Vaccination with replicon particles and oily adjuvants

Abstract

To provide vaccination against animal pathogens using alphavirus-replicon RNA particles and oil adjuvants, and methods for immunizing animals.SOLUTION: A vaccine comprises an alphavirus RNA replicon particle encoding an antigen originating from an animal pathogen, where the vaccine also comprises an oil adjuvant.SELECTED DRAWING: Figure 6

Description

Technical Field

[0001] The present invention relates to vaccination against animal pathogens using alphavirus-replicon RNA particles and an oil adjuvant. It relates to vaccines, and to kits of parts comprising replicon particles and an oil adjuvant. It also relates to methods of making and using the vaccines and their kit components and uses thereof. For the purpose of protecting against animal pathogens, many vector-based strategies have been employed for many years for vaccines. 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:10.101 7 / S1466252312000011, Kamrud et al., J Gen

Background Art

[0002] For the purpose of protecting against animal pathogens, many vector-based strategies have been employed for many years for vaccines. 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:10.101 7 / S1466252312000011, Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727(2010)], which includes the use of replicon RNA particles (RPs) derived from alphaviruses such as Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virology 239:389-401(1997)], Sindbis virus [Bredenbeek et al., Journal of Virology 67:6439-6446( 1993)] and Semliki Forest virus [Liljestrom and Ga roff, Biotechnology(NY) 9:1356-361(1991)]. After infection of target human or animal cells, the pathogenic antigen encoded by the replicon particles is expressed. animal cells, the pathogenic antigen encoded by the replicon particles is expressed. roff, Biotechnology(NY) 9:1356-361(1991)]. After infection of target human or animal cells, the pathogenic antigen encoded by the replicon particles is expressed. animal cells, the pathogenic antigen encoded by the replicon particles is expressed. As a result, protective antibodies against the expressed antigen are induced. RP has attractive safety and effective properties compared to some traditional vaccine formulations [Vander Vee n, et al., Anim Health Res Rev. 13(1):1-9(2 012)]. The RP platform is based on several USDA-approved vaccines, including a porcine epidemic diarrhea vaccine, RNA particles (product code 19U5P1 ), a porcine influenza vaccine, RNA (product code 19A5D0), an avian influenza vaccine, RNA (product code 19O5D0) and a pharmaceutical, RNA particles (product co de 9PP000).

[0003] Replicon RNA particles derived from alphavirus lack the alphavirus structural protein gene, but maintain the replication elements necessary for cytoplasmic RNA self-amplification and the expression of inserted heterologous nucleic acids driven by the highly active 26S alphavirus subgenomic promoter. Thus, RP is a single-cycle infectious particle that is replication-defective due to the lack of the structural protein gene [Lundstrom, Vaccines 6: 2392-2415(2014)]. Therefore, the structural proteins required for the packaging and production of replicon particles must be provided between different molecules in a suitable host cell for producing RP [see Vajdy et al., Immunol. and Cell Biol. 82:617-627(2004)]. The structural proteins are generally provided by transiently co-transfecting one or more "helper" RNAs encoding the replicon RNA and the structural proteins. Alternative ​​​​​​​ In terms of RP, it can be produced from a packaging cell line that constitutively or transiently expresses one or more DNA expression cassettes to express viral structural proteins. In this way, the product of the replicon particle does not contain structural proteins in the resulting RP genome, thus maintaining the replication-defective nature of the vector [Polo et al. Dev. Biol., 104: 181-185 (2000)]. Such replication-defective alphavirus RNA replicon particles induce a protective immune response in vivo when used for immunization of target humans or animals. For example, alphavirus vectors based on VEE induce strong mucosal and systemic immune responses after systemic immunization of mice and larger animals [Davis et al., IUBMB Life 53: 209-211 (2002)].

[0004] An adjuvant is a known compound capable of providing a non-specific stimulus to the immune system of the target human or animal. The use of standard adjuvants is in vaccines based on inactivated or subunit antigens. There are various types and compositions of adjuvants: for example, aluminum salts such as aluminum hydroxide or aluminum phosphate, liposomes, glucans, alginates, bacterial components such as cell wall components, mineral oils or non-mineral oils, synthetic adjuvants such as non-ionic block polymers, polyamines such as dextran sulfate, Carbopol (trademark), pyran and Quil A (trademark), saponins such as Q-vac (trademark). In ISCOM (trademark), saponin and vaccine components may be combined.

[0005] Furthermore, peptides such as muramyl dipeptide, dimethylglycine, and tuftsin are often used as adjuvants. Similarly, combination products such as ISA (trademark) compositions (Seppic, France) are used.

[0006] A handbook on adjuvants and their use and effects is "Vaccine ad juvants" (Methods in molecular medicine, v ol. 42, D. O'Hagan ed., 2000, Humana press, NJ , ISBN: 0896037355).

[0007] Any citation of a reference in this specification should not be construed as an approval that such reference is available as "prior art" for this application.

Prior art documents

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[0008]

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[0009] The present invention provides alphavirus RNA sequences encoding antigens derived from animal pathogens. A vaccine comprising a precon particle is provided, wherein the vaccine also comprises an oil-based adjuvant. nothing.

[0010] In one embodiment of the vaccine of the invention, the oil-based adjuvant is selected from mineral oil and non-mineral oil. The composition further comprises at least one oil that is

[0011] In one embodiment of the vaccine of the invention, the oil-based adjuvant comprises mineral oil, preferably The oil is liquid paraffin oil.

[0012] In one embodiment of the vaccine of the present invention, the oily adjuvant contains non-mineral oil, preferably the non-mineral oil is selected from synthetic oils, semi-synthetic oils, animal oils and vegetable oils. More preferably, the non- mineral oil is selected from squalane, squalene, tocopherol and vegetable oils. In one embodiment the tocopherol is alpha-tocopherol, and more preferably, the alpha- tocopherol is selected from vitamin E and vitamin E-acetate. In one embodiment, the vegetable oil is oleate, and more preferably ethyl-oleate.

[0013] More preferably, the non-mineral oil is squalane.

[0014] In a preferred embodiment of the vaccine of the present invention, the oily adjuvant contains two or more oils .

[0015] In one embodiment of the oily adjuvant containing two or more oils, the adjuvant contains mineral oil and one or more non-mineral oils. More preferably, the oily adjuvant contains liquid paraffin oil as the mineral oil and one or more non-mineral oils selected from squalane, squalene, vitamin E, vitamin E-acetate, oleate and ethyl-oleate. Even more preferably the oily adjuvant contains liquid paraffin oil and vitamin E-acetate. Most preferably, the oily adjuvant is XSolve (trademark).

[0016] In an alternative embodiment of the oily adjuvant containing two or more oils, the adjuvant contains two or more non-mineral oils. Preferably, the oily adjuvant contains squalane, squalene, vitamin Two or more non-mineral oils selected from vitamin E, vitamin E-acetate, oleate, and ethyl-oleate are included. Even more preferably, the oily adjuvant includes squalane and vitamin E-acetate. Most preferably, the oily adjuvant is SVEA (trademark). In one embodiment of the vaccine of the present invention, the amount of mineral oil in the oily adjuvant is 1 to 70% v / v of the oily adjuvant. Preferably, the oily adjuvant contains mineral oil in an amount of 5 to 60% v / v of the oily adjuvant. In one embodiment of the vaccine of the present invention, the total amount of non-mineral oil is 0.1 to 30% w / v of the oily adjuvant. Preferably, the oily adjuvant contains a total amount of non-mineral oil of 0.5 to 25% v / v of the oily adjuvant. In one embodiment, when the non-mineral oil includes squalane, the oily adjuvant contains squalane at 0.5 to 30% w / v of the oily adjuvant; more preferably, the oily adjuvant contains squalane at 1 to 25% w / v, 2 to 15% w / v, or 3 to 10% w / v of the oily adjuvant.

[0017] In one embodiment of the vaccine of the present invention, the amount of mineral oil in the oily adjuvant is 1 to 70% v / v of the oily adjuvant. Preferably, the oily adjuvant contains mineral oil in an amount of 5 to 60% v / v of the oily adjuvant. In one embodiment of the vaccine of the present invention, the total amount of non-mineral oil is 0.1 to 30% w / v of the oily adjuvant. Preferably, the oily adjuvant contains a total amount of non-mineral oil of 0.5 to 25% v / v of the oily adjuvant. In one embodiment, when the non-mineral oil includes squalane, the oily adjuvant contains squalane at 0.5 to 30% w / v of the oily adjuvant; more preferably, the oily adjuvant contains squalane at 1 to 25% w / v, 2 to 15% w / v, or 3 to 10% w / v of the oily adjuvant.

[0018] In one embodiment of the vaccine of the present invention, the amount of mineral oil in the oily adjuvant is 1 to 70% v / v of the oily adjuvant. Preferably, the oily adjuvant contains mineral oil in an amount of 5 to 60% v / v of the oily adjuvant. In one embodiment of the vaccine of the present invention, the total amount of non-mineral oil is 0.1 to 30% w / v of the oily adjuvant. Preferably, the oily adjuvant contains a total amount of non-mineral oil of 0.5 to 25% v / v of the oily adjuvant. In one embodiment, when the non-mineral oil includes squalane, the oily adjuvant contains squalane at 0.5 to 30% w / v of the oily adjuvant; more preferably, the oily adjuvant contains squalane at 1 to 25% w / v, 2 to 15% w / v, or 3 to 10% w / v of the oily adjuvant.

[0019] In one embodiment, when the non-mineral oil includes squalane, the oily adjuvant contains squalane at 0.5 to 30% w / v of the oily adjuvant; more preferably, the oily adjuvant contains squalane at 1 to 25% w / v, 2 to 15% w / v, or 3 to 10% w / v of the oily adjuvant. Alternatively or additionally, in one embodiment when the non-mineral oil includes vitamin E-acetate, the oily adjuvant contains vitamin E-acetate at 0.1 to 30% w / v of the oily adjuvant, more preferably the oily adjuvant contains vitamin E-acetate at 0.5 to 20% w / v, 1 to 16% w / v, or 2 to 10% w / v of the oily adjuvant. In one embodiment, when the non-mineral oil includes squalane, the oily adjuvant contains squalane at 0.5 to 30% w / v of the oily adjuvant; more preferably, the oily adjuvant contains squalane at 1 to 25% w / v, 2 to 15% w / v, or 3 to 10% w / v of the oily adjuvant. Alternatively or additionally, in one embodiment when the non-mineral oil includes vitamin E-acetate, the oily adjuvant contains vitamin E-acetate at 0.1 to 30% w / v of the oily adjuvant, more preferably the oily adjuvant contains vitamin E-acetate at 0.5 to 20% w / v, 1 to 16% w / v, or 2 to 10% w / v of the oily adjuvant.

[0020] Alternatively or additionally, in one embodiment when the non-mineral oil includes vitamin E-acetate, the oily adjuvant contains vitamin E-acetate at 0.1 to 30% w / v of the oily adjuvant, more preferably the oily adjuvant contains vitamin E-acetate at 0.5 to 20% w / v, 1 to 16% w / v, or 2 to 10% w / v of the oily adjuvant. In one embodiment, when the non-mineral oil includes squalane, the oily adjuvant contains squalane at 0.5 to 30% w / v of the oily adjuvant; more preferably, the oily adjuvant contains squalane at 1 to 25% w / v, 2 to 15% w / v, or 3 to 10% w / v of the oily adjuvant. Alternatively or additionally, in one embodiment when the non-mineral oil includes vitamin E-acetate, the oily adjuvant contains vitamin E-acetate at 0.1 to 30% w / v of the oily adjuvant, more preferably the oily adjuvant contains vitamin E-acetate at 0.5 to 20% w / v, 1 to 16% w / v, or 2 to 10% w / v of the oily adjuvant. In one embodiment, when the non-mineral oil includes squalane, the oily adjuvant contains squalane at 0.5 to 30% w / v of the oily adjuvant; more preferably, the oily adjuvant contains squalane at 1 to 25% w / v, 2 to 15% w / v, or 3 to 10% w / v of the oily adjuvant. Alternatively or additionally, in one embodiment when the non-mineral oil includes vitamin E-acetate, the oily adjuvant contains vitamin E-acetate at 0.1 to 30% w / v of the oily adjuvant, more preferably the oily adjuvant contains vitamin E-acetate at 0.5 to 20% w / v, 1 to 16% w / v, or 2 to 10% w / v of the oily adjuvant.

[0021] In one embodiment of the vaccine of the present invention, the oily adjuvant is an emulsion of an oil phase and an aqueous phase. Preferably, the oily adjuvant is formulated as an oil-in-water (O / W) emulsion.

[0022] In one embodiment, the aqueous phase contains water of pharmaceutically acceptable quality.

[0023] In one embodiment, the emulsion of the oily adjuvant is formulated as a microemulsion, where the droplets of the internal phase are less than 1 micrometer. Preferably, the microemulsion is an O / W emulsion, and more preferably, the O / W microemulsion is prepared using high-energy homogenization, and even more preferably, it is prepared by the method of microfluidization.

[0024] In one embodiment of the vaccine of the present invention, the emulsion of the oily adjuvant contains an emulsifier. Preferably, the emulsifier contains polysorbate, and more preferably, the emulsifier contains polysorbate 80.

[0025] In one embodiment, the vaccine of the present invention contains an emulsion of the oily adjuvant. Preferably, the vaccine contains an oily adjuvant formulated as an O / W emulsion.

[0026] In one embodiment of the vaccine of the present invention, the vaccine is formulated as an oil-in-water (O / W) emulsion.

[0027] In one embodiment of the vaccine of the present invention, the alphavirus RNA replicon particles are Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles. In a more specific embodiment ​​​​​​​​​​In one form, the VEE alphavirus RNA replicon particle is a TC-83VEE alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Sindbis alphavirus RNA replicon particle. In still other embodiments, the alphavirus RNA replicon particle is a Semliki Forest virus alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Sindbis alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Sindbis alphavirus RNA replicon particle. In still other embodiments, the alphavirus RNA replicon particle is a Semliki Forest virus alphavirus RNA replicon particle. In still other embodiments, the alphavirus RNA replicon particle is a Semliki Forest virus alphavirus RNA replicon particle. In still other embodiments, the alphavirus RNA replicon particle is a Semliki Forest virus alphavirus RNA replicon particle.

[0028] In one embodiment of the vaccine of the present invention, for an antigen encoded from an animal pathogen, the animal pathogen is selected from viruses, bacteria, parasites, protozoa, fungi, rickettsia, and prions. More preferably, the antigen encoded from the animal pathogen is an antigen derived from a virus or a bacteria. Most preferably, the antigen is derived from a virus. In one embodiment of the vaccine of the present invention, for an antigen encoded from an animal pathogen, the animal pathogen is selected from viruses, bacteria, parasites, protozoa, fungi, rickettsia, and prions. More preferably, the antigen encoded from the animal pathogen is an antigen derived from a virus or a bacteria. Most preferably, the antigen is derived from a virus. In one embodiment of the vaccine of the present invention, for an antigen encoded from an animal pathogen, the animal pathogen is selected from viruses, bacteria, parasites, protozoa, fungi, rickettsia, and prions. More preferably, the antigen encoded from the animal pathogen is an antigen derived from a virus or a bacteria. Most preferably, the antigen is derived from a virus. In one embodiment of the vaccine of the present invention, for an antigen encoded from an animal pathogen, the animal pathogen is selected from viruses, bacteria, parasites, protozoa, fungi, rickettsia, and prions. More preferably, the antigen encoded from the animal pathogen is an antigen derived from a virus or a bacteria. Most preferably, the antigen is derived from a virus.

[0029] In one embodiment of the vaccine of the present invention, the RP encodes an antigen derived from an animal pathogen, whereby the animal is an animal related to veterinary science. Preferably, the animal is selected from fish, birds, and mammals. More preferably, the animal is a wild animal, a domestic animal, or a pet animal. The domestic animal is a fish, a bird, a pig, or a ruminant; preferably, the porcine is a pig; preferably, the bird is a chicken, a duck, a goose, a turkey, a quail, or an ostrich; preferably, the ruminant is a cow, a sheep, a goat, a buffalo, a camel, or a deer; preferably, the fish is a bony fish with fins, more preferably, a salmonid fish or a cichlid fish (i.e., In one embodiment of the vaccine of the present invention, the RP encodes an antigen derived from an animal pathogen, whereby the animal is an animal related to veterinary science. Preferably, the animal is selected from fish, birds, and mammals. More preferably, the animal is a wild animal, a domestic animal, or a pet animal. The domestic animal is a fish, a bird, a pig, or a ruminant; preferably, the porcine is a pig; preferably, the bird is a chicken, a duck, a goose, a turkey, a quail, or an ostrich; preferably, the ruminant is a cow, a sheep, a goat, a buffalo, a camel, or a deer; preferably, the fish is a bony fish with fins, more preferably, a salmonid fish or a cichlid fish (i.e., In one embodiment of the vaccine of the present invention, the RP encodes an antigen derived from an animal pathogen, whereby the animal is an animal related to veterinary science. Preferably, the animal is selected from fish, birds, and mammals. More preferably, the animal is a wild animal, a domestic animal, or a pet animal. The domestic animal is a fish, a bird, a pig, or a ruminant; preferably, the porcine is a pig; preferably, the bird is a chicken, a duck, a goose, a turkey, a quail, or an ostrich; preferably, the ruminant is a cow, a sheep, a goat, a buffalo, a camel, or a deer; preferably, the fish is a bony fish with fins, more preferably, a salmonid fish or a cichlid fish (i.e., In one embodiment of the vaccine of the present invention, the RP encodes an antigen derived from an animal pathogen, whereby the animal is an animal related to veterinary science. Preferably, the animal is selected from fish, birds, and mammals. More preferably, the animal is a wild animal, a domestic animal, or a pet animal. The domestic animal is a fish, a bird, a pig, or a ruminant; preferably, the porcine is a pig; preferably, the bird is a chicken, a duck, a goose, a turkey, a quail, or an ostrich; preferably, the ruminant is a cow, a sheep, a goat, a buffalo, a camel, or a deer; preferably, the fish is a bony fish with fins, more preferably, a salmonid fish or a cichlid fish (i.e., In one embodiment of the vaccine of the present invention, the RP encodes an antigen derived from an animal pathogen, whereby the animal is an animal related to veterinary science. Preferably, the animal is selected from fish, birds, and mammals. More preferably, the animal is a wild animal, a domestic animal, or a pet animal. The domestic animal is a fish, a bird, a pig, or a ruminant; preferably, the porcine is a pig; preferably, the bird is a chicken, a duck, a goose, a turkey, a quail, or an ostrich; preferably, the ruminant is a cow, a sheep, a goat, a buffalo, a camel, or a deer; preferably, the fish is a bony fish with fins, more preferably, a salmonid fish or a cichlid fish (i.e., In one embodiment of the vaccine of the present invention, the RP encodes an antigen derived from an animal pathogen, whereby the animal is an animal related to veterinary science. Preferably, the animal is selected from fish, birds, and mammals. More preferably, the animal is a wild animal, a domestic animal, or a pet animal. The domestic animal is a fish, a bird, a pig, or a ruminant; preferably, the porcine is a pig; preferably, the bird is a chicken, a duck, a goose, a turkey, a quail, or an ostrich; preferably, the ruminant is a cow, a sheep, a goat, a buffalo, a camel, or a deer; preferably, the fish is a bony fish with fins, more preferably, a salmonid fish or a cichlid fish (i.e., In one embodiment of the vaccine of the present invention, the RP encodes an antigen derived from an animal pathogen, whereby the animal is an animal related to veterinary science. Preferably, the animal is selected from fish, birds, and mammals. More preferably, the animal is a wild animal, a domestic animal, or a pet animal. The domestic animal is a fish, a bird, a pig, or a ruminant; preferably, the porcine is a pig; preferably, the bird is a chicken, a duck, a goose, a turkey, a quail, or an ostrich; preferably, the ruminant is a cow, a sheep, a goat, a buffalo, a camel, or a deer; preferably, the fish is a bony fish with fins, more preferably, a salmonid fish or a cichlid fish (i.e., In one embodiment of the vaccine of the present invention, the RP encodes an antigen derived from an animal pathogen, whereby the animal is an animal related to veterinary science. Preferably, the animal is selected from fish, birds, and mammals. More preferably, the animal is a wild animal, a domestic animal, or a pet animal. The domestic animal is a fish, a bird, a pig, or a ruminant; preferably, the porcine is a pig; preferably, the bird is a chicken, a duck, a goose, a turkey, a quail, or an ostrich; preferably, the ruminant is a cow, a sheep, a goat, a buffalo, a camel, or a deer; preferably, the fish is a bony fish with fins, more preferably, a salmonid fish or a cichlid fish (i.e., , a member of the Cyprinidae family). Salmonidae fish are preferably selected from rainbow trout, steelhead trout , masu salmon, ginza salmon, chum salmon, white salmon and pink salmon, rainbow trout, masu salmon , lake trout and brown trout and char. Cy rid fish are preferably tilapia. The pet animals are preferably selected from cats, dogs and horses. More preferably, the animal is tilapia, chicken or pig .

[0030] In one embodiment of the vaccine of the present invention, the nucleotide sequence of the gene encoding the antigen derived from the animal pathogen of the present invention is optimized for expression in the cells of the target animal species for vaccine purposes . In one embodiment, the nucleotide sequence optimization is codon optimization. One embodiment, the nucleotide sequence optimization is to optimize the secondary structure of RNA transcription .

[0031] In a preferred embodiment, the nucleotide sequence optimization of the gene encoding the antigen derived from the animal pathogen of the present invention is related to both codon usage and the secondary structure of RNA transcription . Preferably, the nucleotide sequence optimization is carried out according to the procedures described in one or more of US Patent No. 7,561,972, US Pat ent No. 7,561,973, US Patent No. 7,805,252 and US Patent No. 8,12 6,653.

[0032] In a detailed preferred embodiment of the vaccine of the present invention, the oily adjuvant contains mineral oil and non-mineral oil, the vaccine is formulated as an O / W emulsion, the alpha virus RNA rep licon particles are VEE alpha virus RNA replicon particles, and the antigen of the animal pathogen is It is a viral antigen, and the virus is a porcine pathogen.

[0033] In a preferred embodiment of the vaccine of the present invention, the encoded antigen derived from an animal pathogen is the hemagglutinin (HA) protein or neuraminidase (NA) protein of influenza virus, or an antigenic fragment of such an HA protein or NA protein is. The HA and / or NA protein is preferably derived from influenza virus A type, more preferably derived from porcine influenza A virus or PEDV is.

[0034] The present invention further provides a multivalent vaccine comprising the alpha virus RNA replicon particles of the present invention, wherein the vaccine comprises two or more RPs encoding an antigen, or one or more RPs each encoding one or more antigens of the present invention.

[0035] The vaccine of the present invention contains an immunologically effective amount of the alpha virus RNA replicon particles for the present invention. In one embodiment, the vaccine contains about 1×10^3 to about 1×10^11 RPs . In a more specific embodiment, the vaccine contains about 1×10^4 to about 1×10^10 R Ps. In an even more specific embodiment, the vaccine contains about 1×10^5 to about 1×10^ 9 RPs.

[0036] The vaccine of the present invention contains an immunologically effective amount of an oily adjuvant for the present invention. In one embodiment , the vaccine contains an oily adjuvant in an amount of about 10% to 90% v / v of the vaccine . More preferably, the vaccine contains about 20% to 80% v / v, 30 to 70% v / It contains an oily adjuvant in an amount of v or even 40 to 60% v / v. Most preferably , the vaccine contains an oily adjuvant in an amount of about 50% v / v of the vaccine.

[0037] In a specific embodiment, the vaccine of the present invention is 0.05 mL to 5 mL per animal dose administered in a volume. In a more specific embodiment, the dose administered per animal is 0.1 m L to 2 mL. In an even more specific embodiment, the dose administered is 0.2 mL to 1 .5 mL. In an even more specific embodiment, the dose administered is 0.3 to 1.0 m L. In an even more specific embodiment, the dose administered per animal is 0.4 mL to 0.8 mL.

[0038] In one embodiment of the vaccine of the present invention, the vaccine contains a further adjuvant. Preferably the further adjuvant is selected from the group of immunostimulatory nucleic acids including bacterial cell wall components, cytokines and unmethylated CpG . In one embodiment, the immunostimulatory nucleic acid is disclosed in International Publication No. 2012 / 089800 (X4 family), International Publication No. 2012 / 160183 ( X43 family) and International Publication No. 2012 / 160184 (X23 family) and one or more are selected therefrom.

[0039] In one embodiment of the vaccine of the present invention, the vaccine contains a further antigen of an animal pathogen. Preferably in an embodiment, the further antigen is selected from the group of attenuated live microorganisms, inactivated microorganisms and microbial sub units.

[0040] In a further aspect, the present invention provides a kit of parts, the kit comprising at least two containers wherein at least one of the containers encodes an antigen derived from an animal pathogen. and at least one of the containers is an oil-based At least two of the containers contain an immunologically effective amount of alphavirus R NA replicon particles or oil-based adjuvant, respectively.

[0041] In a preferred embodiment of the kit-of-parts of the present invention, an alphavirus RNA replicon is one or more of the following: serotype particle, encoded antigen, animal pathogen, and oil-based adjuvant, or all of the above: is as defined in any one or more embodiments described herein. do.

[0042] In a preferred embodiment, at least one of the containers containing the RP contains the RP as a lyophilisate. include.

[0043] In an alternative embodiment, at least one of the containers containing the RP contains the RP in an aqueous solution; The aqueous solution preferably contains a buffer, and the aqueous solution is preferably kept cooled or frozen. In one embodiment, the aqueous solution is a reconstituted RP solution, which is a RP lyophilized The composition is prepared by mixing the composition with a suitable aqueous diluent.

[0044] In one embodiment in which at least one container contains RP as a lyophilisate, the kit of the present invention The second part contains an additional diluent containing the appropriate diluent for reconstituting the lyophilized RP. In a preferred embodiment, the diluent is an aqueous solution, preferably a buffer. Contains buffer and / or stabilizers and water of pharma- ceutically acceptable quality.

[0045] In a preferred embodiment, the container containing the oily adjuvant contains an oily adjuvant formulated as an emulsion of an oil phase and an aqueous phase. Preferably, the emulsion is an oil-in-water emulsion.

[0046] In one embodiment of the kit of parts, the kit includes instructions regarding the use of the kit and / or its components. In a preferred embodiment, the instructions regarding use are provided on or attached to one or more kit components; or are provided by reference to instructions in electronic form, such as information viewable or downloadable from an Internet website of the manufacturer or seller of the kit.

[0047] In one embodiment, the kit of parts includes a box containing at least two containers and instructions regarding use displayed on an information medium (such as a card or leaflet) included with or within the box.

[0048] In one embodiment of the kit of parts, the kit may also include the provision of components (related to commerce) on, for example, an Internet website for use in the method of immunization of the present invention.

[0049] In one embodiment of the kit of parts, one or more containers may contain a further adjuvant as described herein, and further or alternatively, one or more containers may contain a further antigen of an animal pathogen as described herein.

[0050] An alphavirus RNA replicon encoding an antigen derived from an animal pathogen and ​​​​​​​​​​The oily adjuvant and the adjuvant, both as defined herein, are administered to the target animal. Such administration can prevent infection or disease caused by animal pathogens. This induces an effective immune defense in the animal against the pathogen. For relevant uses of medicines, e.g. European Medicines Agency's Committee for Veterinary Medicinal Products (EMA-CVMP) Administration can be carried out in accordance with the guidelines.

[0051] Thus, in a further aspect, the present invention provides an immunologically effective amount of a virulent antigen derived from an animal pathogen. The antigen-encoding alphavirus RNA replicon particles and an oil-based adjuvant are The present invention also provides a method of immunizing an animal comprising administering to the animal a method for ....

[0052] In a preferred embodiment of the method of immunizing an animal of the invention, the method comprises administering to the animal a vaccine of the invention. This includes administering a chin.

[0053] In a preferred embodiment of the method of immunizing an animal of the present invention, the alphavirus RNA replica is one or more of a recombinant particle, an encoded antigen, an animal pathogen, and an oil-based adjuvant. or all are as defined in any one or more embodiments described herein. do.

[0054] In a preferred embodiment of the method of immunizing an animal of the present invention, a coimmunoglobulin derived from an animal pathogen is used. The antigens tested were from fish, cichlids, tilapia, mammals, birds and chickens. It is an antigen derived from these pathogens.

[0055] In one embodiment of the method of immunizing an animal of the present invention, an alphavirus RNA replicon The particles and the oily adjuvant are administered, either simultaneously or in parallel, into or onto the body of the target animal.

[0056] In a preferred embodiment of the method of immunizing an animal of the present invention, the alpha virus RNA rep licon particles and the oily adjuvant are administered, by simultaneous use (i.e., as a single composition), into or onto the body of the target animal.

[0057] In a preferred embodiment, the single composition is the vaccine of the present invention.

[0058] In a preferred embodiment, the single composition is more preferably prepared by mixing a composition containing RP and a composition containing an oily adjuvant, both as described for use in the present invention, and even more preferably by mixing the contents of the containers of the kit of parts of the present invention, and even more preferably by mixing an aqueous solution containing RP and a composition containing an O / W emulsion of the oily adjuvant immediately prior to administration to the target animal. In an alternative even more preferred embodiment, the single composition is prepared by reconstituting the RP lyophilisate with an O / W emulsion of the oily adjuvant, both as described herein for use in the present invention. Effectively, the vaccine of the present invention is produced by the preparation of the single composition. Preferably, "immediately prior to administration to the target animal" means, in a desired order, within 24 hours before administration to the target animal,

[0059] more preferably within 16 hours before, within 12 hours before, within 8 hours before, within 4 hours before, or even within 2 hours before administration to the target animal. In an alternatively preferred embodiment of the method of immunizing an animal of the present invention, the alpha virus R

[0060] NA replicon particles and the oily adjuvant are administered, either simultaneously or in parallel, into or The NA replicon particles and the oily adjuvant are administered into or onto the body of the target animal by concurrent use (i.e., contained within separate compositions such that they are administered at separate locations and / or times).

[0061] In a preferred embodiment, the concurrent use involves administering the alphavirus RNA replicon particles and the oily adjuvant contained within the kit of parts of the present invention into or onto the body of the target animal at separate locations and / or times.

[0062] In a preferred embodiment of the concurrent use of the present invention, the separate compositions are administered to separate sites within or on the body of the target animal via the same or different administration routes within a limited time period relative to each other; preferably, the "limited time period" is, in descending order of desirability, within 2 weeks of each other, more preferably within 1 week of each other, even more preferably within 1 day, within 16 hours, within 12 hours, within 8 hours, within 4 hours, within 2 hours, within 1 hour, within 30 minutes, or even within 10

[0063] minutes of each other. Most preferably, the administration by concurrent use is substantially simultaneous use. In a preferred embodiment of the concurrent use of the present invention, the separate compositions are administered into or onto the body of the target animal via the same or different administration routes at separate sites within a limited time period

[0064] In a preferred embodiment of the concurrent use of the present invention, the separate compositions are administered, by the same or different routes of administration, into or onto the body of the target animal at substantially the same site within or on the body of the target animal, provided that the compositions are not mixed at the site of administration, and the times of administration are sufficiently separated from each other. In the present invention, "sufficiently separating the times" to prevent mixing means that, in the desired order, they must not be within 2 hours of each other, preferably not within 6 hours of each other, not within 12 hours of each other, not within 1 day of each other,

[0065] not within 2 days of each other, or not even within 1 week of each other. In one embodiment of the method of immunizing an animal of the present invention, the administration into or onto the body of the target animal is effected by parenteral administration. In

[0066] an alternative embodiment, the administration is by the method of mucosal administration. In a further alternative embodiment, the vaccine is administered by the method of topical administration. Preferred methods of administration are selected from intradermal, intramuscular,

[0067] intraperitoneal, subcutaneous, dipping and spraying. The intradermal administration method is preferably by administration without using a needle, more preferably using an IDAL (registered trademark) device (Intra-Dermal Application of Liquids). In one embodiment of the vaccine administration of the present invention, the vaccine is

[0067] administered as a primer vaccine and / or as a booster vaccine. In a particular embodiment, the vaccine of the present invention is administered as a one-shot vaccination that does not require a subsequent booster administered. In certain embodiments, both the primer vaccine and the booster vaccine are administered. When administered, the primer vaccine and the booster vaccine are administered by the same route. In alternative embodiments, when both the primer vaccine and the booster vaccine are administered, administration of the primer vaccine is effected by one route and administration of the booster vaccine is effected by a different route.

[0068] In one embodiment of vaccine administration of the present invention, the vaccine is administered to pigs, and both the primer vaccine and the booster vaccine are administered by intradermal injection. In alternative embodiments, the primer vaccine is administered by intradermal injection and the booster vaccine is administered by a

[0069] different route. In a further aspect, the present invention provides a method for manufacturing a vaccine of the present invention, the method comprising mixing an alpha virus RNA replicon particle encoding an antigen derived from an animal pathogen with an oil adjuvant. Both the alpha virus RNA replicon particle and the oil

[0070] adjuvant are mixed in an immunologically effective amount. In a preferred embodiment of the method for manufacturing a vaccine of the present invention, one or more or all of the vaccine, the alpha virus RNA replicon particle, the encoded antigen, the animal pathogen and the oil adjuvant are as defined in any one

[0071] or more of the embodiments described herein. In one embodiment of the method for manufacturing a vaccine of the present invention, the alpha virus RNA replicon

[0072] In a preferred embodiment of the method for producing the vaccine of the present invention, the alphavirus R NA replicon particles, an aqueous solution containing alphavirus RNA replicon particles respectively, and the oil adjuvant are mixed in a volume ratio of 1:10 to 10:1, more preferably in a volume ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1 or even 1:2 to 2:1 so that the mixing is carried out in such a volume ratio. Most preferably, the alphavirus RNA replicon particles, an aqueous solution containing alphavirus RNA replicon particles respectively, and the oil adjuvant are mixed in a volume ratio of about 1:1.

[0073] In one embodiment, the method for producing the vaccine of the present invention includes mixing the contents of the containers of the kit of parts of the present invention.

[0074] In one embodiment of the method for producing the vaccine of the present invention, the alphavirus RNA re plicon particles, an aqueous solution containing alphavirus RNA replicon particles respectively, are mixed with the oil adjuvant contained in another O / W emulsion containing the antigen of the pathogen defined for the present invention. Preferably, the other O / W emulsion is a vaccine containing inactivated virus and / or bacterial pathogen. In a more preferred embodiment, RP encoding an antigen derived from SIV or PEDV is mixed with an O / W emulsion vaccine containing porcine circovirus (PCV) and / or Mycoplasma hyopneumoniae such as Circumvent® PCVM.

[0075] In a further aspect, the present invention relates to a vaccine for preventing or treating an infectious disease or disorder caused by an animal pathogen alpha-antigens encoding antigens derived from animal pathogens for use in the protection of animals The present invention provides a method for producing an alphavirus RNA replicon particle comprising the steps of: The drug is administered simultaneously or in parallel with an oil-based adjuvant into the body of the target animal. Alphavirus RNA replicon particles and oil-based adjuvants When used to protect an animal, both are included in an immunologically effective amount.

[0076] Preferred embodiments of alphavirus RNA replicons for use in the protection of the present invention In, alphavirus RNA replicon particles, encoded antigens, animal pathogens and oils One or more or all of the sexual adjuvants may be any one or more of those described herein. As defined in the embodiment.

[0077] Preferred embodiments of alphavirus RNA replicons for use in the protection of the present invention So, use includes use of the vaccine of the present invention.

[0078] In one embodiment of an alphavirus RNA replicon for use in the protection of the present invention, Protection is effective against target animals of different ages and species.

[0079] In one embodiment, the use is for the protection of young animals. Preferably, for pigs up to 3 weeks old, or chickens up to 1 week old, or chickens up to 14 months old It is a fish of the salmon family.

[0080] In a further embodiment, the use is for the protection of adolescent animals. The animals in the adult stage are pigs aged 3 weeks to 8 months, or chickens aged 1 to 22 weeks, or or a salmonid fish at 14 to 24 months of age.

[0081] In a further embodiment, the use is for the defense of adult animals. Preferably, the adult animal is a pig at 8 months of age or older, or a chicken at 22 weeks of age or older, or a salmonid fish at 24 months of age or older.

[0082] The preferred period for using the present invention for the defense of tilapia is usually not by age, but is represented by indicating the range of the whole body weight; vaccination by medicated bath treatment is preferably carried out when the weight of tilapia is 0.5 g to 5 g. Vaccination by parenteral injection is preferably carried out when the weight of tilapia is 10 g to 100 g, and more preferably when the weight of tilapia is 20 g to 25 g.

[0083] In one embodiment of the alphavirus RNA replicon for use in the defense of the present invention, the target animal can be positive or negative in serum reaction against an antibody to an animal pathogen or against each of the antigens derived from the animal pathogen.

[0084] In one embodiment of the alphavirus RNA replicon for use in the defense of the present invention, the target animal is an animal positive for MDA (maternally derived antibodies), whereby MDA reacts with the animal pathogen of the target animal for which the defense is intended. More preferably, the animal positive for MDA is a bird, a ruminant or a pig. Even more preferably, the animal positive for MDA is a pig.

[0085] ​​One embodiment of an alphavirus RNA replicon for use in the defense of the present invention In this case, the target animal is a pregnant animal. More preferably, the pregnant animal is a ruminant or a pig. Even more preferably, the pregnant animal is a pig.

[0086] One embodiment of an alphavirus RNA replicon for use in the defense of the present invention In this case, the defense is for livestock animals. Preferably, the livestock animal is a pig raised for fattening or a broiler or laying hen, or a ruminant raised for milk or meat production, or a salmon, or a tilapia.

[0087] In a further embodiment, the defense is for animals for breeding a population. Preferably , the animals for breeding a population are the parental or grandparental lines of livestock animals.

[0088] In a further aspect, the present invention provides the use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for the manufacture of a vaccine for the defense of animals against an infection or disease caused by the animal pathogen, including co-use or concurrent use with an oil adjuvant of the alphavirus RNA replicon particle. Both the alphavirus RNA replicon particle and the oil adjuvant are used in immunologically effective amounts.

[0089] In a preferred embodiment of the use for manufacturing the vaccine of the present invention, one or more or all of the alphavirus R NA replicon particle, the encoded antigen, the animal pathogen and the oil adjuvant are as defined in any one or more of the embodiments described herein ​​​​​​

[0090] In a further aspect, the present invention relates to a kit-of-parts for the invention as defined herein. Alphaviruses encoding antigens derived from animal pathogens for the production of components The kit provides for the use of RNA replicon particles, whereby the components of the kit can be integrated together. The simultaneous or concurrent use of Alphavirus RNA replicon particles for protecting animals against disease. Both the aqueous and oily adjuvants are used in immunologically effective amounts.

[0091] In a preferred embodiment of the use for the manufacture of a component of the kit-of-parts of the present invention, Rufavirus RNA replicon particle, encoded antigen, animal pathogen, oil-based adjuvant One or more or all of the kits and kits of parts may be as described herein. As defined in any one or more embodiments.

[0092] In a further aspect, the present invention provides a method for treating an infection or disease caused by an animal pathogen. Alphavirus R encoding antigens derived from animal pathogens for protection of animals infected with the virus The present invention provides a method for the preparation of an alphavirus RNA replicon particle, comprising: The use of precon particles in combination with or in parallel with an oil-based adjuvant. Both the RNA replicon particles and the oil-based adjuvant are used in immunologically effective amounts. do.

[0093] In a preferred embodiment of the use of the present invention for the protection of animals, the alphavirus RNA One or more of the following: a precon particle, an encoded antigen, an animal pathogen, and an oil-based adjuvant. or all of them are as defined in any one or more of the embodiments described herein is the case.

[0094] These and other aspects of the present invention will be better understood with reference to the following figures and detailed description. will be better understood.

Brief Description of the Drawings

[0095]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0096] The present invention provides a vaccine encoding one or more antigens of an animal pathogen and comprising an immunologically effective amount of one or more alphavirus RNA replicon particles and an oil adjuvant. The alphavirus RNA replicon particles are substantially similar to live viruses in that they can infect the target human or animal host cells and can express the genes they contain. This is also demonstrated by the fact that RPs are generally quantified by infectivity-titration on cells. Therefore, such RPs are pharmaceutical viral vectors that can be used as vaccines. infection-susceptible and can express the genes they contain. This is also demonstrated by the fact that RPs are generally quantified by infectivity-titration on cells. Therefore, such RPs are pharmaceutical viral vectors that can be used as vaccines. infection-susceptible and can express the genes they contain. This is also demonstrated by the fact that RPs are generally quantified by infectivity-titration on cells. Therefore, such RPs are pharmaceutical viral vectors that can be used as vaccines. is a single component of a generally effective vaccine in an acceptable carrier and is a live (attenuated) virus vaccine similar to that. Some vaccines based on non - adjuvant RP have been developed and commercialized.

[0097] Adjuvants are mainly used in combination only with killed bacteria or subunit vaccine antigens. Furthermore, oily adjuvants can be extremely active against other vaccine components, so that oily adjuvants generally are not combined with live vaccines. Furthermore, vaccines of alphavirus RNA RP, especially those based on VEE alphavirus are known to induce existing strong antiviral responses from the target innate immune system alone, compared to the adaptive immune response. Thus, it effectively prevents any need for additional immune stimulation.

[0098] Nevertheless, surprisingly, it has been found that oily adjuvants can significantly enhance the immunogenic effect of alphavirus RNA replicon particles encoding antigens derived from animal pathogens. This is in contrast to aluminum - based adjuvants such as aluminum hydroxide. When RP and oily adjuvants are applied, an enhancing effect can be obtained both when combined in a single composition (i.e., used simultaneously) or when administered as separate compositions (i.e., used in parallel). When compared to the case without (oily) adjuvant, vaccination with RP and oily adjuvant can reduce the minimum effective dose of RP by orders of magnitude, so that the oily adjuvant enables a significant reduction in the minimum effective dose of RP. The degree of enhancement by this could not be predicted either. Furthermore, by using an oily adjuvant it was possible to increase the duration of the immune response by inoculating the RP vaccine . Furthermore, by combining an oily adjuvant and RP, it was possible to provide an excellent immune response even when RP components alone did not induce any immunity .

[0099] To more fully understand the present invention, the following definitions are provided.

[0100] "Vaccine" is a well-known composition having a medical effect and includes an immunologically active component and a pharmaceutically acceptable carrier. An aqueous solution and / or an oily adjuvant can function as a "carrier" for the vaccine. For the vaccine of the present invention , the "immunologically active component" is a coded antigen derived from an animal pathogen which is delivered and expressed using RP. The vaccine stimulates the immune system of the vaccinated target animal and induces a defensive immunological response. The response may be derived from the innate immune system and / or the acquired immune system of the animal and may be of the cellular type and / or the humoral type .

[0101] The vaccine provides "protection against" an "infection or disease" by, for example, reducing the number of pathogens, or shortening the period during which the pathogen multiplies in or on the animal, and reducing the number, intensity or severity of the lesions caused by the infection or invasion, thereby reducing the severity of subsequent infections or invasions in the vaccinated animal. Furthermore or as a result, such infections, invasions or replications, and also is effective in reducing or ameliorating the (clinical) symptoms due to a disease that can be caused by a targeted response to its infection, invasion or replication. References to such diseases and clinical signs are found in “The Merck veterinary manual” (10th ed., 2010, C.M. Kahn edt., ISBN: 091191093X). Such vaccines are colloquially referred to as vaccines “against” a particular pathogen. When used herein, the term “comprising” (and variations such as “comprise”, “comprises” and “comprised”) refers to all elements within the scope of, or included in, a portion of text, paragraph, claim etc. in which the term is used, whether or not such elements or combinations are explicitly listed, and to any optional possibilities contemplated for the present invention, and does not refer to the exclusion of any such element(s) or combination(s). Therefore, any portion of text, paragraph, claim etc. is also

[0102]

[0103] When used herein, the term “replicon” refers to one or more elements (e.g., structural proteins) in a cell culture or animal host that enable the normal growth of the parental virus. ​​​​​​​​​​​​​​​ If there are coding sequences for the proteins), the modified RNAs lacking them refers to the viral genome. In appropriate cellular contexts, the replicon can amplify itself and produce one or more subgenomic RNA species.

[0104] As used herein, the term "alpha virus RNA replicon particle", abbreviated as "RP", is an RNA replicon derived from an alpha virus, such as capsid and glycoprotein, packaged within viral structural proteins, and is also derived from an alpha virus as described, for example, by Pushko et al. [Virology 239(2):389-401(1997) . RP infects appropriate target cells and then expresses the inserted heterologous gene(s), but cannot be propagated (without a helper plasmid or similar component) in cell culture or an animal host because the replicon does not encode alpha virus structural components (e.g., capsid and viral glycoproteins). RP that "encodes" an antigen refers to the transcription of the nucleic acid and / or the translation of the nucleic acid for the protein antigen contained in the RP, resulting in the expressed protein antigen in cell culture or an animal host, (without a helper plasmid or similar component), because the replicon does not encode alpha virus structural components (e.g., capsid and viral glycoproteins). .

[0105] The use of singular terms for convenience of explanation is not intended to limit in that way. Thus, for example, a reference to "alpha virus RNA replicon particle" includes references to such multiple alpha virus RNA replicon particles, unless otherwise indicated. .

[0106] RP that "encodes" an antigen refers to the transcription of the nucleic acid and / or the translation of the nucleic acid for the protein antigen contained in the RP, resulting in the expressed protein antigen producing the expressed protein antigen . Generally, such nucleic acids encoding a protein are open reading frames ( ORFs), meaning that there are no unwanted stop codons that can prematurely terminate protein translation. The nucleic acid may be a complete gene encoding a complete protein or a gene fragment, which may encode, for example, only the mature or secreted form of the protein, i.e., a part of the protein without a "leader", "anchor" or "signal sequence". The nucleotide sequence may be of natural or synthetic origin. The construction and manipulation of heterologous nucleic acid sequences expressing the antigen for the present invention can be carried out by well-known molecular biological techniques including cloning, transfection, recombination, selection and amplification. Such techniques and other techniques are described in "Molecular cloning: a laboratory manual" by Sambrook & Russell

[0107] (2001, Cold Spring Harbour Laboratory Press; ISBN: 0879695773), "Current Protocols in Molecular Biology" by Ausubel et al. (J. Wiley and Sons Inc., NY, 2003, ISBN: 04715033 8X), "PCR primers: a laboratory manual" by C. Dieffenbach & G. Dveksler (CSHL Press, ISBN 0879696540) and "Molecular Biology, Essential Techniques" by J. Bartlett and D. Stirling (2003, Oxford University Press, ISBN: 0199254947). (Humana press, ISBN: 0896036421) such as "PCR pr otocols" are described in more detail in standard texts.

[0108] In the present invention, "protein" is a molecular chain of amino acids. Proteins can be natural or mature proteins, pre- or pro-proteins, or parts of proteins. For example, peptides, oligopeptides, and polypeptides are included within the definition of proteins. For example, peptides, oligopeptides, and polypeptides are included within the definition of proteins. For example, peptides, oligopeptides, and polypeptides are included within the definition of proteins. are included.

[0109] The "antigen" in the present invention refers to a protein that can induce a defensive immune response in a target animal within an appropriate environment. The "antigen" in the present invention refers to a protein that can induce a defensive immune response in a target animal within an appropriate environment.

[0110] The terms "originate from", "originates from", and "originating from" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it. As used herein, these terms indicate that the amino acid sequence of the given protein antigen, unmodified and / or modified, is encoded by that pathogen or strain of pathogen. The coding sequence within the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may be genetically engineered to modify, shorten, and / or extend the amino acid sequence of the expressed protein antigen related to the corresponding coding sequence of that protein antigen within the pathogen or strain of pathogen (including naturally attenuated strains). inates from", and "originating fr om" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it. As used herein, these terms indicate that the amino acid sequence of the given protein antigen, unmodified and / or modified, is encoded by that pathogen or strain of pathogen. The coding sequence within the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may be genetically engineered to modify, shorten, and / or extend the amino acid sequence of the expressed protein antigen related to the corresponding coding sequence of that protein antigen within the pathogen or strain of pathogen (including naturally attenuated strains). om" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it. As used herein, these terms indicate that the amino acid sequence of the given protein antigen, unmodified and / or modified, is encoded by that pathogen or strain of pathogen. The coding sequence within the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may be genetically engineered to modify, shorten, and / or extend the amino acid sequence of the expressed protein antigen related to the corresponding coding sequence of that protein antigen within the pathogen or strain of pathogen (including naturally attenuated strains). om" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it. As used herein, these terms indicate that the amino acid sequence of the given protein antigen, unmodified and / or modified, is encoded by that pathogen or strain of pathogen. The coding sequence within the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may be genetically engineered to modify, shorten, and / or extend the amino acid sequence of the expressed protein antigen related to the corresponding coding sequence of that protein antigen within the pathogen or strain of pathogen (including naturally attenuated strains). om" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it. As used herein, these terms indicate that the amino acid sequence of the given protein antigen, unmodified and / or modified, is encoded by that pathogen or strain of pathogen. The coding sequence within the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may be genetically engineered to modify, shorten, and / or extend the amino acid sequence of the expressed protein antigen related to the corresponding coding sequence of that protein antigen within the pathogen or strain of pathogen (including naturally attenuated strains). om" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it. As used herein, these terms indicate that the amino acid sequence of the given protein antigen, unmodified and / or modified, is encoded by that pathogen or strain of pathogen. The coding sequence within the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may be genetically engineered to modify, shorten, and / or extend the amino acid sequence of the expressed protein antigen related to the corresponding coding sequence of that protein antigen within the pathogen or strain of pathogen (including naturally attenuated strains). om" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it. As used herein, these terms indicate that the amino acid sequence of the given protein antigen, unmodified and / or modified, is encoded by that pathogen or strain of pathogen. The coding sequence within the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may be genetically engineered to modify, shorten, and / or extend the amino acid sequence of the expressed protein antigen related to the corresponding coding sequence of that protein antigen within the pathogen or strain of pathogen (including naturally attenuated strains). om" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it. As used herein, these terms indicate that the amino acid sequence of the given protein antigen, unmodified and / or modified, is encoded by that pathogen or strain of pathogen. The coding sequence within the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may be genetically engineered to modify, shorten, and / or extend the amino acid sequence of the expressed protein antigen related to the corresponding coding sequence of that protein antigen within the pathogen or strain of pathogen (including naturally attenuated strains). om" are used interchangeably with respect to a given protein antigen and the pathogen or strain of pathogen that naturally encodes it. As used herein, these terms indicate that the amino acid sequence of the given protein antigen, unmodified and / or modified, is encoded by that pathogen or strain of pathogen. The coding sequence within the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may be genetically engineered to modify, shorten, and / or extend the amino acid sequence of the expressed protein antigen related to the corresponding coding sequence of that protein antigen within the pathogen or strain of pathogen (including naturally attenuated strains).

[0111] "Animal pathogens" include pathogens that are present in veterinary medicine, such as wild animals, livestock animals, or companion animals. Any organism capable of causing infection and / or disease in the relevant animal Refers to the target entity.

[0112] In the present invention, the animal pathogen is a natural pathogen of the target animal receiving the vaccine of the present invention. But it's not necessary.

[0113] "Oil" is used herein in a general sense and is typically a relatively viscous liquid. A non-polar, hydrophobic, and lipophilic material having a relatively high hydrocarbon content, with a density lighter than water. Oils can be of mineral or mineral origin, such as synthetic, semi-synthetic, animal or vegetable origin. can be of "non-mineral" origin. Some of the oils are metabolizable.

[0114] The term "mineral" indicates that the respective oil is derived from a mineral source, typically petroleum. show.

[0115] "Semi-synthetic oil" means an oil that is non-mineral in origin, such as an animal or vegetable oil, but which They are modified in structure and / or composition by chemical or physical processes.

[0116] The term "adjuvant" is used herein to refer to an agent that stimulates an immune response in a target animal in a non-specific manner. It is used in the composition in a general sense where possible.

[0117] "Liquid paraffin oil" is a type of mineral oil also known as white (mineral) oil. or light liquid paraffin oil, which has the CAS number 8042-47-5. They are commonly available and of pharmaceutical grade quality. Examples include Drakeol (registered trademark) Recorded trademarks) 6VR (Penreco), Marcol (registered trademark) 52 (Exxon Mo bile) and Klearol (registered trademark) (Sonneborn).

[0118] "Vitamin E - acetate" refers to a chemical compound having the CAS number 58 - 95 - 7 . Some other names are tocopheryl acetate or alpha - tocopherol - acetate . Vitamin E - acetate is the acetate ester of vitamin E (tocopherol) and can be derived from plant materials such as seeds, nuts, fruits or leaves, or from fats , but may also be manufactured synthetically. Therefore, the definition of vitamin E - acetate includes natural, synthetic or semi - synthetic forms, or mixtures thereof. Vitamin E - acetate is commercially available in different degrees of purity.

[0119] "Squalane" refers to a chemical compound having the CAS number 111 - 01 - 3. Some other names are hydrogenated shark liver oil, hexamethyltetracosane or perhydrosqualene . This is a polyunsaturated C30 oil and should not be confused with squalene (CAS number 111 - 02 - 4) which is a compound of the cholesterol pathway and is metabolizable.

[0120] Originally, the precursor of squalane was obtained from shark liver, but due to environmental concerns , this has shifted to other natural sources such as olive oil or to chemical synthesis. Therefore, the definition of squalane includes natural, synthetic or semi - synthetic forms, or mixtures thereof. Squalane is, for example, derived from plant sources, from Worlee (Squalane, plant) or Cro da (Pripure Squalane), or from, for example, Kuraray (Squ It is commercially available in various purities from compositions by alane-PE). In the present invention, high-purity squalane is preferred. In order of preference, preferably a purity of more than 75%, more preferably 80, 90 or even more preferably a purity of more than 95%.

[0121] An "emulsion" is a mixture of at least two immiscible liquids, whereby one is dispersed in the other. Typically, the droplets of the dispersed phase are less than micrometers in size and are very small. In the present invention, the emulsion comprises an oil phase and an aqueous phase.

[0122] Procedures and equipment for preparing emulsions on any scale are well known in the art, for example, in handbooks such as "Remington: the science and prac tice of pharmacy" (2000, Lippincot, USA, ISB N: 683306472) and "Veterinary vaccinology" ( P. Pastoret et al. ed., 1997, Elsevier, Amste rdam, ISBN 0444819681). .

[0123] When the oily adjuvant in the present invention is an emulsion, the emulsion can be a water-in-oil ( W / O) emulsion, where the oil is the continuous outer phase. Alternatively, the emul sion can be an "oil-in-water" (O / W) emulsion, where the oil is the dispersed inner phase.

[0124] By selecting a suitable type and concentration of emulsifier(s), such emulsions can be formed and maintained stably.

[0125] The emulsifier is present at the position which is the interfacial phase between water and oil and stabilizes the droplets of the internal dispersed phase. Many different emulsifiers are known and are suitable for pharmaceutical uses such as vaccines. A preferred emulsifier for the oily adjuvant of the present invention is polysorbate 80, which is also known as polyoxyethylene sorbitan monooleate and is commercially available as Tween (registered trademark) 80. Tween 80 is used in the oily adjuvant for the present invention in an amount of 0.1 to 10% w / v.

[0126] In the present invention, when in the form of an O / W emulsion, the oily adjuvant therefore consists of an outer aqueous phase and a dispersed internal oil phase. This facilitates the mixing of the RP encoding the antigen derived from the animal pathogen in the present invention with the oily adjuvant as an O / W emulsion. For example, by mixing an aqueous composition containing RP with the oily adjuvant O / W emulsion. Then, with a simple hand stirring over about 1 minute, the two aqueous compositions are properly mixed well.

[0127] Alternatively and highly advantageously, the oily adjuvant as an O / W emulsion can be used directly to reconstitute RP from the lyophilized form. This means that RP can be provided in a highly stable form as a lyophilized product and the vaccine of the present invention can be prepared by mixing at the site at a convenient time before administration to the target animal.

[0128] As used herein, the term "about" means the term "approx is used interchangeably with “approximately” and the value is within 50 percent of the stated value indicating that a composition containing “approximately” 1 ×10^8 alpha virus RNA replicon particles per milliliter contains 5×10^7 to 1.5×10^8 alpha virus RNA replicon particles per milliliter .

[0129] An example of an O / W emulsion oil adjuvant for use in the vaccines of the present invention is XSolv e (trademark). XSolve is a combination of two O / W emulsion adjuvant components, Diluvac Forte (trademark) based on vitamin E acetate (see European Patent No. 38 2,271), and Micr osol (trademark) based on liquid paraffin oil (see International Publication No. 2009 / 144,088).

[0130] In such emulsions, the volume average size of the oil droplets, whether mineral oil or non-mineral oil, may be different . Preferably, the droplets of mineral oil are submicron in size

[0131] Advantageously, the oil adjuvant emulsion is prepared separately from the RP of the present invention . Therefore, when the RP is present in the oil adjuvant, methods and equipment for emulsifying an oil adjuvant that is compatible with maintaining its properties can be used. An example is the high shear emulsification method used to obtain submicron emulsions by high pressure homogenization such as that of a Microfluidics processor (Microfluidics, MA, USA).

[0132] ​​​A further example of an O / W emulsion oil adjuvant for use in the vaccines of the present invention is SVE A™, which contains squalane and vitamin E-acetate and is described in International Publication No. 2018 / 115,435.

[0133] In the present invention, names of microorganisms or pathogens such as, for example, Venezuelan equine encephalitis virus (VEE) and avian influenza virus refer to each taxonomic division of the aforementioned microorganisms that is currently applicable. However, such names may change over time as new insights may reclassify them into new or different taxa. Nevertheless, this does not change the microorganisms themselves and the antigen repertoire, but only the scientific names or classifications change, and the reclassified microorganisms continue to be within the scope of the present invention.

[0134] References to taxonomic families include any microorganism that is a species, subtype, variant, biotype, serotype, or genotype within that family.

[0135] In the present invention, "swine" refers to animals of the family Suidae, preferably, for example, wild or domestic swine, wild boars, babirusas, or warthogs of the genus Sus. This also includes swine indicated by any name referring to their sex, age, or size (e.g., female swine, male swine, domestic swine, young female swine, weaned swine, or piglets).

[0136] As used herein, the term "avian" refers to, for example, chickens, turkeys, ducks, geese, quails, peacocks, pheasants, pigeons, partridges, butterflies, or damselflies ​​​​​​​​​​ Refers to agriculturally relevant birds such as chickens. Preferably, the birds are chickens, turkeys, ducks, geese or ostriches. More preferably, the birds are chickens or turkeys. Most preferably, the birds are chickens.

[0137] The birds can be of any kind, such as laying hens, breeding hens, broilers, hybrids or parental lines of any such varieties. A preferred type of bird is a broiler.

[0138] As used herein, the term "tilapia" includes those that are of the family Cichlidae and have fins among nearly a hundred species of bony fish. Tilapia are mainly freshwater fish that inhabit shallow streams, ponds, rivers and lakes and are rarely found in brackish water.

[0139] The "kit of parts" for the present invention is typically a container packed and combined with a predetermined amount of a specific composition, and this kit may include or refer to instructions for practicing the preparation and vaccination of the present invention.

[0140] The vaccines, immunization methods and compounds and uses of the present invention for protecting animals are for animals that need to be vaccinated against an infectious disease or disorder caused by a specific pathogen from which the encoded antigen is derived. It is clearly preferable to vaccinate healthy, non-infected targets and to vaccinate as early as possible, although age, weight, sex, immunological status and other parameters of the target to be vaccinated / protected / immunized are not important.

[0141] As used herein, a "phylogenetic cluster" refers to a phylogenetic tree or a set of the same (homologous) A series of influenza viruses that have been grouped together (on the same branch) on the evolutionary tree dating back to their ancestors It is a viral neuraminidase. IAV-S neuraminidase discovered in the United States ( Regarding N1, there are two dominant phylogenetic clusters, N1-classical and N1 - pandemic, as well as the two dominant phylogenetic clusters of N2, N2-19 The N1 classical phylogenetic cluster consists of H1N1, N2-2002 and N2-2003. It contains NA that has been classified together with NA derived from classical swine influenza viruses. The N1 pandemic phylogenetic cluster is the H1N1 pandemic influenza virus Contains NAs classified together with NAs originating from N2-1998 phylogenetic cluster. - is a strain of N derived from a human H3N2 influenza virus that infected pigs in 1998. The N2-2002 phylogenetic cluster contains NA, which is classified with A, whereas the N2-2002 phylogenetic cluster contains 20 Along with NA, which is derived from the human H3N2 influenza virus that infected pigs in 2002, Contains classified NA. [Anderson et al., Influenza and other Respiratory Viruses 7(Suppl.4) :42-51(2013).

[0142] The term "non-IAV-S" means that the respective pathogen and / or antigen (or immunogen) is not an IAV -S is neither a pathogen nor an IAV-S antigen (immunogen), and is a non-IAV-S protein antigen (also Pathogens and / or antigens (also immunogens) that indicate that the virus is not derived from IAV-S The term is used to modify terms such as antigen-specific antigen (e.g., antigen-specific antigen or immunogen).

[0143] As used herein, a multivalent vaccine is a vaccine that contains two or more different antigens such that the differences can be at any of many biological levels such as genus, species, serotype, etc. In a specific embodiment of this type, the multivalent vaccine stimulates the immune system of the target animal against two or more different animal pathogens or variants of the same pathogen that are immunologically heterogeneous isotypes.

[0144] 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 the term is used to describe an excipient in a pharmaceutical vaccine, the excipient is characterized as being compatible with the other components of the composition and not having any toxicity such that it is disadvantageous to the intended recipient animal (e.g., a pig).

[0145] "Administration" of the vaccine, which is a component of each kit of the present invention, to an animal target can be carried out using any practicable method and route. Typically, the optimal method of administration will be determined by the type of vaccine / compound being applied, the characteristics of the target, and the disease for which protection is intended such that different administration techniques may be applicable depending on how the vaccine / compound is formulated. For example, in the case of the O / W emulsion vaccine / compound of the present invention, it can be administered via the enteral or mucosal route, i.e., as eye drops, nasal drops, oral preparations, enteric preparations, oro-nasal drops, sprays. Other options include mass administration methods such as via drinking water, wide-area spraying, atomization, taking food, etc. is to be mediated.

[0146] "Parenteral administration" includes subcutaneous injection, submucosal injection, intravenous injection, intramuscular injection, intradermal injection and also infusion.

[0147] "Mucosal administration" includes ocular, nasal, oral, ocular-nasal, intratracheal, intraintestinal, anal and intravaginal administration routes and the like.

[0148] "Topical administration" includes skin and transdermal administration routes.

[0149] The method, timing and volume of administering each component of the vaccine and the kit of the present invention are preferably unified with the vaccination schedule of other existing vaccinations that the target animal may need for the purpose of reducing the stress of the target and reducing labor costs. These other immunizations themselves can be administered by related usage methods in a manner compatible with the registered uses. methods.

[0150] As used herein, the term "antigen fragment" with respect to a specific protein (e.g., protein antigen) is a fragment of a protein that is antigenic, i.e., can specifically interact with antigen recognition molecules of the immune system such as immunoglobulins (antibodies) or T cell antigen receptors. For example, the IAV-S neuraminidase (NA) antigen fragment is a fragment of the antigenic NA protein. Preferably, the antigen fragment of the present invention is immunodominant with respect to the recognition of antibodies and / or T cell receptors. In a specific embodiment, the antigen fragment with respect to a given protein antigen retains at least 25% of the antigenicity of the full-length protein. In a preferred embodiment the antigen fragment retains at least 50% of the antigenicity of the full-length protein. In a more preferred embodiment In an embodiment, the antigen fragment retains at least 75% of the antigenicity of the full-length protein. The antigen fragment can be as small as about 12 amino acids, or on the other hand, can be a large fragment that is missing as little as a single amino acid from the full length protein. In a specific embodiment, the antigen fragment contains 25 to 150 amino acid residues. In other embodiments, the antigen fragment contains 50 to 250 amino acid residues.

[0151] As used herein, an amino acid sequence is 100% "identical" or has 100% "identity" to a second amino acid sequence if the amino acid residues of both sequences are identical to each other. Thus, if 50% of the amino acid residues of two amino acid sequences are identical to each other, an amino acid sequence is 50% "identical" to a second amino acid sequence. Sequence comparison is performed over adjacent blocks of amino acid residues contained by a given protein, such as a protein, or a portion of the polypeptide being compared. In a specific embodiment, selected deletions or insertions, which can otherwise vary the correspondence between two amino acid sequences, are considered.

[0152] As used herein, percent identity of nucleotide and amino acid sequences is , C, MacVector (MacVector, Inc. Cary, NC 27519 )、Vector NTI (Informax, Inc. MD)、Oxford Mol ecular Group PLC (1996), as well as the Clustal W algorithm using alignment default pa rameters and default parameters for identity can be determined using. Such commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively for example, the GCG Package v.7 (Gen etics Computer Group, Madison, Wisconsin) P ileup program can be used to perform an Advanced B last search under default filter conditions.

[0153] Although alphavirus RNA replicon particles are stored individually, if mixing with other vaccine components is intended prior to administration, the alphavirus RNA replicon particles can be stored in the same aqueous stabilizing solution as the solution of the other components, such as a buffer or a high sucrose solution. obtained.

[0154] The vaccines of the present invention can be readily administered by any standard "method of immunizing an animal". One skilled in the art will understand that the route of administration will be selected taking into account the target animal to be administered and the characteristics of the vaccine. Preferably, the vaccine composition is appropriately formulated for various target animals and routes of administration. target animals and routes of administration. In the present invention, a "subunit of a microorganism" can be a biological or synthetic molecule such as a protein, carbohydrate, lipopolysaccharide, lipid or nucleic acid molecule.

[0155] Further optimization of the vaccines, kits, methods or uses of the present invention is well within the reach of one skilled in the art. Generally this is for the purpose of further enhancing the provided immune defense and is

[0156] within the capabilities of those skilled in the art. Generally this is within the capabilities of those skilled in the art and is for the purpose of further enhancing the provided immune defense Including fine-tuning the effects of vaccination / immunization. This can be achieved by applying the dose, volume, adjuvant or antigen content of the substance to be administered, or by administration via different routes, methods or regimens. All of these are within the scope of the present invention.

[0157] Since such configurations, process steps and substances can be varied to some extent, the present invention is not limited to specific configurations, process steps, substances disclosed herein. It should also be understood that the scope of the present invention is limited only by the appended claims and their equivalents. Therefore, the technical terms used herein are for the purpose of describing only specific embodiments and are not intended to be limiting. [Table 1]

[0158] The following non-limiting examples are useful for providing a further understanding of the present invention, but do not mean to limit the effective scope of the present invention in any way.

[0159] [Examples] [Example 1] The oily adjuvant improves the magnitude and duration of the antibody response in pigs against alphavirus RNA replicon particles encoding porcine influenza virus hemagglutinin. Substances and Methods The HA gene insertion is subjected to sequence optimization (ATUM, CA, USA) modification to express the hemagglutinin (HA) gene, and the VEE replicon vector designed as such is as previously described. ​configured as such [for reference, incorporated herein by reference is U.S. Pat. No. 9,441,247B2]. The Tc-83-derived replicon vector "pVEK" [disclosed and described in U.S. Pat. No. 9,441,247B2] was digested with the restriction enzymes AscI and PacI. A DNA plasmid containing an open reading frame sequence with codon-optimized N1 or N2 gene having a 5' flanking sequence (5 '-GGCGCGCCGCACC-3') [SEQ ID NO: 1] and a 3' flanking sequence (5'-TTAATTAA-3') [SEQ ID NO: 2] was similarly digested with the restriction enzymes AscI and PacI. Then, a synthetic gene cassette set was ligated into the digested pVEK vector, and the resulting clones were re-named "pVHV-N1 -pandemic", "pVHV-N1-classical", "pVHV-N2-2002" and "pVHV-N2-1998". The "pVHV" vector nomenclature was selected to refer to a pVEK-derived replicon vector containing the cloned transgene cassette via the AscI and PacI sites at multiple cloning sites of pVEK as such.

[0160] According to the method described previously [for reference, incorporated herein by reference are U.S. Pat. No. 9,441,247B2 and U.S. Pat. No. 8,460,913B2], the production of TC-83 RNA replicon particles was carried out. Briefly, prior to in vitro transcription using the MegaScrip t (trademark) T7 RNA polymerase and a cap analog (Promega, Madison, WI), the pVHV replicon vector DNA and The call helper DNA plasmid was linearized with NotI restriction enzyme. Importantly , the helper RNA used for production lacks the VEE subgenomic pro motor sequence as previously described [Kamrud et al., J Gen Virol. 9 1(Pt 7):1723-1727(2010)]. The purified RNA for the replicon and helper components was combined and mixed with a suspension of Vero cells and electroporated in a 4 mm cuvette and then returned to OptiPro™ SFM cell medium (Thermo Fisher, Walt am, MA). After overnight incubation, alphavirus RNA repl icon particles were purified and formulated in phosphate-buffered saline with 5% w / v sucrose and 1% v / v porcine serum and sterilized by passing through a 0.22 micron membrane filter and dispersed into aliquots for storage. The titer of functional RP was determined by infection immunofluorescence assay on a Vero cell monolayer. The batches of RP were identified by the antigen encoded by the packaged replicon.

[0161] Ten piglets negative for antibodies against porcine influenza virus were randomized into groups of five pigs. An RP vaccine expressing the hemagglutinin antigen of the H3N2 porcine influenza strain was prepared at a titer of 5×10^5 RP / dose. Immediately before vaccination, the vaccine of the RP-only group was diluted 1:1 (v / v) with sterile PBS diluent, while the vaccine of the RP+adjuvant group was diluted 1 :1 (v / v) with XSolve adjuvant. Then, the pigs were vaccinated intramuscularly with 2.0 mL of the appropriate substance. :1 (v / v). Thereafter, the pigs were vaccinated intramuscularly with 2.0 mL of the appropriate substance. ​​​​​Inoculated with Kutine. The vaccination process was carried out on a test schedule of 0 to 21 days, and at that time the vaccine was freshly prepared every time. The sera collected during the test were assayed for hemagglutination inhibition (HI) activity using the H3N2 swine influenza virus antigen. The results were reported as the maximum dilution with inhibitory activity, and titers less than 1:10 were reported as 1:9, and titers over 640 were reported as 1:641. The geometric mean titers are shown in Table 1.

Table 2

[0162] At a relatively low dose of 5×10^5 RP, the non - adjuvant vaccine induced a low - scale, short - term, transient HI titer. In comparison, the adjuvant vaccine induced high HI titers after the booster vaccine inoculation, and those HI titers remained elevated until the end of the 84 - day test.

[0163] This discovery first clearly showed that XSolve can have a very significant effect in swine RP immunization at a very low dose (5×10^5 RP). Previous studies using RP vaccines without adjuvants had to use very high amounts of RP components per dose, which was, for example, 1×10^9 for FMD RP and 5×10^7 for SIV RP.

[0164] [Example 2] The oil - based adjuvant improves the magnitude and duration of the antibody response in pigs against the multivalent alphavirus RNA replicon particles encoding the swine influenza virus antigen. A group of piglets separated from their mothers, negative for swine influenza, is shown in Table 2 from the group that was negative for swine influenza.​ Randomized into treatment groups as follows. Multivalent alpha virus RNA replicon particle vaccines for swine influenza were formulated with eight individual RNA particle antigens, each expressing either HA or NA from various strains of swine influenza, at approximately 1 x 10^7 RP / dose. Two H3, four H1, one N1, and one N2 antigens were included, and a pair of serological assays (HI or NI) were used to evaluate the antibody response to each antigen. The study was conducted as a replicated design, with half of the animals challenged with H1N1 virus and the other half [Table 3]

[0165] The use of XSolve oil adjuvant was found to significantly increase the magnitude of the serum response to all eight vaccine fractions. Both vaccine formulations protected against lung lesions (Figures 1A - 1B), but the addition of XSolve improved the vaccine efficacy as measured by nasal shedding of H1N1 and H1N2 influenza viruses (Figures 1C - 1D). Figures 1 - 1F represent the corresponding NI titer scores. The effect

[0166] of adjuvant on HI titers is shown in Figures 2A - 2F. This study demonstrated that non - adjuvant multivalent SIV RP induced antibodies against all fractions, but the addition of an oil adjuvant or represents an important clinical advantage related to restricting the horizontal spread of infectious diseases within a population Yes.

[0167] [Example 3] Effect of a four - adjuvant - combined NA - RP vaccine against H1N1 infection in weaned pigs with N1 antibodies at the time of the first vaccination Effect of a four - adjuvant - combined NA - RP vaccine Effect and immunogenicity at two dose levels of a four - adjuvant - combined NA - RP vaccine To determine this, a vaccination - challenge test was conducted. The adjuvant vaccine contains four RP constructs, each of which individually encodes a different NA gene from modern U.S. IAV - S isolates Along with these NA genes, two N1 phylogenetic clusters (N1 - classical and N1 - pandemic) and two N 2 clusters (N2 - 1998 and N2 - 2002) are represented (see Table 3). Weaned pigs that were antibody - positive for N1 - classical antigen at the time of the first vaccination were administered the adjuvant vaccine by intramuscular (IM) vaccination at 1 m L / dose. The effect of the four - adjuvant - combined NA - RP vaccine was tested against a heterologous N1 (H1N1 virus challenge infection).

[0168] Substances and Methods Construction of NA - RP antigen: Alpha - virus RNA replicon particles (RP) defective in replication encode the neuraminidase (NA) gene and were prepared essentially as described in Example 1 of this specification This was done.

Table 4

[0169] Virus The challenge virus was obtained from the USDA's National Veterinary Services Laboratories. A / Swine / Illinois / A01554351 / 2015 (H1N1) carries the HA gene of the H1-gamma cluster and the NA gene of the N1-classical cluster. The virus was propagated in MDCK cell culture. Confluent cells were infected for approximately 48 hours until a cytopathic effect was evident in more than 70% of the cell monolayer. At the time of collection, the supernatant was removed from the container and clarified by centrifugation before storing the virus frozen.

[0170] Animal Weaned piglets were selected from a highly healthy herd based on serum screening to confirm the absence of existing HI or NI (neuraminidase inhibition) antibodies against the vaccine and the challenge strain. The animals were a mixture of males and females and were approximately 3 weeks old at the time of the first vaccination.

[0171] Vaccination and Challenge The treatment groups are summarized in Table 4. The quadrivalent NA-RP vaccine was formulated at 10^6 copies per RP / dose, which was based on an immunofluorescence-based potency assay to quantify functional RP. The NA-RP antigen was formulated in a stabilizer consisting of 1% porcine serum and 5% sucrose. The placebo vaccine consisted of the same stabilizer but did not contain antigen. The vaccine was administered IM to pigs at 3 and 7 weeks of age immediately prior to administration by the XSolve oil adjuvant (1:1 v / v, 1 mL). route immediately before administration to pigs at 3 and 7 weeks of age with the XSolve oil adjuvant (1:1 v / v, 1 mL). quantity) and mixed. The dosage level was determined by performing an IFA test on the vaccine substance stored after vaccination. Serum samples were collected on the day of the first vaccination, the day of the second vaccination, and the day of challenge infection. All pigs were weighed one day before the first vaccination, and pigs in the N1-classical antibody-positive group were subcutaneously injected with 2 mL / kg of N1-classical hyperimmune serum (anti-NI antibody titer of 1:2560 against N1-classical antigen). The challenge infection was administered to the pigs three weeks after the second vaccination. The challenge substance for H1N1 (H1-gamma-N1-classical) was formulated to a target dosage of 10^6.5 TCID50 / pig (6 mL volume). The challenge substance was administered via the intratracheal route. The challenge virus dosage was confirmed by reverse titration of the stored challenge substance. Nasal swabs were collected from all pigs at the time points of the day before challenge, 1 day, 3 days, and 5 days after challenge.

[0172] All pigs were weighed one day before the first vaccination, and pigs in the N1-classical antibody-positive group were subcutaneously injected with 2 mL / kg of N1-classical hyperimmune serum (anti-NI antibody titer of 1:2560 against N1-classical antigen). quantity) and mixed. The dosage level was determined by performing an IFA test on the vaccine substance stored after vaccination. Serum samples were collected on the day of the first vaccination, the day of the second vaccination, and the day of challenge infection. quantity) and mixed. The dosage level was determined by performing an IFA test on the vaccine substance stored after vaccination. Serum samples were collected on the day of the first vaccination, the day of the second vaccination, and the day of challenge infection.

Table 5

[0173] The challenge infection was administered to the pigs three weeks after the second vaccination. The challenge substance for H1N1 (H1-gamma-N1-classical) was formulated to a target dosage of 10^6.5 TCID50 / pig (6 mL volume). The challenge substance was administered via the intratracheal route. The challenge virus dosage was confirmed by reverse titration of the stored challenge substance. Nasal swabs were collected from all pigs at the time points of the day before challenge, 1 day, 3 days, and 5 days after challenge. The challenge infection was administered to the pigs three weeks after the second vaccination. The challenge substance for H1N1 (H1-gamma-N1-classical) was formulated to a target dosage of 10^6.5 TCID50 / pig (6 mL volume). The challenge substance was administered via the intratracheal route. The challenge virus dosage was confirmed by reverse titration of the stored challenge substance. Nasal swabs were collected from all pigs at the time points of the day before challenge, 1 day, 3 days, and 5 days after challenge. The challenge infection was administered to the pigs three weeks after the second vaccination. The challenge substance for H1N1 (H1-gamma-N1-classical) was formulated to a target dosage of 10^6.5 TCID50 / pig (6 mL volume). The challenge substance was administered via the intratracheal route. The challenge virus dosage was confirmed by reverse titration of the stored challenge substance. Nasal swabs were collected from all pigs at the time points of the day before challenge, 1 day, 3 days, and 5 days after challenge. The challenge infection was administered to the pigs three weeks after the second vaccination. The challenge substance for H1N1 (H1-gamma-N1-classical) was formulated to a target dosage of 10^6.5 TCID50 / pig (6 mL volume). The challenge substance was administered via the intratracheal route. The challenge virus dosage was confirmed by reverse titration of the stored challenge substance. Nasal swabs were collected from all pigs at the time points of the day before challenge, 1 day, 3 days, and 5 days after challenge. The challenge infection was administered to the pigs three weeks after the second vaccination. The challenge substance for H1N1 (H1-gamma-N1-classical) was formulated to a target dosage of 10^6.5 TCID50 / pig (6 mL volume). The challenge substance was administered via the intratracheal route. The challenge virus dosage was confirmed by reverse titration of the stored challenge substance. Nasal swabs were collected from all pigs at the time points of the day before challenge, 1 day, 3 days, and 5 days after challenge. The challenge infection was administered to the pigs three weeks after the second vaccination. The challenge substance for H1N1 (H1-gamma-N1-classical) was formulated to a target dosage of 10^6.5 TCID50 / pig (6 mL volume). The challenge substance was administered via the intratracheal route. The challenge virus dosage was confirmed by reverse titration of the stored challenge substance. Nasal swabs were collected from all pigs at the time points of the day before challenge, 1 day, 3 days, and 5 days after challenge.

[0174] Necropsy was performed 5 days after challenge. Under the supervision of a qualified veterinarian, the pigs were euthanized by overdose administration of barbiturates at 5 DPC. The lungs were collected and observed to record the surface area of each lobe invaded by grossly visible lesions, and a comprehensive percent lung lesion score was obtained. Bronchoalveolar lavage fluid and nasal swabs were collected from all pigs, and the virus titer was measured. Lung sections were collected for histopathological analysis of microscopic lesions. Necropsy was performed 5 days after challenge. Under the supervision of a qualified veterinarian, the pigs were euthanized by overdose administration of barbiturates at 5 DPC. The lungs were collected and observed to record the surface area of each lobe invaded by grossly visible lesions, and a comprehensive percent lung lesion score was obtained. Bronchoalveolar lavage fluid and nasal swabs were collected from all pigs, and the virus titer was measured. Lung sections were collected for histopathological analysis of microscopic lesions. Necropsy was performed 5 days after challenge. Under the supervision of a qualified veterinarian, the pigs were euthanized by overdose administration of barbiturates at 5 DPC. The lungs were collected and observed to record the surface area of each lobe invaded by grossly visible lesions, and a comprehensive percent lung lesion score was obtained. Bronchoalveolar lavage fluid and nasal swabs were collected from all pigs, and the virus titer was measured. Lung sections were collected for histopathological analysis of microscopic lesions. Necropsy was performed 5 days after challenge. Under the supervision of a qualified veterinarian, the pigs were euthanized by overdose administration of barbiturates at 5 DPC. The lungs were collected and observed to record the surface area of each lobe invaded by grossly visible lesions, and a comprehensive percent lung lesion score was obtained. Bronchoalveolar lavage fluid and nasal swabs were collected from all pigs, and the virus titer was measured. Lung sections were collected for histopathological analysis of microscopic lesions. Necropsy was performed 5 days after challenge. Under the supervision of a qualified veterinarian, the pigs were euthanized by overdose administration of barbiturates at 5 DPC. The lungs were collected and observed to record the surface area of each lobe invaded by grossly visible lesions, and a comprehensive percent lung lesion score was obtained. Bronchoalveolar lavage fluid and nasal swabs were collected from all pigs, and the virus titer was measured. Lung sections were collected for histopathological analysis of microscopic lesions. Necropsy was performed 5 days after challenge. Under the supervision of a qualified veterinarian, the pigs were euthanized by overdose administration of barbiturates at 5 DPC. The lungs were collected and observed to record the surface area of each lobe invaded by grossly visible lesions, and a comprehensive percent lung lesion score was obtained. Bronchoalveolar lavage fluid and nasal swabs were collected from all pigs, and the virus titer was measured. Lung sections were collected for histopathological analysis of microscopic lesions.

[0175] Immune response analysis: IAV-S specific antibodies in porcine serum samples were determined by the NI test. The sera were heat-inactivated at 56 °C for 30 - 60 minutes. The NI test was performed with a slight modification of the method of Sandbulte & Eiche lberger (Methods Mol Biol 1161:337 - 45, 201 4). Briefly, two-fold serial dilutions of the sera were mixed on 96-well plates coated with fetuin at an equal amount to the expressed protein antigen and incubated overnight at 37 °C. Peanut agglutinin-horseradish peroxidase conjugate was added for 2 hours at room temperature to bind to the fetuin moiety from which sialic acid had been removed. Signals were obtained with a TMB chromogenic substrate and the results were read at 650 nm. The mean optical density (OD) of the negative control lacking the NA antigen was subtracted from all wells. Thereafter, the OD values of the test samples were normalized on a 0 - 100% scale, where the mean OD of the positive control well (containing the NA antigen but not the sera) was defined as 100%. The NI antibody titer was defined as the maximum dilution of the sample that inhibited more than 50% of the neuraminidase activity.

[0176] Lung pathological examination Macroscopically observable lesions (purple to pinkish, clearly demarcated infiltration shadows) observed on the outer sides of all lung lobes were recorded on grid diagrams of the anterior and posterior parts of the lungs. The overall score ([% lung lesions) for each pig was calculated according to the number of grids invaded by the lesions.

[0177] Virus excretion ​​​​​​​​Nasal swabs and BAL fluids were serially diluted 10-fold in infected medium [Dulbecco's Minimum Essential Medium (DMEM) supplemented with 0.3% bovine serum albumin (fraction V ), 2 mM L-glutamine, 25 μg / mL gentamicin, and 2 μg / mL trypsin IX], and 100 μ L of each dilution was added to quadruplicate wells of confluent MDCK cells in a 96-well plate. The plates were incubated at 37 °C in 5% CO2, and the presence of infectious virus was observed after 72 h by hemagglutination assay of the supernatant from each well. The I AV-S titer was calculated using the Spearman-Karber method and expressed as Log10 TCID50 per mL.

[0178] Results The immune responses of pigs vaccinated with the multivalent NA-RP are shown in Figure 3. The following points are notable.

[0179] - Pigs passively primed with N1-classical hyperimmune serum on the day before the first vaccination had N1-classical antibody titers of 40 - 80 at the time of vaccination.

[0180] - After two vaccinations with a four-component mixed NA-RP vaccine with the XSolve adjuvant, both seronegative and N1-antibody serum-positive pigs showed a marked increase in their N1-classical antibody titers. This indicates that vaccination with RP vaccines containing oil adjuvants is effective even in target animals that are positive for antibodies against RP-encoded antigens.

[0181] - The NI titers of pigs in the N1-classical negative / placebo-vaccinated group remained seronegative. ​​​​​​​​​​

[0182] Regarding the lung lesions, the effect of the four-component NA-RP vaccine against the challenge infection is shown in Fig. 4. It should be noted that the vaccination with the four-component NA-RP vaccine significantly reduced the lung lesions in both groups of pigs vaccinated at their first vaccination, whether they had N1-classical antibodies or not. The percentage of lung lesions in both groups was significantly reduced compared to the group vaccinated with the placebo vaccine. When pigs with or without N1-classical antibodies were vaccinated, the four-component NA-RP vaccine was highly effective in reducing lung lesions in both cases. In both groups, the percentage of lung lesions was significantly reduced compared to the group vaccinated with the placebo vaccine.

[0183] [Example 4] The oil adjuvant improves the magnitude and duration of the antibody response in pigs against alphavirus RNA replicon particles encoding porcine epidemic diarrhea virus antigen compared to aluminum adjuvant or water. Nine piglets at about 3 weeks of age were randomly assigned to three groups of three animals each. An alphavirus RNA replicon particle vaccine expressing the spike glycoprotein of porcine epidemic diarrhea virus (PEDV) was prepared and lyophilized in 20-dose vials. On day 0 of the study, the pigs were vaccinated intramuscularly with 1.0 ml of the alphavirus RNA replicon particle vaccine rehydrated with either water, aluminum hydroxide adjuvant, or XSolve adjuvant. On day 21 of the study, the process was repeated with a new vial of the vaccine. The final titer of alphavirus RNA replicon particles per dose after rehydration was determined by immunofluorescence assay and was approximately 7×10^6 RP / dose for all groups. Serum collected during the study was assayed for PEDV neutralizing antibodies. See Table 5.

Table 6

[0184] Pigs treated with the non - adjuvant vaccine had low or undetectable neutralizing antibodies after two doses of the vaccine. Pigs treated with the aluminum hydroxide - adjuvanted vaccine all expressed neutralizing antibodies and were just above the detection limit. In contrast, the XSol ve - adjuvanted vaccine induced higher neutralizing antibodies after two doses of the vaccine, and all three pigs reached a minimum antibody titer higher than the peak titers observed in the other test groups. Therefore, the use of aluminum hydroxide as an adjuvant was not effective, and substantially, the oil - based adjuvant was more effective. ve titer effective and substantially, the oil - based adjuvant was more effective.

[0185] [Example 5] The oil - based adjuvant improves the vaccination effect of alphavirus RNA replicon particles encoding influenza antigens in chickens. improves the vaccination effect of alphavirus RNA replicon particles encoding influenza antigens in chickens. Newly hatched chicks were randomized into groups of 10 birds. An alphavirus RNA replicon particle vaccine expressing the hemagglutinin antigen of the H3N2 swine influenza strain was prepared at a titer of 1 × 10^8 RP / dose. Immediately before vaccination, the vaccine for the group receiving only RP was diluted 1:1 (v / v) with sterile PBS diluent, while the vaccine for the group of alphavirus RNA replicon particles with adjuvant was diluted 1:1 (v / v) with X Solve adjuvant. Serum from the chicks was assayed for hemagglutination inhibition (HI) titers on days 0, 7, and 14 after vaccination. group adjuvant Solve ated assayed.

Table 7

[0186] In contrast to the non - adjuvant vaccine and placebo vaccine, addition of the XSolve adjuvant to the alphavirus RNA replicon particle vaccine induced a clear increase in the HI titer in birds after a single vaccination.

[0187] [Example 6] The oil - based adjuvant improves the effect of the alphavirus RNA replicon particle vaccine expressing the IBDV antigen in chickens. This vaccine - inoculation challenge test examined the effect of the oil - based adjuvant on the vaccine - inoculation effect of the RP expressing the VP2 - 4 - 3 polyprotein antigen derived from the very virulent infectious bursal disease virus (IBDV) strain Faragher 52 / 70. The test animals were specific - pathogen - free (SPF) chickens that were antibody - negative to IBDV. The group sizes were 5 or 10 birds. Vaccination was performed subcutaneously at the day of age (hatch). One group (n = 5) was sham - vaccinated with phosphate - buffered saline (PBS). One vaccinated group received the RP vaccine in an aqueous buffer, and the other vaccinated groups received the RP vaccine mixed with the XSolve adjuvant at a ratio of 1:1. On the 28th day after vaccination, all groups were challenged with the highly virulent IBDV strain CS89 by the ocular route. At necropsy performed 10 days after challenge, the bursa - like tissues for the overall lesions were scored, and the vaccine - inoculation effect was monitored histopathologically according to well - known criteria

Table 8

[0188] By adding an oily adjuvant to the RP vaccine, the vaccination effect against a severe challenge infection that clearly improves the immune response of chickens from zero to complete protection was significantly effective. in the

[0189] [Example 7] The oily adjuvant also improves the effect of the alphavirus RNA replicon particle vaccine in fish An alphavirus RNA replicon particle vaccine expressing the major capsid protein of red sea bream iridovirus (RSIV) was prepared by the standard method as described herein. Tilapia were vaccinated intramuscularly with 0.05 ml of the vaccine and then challenged at different time points after vaccination. In this study, 30 fish were used for each treatment and each challenge time. Immediately before vaccination, the alphavirus RNA replicon particle vaccine was mixed with PBS or SVEA, a non-mineral oil adjuvant containing two substances, at a ratio of 1:1 (v / v). The titer of RP for each treatment was 1×10 ^7 RP / dose. Control fish were vaccinated with a placebo vaccine and then challenged at different time points after vaccination. In this study, 30 fish were used for each treatment and each challenge time. Immediately before vaccination, the alphavirus RNA replicon particle vaccine was mixed with PBS or SVEA, a non-mineral oil adjuvant containing two substances, at a ratio of 1:1 (v / v). The titer of RP for each treatment was 1×10 ^7 RP / dose. Control fish were vaccinated with a placebo vaccine ^7 RP / dose. Control fish were vaccinated with a placebo vaccine

Table 9

Table 10

[0190] For both the "3-week low-dose" and "6-week high-dose" challenges, the adjuvant ​​​​Survival was significantly improved in fish vaccinated with the RP vaccine compared to two other challenges and equivalent results were obtained with the other two treatments. Thus, the alphavirus RNA RP vaccine containing an adjuvant was significantly more effective than the adjuvant-free RP vaccine and provided effective vaccination using only a single-dose vaccination

[0191] [Example 8] Efficacy of simultaneous and concurrent use of RP and oil adjuvants Trials were conducted in pigs using a quadrivalent porcine influenza virus NA vaccine as described in Example 3. This was useful for showing the effect of using RP, a component of the vaccine and an oil adjuvant in different types

[0192] Substances and Methods Pigs were vaccinated at approximately 4 and 7 weeks of age. The quadrivalent NA vaccine was a mixture of the dual gene N1 and the dual gene N2 RP construct and was administered at approximately 2×10^6 RP / dose each. Each group consisted of 4 pigs. The composition of the test protocol is shown in Table 9. Group 2 received NA RP mixed with the XSolve adjuvant simultaneously

[0193] Groups 3 and 4 tested the effect of concurrent use of RP and an oil adjuvant; administration was performed on the same side of the neck (more than 5 cm apart) or on opposite sides of the neck within approximately 10 minutes of each other

[0194] ​​​​​​​One control group, group 5, used an RP encoding porcine influenza HA H1 antigen. This was administered at approximately 1×10^7 RP / dose. Serum was collected on the day of the first vaccination, the day of the second vaccination, and 7 days after the second vaccination. These sera were each tested on separate homologous NI assays for 4 NA types. administered.

[0195] The NI titers obtained were measured and are shown in FIGS. 5 and 6, where FIG. 5 represents the NI titers against the N1 classical NA antigen at 3 time points including the standard deviation measured over time. FIG. 6 represents the group mean NI titers of the 4 bound NA types at 7 days post - vaccination 2. The NI titers obtained were measured and are shown in FIGS. 5 and 6, where FIG. 5 represents the NI titers against the N1 classical NA antigen at 3 time points including the standard deviation measured over time. FIG. 6 represents the group mean NI titers of the 4 bound NA types at 7 days post - vaccination 2. assays for 4 NA types.

[0196] The NI titers obtained were measured and are shown in FIGS. 5 and 6, where FIG. 5 represents the NI titers against the N1 classical NA antigen at 3 time points including the standard deviation measured over time. FIG. 6 represents the group mean NI titers of the 4 bound NA types at 7 days post - vaccination 2. The NI titers obtained were measured and are shown in FIGS. 5 and 6, where FIG. 5 represents the NI titers against the N1 classical NA antigen at 3 time points including the standard deviation measured over time. FIG. 6 represents the group mean NI titers of the 4 bound NA types at 7 days post - vaccination 2. The NI titers obtained were measured and are shown in FIGS. 5 and 6, where FIG. 5 represents the NI titers against the N1 classical NA antigen at 3 time points including the standard deviation measured over time. FIG. 6 represents the group mean NI titers of the 4 bound NA types at 7 days post - vaccination 2. The NI titers obtained were measured and are shown in FIGS. 5 and 6, where FIG. 5 represents the NI titers against the N1 classical NA antigen at 3 time points including the standard deviation measured over time. FIG. 6 represents the group mean NI titers of the 4 bound NA types at 7 days post - vaccination 2. [Table 11]

[0197] Results and Conclusions From the results measured in Example 8, several effects could be observed: - The pattern of NI titers against the 4 NA types at the 3 measured time points was nearly identical, and thus the NA N1 classical results shown in FIG. 5 represent the titer patterns against the other 3 NA types. - The pattern of NI titers against the 4 NA types at the 3 measured time points was nearly identical, and thus the NA N1 classical results shown in FIG. 5 represent the titer patterns against the other 3 NA types. - The pattern of NI titers against the 4 NA types at the 3 measured time points was nearly identical, and thus the NA N1 classical results shown in FIG. 5 represent the titer patterns against the other 3 NA types.

[0198] - Furthermore, the pattern of NI titer responses against the 4 NA types at 7 dpv2 is mostly the same as shown in FIG. 6. - As expected, the NI titer results for the HA H1 RP control (group 5) were N

[0199] - As expected, the NI titer results for the HA H1 RP control (group 5) were N No titer was induced; thereby, a titer threshold for specific response is set.

[0200] - Except for some test variations, most of the NI titers in Groups 2-4 were significantly higher than the titers of the group that received only RP (Group 1) when receiving RP and an oil adjuvant. This indicates that the oil adjuvant strongly enhances immunization by RP. - The group that received RP and an oil adjuvant simultaneously (Group 2) confirmed the strong effect of the oil adjuvant already observed in the previous examples. - The immunization effect of using RP and an oil adjuvant in parallel (Groups 3-4) was in a state very close to that of simultaneous use and was a strong stimulation by the RP immune response. There was no clear influence due to the difference in the administration site.

[0201]

[0202]

[0203] The present invention should not be limited within the scope by specific embodiments as described herein. In fact, in addition to the changes as described herein, various changes of the present invention will be apparent to those skilled in the art from the foregoing description. Such changes are intended to be within the scope of the appended claims.

[0204] All base sizes or amino acid sizes, all molecular weights or molecular mass values given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. It will be further understood.

Claims

1. A vaccine comprising alphavirus RNA replicon particles encoding an antigen derived from an animal pathogen, wherein the vaccine further comprises an oily adjuvant, wherein the oily adjuvant comprises at least two non-mineral oils selected from the group consisting of squalane and tocopherol.

2. The vaccine according to claim 1, wherein the tocopherol is selected from the group consisting of vitamin E, vitamin E-acetate, and combinations thereof.

3. The vaccine according to claim 1 or 2, wherein the oily adjuvant comprises squalane and vitamin E-acetate.

4. The vaccine according to any one of claims 1 to 3, wherein the total amount of non-mineral oil in the oily adjuvant is 0.1 to 30% w / v of the oily adjuvant.

5. The vaccine according to any one of claims 1 to 4, wherein the oily adjuvant is formulated as an oil-in-water emulsion, and wherein the emulsion of the oily adjuvant comprises an emulsifier.

6. The vaccine according to any one of claims 1 to 5, wherein the alphavirus RNA replicon particles are Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles.

7. The vaccine according to any one of claims 1 to 6, wherein the animal pathogen is a pathogen of an animal that is a fish.

8. The vaccine according to claim 7, wherein the fish is a member of the Cichlidae family.

9. The vaccine according to claim 8, wherein the Cichlidae fish is tilapia.

10. A kit comprising at least two containers, wherein at least one container contains Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles encoding an antigen derived from an animal pathogen, and at least one container contains an oily adjuvant comprising at least two non-mineral oils selected from the group consisting of squalane and tocopherol. **Claim 11**: A method of immunizing a non-human animal, the method comprising administering an immunologically effective amount of a vaccine according to any one of claims 1 to 9. **Claim 12** The vaccine according to claim 1, for use in protecting a non-human animal against an infection or disease caused by an animal pathogen, wherein the alphavirus RNA replicon particles are administered into or onto the body of the target non-human animal by co-use or concurrent use with an oily adjuvant comprising at least two non-mineral oils selected from the group consisting of squalane and tocopherol. **Claim 13**: A method of immunizing a non-human animal, the method comprising administering an immunologically effective amount of a vaccine comprising Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particles encoding an antigen derived from an animal pathogen of the non-human animal into or onto the body of the non-human animal, wherein the alphavirus RNA replicon particles are administered into or onto the body of the non-human animal by co-use or concurrent use with an oily adjuvant comprising at least two non-mineral oils selected from the group consisting of squalane and tocopherol.

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