, the immunogenic composition comprises a first set of antigens comprising a first nucleic acid construct. lufavirus RNA replicon particles, another set of alphaviruses containing a second nucleic acid construct a third set of alphavirus RNA replicon particles, comprising a third nucleic acid construct; Plicon particles, and a fourth set of alphavirus RNA replicons comprising a fourth nucleic acid construct. In a particular embodiment of this type, the first nucleic acid construct comprises a rabies virus particle. the second nucleic acid construct encodes a virulent strain G antigen or an antigenic fragment thereof; A feline calicivirus ( The third nucleic acid construct encodes a classical (F9-like) feline calicivirus antigen. or an antigenic fragment thereof. and a fourth nucleic acid construct encoding a FeLV antigen or an antigenic fragment thereof. .

[0021] In yet another embodiment, the immunogenic composition comprises a first set of antigens comprising a first nucleic acid construct. lufavirus RNA replicon particle, alphavirus RNA containing second nucleic acid construct Another set of replicon particles, a third set of alphavirus RNA replicon particles containing 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 sequence 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 compositions of the present invention induce protective immunity against non-rabies viral pathogens. a nucleic acid construct encoding at least one non-rabies virus antigen for use in the manufacture of an alpha-rabies virus vaccine; In a particular embodiment of this type, the vector may contain a viral RNA replicon particle. The non-rabies virus antigen is a protein derived from feline herpesvirus (FHV). In another embodiment, the non-rabies virus antigen is a feline calicivirus (F In yet another embodiment, the protein antigen is derived from a non-rabies virus. The serotype antigen is a protein antigen derived from feline pneumovirus (FPN). In another embodiment, the non-rabies viral antigen is derived from feline parvovirus (FPV). It is a protein antigen.

[0023] The present invention further provides a method for detecting rabies virus-derived antigens or antigenic fragments thereof. Alphavirus RNA replicon particles that contain one or more modified live (e.g., attenuated) or and a killed mammalian pathogen. do.

[0024] In a particular embodiment of the invention, the rabies virus antigen is rabies virus G. In a particular embodiment of this type, the rabies virus G has the amino acid sequence of SEQ ID NO:2. In a more specific embodiment of this type, the amino acid sequence has an identity of 95% or more. Thus, rabies virus G comprises the amino acid sequence of SEQ ID NO: 2. In a specific embodiment, the rabies virus G has the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4. It is encoded by the code sequence.

[0025] The present invention further includes vaccines and multivalent vaccines comprising the immunogenic compositions of the present invention. In certain embodiments, the vaccine is a non-adjuvanted vaccine. The compound can aid in the prevention of disease associated with the rabies virus. In this case, antibodies are induced in a mammalian subject when the mammal is immunized with a vaccine. In certain embodiments, the mammal is a dog. In yet another embodiment, the mammal is a horse. In a particular embodiment of this type, the mammal is a Mustelidae. In yet another embodiment, the mammal is a bovine. In certain embodiments of the present invention, the bovine animal is a cow. The Bovidae family includes sheep.

[0026] The present invention also includes administering to a mammal an immunologically effective amount of a vaccine of the present invention, Methods for immunizing a mammal against rabies virus are provided. In another embodiment, the vaccine is administered by intramuscular injection. In another embodiment, the vaccine is administered by intravenous injection. In yet other embodiments, the vaccine is administered by intradermal injection. In embodiments, the vaccine is administered by oral administration. In certain embodiments, the mammal is administered intranasally. In another specific embodiment, the mammal is a dog. In the present invention, the mammal is a horse.

[0027] The vaccines of the present invention (including multivalent vaccines) may be used as a primer vaccine and / or a booster vaccine. In certain embodiments, the vaccines of the present invention can be administered as It is administered as a one-shot vaccine (one dose) without the need for subsequent administration. In some embodiments, both a primer vaccine and a booster vaccine are administered. If necessary, the primer and booster vaccines can be administered by the same route. In other embodiments of this type, the primer vaccine and the booster vaccine are both Both are administered by subcutaneous injection. In an alternative embodiment, administration of the primer vaccine is A booster vaccination can be given by one route and a booster vaccination by another route. In certain embodiments, the primer vaccine may be administered by subcutaneous injection, and the booster vaccine may be administered by subcutaneous injection. - The vaccine may be administered orally.

[0028] The present invention further comprises injecting an immunologically effective amount of a vaccine of the present invention into a mammal. In certain embodiments, a method of immunizing a mammal against rabies virus is provided. The vaccine is approximately 1 x 10 5 ~Approx. 1×10 10 or more RPs In a more particular embodiment, the vaccine is about 1 x 10 6 ~Approx. 1×10 9 Contains RP In a more specific embodiment, the vaccine comprises about 1 x 10 7 ~Approx. 1×10 8 It can contain RPs.

[0029] In one embodiment, the vaccine of the present invention is administered in a dose of 0.03 mL to 5 mL. In certain embodiments, the vaccines of the present invention are administered in doses of 0.05 mL to 3 mL. In a more particular embodiment, the administered dose is 0.1 mL to 2 mL. In a very particular embodiment, the administered dose is between 0.2 mL and 1.5 mL. In certain embodiments, the administered dose is 0.3 to 1.0 mL. In this case, the administered dose is 0.4 mL to 0.8 mL.

[0030] These and other aspects of the present invention are better understood by reference to the following detailed description. It will be understood. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides a vaccine composition comprising an immunologically effective amount of an antigen from one or more strains of rabies virus. The composition provides a vaccine that induces protective immunity in vaccinated recipient animals. In one aspect of the invention, the vaccine is directed against Venezuelan equine encephalitis virus (VEE). E) capsid protein and glycoprotein, and rabies glycoprotein (G) or its equivalent It contains alphavirus RNA replicon particles (RP) encoding antigenic fragments of In an even more specific embodiment, the vaccine comprises the avirulent TC-83 strain of VEE. The capsid protein and glycoprotein of rabies glycoprotein (G) or its antigen The alphavirus RNA replicon particle (RP) encodes a viral fragment. In another embodiment of the invention, the vaccine comprises a naked DNA vector encoding the rabies glycoprotein (G). Contains an alphavirus RNA replicon encoding the rabies glycoprotein (G). Vaccines containing HIV particles can be administered in the absence of adjuvant and still prevent rabies. Effective in inducing protective immunity in mammals vaccinated against canine disease viruses Useful.

[0032] Thus, one aspect of the present invention is to provide an improved, safe, non-adjuvanted rabies virus vaccine. In a related aspect, the vaccine of the present invention does not induce injection site sarcoma, Nevertheless, vaccines against the debilitating disease caused by rabies virus infection are provides protection to vaccinated mammals with efficacy equal to or greater than that of the corresponding adjuvanted vaccine Has power.

[0033] In order to more fully understand the present invention, the following definitions are provided.

[0034] The use of singular terms for convenience of description in no way implies such limitation. Thus, for example, reference to a composition comprising a "polypeptide" is "Alphavirus RNases" includes reference to one or more of such polypeptides. References to "alphavirus R replicon particles" refer to such alphavirus R replicon particles unless otherwise indicated. Contains reference to multiple NA replicon particles.

[0035] As used herein, the term "approximately" is used interchangeably with the term "about" and refers to a value is within 50 percent of the indicated value, i.e., "approximately" 1 x 10 8 Al A composition containing phagocytic RNA replicon particles / milliliter is 5x10 7~ 1.5x10 8 Contains alphavirus RNA replicon particles.

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

[0037] As used herein, the term "dog" refers to all domestic dogs, Cali breeds, and all other domesticated dogs, unless otherwise indicated. Includes the Canis lupus family or the Canis family.

[0038] As used herein, a "ferret" is a mammalian member of the Mustelidae family. They are mammals, one of them.

[0039] As used herein, "bovids" includes antelopes, sheep (sheep), goats, muscovy ducks, and other domesticated animals. Cattle and cows, such as bison, African buffalo, water buffalo, and cots The mammalian family of cloven-hoofed, cud-chewing mammals includes the hoofed and cloven-hoofed mammals.

[0040] As used herein, the term "replicon" refers to a gene that, if present, is a cellular One or more factors that will allow successful propagation of the parent virus in cell culture or in an animal host. A modified RNA viral genome lacking structural proteins (e.g., coding sequences for structural proteins). In the appropriate cellular context, the replicon amplifies itself and produces one or more subgenomic RNAs. Seeds can be generated.

[0041] As used herein, the term "alphavirus RNA replicon particle," abbreviated as " RP" is described, for example, in Pushko et al., Virology, 239(2):389-401 (1997)], structural proteins, e.g., also alphaviruses Alphavirus-derived proteins packaged in capsids and glycoproteins also derived from RPs are RNA replicons. They can be expressed in cell culture or animal hosts (with helper plasmids or without similar components) because the replicon is an alphavirus. This is because they do not encode the structural components of the virus (e.g., capsid or glycoproteins).

[0042] The term "non-rabies virus" modifies terms such as pathogen and / or antigen (or immunogen). Each pathogen and / or antigen (or immunogen) is used to decorate the rabies virus. It is neither a virus nor a rabies virus antigen (or immunogen), and is a non-rabies virus protein. This means that the protein antigen (or immunogen) is not derived from the rabies virus.

[0043] The terms "originate from," "originates from," and "origin" "originating from" refers to a given protein antigen and the pathogen or organism that naturally encodes it. are used interchangeably with respect to that strain, and as used herein, refers to that given protein. The unmodified and / or truncated amino acid sequence of the protein antigen is specific to the pathogen or strain of the pathogen. The present invention relates to a protein antigen derived from a pathogen. The coding sequence within the nucleic acid construct of interest may be expressed in the pathogen or pathogen strain (including naturally attenuated strains) from which it is derived. the expressed protein antigen by comparing it with the corresponding sequence of the protein antigen in may be genetically engineered to result in alterations and / or truncations of the amino acid sequence of .

[0044] As used herein, the terms "protection" or "providing protection" or "eliciting protective immunity" refer to "Aid in the prevention of disease" and "Aid in protection" means complete protection from any manifestation of infection. For example, "aided protection" means that after challenge, the underlying infection is eliminated. The symptoms of the condition are at least reduced and / or the underlying cellular, physiological, and or biochemical causes or mechanisms are reduced and / or eliminated. In this context, "reduced" refers to the physiological state of infection only. It is understood to mean anything related to the symptoms of an infection, including the molecular state of the infection, rather than just the symptoms.

[0045] As used herein, a "vaccine" refers to a vaccine administered to an animal, e.g., a dog (in certain embodiments). and a water-containing composition suitable for application to humans (including humans, but in other embodiments specifically excluding humans). The formulation typically contains one or more antigens combined with a pharmaceutically acceptable carrier, such as a liquid. and a composition comprising the compound of formula (I) and (II) which, when administered to an animal, prevents disease caused by infection with a wild-type microorganism. sufficient to minimally assist in the protection of the patient, i.e., to assist in the prevention of disease and / or to prevent disease. Induce an immune response strong enough to prevent, ameliorate or treat.

[0046] As used herein, a multivalent vaccine is a vaccine that contains two or more different antigens. In certain embodiments of this type, the multivalent vaccine is a vaccine for two or more different diseases. Stimulates the recipient's immune system against the drug.

[0047] The terms "adjuvant" and "immunostimulant" are used interchangeably herein. is defined as one or more substances that cause stimulation of the immune system. Incubant is used to enhance the immune response to one or more vaccine antigens / isolates. Therefore, an "adjuvant" is a substance that nonspecifically increases the immune response to a specific antigen. and thus the amount of antigen required for any given vaccine and / or the antigen of interest. This reduces the frequency of injections required to generate a sufficient immune response to the In this context, adjuvants are used to enhance the immune response to one or more vaccine antigens / isolates. It is used for this purpose.

[0048] As used herein, a "non-adjuvanted vaccine" refers to a vaccine that does not contain an adjuvant. It is a quinine or polyvalent vaccine.

[0049] As used herein, the term "pharmaceutically acceptable" means that the modified noun is Used adjectively to mean suitable for use in a product, e.g., medical When used to describe an additive to a pharmaceutical vaccine, it refers to the fact that the excipient is not part of any other component of the composition. It is particularly important that the ingredients are compatible with the intended recipient animal, e.g., dogs, and that they are not adversely harmful. To mark.

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

[0051] As used herein, a protein antigen refers to a specific protein (e.g., a protein antigen). The term "antigenic fragment" refers to a fragment that is antigenic, i.e., an immunoglobulin (antibody) or T-cell capable of specifically interacting with antigen-recognizing molecules of the immune system, such as cellular antigen receptors, Preferably, the antigenic fragment of the present invention is an antigen. In certain embodiments, a given An antigenic fragment of a protein antigen has at least the antigenicity of the full-length protein. In a preferred embodiment, the antigenicity of the protein is maintained at 25%. The fragment retains at least 50% of the antigenicity of the full-length protein. In embodiments, an antigenic fragment retains at least 75% of the antigenicity of the full-length protein. The antigenic fragment may be as small as 20 amino acids. Conversely, a large fragment of the full-length protein lacking just one amino acid In certain embodiments, the antigenic fragment may be 25 to 150 amino acids. In another embodiment, the antigenic fragment contains 50 to 250 amino acid residues. nothing.

[0052] As used herein, when the amino acid residues of two sequences are identical, they are considered to be one amino acid. An amino acid sequence may be 100% "identical" to a second amino acid sequence, or 100% "identical" to a second amino acid sequence. Therefore, if 50% of the amino acid residues in two amino acid sequences are identical, If the amino acid sequence is 50% identical to the second amino acid sequence, the amino acid sequence is 50% "identical" to the second amino acid sequence. Consisting of a portion of a given protein, e.g., a protein or polypeptide to be compared In certain embodiments, the amino acid sequence is carried out over a contiguous block of amino acid residues that is not Selected deletions or insertions that would otherwise alter the correspondence between the two amino acid sequences are considered. .

[0053] As used herein, nucleotide and amino acid sequence percent identity is expressed as C, MacVector (MacVector, Inc. Cary, NC27519), Ve ctor NTI (Informax, Inc. MD), Oxford Molecule Using the ar Group PLC (1996) and Clustal W algorithms, Determined by the default parameters for alignment and identity. These commercially available programs can be used with the same or similar default parameters. Alternatively, you can use the default filter criteria to determine sequence similarity. Under the conditions below, for example, use the default parameters to generate a GCG (Genetics Co. mputer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) Pailua Using the POP program, an Advanced Blast search can be used.

[0054] For purposes of this invention, an "inactivated" microorganism is one that is capable of inducing an immune response in an animal. These are microorganisms that can infect animals but cannot infect animals. For example, inactivated rabies The disease virus is caused by binary ethyleneimine, formalin, β-propiolactone, thimerosin, It can be inactivated by a substance selected from the group consisting of sarcoid or heat.

[0055] The alphavirus RNA replicon particles of the present invention are lyophilized and rehydrated with a sterile water diluent. On the other hand, alphavirus RNA replicon particles can be stored separately. However, if it is intended to be mixed with other vaccine components before administration, The RNA replicon particles are stored in a solution that stabilizes their structure, e.g., a high sucrose solution. It can be stored.

[0056] The vaccines of the present invention may be administered intravenously, intramuscularly, subcutaneously, orally, intranasally, intradermally, and / or intraperitoneally. It can be readily administered by any standard route, including vaccination. The cutin composition is preferably formulated appropriately for each type of recipient animal and route of administration. It will be understood that the present invention also provides a method for treating rabies and / or other mammalian diseases. Methods for immunizing a mammal against a pathogen are provided. One such method comprises administering a mammalian an immunologically effective amount of rabies virus glycoprotein (G) so that the subject produces appropriate rabies virus glycoprotein (G) antibodies. This involves injecting the mammal with the vaccine of the present invention.

[0057] Multivalent vaccines The present invention also provides a multivalent vaccine. Any antigen useful in a mammalian vaccine. or a combination of such antigens in a vaccine, such as rabies virus [e.g., rabies glycoprotein Replication-defective alphavirus RNA replicon particles encoding mammalian antigens of protein (G) Such a multivalent vaccine can be added to the parenteral parenteral (RP). Included.

[0058] [Table 1]

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

[0060] TIFF2025169249000002.tif83165

[0061] TIFF2025169249000003.tif48166

[0062] TIFF2025169249000004.tif86168 [Example]

[0063] The following examples serve to provide a further understanding of the present invention, but do not limit the scope of the invention. It is not a restriction.

[0064] Example 1 Delivery of the rabies virus glycoprotein coding sequence into alphavirus RNA replicon particles. Built-in introduction RNA viruses are vectors for introducing genetically engineered vaccine antigens into their genomes. However, their use to date has been limited to viruses. by incorporating the virus antigen into an RNA virus and then introducing the virus into a recipient host. As a result, protective antibodies against the ingested viral antigens are induced. Alphavirus RNA replicon particles are used to encode pathogenic antigens. Such alphavirus replicon platforms have been used in 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 contents of which are incorporated herein. ], and Semliki Forest virus (SFV) [Liljestrom and G aroff, Biotechnology (NY), 9:1356-1361 (1991 ), the contents of which are incorporated herein in their entirety]. Additionally, alphavirus RNA replicon particles have been developed for use in pigs and poultry. These include the porcine epidemic diarrhea vaccine, Cutin, RNA particles (product code 19U5.P1), swine flu vaccine, RNA (Product Code 19A5.D0), Avian Influenza Vaccine, RNA (Product Code 19O 5.D0), and a prescription product, RNA Particles (product code 9PP0.00).

[0065] Alphavirus RNA replicon construction Capsid proteins and glycoproteins of the avirulent TC-83 strain of Venezuelan equine encephalitis virus Encodes the rabies virus glycoprotein (G) from rabies virus packaged in a protein A vaccine containing alphavirus RNA replicon particles was prepared. The nucleotide sequence of the G protein was codon-optimized for human. The resulting sequence was Despite having 0.000% amino acid identity with the live rabies virus glycoprotein (G) The vaccine has only about 85% nucleotide identity to the sequence. as a single dose administered subcutaneously to, for example, cats and dogs 12 weeks of age or older, or alternatively Alternatively, it may be administered as multiple doses comprising a primary dose followed by one or more booster doses.

[0066] The amino acid sequence of the rabies virus glycoprotein (G) was used to codon-optimize (human The nucleotide sequence (codon usage) was generated in silico. Optimized sequences were obtained from a commercial supplier (ATUM, Newark, CA, USA). The synthetic gene [SEQ ID NO: 1] was prepared as synthetic DNA by the United States. The construct (RABV) was designed based on the amino acid sequence of the rabies virus glycoprotein. -G) is a frankinone suitable for cloning into alphavirus replicon plasmids. The wild-type amino acid sequence was codon-optimized for humans [SEQ ID NO: 2] with the following sequence: .

[0067] The VEE replicon vector designed to express rabies virus G has the following modifications: Modifications were made as previously described [see U.S. Pat. No. 9,441,247 B2; The contents of which are incorporated herein by reference.] TC-83 derived replicon vector "p VEK" [disclosed and described in U.S. Pat. No. 9,441,247 B2] as a restriction enzyme. The 5' flanking sequence (5'-GGCGCGCCGC ACC-3') [SEQ ID NO: 3] and the 3' flanking sequence (5'-TTAATTAA-3 A codon-optimized open reading frame (OPF) of the rabies G gene containing the nucleotide sequence The DNA plasmid containing the nucleotide sequence 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 The vector was renamed "pVHV-RABV-G." The "pVHV" vector nomenclature is Cloning via the AscI and PacI sites in the multiple cloning site of VEK was chosen to refer to a pVEK-derived replicon vector containing a transgene cassette Ta.

[0068] TC-83 RNA replicon particles (RP) were produced according to a previously described method [U.S. Pat. No. 9,441,247 B2 and U.S. Pat. No. 8,460,913 B2; the contents of which are incorporated herein by reference.

[0043] Briefly, the MegaScr ipt T7 RNA polymerase and cap analog (Promega, Madison, Wisconsin) pVHV replicon was prepared using PBS (Columbus, OH, USA) prior to in vitro transcription. The vector DNA and helper DNA plasmids were linearized with NotI restriction enzyme. In addition, the helper RNA used for production is expressed by the VEE subgenomic promoter as previously described. Lacking sequence [Kamrud et al., J Gen Virol. 91(Pt7):17 23-1727 (2010)]. Purified RNA for replicon and helper components was synthesized. The mixture was mixed with a suspension of Vero cells and electroporated in a 4 mm cuvette. , and OptiPro® SFM cell culture medium (Thermo Fishe r, Waltham, Massachusetts, USA). After incubation, the alphavirus RNA replicon particles were transferred to the ZetaPlus suspension. BioCap® depth filters (3M, Maplewood, Minnesota, USA) and washed with phosphate-buffered saline containing 5% sucrose (w / v). Finally, the retained RP was eluted with 400 mM NaCl buffer to separate it from the cells and medium. The eluted RP was finally formulated in 5% sucrose (w / v) and diluted to 0.22 The solution was passed through a micron membrane filter and dispensed into aliquots for storage. P titers were measured by immunofluorescence assay on infected Vero cell monolayers.

[0069] Example 2 A virus containing alphavirus RNA replicon particles encoding viral glycoproteins Administration of cutin to dogs To evaluate the safety and serological response in dogs after RP-rabies virus G vaccination The RP rabies virus G vaccine for research purposes was formulated with The liquid vaccine is formulated in 5% sucrose and 1% canine serum and 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 The currently licensed commercial rabies vaccine, DEFENSOR, is marketed by Dogs in group 5 received an unrelated RP construct, canine non-rabies virus 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 1 day 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 virus 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 a 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. 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] TIFF2025169249000008.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 The currently licensed commercial rabies vaccine, DEFENSOR®, is sold by )3, which was administered with chemically inactivated rabies virus along with aluminum hydroxide adjuvant. Includes canine disease virus.

[0081] After vaccination, cats were clinically evaluated 4-8 hours after vaccination and daily for 7 days after vaccination. The cats were examined for any adverse reactions to the vaccine, and the injection site was examined by palpation. Cats in groups 1, 2, and 3 were observed for 10 to 15 minutes immediately after insemination. Systemic reactions have been observed, with cats experiencing a stinging or painful reaction upon injection. These reactions did not last more than 5 minutes. These injection reactions were This was due to the composition of the stable formulation. No local side effects were observed after vaccination. Serum was collected the day before vaccination and at monthly intervals after vaccination for the first 3 months of the study. Test serum antibody titers against rabies virus using the rapid fluorescent focus inhibition test (RFFIT) The anti-rabies virus serological results are shown in Table 5 below. The titers are expressed in international units / mL (IU / mL), with 0.5 IU / mL considered a protective titer.

[0082] TIFF2025169249000009.tif240167 TIFF2025169249000010.tif30166

[0083] A single dose of RP-rabies virus vaccine induces high serum antirabies titers in cats Notably, RFFIT titers in cats were higher than those observed in dogs vaccinated with similar doses. A titer of 0.5 IU / mL in the RFFIT test is considered a protective titer. However, cats with serological antibody titers below this level have not shown toxicity in long-term immunological studies. It is known that RP rabies virus vaccines often protect against attack by All three groups were vaccinated with the appropriate doses of currently licensed commercial rabies products. have higher group geometric mean anti-rabies virus RFFIT titers than the vaccinated group. The immunity period is indicated as 3 years.

[0084] This first study was followed up with a second study (currently ongoing) that yielded similar results. At least six months have passed.

[0085] 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. Such modifications will be apparent to those skilled in the art from the above description. is intended to be included in the scope.

[0086] All base sizes or amino acid sizes given for nucleic acids or polypeptides and It is further understood that all molecular weights or molecular weight values ​​are approximate and are provided for purposes of description. Let's do it.

Claims

1. 1. An immunogenic composition comprising Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles comprising a nucleic acid construct encoding a rabies virus antigen, An immunogenic composition wherein the rabies virus antigen is glycoprotein (G) or an antigenic fragment thereof.

2. The immunogenic composition of claim 1, wherein the rabies virus antigen is of a first strain, and the immunogenic composition further comprises one or more additional alphavirus RNA replicon particles encoding a second rabies virus antigen derived from a second strain of rabies virus.

3. 3. The immunogenic composition of claim 2, wherein the second rabies virus antigen is glycoprotein (G) or an antigenic fragment thereof of a second strain of rabies virus.

4. 4. The immunogenic composition of claim 2 or 3, wherein said one or more additional alphavirus RNA replicon particles are VEE alphavirus RNA replicon particles.

5. 5. The immunogenic composition of claim 1, 2, 3 or 4, wherein the rabies virus glycoprotein (G) comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

2.

6. An immunogenic composition comprising a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle encoding a rabies virus glycoprotein (G) comprising an amino acid sequence comprising at least 95% identity to the amino acid sequence of SEQ ID NO:

2.

7. The immunogenic composition of any one of claims 1 to 6, further comprising an alphavirus RNA replicon particle comprising a nucleotide sequence encoding at least one protein antigen or an antigenic fragment thereof derived from a non-rabies viral pathogen.

8. A vector comprising a nucleotide sequence encoding a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle and a nucleic acid sequence encoding a rabies virus antigen.

9. 9. The vector of claim 8, wherein the nucleic acid sequence encoding a VEE alphavirus RNA replicon particle comprises a TC-83 derived replicon vector.

10. The vector according to claim 8 or 9, wherein the rabies virus antigen is rabies virus glycoprotein (G).

11. 11. The vector of claim 10, wherein the rabies virus glycoprotein (G) comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

2.

12. 12. The vector of claim 8, 9, 10, or 11, comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:

4.

13. A host cell comprising a nucleic acid sequence encoding a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle and a nucleic acid sequence encoding a rabies virus antigen.

14. 14. The host cell of claim 13, wherein the nucleic acid sequence encoding the VEE alphavirus RNA replicon particle is transcribed from a TC-83 derived replicon vector.

15. The host cell of claim 14, wherein the TC-83-derived replicon vector is a pVHV replicon vector.

16. 16. The host cell of claim 13, 14 or 15, wherein the rabies virus antigen is rabies virus glycoprotein (G).

17. 17. The host cell of claim 16, wherein the rabies virus glycoprotein (G) comprises an amino acid sequence comprising at least 95% identity to the amino acid sequence of SEQ ID NO:

2.

18. 18. The host cell of claim 13, 14, 15, 16, or 17, comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:

4.

19. 19. The host cell of any one of claims 13 to 18, wherein the nucleic acid sequence encoding the VEE alphavirus RNA replicon particle and the nucleic acid sequence encoding the rabies virus antigen are in the same nucleic acid molecule.

20. 20. The host cell of claim 19, wherein the nucleic acid molecule is RNA.

21. 21. The host cell of claim 20, wherein the nucleic acid molecule comprises a VEE subgenomic promoter sequence.

22. The host cell of any one of claims 13 to 21, wherein the cell is a Vero cell.

23. 23. The host cell of any one of claims 13 to 22, further comprising helper nucleic acid encoding alphavirus structural components.

24. 24. The host cell of claim 23, wherein the helper nucleic acid is RNA.

25. 25. The host cell of claim 24, wherein the helper nucleic acid lacks a VEE subgenomic promoter sequence.

26. 1. A method for producing VEE alphavirus RNA replicon particles, comprising: Cultivating a host cell according to any one of claims 23 to 25 under conditions suitable for protein expression, and purifying VEE alphavirus RNA replicon particles from said host cells and cultures.