Vaccines with replicon particles and oil adjuvant

Abstract

The present invention relates to vaccination against animal pathogens using alphavirus-replicon RNA particles and an oil adjuvant. The invention also relates to vaccines and kits comprising the replicon particles and the oil adjuvant. The invention also relates to methods and uses of the components of the vaccine and the kits.

Description

Technical Field

[0001] The present invention relates to vaccination against animal pathogens using alphavirus-replicon RNA particles and an oil adjuvant. The invention also relates to vaccines and kits of parts comprising the replicon particles and the oil adjuvant. The invention also relates to methods and uses of the vaccines and components of the kits. Background Art

[0002] Over the years, a variety of vector strategies have been used in vaccines in an effort to provide protection against animal pathogens. One such vector strategy involves the use of alphavirus-derived replicon RNA particles (RPs) [Vander Veen et al., Anim Health Res Rev. 13(1):1-9 (2012) doi:10.1017 / S1466252312000011; Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)], which were developed from several different alphaviruses, including 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 [Veen et al., Anim Health Res Rev. 13(1):1-9 (2012) doi:10.1017 / S1466252312000011; Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)]. virus) [Liljestrom and Garoff, Biotechnology (NY) 9: 1356-361 (1991)]. The encoded pathogen antigen is expressed by the replicon particles after they infect human or animal target cells. The result is the induction of protective antibodies against the expressed antigen. When compared with some traditional vaccine formulations, RP has attractive safety and efficacy properties [Vander Veen et al., Anim Health Res Rev. 13 (1): 1-9 (2012)]. The RP platform is the basis of several USDA-licensed vaccines, including: porcine epidemic diarrhea vaccine, RNA particles (product code 19U5.P1), swine influenza vaccine, RNA (product code 19A5.D0), avian influenza vaccine, RNA (product code 19O5.D0) and a prescription product, RNA particles (product code 9PP0.00).

[0003] Alphavirus-derived replicon RNA particles lack alphavirus structural protein genes, but retain the replication elements necessary for self-amplification of cytoplasmic RNA and express the inserted heterologous nucleic acid under the drive of the highly active 26S alphavirus subgenomic promoter. Therefore, RP is a single-cycle infectious particle that is replication-defective due to the lack of structural protein genes. [Lundstrom, Vaccines 6:2392-2415 (2014)]. Therefore, the structural proteins necessary for packaging and production of replicon particles must be provided in a suitable host cell to produce RP [see Vajdy et al., Immunol. and Cell Biol. 82:617-627 (2004)]. Structural proteins are typically provided by transient co-transfection of replicon RNA and one or more "helper" RNAs encoding the structural proteins. Alternatively, RP can be produced by a packaging cell line that constitutively or transiently expresses viral structural proteins from one or more DNA expression cassettes. In this way, the replicon particles produced retain the replication-defective nature of the vector because the structural proteins are not included in the resulting RP genome [Polo et al., Dev. Biol., 104: 181–185 (2000)]. When used to immunize target humans or animals, these replication-defective alphavirus RNA replicon particles induce a protective immune response in vivo. For example, after systemic immunization of mice and large animals, VEE-based alphavirus vectors elicited strong mucosal and systemic immune responses [Davis et al., IUBMB Life 53: 209-211 (2002)].

[0004] Adjuvants are known compounds that provide nonspecific stimulation to the immune system of a target human or animal. The standard use of adjuvants is in vaccines based on inactivated or subunit antigens. There are many types of adjuvants and components, such as aluminum salts (aluminum hydroxide or aluminum phosphate), liposomes, dextran, alginates, bacterial components (such as cell wall components), mineral or non-mineral oils, synthetic adjuvants (such as nonionic block polymers), polyamines (such as dextran sulfate), carbopol TM , pyrans and saponins (such as Quil A TM or Q-vac TM ). Saponin and vaccine components can be combined in ISCOM TM middle.

[0005] In addition, peptides such as muramyl dipeptide, dimethylglycine, and tuftsin are often used as adjuvants. Similarly, combination products such as ISA TM composition (Seppic, France).

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

[0007] The citation of any reference herein should not be construed as an admission that such reference is available as "prior art" to the present application. Summary of the Invention

[0008] The present invention provides a vaccine comprising alphavirus RNA replicon particles encoding antigens derived from animal pathogens, wherein the vaccine further comprises an oil adjuvant.

[0009] In one embodiment of the vaccine of the present invention, the oil adjuvant comprises at least one oil selected from the group consisting of mineral oil and non-mineral oil.

[0010] In one embodiment of the vaccine of the present invention, the oil adjuvant comprises mineral oil; preferably, the mineral oil is liquid paraffin oil.

[0011] In one embodiment of the vaccine of the present invention, the oil adjuvant comprises a 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; more preferably, the alpha tocopherol is selected from vitamin E and vitamin E-acetate. In one embodiment, the vegetable oil is an oleate, more preferably ethyl oleate.

[0012] More preferably, the non-mineral oil is squalane.

[0013] In a preferred embodiment of the vaccine of the present invention, the oil adjuvant comprises more than one oil.

[0014] In one embodiment of the oil adjuvant comprising more than one oil, the adjuvant comprises mineral oil and one or more non-mineral oils. More preferably, the oil adjuvant comprises liquid paraffin oil as the mineral oil and one or more non-mineral oils selected from the group consisting of squalane, squalene, vitamin E, vitamin E-acetate, oleate, and ethyl oleate. More preferably, the oil adjuvant comprises liquid paraffin oil and vitamin E-acetate. Most preferably, the oil adjuvant is XSolve TM .

[0015] In an alternative embodiment of the oil adjuvant comprising one or more oils, the adjuvant comprises one or more non-mineral oils. Preferably, the oil adjuvant comprises one or more non-mineral oils selected from the group consisting of squalane, squalene, vitamin E, vitamin E-acetate, oleate, and ethyl oleate. More preferably, the oil adjuvant comprises squalane and vitamin E acetate. Most preferably, the oil adjuvant is SVEA. TM .

[0016] In one embodiment of the vaccine of the present invention, the amount of mineral oil in the oil adjuvant is 1-70% v / v of the oil adjuvant. Preferably, the amount of mineral oil contained in the oil adjuvant is 5-60% v / v of the oil adjuvant.

[0017] In one embodiment of the vaccine of the present invention, the total amount of non-mineral oil is 0.1-30% w / v of the oil adjuvant. Preferably, the total amount of non-mineral oil contained in the oil adjuvant is 0.5-25% v / v of the oil adjuvant.

[0018] In one embodiment, when the non-mineral oil comprises squalane, the oil adjuvant comprises 0.5-30% w / v of the oil adjuvant; more preferably, the oil adjuvant comprises 1-25% w / v, 2-15% w / v of squalane, or even 3-10% w / v of the oil adjuvant.

[0019] Alternatively or additionally, in one embodiment, when the non-mineral oil comprises vitamin E acetate, the oil adjuvant comprises vitamin E acetate at 0.1-30% w / v of the oil adjuvant; more preferably, the oil adjuvant comprises 0.5-20% w / v, 1-16% w / v vitamin E acetate, or even 2-10% w / v of the oil adjuvant.

[0020] In one embodiment of the vaccine of the present invention, the oil adjuvant is formulated into an emulsion of an oil phase and an aqueous phase. Preferably, the oil adjuvant is formulated into an oil-in-water (O / W) emulsion.

[0021] In one embodiment, the aqueous phase comprises water of pharmaceutically acceptable quality.

[0022] In one embodiment, the emulsion of oil adjuvant is formulated as a microemulsion in which the droplets of the internal phase are less than 1 micron. Preferably, the microemulsion is an O / W emulsion, more preferably, the O / W microemulsion is prepared using high energy homogenization, even more preferably, by microfluidization.

[0023] In one embodiment of the vaccine of the present invention, the emulsion of the oil adjuvant comprises an emulsifier, preferably the emulsifier comprises polysorbate, more preferably the emulsifier comprises polysorbate 80.

[0024] In one embodiment, the vaccine of the present invention comprises an emulsion of an oil adjuvant, preferably the vaccine comprises an oil adjuvant formulated as an O / W emulsion.

[0025] In one embodiment of the vaccine of the present invention, the vaccine is formulated as an oil-in-water (O / W) emulsion.

[0026] In one embodiment of the vaccine of the present invention, the alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle. In a more specific embodiment, the VEE alphavirus RNA replicon particle is a TC-83 VEE alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Sindbis alphavirus RNA replicon particle. In yet other embodiments, the alphavirus RNA replicon particle is a Semliki Forest virus alphavirus RNA replicon particle.

[0027] In one embodiment of the vaccine of the present invention, for the encoded antigen derived from an animal pathogen, the animal pathogen is selected from viruses, bacteria, parasites, protozoa, fungi, rickettsiae and prions. More preferably, the encoded antigen derived from an animal pathogen is an antigen derived from a virus or bacteria. Most preferably, the antigen is from a virus.

[0028] In one embodiment of the vaccine of the present invention, RP encodes an antigen derived from an animal pathogen, so the animal is an animal related to veterinary science. Preferably, the animal is selected from fish, poultry and mammals. More preferably, the animal is a wild animal, livestock or companion animal. Livestock animals are fish, poultry, pigs or ruminants; preferably, the porcine is a pig; preferably, the poultry is a chicken, turkey, duck, goose, quail or ostrich; preferably, the ruminant is a cow, sheep, goat, buffalo, camel or deer; preferably, the fish is a bony fin fish, more preferably a fish of the salmon family or the cichlid family (i.e., a member of the cichlid family). The fish of the salmon family is preferably selected from Atlantic salmon, steelhead salmon, Chinook salmon, coho salmon, pink salmon, dog salmon and sockeye salmon, rainbow trout, brook salmon, lake trout and brown trout, and salmon (char). The fish of the cichlid family is preferably tilapia. The companion animal is preferably selected from cats, dogs and horses. More preferably, the animal is tilapia, chicken or pig.

[0029] In one embodiment of the vaccine of the present invention, the nucleotide sequence encoding the antigen gene derived from the animal pathogen of the present invention is optimized so that it is expressed in the cells of the vaccine target animal species. In one embodiment, the nucleotide sequence optimization is codon optimization. In one embodiment, the nucleotide sequence optimization is the optimization of the secondary structure of the RNA transcript.

[0030] In a preferred embodiment, the nucleotide sequence encoding the antigen gene derived from the animal pathogen of the present invention is optimized taking into account codon usage and the secondary structure of the RNA transcript. Preferably, the nucleotide sequence is optimized according to the procedures described in one or more of US 7,561,972, US 7,561,973, US 7,805,252 and US 8,126,653.

[0031] In a particularly preferred embodiment of the vaccine of the present invention, the oil adjuvant comprises mineral oil and non-mineral oil, the vaccine is formulated as an O / W emulsion, the alphavirus RNA replicon particles are VEE alphavirus RNA replicon particles, the animal pathogen antigen is a viral antigen, and the virus is a porcine pathogen.

[0032] In a preferred embodiment of the vaccine of the present invention, the encoded antigen derived from an animal pathogen is an influenza virus hemagglutinin (HA) or neuraminidase (NA) protein, or an antigenic fragment of such HA or NA protein. The HA and / or NA protein is preferably derived from influenza A virus, more preferably from swine influenza A virus or PEDV.

[0033] The invention also provides multivalent vaccines comprising the alphavirus RNA replicon particles of the invention, wherein the vaccine comprises more than one RP encoding an antigen, or the vaccine comprises one or more RPs, each encoding one or more antigens of the invention.

[0034] The vaccines of the present invention comprise an immunologically effective amount of alphavirus RNA replicon particles of the present invention. In one embodiment, the vaccine comprises about 1 x 10^3 to about 1 x 10^11 RPs. In a more specific embodiment, the vaccine comprises about 1 x 10^4 to about 1 x 10^10 RPs. In an even more specific embodiment, the vaccine comprises about 1 x 10^5 to about 1 x 10^9 RPs.

[0035] Vaccine of the present invention comprises the oil adjuvant of the present invention of immune effective amount.In one embodiment, vaccine comprises the oil adjuvant of the amount that accounts for vaccine about 10%-90%v / v.More preferably, vaccine comprises the oil adjuvant of the amount that accounts for vaccine about 20%-80%v / v, 30-70%v / v or even 40-60%v / v.Most preferably, vaccine comprises the oil adjuvant of the amount that accounts for vaccine about 50%v / v.

[0036] In a specific embodiment, the vaccine of the present invention is administered in a volume of 0.05 mL to 5 mL per animal dose. In a more specific embodiment, the administered dose per animal is 0.1 mL to 2 mL. In an even more specific embodiment, the administered dose is 0.2 mL to 1.5 mL. In an even more specific embodiment, the administered dose is 0.3 to 1.0 mL. In an even more specific embodiment, the administered dose per animal is 0.4 mL to 0.8 mL.

[0037] In one embodiment of the vaccine of the present invention, the vaccine comprises other adjuvants. Preferably, the other adjuvants are selected from the following: bacterial cell wall components, cytokines and immunostimulatory nucleic acids containing unmethylated CpG. In one embodiment, the immunostimulatory nucleic acid is selected from one or more of the following: WO 2012 / 089.800 (X4 family), WO 2012 / 160.183 (X43 family) and WO 2012 / 160.184 (X23 family).

[0038] In one embodiment of the vaccine of the present invention, the vaccine comprises other antigens of animal pathogens. In a preferred embodiment, the other antigens are selected from the group consisting of live attenuated microorganisms, inactivated microorganisms and subunits of microorganisms.

[0039] In a further aspect, the present invention provides a kit comprising at least two containers, wherein at least one container comprises an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen, and at least one container comprises an oil adjuvant. The at least two containers each comprise an immunologically effective amount of the alphavirus RNA replicon particle or the oil adjuvant.

[0040] In a preferred embodiment of the kit of the present invention, one or more or all of the alphavirus RNA replicon particles, the encoded antigen, the animal pathogen and the oil adjuvant are as defined in any one or more of the embodiments described herein.

[0041] In a preferred embodiment, at least one container comprising RP comprises the RP in the form of a lyophilisate.

[0042] In an alternative embodiment, at least one container comprising RP comprises RP in an aqueous solution; the aqueous solution preferably comprises a buffer; the aqueous solution is preferably kept chilled or frozen. In one embodiment, the aqueous solution is a reconstituted RP solution produced by mixing a lyophilisate of RP with a suitable aqueous diluent.

[0043] In embodiments where at least one container comprises RP in lyophilisate form, the kit of the invention may comprise an additional container containing a suitable diluent for reconstitution of the lyophilized RP. In a preferred embodiment, the diluent is an aqueous solution, preferably comprising a buffer and / or stabilizer of pharmaceutically acceptable quality and water.

[0044] In a preferred embodiment, the container comprising the oil adjuvant comprises the oil adjuvant formulated in the form of an emulsion of oil and water phases; preferably the emulsion is an oil-in-water emulsion.

[0045] In an embodiment of the kit, the kit comprises instructions for use of the kit and / or its components. In a preferred embodiment, the instructions are provided on or with one or more components of the kit, or by reference to electronic instructions, such as information that can be viewed or downloaded from the internet website of the kit manufacturer or distributor.

[0046] In one embodiment, the kit comprises a box of at least two containers, and the instructions for use are displayed on the box or on an information carrier (eg, a card or instructions) within the box.

[0047] In one embodiment of the kit, the kit may also provide its components (in connection with commercial sales) on, for example, an internet website relating to the use of the immunization method of the invention.

[0048] In one embodiment of the kit, one or more containers may contain other adjuvants as described herein; similarly or alternatively, one or more containers may contain other antigens of animal pathogens as described herein.

[0049] The alphavirus RNA replicon and oil adjuvant, both encoding antigens derived from animal pathogens as defined herein, can be administered to a target animal. Such administration will induce effective immune protection in the animal against infection or disease caused by the animal pathogen. For example, administration can be performed according to the EMA-CVMP guidelines for use in connection with immunological veterinary medicinal products.

[0050] Thus, in a further aspect, the present invention provides a method of immunizing an animal comprising administering to the animal an immunologically effective amount of alphavirus RNA replicon particles encoding an antigen derived from an animal pathogen and an oil adjuvant.

[0051] In a preferred embodiment of the method for immunizing an animal of the present invention, the method comprises administering the vaccine of the present invention to the animal.

[0052] In a preferred embodiment of the method for immunizing animals of the present invention, one or more or all of the alphavirus RNA replicon particles, the encoded antigen, the animal pathogen and the oil adjuvant are as defined in any one or more of the embodiments described herein.

[0053] In a preferred embodiment of the method for immunizing animals of the present invention, the encoded antigen derived from an animal pathogen is an antigen derived from the following pathogens: fish, members of the family Cichlidae, tilapia, mammals, poultry and chicken.

[0054] In one embodiment of the method for immunizing an animal of the present invention, the alphavirus RNA replicon particles and the oil adjuvant are administered simultaneously or concurrently in or on the target animal.

[0055] In a preferred embodiment of the method of immunizing an animal of the present invention, the alphavirus RNA replicon particles and the oil adjuvant are administered simultaneously (ie, as a single composition) in or on the target animal.

[0056] In a preferred embodiment, the single composition is a vaccine of the present invention.

[0057] In a preferred embodiment, a single composition is prepared immediately prior to administration to a target animal by mixing a composition comprising RP, both as described herein, with a composition comprising an oil adjuvant; more preferably, by mixing the contents of a container of a kit of the present invention; even more preferably, by mixing an aqueous solution comprising RP with a composition comprising an oil adjuvant O / W emulsion. In an alternative, even more preferred embodiment, a single composition is prepared by reconstituting a lyophilisate of RP, both as defined herein, with an O / W emulsion of an oil adjuvant. Effectively, preparing a single composition yields the vaccine of the present invention.

[0058] Preferably, "immediately before administration to the target animal" is within 24 hours before administration to the target animal, more preferably within 16 hours, within 12 hours, within 8 hours, within 4 hours or even within 2 hours before administration to the target animal, in this order of preference.

[0059] In an alternative preferred embodiment of the method for immunizing animals of the present invention, the alphavirus RNA replicon particles and the oil adjuvant are administered in or on the target animal in a concurrent manner, i.e., contained in separate compositions that are administered separately in location and / or time.

[0060] In a preferred embodiment, concurrent use comprises administering the alphavirus RNA replicon particles and the oil adjuvant contained in the kit of the present invention in or on the target animal, but separately in location and / or time.

[0061] In preferred embodiments of the present invention for concurrent use, the separate compositions are administered to separate sites in or on the body of the target animal by the same or different routes of administration within a limited amount of time of each other; preferably, "limited amount of time" is 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 minutes of each other, in that order of preference. Most preferably, the concurrent use administration is substantially simultaneous.

[0062] In a preferred embodiment of the present invention for concurrent use, the separate compositions are administered to separate sites in or on the body of a target animal, by the same or different routes of administration, within a limited amount of time of each other. For the present invention, the separate sites of administration are separated from each other by at least 1 cm; preferably, by at least 2 cm, at least 5 cm, at least 10 cm, or even at least 25 cm, in that order of preference.

[0063] In a preferred embodiment of the present invention for concurrent use, the separate compositions are administered within a limited amount of time of each other in or on the body of a target animal, by the same or different routes of administration, at substantially the same location in or on the body of a target animal, but sufficiently separated in time from each other to prevent mixing of the compositions at the site of administration. For the present invention, sufficient separation in time to prevent mixing is not 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, not within 2 days of each other, or even not within 1 week of each other, in that order of preference.

[0064] In an embodiment of the method of immunizing an animal of the present invention, administration in or on the body of the target animal is performed by parenteral administration. In an alternative embodiment, administration is by mucosal administration. In yet another alternative embodiment, the vaccine is administered by topical administration.

[0065] The preferred administration method is selected from the group consisting of intradermal, intramuscular, intraperitoneal, subcutaneous, immersion and spray. The intradermal administration method is preferably administered by needle-free means, more preferably by using Device (intradermal application of liquid).

[0066] In one embodiment of using vaccine of the present invention, vaccine is used as initial exemption vaccine and / or as booster vaccine.In a specific embodiment, vaccine of the present invention is once (single injection) inoculation and is used, does not need subsequent booster administration.In some embodiments, when using initial exemption vaccine and booster vaccine, initial exemption vaccine and booster vaccine can be used with the same approach.In an alternative embodiment, when using initial exemption vaccine and booster vaccine, the use of initial exemption vaccine can be carried out by a kind of approach and booster vaccine can be used by another approach.

[0067] In certain embodiments of administering the vaccines of the present invention, the vaccines are administered to pigs and both the priming vaccine and the boosting vaccine are administered by intradermal injection. In an alternative embodiment, the priming vaccine is administered by intradermal injection and the boosting vaccine is administered by another route.

[0068] In a further embodiment, the present invention provides a method for producing the vaccine of the present invention, comprising the step of mixing alphavirus RNA replicon particles encoding an antigen derived from an animal pathogen with an oil adjuvant. The alphavirus RNA replicon particles are mixed with the oil adjuvant in an immunologically effective amount.

[0069] In a preferred embodiment of the method for producing a vaccine of the invention, one or more or all of the vaccine, alphavirus RNA replicon particles, encoded antigen, animal pathogen and oil adjuvant are as defined in any one or more of the embodiments described herein.

[0070] In one embodiment of the method for producing a vaccine of the invention, the alphavirus RNA replicon particles are contained in an aqueous solution.

[0071] In a preferred embodiment of the method for producing the vaccine of the present invention, mixing is performed such that the alphavirus RNA replicon particles, respectively, the aqueous solution containing the alphavirus RNA replicon particles, and the oil adjuvant are mixed in a volume ratio of 1:10 to 10:1; more preferably, in a volume ratio of between 1:5 and 5:1, between 1:4 and 4:1, between 1:3 and 3:1, or even between 1:2 and 2:1, in this order of preference. Most preferably, the alphavirus RNA replicon particles, respectively, the aqueous solution containing the alphavirus RNA replicon particles, and the oil adjuvant are mixed in a volume ratio of about 1:1.

[0072] In one embodiment, the method for producing the vaccine of the present invention comprises mixing the contents of the containers of the kit of the present invention.

[0073] In one embodiment of the method for producing a vaccine according to the invention, alphaviral RNA replicon particles, respectively an aqueous solution comprising the replicon particles, are mixed with an oil adjuvant as defined herein, said oil adjuvant being contained in another O / W emulsion containing pathogen antigens. Preferably, the other O / W emulsion is a vaccine comprising an inactivated viral and / or bacterial pathogen. In a more preferred embodiment, RP encoding antigens from SIV or from PEDV are mixed with an O / W emulsion vaccine comprising porcine circovirus (PCV) and / or Mycoplasma hyopneumoniae, such as PCVM.

[0074] In a further embodiment, the present invention provides an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for use in protecting an animal from infection or disease caused by the animal pathogen, wherein the alphavirus RNA replicon particle and an oil adjuvant are administered simultaneously or concurrently to or on the target animal. Immunologically effective amounts of both the alphavirus RNA replicon particle and the oil adjuvant are included in the use for protecting an animal.

[0075] In a preferred embodiment of the alphavirus RNA replicon for use in protection according to the present invention, one or more or all of the alphavirus RNA replicon particles, the encoded antigen, the animal pathogen and the oil adjuvant are as defined in any one or more of the embodiments described herein.

[0076] In a preferred embodiment of the invention wherein the alphavirus RNA replicon is used for protective purposes, the use comprises use as a vaccine according to the invention.

[0077] In one embodiment of the invention wherein the alphavirus RNA replicon is used for protective purposes, protection is effective in target animals of varying ages and types.

[0078] In one embodiment, the use is for protecting young animals. Preferably, the young animals are pigs up to 3 weeks old, or chickens up to 1 week old, or salmon up to 14 months old.

[0079] In a further embodiment, the use is for protecting young animals. Preferably, the young animals are pigs between 3 weeks and 8 months old, or chickens between 1 week and 22 weeks old, or salmon between 14 months and 24 months old.

[0080] In a further embodiment, the use is for protecting an adult animal. Preferably, the adult animal is a pig over 8 months old, or a chicken over 22 weeks old, or a salmon over 24 months old.

[0081] For tilapia, the preferred period for the protection purposes of the present invention is generally not expressed by age, but rather by the entire weight range: when the tilapia weighs between 0.5 g and 5 g, it is preferably immunized by immersion treatment. When the tilapia weighs between 10 g and 100 g, and more preferably when the tilapia weighs between 20 g and 25 g, it is preferably immunized by parenteral injection.

[0082] In one embodiment of the invention where the alphavirus RNA replicon is used for protective purposes, the target animal may be seropositive or seronegative for antibodies to the animal pathogen or, in the case of an animal pathogen, for antigens from the animal pathogen, respectively.

[0083] In one embodiment of the present invention, wherein the alphavirus RNA replicon is used for protective purposes, the target animal is an MDA (maternally derived antibody)-positive animal, whereby the MDA reacts with the animal pathogen being protected. More preferably, the MDA-positive animal is poultry, ruminants, or pigs. Even more preferably, the MDA-positive animal is porcine.

[0084] In one embodiment of the present invention, the target animal is a pregnant animal. More preferably, the pregnant animal is a ruminant or a pig. Still more preferably, the pregnant animal is a pig.

[0085] In one embodiment of the invention, the alphavirus RNA replicon is used for protection purposes, the protection is to protect production animals. Preferably, the production 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.

[0086] In a further embodiment, the protection is protection of restocking animals. Preferably, the animals used to restock the population are the parental or grand-parental lines of producing animals.

[0087] In a further embodiment, the present invention provides a use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for preparing a vaccine to protect an animal from infection or disease caused by the animal pathogen, comprising administering the alphavirus RNA replicon particle simultaneously or concurrently with an oil adjuvant. The alphavirus RNA replicon particle and the oil adjuvant are both administered in an immunologically effective amount.

[0088] In a preferred embodiment of the use for preparing a vaccine of the present invention, one or more or all of the alphavirus RNA replicon particles, the encoded antigen, the animal pathogen and the oil adjuvant are as defined in any one or more of the embodiments described herein.

[0089] In a further embodiment, the present invention provides the use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for the preparation of components of a kit as defined herein for protecting an animal from infection or disease caused by the animal pathogen by simultaneous or concurrent use of the components of the kit. The alphavirus RNA replicon particle and the oil adjuvant are both used in an immunologically effective amount.

[0090] In a preferred embodiment of the use for preparing the kit components of the present invention, one or more or all of the alphavirus RNA replicon particles, the encoded antigen, the animal pathogen, the oil adjuvant and the kit are as defined in any one or more of the embodiments described herein.

[0091] In a further embodiment, the present invention provides a use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for protecting an animal from infection or disease caused by the animal pathogen, wherein the use comprises administering the alphavirus RNA replicon particle simultaneously or concurrently with an oil adjuvant. The alphavirus RNA replicon particle and the oil adjuvant are both administered in an immunologically effective amount.

[0092] In a preferred embodiment of the use of the present invention for protecting animals, one or more or all of the alphavirus RNA replicon particles, the encoded antigen, the animal pathogen and the oil adjuvant are as defined in any one or more of the embodiments described herein.

[0093] These and other aspects of the invention will be better understood with reference to the following drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 and Figure 2 : The results in Example 2:

[0095] Figures 1A-1B : Lung lesions

[0096] Figures 1C-1D : Nose loss

[0097] Figures 1E-1F :NI titer

[0098] Figure 2 A-2F: HI titer results (Figures are numbered from left to right, first the top row, then the bottom row)

[0099] Figure 3 : Serum antibody responses against N1-classical antigen in pigs vaccinated with polyvalent NA-RP in Example 3:

[0100] Shown are serum neuraminidase inhibition (NI) antibody responses specific for the N1 classical strain of the vaccine composition as described in Example 3. Serum samples were collected before the first immunization (3 weeks of age), before the second immunization (7 weeks of age), and before challenge (10 weeks of age).

[0101] Figure 4 : Efficacy of the 4-way NA-RP vaccine against challenge infection as determined in Example 3, involving macroscopic lung lesion scoring 5 days after infection with H1N1 virus:

[0102] Shown are lung lesion scores of pigs administered the immune composition described in Example 3 and challenged with H1-γ-N1-classical virus.

[0103] Figure 5 and 6 : NI titer results in Example 8:

[0104] Figure 5 NI titers against the N1 classical NA antigen at three time points measured over time are shown, including standard deviations. This represents the NI titer profile measured against the other three NA types.

[0105] Figure 6 Shown are group mean NI titers of the four NA types combined 7 days after the second immunization. DETAILED DESCRIPTION

[0106] The present invention provides a vaccine comprising an immunologically effective amount of one or more alphavirus RNA replicon particles encoding one or more antigens derived from animal pathogens and an oil adjuvant. Alphavirus RNA replicon particles are in fact similar to live viruses in that they are able to infect target human or animal host cells and express the genes they contain. This is also evidenced by the fact that RP is typically quantified by cell infectivity titration. Therefore, similar to live (attenuated) virus vaccines, such RP in a pharmaceutically acceptable carrier is typically the only component of an effective vaccine. Several vaccines based on unadjuvanted RP have been developed and commercialized.

[0107] Adjuvants are primarily used in combination with inactivated or subunit vaccine antigens. Furthermore, oil adjuvants can be very aggressive towards other vaccine components and are therefore not typically used in combination with live vaccines. Furthermore, known alphavirus RNA RP vaccines (particularly those based on VEE alphaviruses) are known to induce a powerful antiviral response from the target's innate immune system itself, following the acquired immune response. Thus, any need for additional immune stimulation is effectively prevented.

[0108] However, surprisingly, it was found that oil adjuvants can significantly enhance the immunogenicity of alphavirus RNA replicon particles encoding antigens derived from animal pathogens. This is different from aluminum-based adjuvants (such as aluminum hydroxide). When RP and oil adjuvant are combined into a single composition (i.e., used simultaneously), or when they are used as separate compositions (i.e., used in parallel), enhanced efficacy can be obtained. The extent to which oil adjuvants enhance efficacy is also unexpected, because compared to vaccines without (oil) adjuvants, immunization using RP and oil adjuvants can reduce the minimum effective dose of RP by several orders of magnitude. In addition, the use of oil adjuvants can increase the duration of the immune response of RP immunizations. Moreover, when the individual RP components do not cause immunity at all, the combination of oil adjuvant and RP can provide an excellent immune response.

[0109] In order that the present invention may be more fully understood, the following definitions are provided.

[0110] A "vaccine" is a well-known composition with medical effects that contains an immunologically active component and a pharmaceutically acceptable carrier. Aqueous solutions and / or oil adjuvants can serve as the "carrier" for the vaccine. The "immunologically active component" of the vaccine of the present invention is an encoded antigen derived from an animal pathogen, which is delivered and expressed via RP. The vaccine stimulates the immune system of the target animal to be vaccinated, inducing a protective immune response. The response can originate from the animal's innate and / or acquired immune systems and can be cellular and / or humoral.

[0111] Vaccine can provide " protection " by reducing the severity of subsequent infection or infection of inoculation animal " for infection or disease ", for example, by reducing the quantity of pathogen, or shortening the persistent period that pathogen replicates in animal body or on animal, and reducing the quantity, intensity or the severity that damage is caused by infection or infection.And, or accordingly, vaccine effectively reduces or alleviates and may be caused by this type of infection, infection or replication, or by target to the (clinical) symptom of the disease caused by the response of infection, infection or replication.The reference book of this type of disease and clinical signs is " Merck Veterinary Manual " (10th edition, 2010, CM Kahn writes, ISBN:091191093X).This vaccine is popularly referred to as: " vaccine for " specific pathogen.

[0112] As used herein, the term "comprising (and its variants comprise, comprises, comprised)" refers to all elements, and in any possible combination conceivable by the present invention, which are covered or included in the text portion, paragraph, claim, etc. in which the term is used, even if such elements or combinations are not explicitly recited; and is not intended to exclude any such elements or combinations. Therefore, any such text portion, paragraph, claim, etc. may also refer to one or more embodiments in which the term "comprising" (or its variants) is replaced by a term such as "consist of, consisting of", or "consisting essentially of".

[0113] As used herein, the term "replicon" refers to a modified RNA viral genome that lacks one or more elements (e.g., coding sequences for structural proteins) that, if present, would enable the parent virus to successfully propagate in cell culture or an animal host. In a suitable cellular environment, the replicon will amplify itself and can produce one or more subgenomic RNA species.

[0114] As used herein, the term "alphavirus RNA replicon particle" (abbreviated as "RP") is an alphavirus-derived replicon packaged in viral structural proteins (e.g., capsid and glycoproteins) also derived from an alphavirus, as described, for example, in Pushko et al. [Virology 239(2):389-401 (1997)]. RP infects appropriate target cells and then expresses one or more inserted heterologous genes, but cannot be propagated in cell culture or animal hosts (in the absence of helper plasmids or similar components) because the replicon does not encode alphavirus structural components (e.g., capsid and viral glycoproteins).

[0115] For ease of description, the use of singular terms is in no way intended to be limiting. Thus, for example, reference to an "alphavirus RNA replicon particle" includes reference to a plurality of such alphavirus RNA replicon particles unless otherwise specified.

[0116] RP "encodes" an antigen means that the transcription and / or translation of the nucleic acid contained in the RP for the protein antigen results in the expression of the protein antigen. Typically, such nucleic acids encoding proteins are open reading frames (ORFs), indicating the absence of undesired stop codons that would prematurely terminate protein translation. The nucleic acid may be a full gene encoding an intact protein, or it may be a gene fragment encoding a portion of a protein, e.g., encoding only the mature or secreted form of the protein, i.e., without a "leader sequence," "anchor sequence," or "signal sequence." The nucleotide sequence may be of natural or synthetic origin.

[0117] Heterologous nucleic acid sequences expressing the antigens of the present invention can be constructed and manipulated by well-known molecular biology techniques, including cloning, transfection, recombination, selection, and amplification. These and other techniques are described in great detail in standard textbooks, such as Sambrook & Russell: "Molecular cloning: a laboratory manual" (2001, Cold Spring Harbour Laboratory Press; ISBN: 0879695773); Ausubel et al., Current Protocols in Molecular Biology (J. Wiley and Sons Inc., NY, 2003, ISBN: 047150338X); C. Dieffenbach & G. Dveksler: "PCR primers: a laboratory manual" (CSHL Press, ISBN 0879696540); and J. Bartlett and D. Stirling, "PCR protocols" (Humana press, ISBN: 0896036421).

[0118] For purposes of the present invention, a "protein" is a molecular chain of amino acids. A protein can be a native or mature protein, a pre- or pro-protein, or a portion of a protein. Peptides, oligopeptides, and polypeptides are included within the definition of protein.

[0119] For the purposes of the present invention, an "antigen" refers to a protein that is capable of inducing a protective immune response in a target animal under appropriate circumstances.

[0120] With respect to a given protein antigen and a pathogen or strain of the pathogen that naturally encodes it, the terms "originate from; originates from; originating from" can be used interchangeably. As used herein, these terms refer to the unmodified and / or modified amino acid sequence of a given protein antigen encoded by the pathogen or strain of the pathogen. The coding sequence in the nucleic acid construct of the present invention for a protein antigen derived from a pathogen may have been genetically manipulated to result in the expressed protein antigen having amino acid sequence modifications, truncations and / or extensions relative to the corresponding coding sequence of the protein antigen in the pathogen or pathogen strain (including naturally attenuated strains) from which it is derived.

[0121] "Animal pathogen" refers to any biological entity capable of causing infection and / or disease in animals of veterinary relevance, such as wildlife, livestock, or companion animals.

[0122] For purposes of the present invention, the animal pathogen may or may not be a natural pathogen of the target animal receiving the vaccine of the present invention.

[0123] "Oil" is used herein in its ordinary sense and refers to a non-polar chemical substance having a relatively high hydrocarbon content, which is generally a relatively viscous liquid, has a density lighter than water, and is hydrophobic and lipophilic. Oils can be of mineral origin, or of "non-mineral" origin, such as synthetic, semi-synthetic, animal, or plant origin. Some oils are metabolizable.

[0124] The term "mineral" indicates that the corresponding oil is derived from a mineral source, typically petroleum.

[0125] "Semi-synthetic oils" are oils of non-mineral origin, such as animal or vegetable oils, but whose structure and / or composition have been modified by chemical or physical methods.

[0126] The term "adjuvant" is used herein in its ordinary sense to refer to a composition capable of stimulating an immune response in a target animal in a nonspecific manner.

[0127] "Liquid paraffin oil" is a type of mineral oil, also known as white (mineral) oil or light liquid paraffin oil, CAS number: 8042-47-5. It is also commonly available in pharmaceutical grade quality. Examples are: 6VR (Penreco), 52 (Exxon Mobile) and (Sonneborn).

[0128] "Vitamin E acetate" refers to the chemical compound with the CAS number 58-95-7. Some alternative names are: tocopheryl acetate or alpha tocopheryl acetate. Vitamin E acetate is the acetate ester of vitamin E (tocopherol), which can be derived from plant materials such as seeds, nuts, fruits or leaves, or from fat, but can also be produced 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 varying degrees of purity.

[0129] "Squalane" refers to the chemical compound with the CAS number 111-01-3. Some alternative names include hydrogenated shark liver oil, hexamethyltetracosane, or perhydrosqualene. It should not be confused with squalene (CAS number 111-02-4), a polyunsaturated C30 oil that is metabolized as a cholesterol pathway compound.

[0130] The precursor of squalane was originally obtained from shark liver, but due to environmental concerns, it has been turned to other natural sources such as olive oil or chemical synthesis. Therefore, the definition of squalane includes natural, synthetic or semi-synthetic forms, or mixtures thereof. Squalane is commercially available in various purities, for example: from plant sources, such as Worlee (Squalane, Plant) or Croda (Pripure Squalane) or synthetically, such as from Kuraray (Squalane-PE). For the present invention, high-purity squalane is preferred: preferably with a purity greater than 75%, more preferably with a purity greater than 80, 90 or even greater than 95%, in this order of preference.

[0131] An "emulsion" is a mixture of at least two immiscible liquids, one of which is dispersed throughout the other. Typically, the dispersed phase droplets are very small, in the micrometer range or smaller. For the purposes of the present invention, an emulsion comprises an oil phase and an aqueous phase.

[0132] Procedures and equipment for preparing emulsions on any scale are well known in the art and are described, for example, in handbooks such as "Remington: The Science and Practice of Pharmacy" (2000, Lippincot, USA, ISBN: 683306472); and "Veterinary Vaccinology" (P. Pastoret et al., eds., 1997, Elsevier, Amsterdam, ISBN 0444819681).

[0133] When the oil adjuvant used in the present invention is an emulsion, the emulsion can be a water-in-oil (W / O) emulsion, in which the oil is the continuous external phase. Alternatively, the emulsion can be an oil-in-water (O / W) emulsion, in which the oil is the dispersed internal phase.

[0134] By selecting the appropriate type and concentration of one or more emulsifiers, such an emulsion can be formed and stably maintained.

[0135] Emulsifiers sit in the middle between water and oil and stabilize the droplets of the inner dispersed phase. Many different emulsifiers are known and suitable for pharmaceutical uses, such as in vaccines. A preferred emulsifier for the oil adjuvant of the present invention is polysorbate 80, also known as polyoxyethylene sorbitan monooleate, and can be used as a Tween. Tween 80 is purchased. The amount of Tween 80 used in the oil adjuvant of the present invention accounts for 0.1-10% w / v of the oil adjuvant.

[0136] The oil adjuvant of the present invention, when in the form of an O / W emulsion, consists of an external aqueous phase and a dispersed internal oil phase. This facilitates mixing the oil adjuvant in the form of an O / W emulsion with the RP encoding an antigen derived from an animal pathogen in the present invention. For example, by mixing an aqueous composition containing the RP with the oil adjuvant O / W emulsion. Simply manually shaking for about 1 minute is sufficient to properly mix the two aqueous compositions.

[0137] Alternatively, and very advantageously, the oil adjuvant in the form of an O / W emulsion can be used directly to reconstitute the RP in lyophilized form. This means that the RP can be provided in a highly stable form as a lyophilisate and the vaccine of the invention can be prepared by mixing on site at a convenient time before administration to the target animal.

[0138] As used herein, the terms "about" and "approximately" are used interchangeably to refer to values ​​within 50 percent of the stated value, i.e., each milliliter contains "approximately" 1 x 10 8 The composition of alphavirus RNA replicon particles contains 5 x 10 7 to 1.5x 10 8 alphavirus RNA replicon particles.

[0139] Examples of O / W emulsion oil adjuvants for use in the vaccines of the present invention are: XSolve TM XSolve is a combination of two O / W emulsion adjuvant components: Diluvac Forte TM , which is based on vitamin E acetate (see EP 382.271), and Microsol TM , which is based on liquid paraffin oil (see WO 2009 / 144.088).

[0140] In these emulsions, the volume average size of the mineral oil and non-mineral oil droplets may be different.Preferably, the mineral oil droplets have a submicron size.

[0141] Conveniently, the oil adjuvant emulsion is prepared separately from the RP of the present invention. Thus, methods and equipment for emulsifying the oil adjuvant that are incompatible with maintaining the quality of the RP present in the oil adjuvant can be used. One example is a high shear emulsification method for obtaining submicron emulsions by high pressure homogenization, such as using a Microfluidiser processor (Microfluidics, MA, USA).

[0142] A further example of an O / W emulsion oil adjuvant for use in the vaccine of the present invention is: SVEA TM , which comprises squalane and vitamin E acetate and is as described in WO 2018 / 115.435.

[0143] For purposes of the present invention, the names of microorganisms or pathogens, such as, for example, Venezuelan equine encephalitis virus (VEE) and avian influenza virus, refer to the currently applicable taxonomic classification of the respective microorganisms. However, these names may change over time as new insights may lead to reclassification into a new or different taxonomic group. However, since this does not change the microorganism itself or its antigenic repertoire, only its scientific name or classification, such reclassified microorganisms remain within the scope of the present invention.

[0144] Reference to a taxonomic family includes any species, subtype, variant, biotype, serotype or genotype of a microorganism within that family.

[0145] For the purposes of the present invention, "pig" refers to an animal of the Suidae family, preferably an animal of the genus Sus, such as a wild or domesticated pig, wild boar, babirusa, or warthog. It also includes any pig designated by sex, age, or size, such as a sow, boar, barrow, gilt, weaner, or piglet.

[0146] As used herein, the term "poultry" refers to poultry associated with agriculture, such as chickens, turkeys, ducks, geese, partridges, peacocks, quail, pigeons, pheasants, guinea fowl, or ostriches. Preferably, the poultry is a chicken, turkey, duck, or goose. More preferably, the poultry is a chicken or turkey. Most preferably, the poultry is a chicken.

[0147] The birds may be of any type, such as layers, breeders, broilers, combined breeds or parent lines of any such breeds. Preferably, the type of bird is a broiler.

[0148] As used herein, the term "tilapia" may include nearly 100 species of bony-finned fish from the family Cichoridae. Tilapia are primarily freshwater fish that inhabit streams, ponds, rivers, and lakes, and rarely live in brackish water.

[0149] A "kit" for use in the present invention is generally a packaged combination of containers having specified ingredients in predetermined quantities, and the kit may include or involve instructions for carrying out the preparation and immunization of the present invention.

[0150] The vaccines, immunization methods, and compounds and uses for protecting animals of the present invention are targeted at animals in need of immunization to protect them from infection or disease caused by the specific pathogen from which the encoded antigen is derived. The age, weight, sex, immunological status, and other parameters of the target to be vaccinated / protected / immunized are not critical, but it is clearly advantageous to vaccinate healthy, uninfected targets and to vaccinate as early as possible.

[0151] As used in this article, " phylogenetic cluster " is a group of influenza virus neuraminidase, which has been grouped together (on the same branch) in a phylogenetic tree or evolutionary tree rooted in similar (homologous) ancestors. For the IAV-S neuraminidase (NA) found in the U.S., there are two main N1 phylogenetic clusters, N1-classical and N1-pandemic, and two main N2 phylogenetic clusters, N2-1998 and N2-2002. The N1 classical phylogenetic cluster comprises the NA combined with the NA of the H1N1 classical swine influenza virus. The N1 pandemic phylogenetic cluster comprises the NA combined with the NA from the H1N1 pandemic influenza virus. The N2-1998 phylogenetic cluster comprises the NA combined with the NA of human H3N2 influenza virus (which jumped into pigs in 1998), and the N2-2002 phylogenetic cluster comprises the NA combined with the NA of human H3N2 influenza virus (which jumped into pigs in 2002). [See, Anderson et al., Influenza and other Respiratory Viruses 7(Suppl. 4):42-51 (2013)].

[0152] The term "non-IAV-S" is used to modify terms such as pathogens and / or antigens (or immunogens) to indicate that the corresponding pathogens and / or antigens (or immunogens) are neither IAV-S pathogens nor IAV-S antigens (or immunogens), and that non-IAV-S protein antigens (or immunogens) are not derived from IAV-S.

[0153] As used herein, a multivalent vaccine is a vaccine comprising two or more different antigens, where the difference can be at any of a number of 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 against immunologically distinct variants of the same pathogen.

[0154] As used herein, the term "pharmaceutically acceptable" is an adjective used to indicate that the modified noun is suitable for use in a pharmaceutical. For example, when used to describe an excipient in a pharmaceutical vaccine, it characterizes that the excipient is compatible with the other ingredients in the composition and has no adverse deleterious effects on the intended recipient animal (e.g., pig).

[0155] Any feasible method and approach can be used to "administer" the vaccine (or components of the kit of the present invention, respectively) to the animal target. Generally, the best mode of administration will be determined by the type of vaccine / compound used, the characteristics of the target, and the disease to be prevented. Depending on the formulation of the vaccine / compound, different administration techniques can be used. For example, a vaccine / compound of the present invention in the form of an O / W emulsion can be administered via the enteral or mucosal route, i.e., via eye drops, nasal drops, oral, enteral, oral and nasal drops, or sprays. Other possible approaches are by mass administration methods, such as by drinking water, coarse spraying, atomization, feeding, etc.

[0156] "Parenteral administration" includes subcutaneous injection, submucosal injection, intravenous injection, intramuscular injection, intradermal injection, and infusion.

[0157] "Mucosal administration" includes ocular, nasal, oral, oculonasal, intratracheal, intestinal, anal and vaginal routes of administration.

[0158] "Topical administration" includes cutaneous and transdermal routes of administration.

[0159] Preferably, the method, time and volume of administration of the vaccine (respectively, the components of the kit of the present invention) are integrated into the existing vaccination schedule of other vaccinations that the target animal may require to reduce stress on the target animal and reduce labor costs. These other vaccinations themselves can be administered by the associated method of use in a manner compatible with the application for which they are registered.

[0160] As used herein, the term "antigenic fragment" for a specific protein (e.g., a protein antigen) refers to a fragment of the protein that is antigenic (including large fragments that are only missing one amino acid from the full-length protein), that is, capable of interacting specifically with antigen recognition molecules of the immune system (such as immunoglobulins (antibodies) or T cell antigen receptors). For example, an antigenic fragment of IAV-S neuraminidase (NA) is a fragment of the NA protein that is antigenic. Preferably, the antigenic fragment of the present invention has an immunoadvantageous effect on antibody and / or T cell receptor recognition. In a specific embodiment, an antigenic fragment for a given protein antigen is a fragment of the protein that retains at least 25% of the antigenicity of the full-length protein. In a preferred embodiment, the antigenic fragment retains at least 50% of the antigenicity of the full-length protein. In a more preferred embodiment, the antigenic fragment retains at least 75% of the antigenicity of the full-length protein. An antigenic fragment can be as small as 12 amino acids, or at the other extreme, it is a large fragment that is only missing one amino acid from the full-length protein. In a specific embodiment, the antigenic fragment comprises 25 to 150 amino acid residues. In other embodiments, the antigenic fragment comprises 50 to 250 amino acid residues.

[0161] As used herein, an amino acid sequence is 100% "identical" or has 100% "identity" to another amino acid sequence when the amino acid residues in the two sequences are the same. Thus, an amino acid sequence is 50% "identical" to another amino acid sequence when 50% of the amino acid residues in the two sequences are identical. Sequence comparisons are performed on contiguous blocks of amino acid residues comprised by a given protein (e.g., a portion of the protein or polypeptide being compared). In a specific embodiment, selected deletions or insertions that may alter the correspondence between two amino acid sequences are contemplated.

[0162] As used in this article, C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and Clustal W algorithms can be used to determine the identity percentages of nucleotide and amino acid sequences using default comparison parameters and the default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or similar default parameters. Alternatively, Advanced Blast searches under the default filter conditions can be used, for example, using the GCG (Genetics Computer Group, GCG Software Package Program Manual, 7th Edition, Madison, Wisconsin) accumulation program with default parameters.

[0163] When the alphavirus RNA replicon particles are stored separately but are intended to be mixed with other vaccine components prior to administration, the alphavirus RNA replicon particles can be stored in a stable aqueous solution similar to the other components, such as a buffer, or a high sucrose solution.

[0164] The vaccines of the present invention can be readily administered by any standard "method of immunizing animals." Those skilled in the art will appreciate that the route of administration is selected taking into account the characteristics of the target animal and the vaccine to be administered. Preferably, the vaccine composition is appropriately formulated for each type of target animal and route of administration.

[0165] For the purposes of the present invention, a "subunit of a microorganism" may be a biological or synthetic molecule, such as a protein, carbohydrate, lipopolysaccharide, lipid or nucleic acid molecule.

[0166] Further optimization of the vaccines, kits, methods, or uses of the present invention is well within the skill of the art. Typically, this involves fine-tuning the efficacy of the vaccination / immunization to further improve the immune protection provided. This can be accomplished by adjusting the dose, volume, adjuvant, or antigen content of the administered material, or by employing different routes, methods, or regimens of administration. All of these are within the scope of the present invention.

[0167] It should also be understood that the present invention is not limited to the specific configurations, process steps, and materials disclosed herein, as such configurations, process steps, and materials may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention, as the scope of the present invention will be limited only by the appended claims and their equivalents.

[0168] Sequence Listing

[0169]

[0170] The following non-limiting examples are intended to provide a further understanding of the present invention, but are not intended to limit the effective scope of the present invention in any way.

[0171] Example

[0172] Example 1

[0173] Oil adjuvants improve the magnitude and duration of pig antibody responses to alphavirus RNA replicon particles encoding swine influenza virus hemagglutinin

[0174] Materials and methods

[0175] A VEE replicon vector designed for expression of the hemagglutinin (HA) gene was constructed as previously described [see US Pat. No. 9,441,247 B2; the contents of which are incorporated herein by reference] with the following modifications: the HA gene insert was sequence optimized (ATUM, CA, USA). The TC-83-derived replicon vector "pVEK" [disclosed and described in US Pat. No. 9,441,247 B2] was digested with restriction endonucleases AscI and Pad. A DNA plasmid containing a codon-optimized open reading frame sequence of the N1 or N2 gene with a 5' flanking sequence (5'-GGCGCGCCGCACC-3') [SEQ ID NO: 1] and a 3' flanking sequence (5'-TTAATTAA-3') [SEQ ID NO: 2] was also digested with restriction endonucleases AscI and Pad. The synthesized gene cassettes were then ligated into the digested pVEK vector and the resulting clones were renamed "pVHV-N1-pandemic," "pVHV-N1-classical," "pVHV-N2-2002," and "pVHV-N2-1998." The "pVHV" vector nomenclature was chosen to refer to replicon vectors derived from pVEK containing the transgene expression cassettes cloned via the AscI and PacI sites in the multiple cloning site of pVEK.

[0176] TC-83 RNA replicon particles were prepared according to previously described methods [US 9,441,247 B2 and US 8,460,913 B2; the contents of which are incorporated herein by reference]. TM Prior to in vitro transcription with T7 RNA polymerase and cap analog (Promega, Madison, WI), the pVHV replicon vector DNA and helper DNA plasmids were linearized with NotI restriction endonuclease. Importantly, the helper RNA used in the preparation lacked the VEE subgenomic promoter sequence, as previously described [Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)]. Purified RNA from the replicon and helper components were combined and mixed with a Vero cell suspension, electroporated in a 4 mm cuvette, and then placed in an OptiPro TM Alphavirus RNA replicon particles were purified and reconstituted in phosphate-buffered saline (PBS) containing 5% w / v sucrose and 1% v / v porcine serum. The particles were sterilized by passing through a 0.22-μm filter and aliquoted for storage. The titer of functional RP was determined using an infection-immunofluorescence assay on Vero cell monolayers. RP batches were identified based on the antigens encoded by the packaged replicon.

[0177] Ten piglets negative for antibodies to swine influenza virus were randomly divided into groups of five pigs each. An RP vaccine expressing the hemagglutinin antigen of the H3N2 swine influenza virus strain was prepared at a titer of 5 x 10^5 RPs / dose. Immediately prior to vaccination, the vaccine in the RP-only group was diluted 1:1 (v / v) with sterile PBS diluent, while the vaccine in the RP+adjuvant group was diluted 1:1 (v / v) with XSolve adjuvant. Pigs were then inoculated intramuscularly with 2.0 mL of the appropriate material. The vaccination procedure was performed on study days 0 and 21, each time using freshly prepared vaccine. Sera collected during the trial were assayed for hemagglutination inhibition (HI) activity using H3N2 swine influenza virus antigen. Results are reported as the highest dilution with inhibitory activity; titers less than 1:10 are reported as 1:9; titers >640 are reported as 1:641. The geometric mean titers are shown in Table 1.

[0178] Table 1: Geometric Mean Hemagglutination Inhibition (HI) Titers

[0179]

[0180] At a relatively low dose of 5 x 10^5 RP, the unadjuvanted vaccine induced low, short-lived, and transient HI titers. In contrast, after the booster vaccination, the adjuvanted vaccine induced higher HI titers, and these HI titers remained elevated until the end of the 84-day trial.

[0181] This finding clearly demonstrates for the first time that XSolve can have such a dramatic effect on pig RP immunity at a very low dose (5 x 10^5 RPs). Previous studies using non-adjuvanted RP vaccines had to use much higher amounts of the RP components per dose, for example, 1 x 10^9 for FMD RP and 5 x 10^7 for SIV RP.

[0182] Example 2

[0183] Oil adjuvants improve the magnitude and duration of pig antibody responses to polyvalent alphavirus RNA replicon particles encoding swine influenza virus antigens

[0184] As shown in Table 2, weaned piglets from a swine influenza-negative herd were randomly divided into treatment groups. A polyvalent alphavirus RNA replicon particle vaccine for swine influenza was prepared using 8 separate RNA particle antigens expressing HA or NA of different swine influenza virus strains, with each antigen mixed to approximately 1x10^7 RP / dose. Two H3 antigens, four H1 antigens, one N1 antigen, and one N2 antigen were included, and paired serological assays (HI or NI) were performed to assess the antibody response to each antigen. The study was conducted in a replicated design, with half of the animals challenged with H1N1 virus and the other half with H1N2 virus.

[0185] Table 2: Experimental plan for Example 2

[0186]

[0187] The use of XSolve oil adjuvant was found to significantly increase the magnitude of serological responses to all eight vaccine components. Although both vaccine formulations produced protection against lung lesions ( Figures 1A-1B ), however, the addition of XSolve was found to improve vaccine efficacy when measured by nasal shedding of H1N1 and N1N2 influenza viruses ( Figures 1C-1D ). Figure 1-1F shows the corresponding NI titer scores. Figure 2 The effect of adjuvant on HI titers is shown in A-2F.

[0188] This study shows that unadjuvanted polyvalent SIV RP induces antibodies against all fractions, but the addition of an oil adjuvant significantly enhances the immune response to all fractions. The reduced nasal shedding and tighter clustering of lung scores represent important clinical advantages, for example in relation to limiting horizontal spread of infection in herds or populations.

[0189] Example 3

[0190] Efficacy of an adjuvanted 4-in-1 NA-RP vaccine against H1N1 infection in weaned piglets with N1 antibodies at the time of first vaccination

[0191] An immunization-challenge study was conducted to determine the efficacy and immunogenicity of a 4-in-1 adjuvanted NA-RP vaccine at two dose levels. The adjuvanted vaccine contains 4 RP constructs, each of which encodes a single, different NA gene of a contemporary USIAV-S isolate. These NA genes collectively represent two N1 phylogenetic clusters (N1-classical and N1-pandemic), and two N2 clusters (N2-1998 and N2-2002) (see Table 3). At the time of the first vaccination, the adjuvanted vaccine was administered twice intramuscularly (IM) at 1 mL / dose in weaned piglets that were antibody-positive for the N1-classical antigen. The efficacy of the 4-in-1 adjuvanted NA-RP vaccine against heterologous N1 (H1N1 virus) challenge infection was tested.

[0192] Materials and methods

[0193] Construction of NA-RP antigen:

[0194] Replication-defective alphavirus RNA replicon particles (RP) encode the neuraminidase (NA) gene and are prepared essentially as described in Example 1 herein.

[0195] Table 3: N1 and N2 genes encoded in the NA-RP construct of Example 3

[0196]

[0197] n = 10 pigs per group

[0198] Virus

[0199] The challenge virus was obtained from the USDA National Veterinary Services Laboratories. A / Swine / Illinois / A01554351 / 2015 (H1N1) virus possesses the HA gene of the H1-γ 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 clear cytopathic effect was observed in more than 70% of the cell monolayer. Upon harvest, the supernatant was removed from the container and clarified by centrifugation before freezing the virus.

[0200] animal

[0201] Weaned piglets were selected from a high health herd based on serological screening to confirm the absence of pre-existing HI or NI (neuraminidase inhibition) antibodies to the vaccine and challenge strains. Animals were mixed male and female and were approximately 3 weeks old at the time of the first vaccination.

[0202] Vaccination and challenge

[0203] The treatment groups are summarized in Table 4. The 4-linked NA-RP vaccine was formulated with 10^6 copies of each RP / dose, and functional RP was quantified based on an immunofluorescence-based titer assay. 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 without the antigen. The vaccine was mixed with XSolve oil adjuvant (1:1 v / v, 1 mL dose) and then immediately administered to 3-week-old and 7-week-old pigs via the IM route. After vaccination, the remaining vaccine material was back-titrated by IFA to determine the dose level. Serum samples were collected on the day of the first vaccination, the day of the second vaccination, and the day of the challenge infection.

[0204] All pigs were weighed 1 day before the first vaccination, and pigs in the N1-classical antibody positive group were subcutaneously injected with 2 mL / kg dose of N1-classical hyperimmune serum (anti-NI antibody titer to N1-classical antigen was 1:2560).

[0205] Table 4: Treatment groups using H1N1 challenge strain as in Example 3

[0206]

[0207] (n = 10 pigs per group)

[0208] Three weeks after the second immunization, the pigs were challenged. The challenge material, H1N1 (H1-γ-N1-classic), was formulated to a target dose of 10^6.5 TCID50 / pig (in a volume of 6 mL). The challenge material was administered intratracheally. The challenge virus dose was confirmed by back titration of the remaining challenge material. Nasal swab samples were taken from all pigs on days -1, 1, 3, and 5 after the challenge.

[0209] Necropsies were performed 5 days after challenge. Under the supervision of a licensed veterinarian, pigs were euthanized with a barbiturate overdose 5 days after challenge. Lungs were harvested and examined to record the surface area of ​​each lobe affected by macroscopic lesions, yielding a comprehensive lung lesion score. Bronchoalveolar lavage fluid and nasal swab samples were collected from all pigs to measure viral titers. Lung sections were collected for histopathological analysis of microscopic lesions.

[0210] Immune response analysis:

[0211] IAV-S-specific antibodies in porcine serum samples were determined by NI assay. The serum was heat inactivated at 56°C for 30-60 minutes. The NI assay was performed using a slightly modified Sandbulte & Eichelberger method (Methods Mol Biol 1161:337-45, 2014). Briefly, two-fold serial dilutions of serum were mixed with equal volumes of the expressed protein antigen on a fetuin-coated 96-well plate and incubated overnight at 37°C. Peanut agglutinin horseradish peroxidase conjugate was added for 2 hours at room temperature to bind to the fetuin molecules stripped of sialic acid. The signal was obtained by TMB chromogenic substrate and the results were read at 650nm. The average optical density (OD) of the negative control without NA antigen was deducted from all wells. The OD values ​​of the test samples were then normalized within the range of 0-100%, where the average OD of the positive control wells (containing NA antigen but no serum) was defined as 100%. The NI antibody titer was defined as the highest dilution of the sample that inhibited neuraminidase activity by ≥50%.

[0212] Lung pathology examination

[0213] Macroscopic lesions (well-defined purple to plum-colored consolidations) observed on the exterior of all lobes were recorded on grid maps of the anterior and posterior lobes. A composite score (percent lung lesions) was calculated for each pig based on the number of grids affected by lesions.

[0214] Viral shedding

[0215] Nasal swab samples and BAL fluid were serially diluted 10-fold using infection medium [Dulbecco's minimal essential medium (DMEM) supplemented with 0.3% bovine serum albumin, component V; 2mM L-glutamine; 25μg / mL gentamicin; 2μg / mL pancreatin IX], and 100μL of each dilution was added to confluent MDCK cells in 96-well plates (quadruplicate wells). The plates were incubated at 37°C, 5% CO2, and the supernatant of each well was examined for the presence of infectious virus by hemagglutination assay after 72 hours. The IAV-S titer was calculated by the immunohistochemistry method and expressed as Log10 TCID50 / mL.

[0216] result

[0217] The immune response of pigs vaccinated with polyvalent NA-RP was as follows Figure 3 It is worth noting that:

[0218] - Pigs that were passively transferred with N1-classical hyperimmune serum one day before receiving their first vaccination had N1-classical antibody titers of 40-80 at the time of vaccination.

[0219] - After two vaccinations with the 4-in-1 NA-RP vaccine with XSolve adjuvant, both seronegative and N1-antibody seropositive pigs showed a significant increase in their N1-classical antibody titers. This demonstrates that RP vaccination with oil adjuvants is effective even in target animals that are positive for antibodies against RP-encoded antigens.

[0220] - In the group vaccinated with N1 classical negative / placebo vaccine, pigs remained seronegative for NI titers.

[0221] Figure 4 The efficacy of the quadruple NA-RP vaccine in terms of lung lesions against challenge infection was demonstrated in the present study. Notably, the quadruple NA-RP vaccination was highly effective in reducing lung lesions, regardless of whether the pigs were vaccinated with or without N1-classical antibodies at the time of the first vaccination. The percentage of lung lesions was significantly reduced in both groups compared to the two placebo-vaccinated groups.

[0222] Example 4

[0223] The extent to which oil adjuvants improve the antibody response of pigs to alphavirus RNA replicon particles encoding porcine epidemic diarrhea virus antigens relative to aluminum adjuvants or water

[0224] Nine piglets, approximately 3 weeks old, were randomized into groups of 3 animals each. An alphavirus RNA replicon particle vaccine expressing the porcine epidemic diarrhea virus (PEDV) spike glycoprotein was prepared and lyophilized in 20-dose vials. On study day 0, pigs were intramuscularly injected with 1.0 ml of the alphavirus RNA replicon particle vaccine, rehydrated with water, aluminum hydroxide adjuvant, or XSolve adjuvant. This process was repeated on study day 21 using a new vaccine vial. The final titer of each rehydrated dose of alphavirus RNA replicon particles was determined by immunofluorescence assay and was approximately 7 x 10^6 RP / dose for all groups. Sera collected during the trial were assayed for PEDV neutralizing antibodies (see Table 5).

[0225] Table 5: Effects of different adjuvants on the production of neutralizing antibodies

[0226]

[0227] Pigs given the unadjuvanted vaccine had low or undetectable neutralizing antibodies after two doses. All pigs given the aluminum hydroxide-adjuvanted vaccine produced neutralizing antibodies just above the limit of detection. In contrast, the XSolve-adjuvanted vaccine induced high levels of neutralizing antibodies after two doses, with all three pigs producing minimum antibody titers that were higher than the peak titers observed in the other groups tested. Therefore, the use of aluminum hydroxide as an adjuvant was ineffective, and oil adjuvants were significantly superior to aluminum hydroxide.

[0228] Example 5

[0229] Oil adjuvant improves vaccination efficacy of alphavirus RNA replicon particles encoding influenza virus antigens in chickens

[0230] One-day-old chickens were randomly divided into groups of 10 birds each. An alphavirus RNA replicon particle vaccine expressing the hemagglutinin antigen of the H3N2 swine influenza virus strain was prepared at a titer of 1 x 10^8 RP particles per dose. The vaccine for the RP-only group was diluted 1:1 (v / v) with sterile PBS, while the vaccine for the alphavirus RNA replicon particle plus adjuvant group was diluted 1:1 (v / v) with XSolve adjuvant prior to vaccination. Hemagglutinin inhibition (HI) titers were measured in chicken sera on study days 0, 7, and 14 post-vaccination.

[0231] Table 6: Effect of oil adjuvants on HI titer

[0232]

[0233] Compared with the unadjuvanted and placebo vaccines, the alphavirus RNA replicon particle vaccine adjuvanted with XSolve induced a significant increase in HI titers in birds after a single vaccination.

[0234] Example 6

[0235] Oil adjuvant improves the efficacy of alphavirus RNA replicon particle vaccines expressing IBDV antigens in chickens

[0236] This vaccination-challenge experiment examined the effect of oil adjuvant on the vaccination-efficacy of RP, which expresses the VP2-4-3 polyprotein antigen from the Faragher 52 / 70 strain of infectious bursal disease virus (IBDV). The test animals were SPF (specific pathogen-free) grade chickens that were negative for antibodies to IBDV. The group size was 5 or 10 chickens. Vaccination was performed subcutaneously on the day of birth (hatching day); one group (n=5) was mock-vaccinated with phosphate buffered saline (PBS). One vaccination group received the RP vaccine in aqueous buffer and the other vaccination group received the RP vaccine mixed 1:1 with XSolve adjuvant. 28 days after vaccination, all groups were challenged with the virulent IBDV strain CS89 via the eye drop route. Vaccination efficacy was monitored by autopsy 10 days after challenge, and gross lesions and histopathological changes in the bursal tissue were scored according to well-known criteria for IBDV infection.

[0237] Table 7: Effect of oil adjuvant on RP vaccination-efficacy against severe IBDV challenge

[0238]

[0239] Clearly, the addition of oil adjuvant to the RP vaccine had a significant positive impact on vaccination efficacy against severe challenge infection: increasing the chickens' immune response from zero to complete protection.

[0240] Example 7

[0241] Oil adjuvants also improve the efficacy of alphavirus RNA replicon particle vaccines in fish

[0242] An alphavirus RNA replicon particle vaccine expressing the major capsid protein of red sea breamiridovirus (RSIV) was prepared by standard methods as described herein. Tilapia were inoculated intramuscularly with 0.05 ml of vaccine and then challenged at different times after vaccination. In the study, 30 fish were used for each treatment and each challenge time. The alphavirus RNA replicon particle vaccine was mixed with PBS or with SVEA (double non-mineral oil adjuvant) at a ratio of 1:1 (v / v) and then inoculated immediately. The RP titer for each treatment was 1x10^7 RP / dose. Control fish were vaccinated with a placebo vaccine.

[0243] Table 8A: Effect of oil adjuvants on the survival percentage of tilapia

[0244]

[0245] Table 8B: Effects of oil adjuvants on relative survival percentage of tilapia

[0246]

[0247] The adjuvant significantly improved the relative survival of RP-vaccinated fish at both the "3-week low-dose" and "6-week high-dose" challenges. The other two challenge treatments produced similar results. Thus, the adjuvanted alphavirus RNA RP vaccine is significantly more effective than the unadjuvanted RP vaccine and provides effective vaccination even with a single administration.

[0248] Example 8

[0249] Efficacy of simultaneous and concurrent use of RP and oil adjuvants

[0250] An experiment was conducted in pigs using a 4-way swine influenza virus NA vaccine using alphavirus RNA replicon particles as described in Example 3. This was used to demonstrate the efficacy of different types of administration of the vaccine components (RP and oil adjuvant).

[0251] Materials and methods

[0252] Pigs were vaccinated at approximately 4 and 7 weeks of age. As described in Example 3, the 4-in-1 NA vaccine was a mixture of the dual-gene N1 and dual-gene N2 RP constructs, each administered at approximately 2 x 10^6 RPs / dose. Each group contained 4 pigs. The assay schedule is listed in Table 9. Group 2 received a mixture of NA RPs concurrently with XSolve adjuvant.

[0253] Groups 3 and 4 examined the effects of concurrent use of RP and oil adjuvant: both were administered within approximately 10 minutes and either on the ipsilateral side of the neck (>5 cm apart) or on the contralateral side of the neck.

[0254] A control group (Group 5) was included and used RP encoding swine influenza HA H1 antigen at a dose of approximately 1 x 10^7 RPs per dose.

[0255] Sera were collected on the day of the first vaccination, the day of the second vaccination, and 7 days after the second vaccination. These sera were tested in separate homologous NI assays for each of the four NA types.

[0256] The NI titer was measured as Figure 5 and Figure 6 As shown in Figure 5 NI titers against the N1 classical NA antigen at three time points measured over time, including standard deviations, are shown. Figure 6 Shown are group mean NI titers of the four NA types combined 7 days after the second vaccination.

[0257] Table 9: Experimental protocol for Example 8

[0258]

[0259] Results and Conclusions

[0260] From the results measured in Example 8, the following effects can be observed:

[0261] - The NI titer patterns of the four NA types were almost identical at the three time points measured, so the classical NI titer for NA Figure 5 The results shown in indicate the titer patterns against the other three NA types.

[0262] - Moreover, Figure 6 As shown in , at 7 dpv2, the NI titer response patterns of the four NA types were basically the same.

[0263] - As expected, the NI titer results for the HA H1 RP control (Group 5) did not induce NI titers; this sets a titer threshold for a specific response.

[0264] - Apart from some experimental variations, most of the NI titers in Groups 2-4 that received RP and oil adjuvant were significantly higher than those in the group that received RP alone (Group 1). This suggests that oil adjuvant can strongly promote RP immunity.

[0265] - The group receiving RP and oil adjuvant in a simultaneous manner (Group 2) confirmed the stronger effect of the oil adjuvant already observed in the previous examples.

[0266] - The effect of concurrent immunization with RP and oil adjuvant (Groups 3-4) was very similar to that of simultaneous immunization: strong stimulation of RP immune response. Different sites of administration had no significant effect.

[0267] The present invention is not limited to the scope of the specific embodiments described herein. In fact, in addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art based on the foregoing description. Such modifications are all intended to fall within the scope of the appended claims.

[0268] It is also to be understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for description.

Claims

1. A vaccine comprising alphavirus RNA replicon particles encoding antigens derived from animal pathogens, wherein the vaccine further comprises an oil adjuvant, wherein the oil adjuvant is Xsolve adjuvant, wherein the animal pathogen is a mammalian pathogen, and wherein the mammal is a pig, The antigen derived from an animal pathogen is swine influenza virus hemagglutinin, swine influenza virus neuraminidase or porcine epidemic diarrhea virus spike glycoprotein.

2. The vaccine according to claim 1, wherein the oil adjuvant is formulated as an oil-in-water emulsion.

3. The vaccine according to any one of claims 1-2, wherein the alphavirus RNA replicon particle is a VEE alphavirus RNA replicon particle.

4. A kit comprising at least two containers, wherein at least one container comprises a VEE alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen, and at least one container comprises an oil adjuvant, wherein the oil adjuvant is Xsolve adjuvant, wherein the animal pathogen is a mammalian pathogen, and wherein the mammal is a pig, The antigen derived from an animal pathogen is swine influenza virus hemagglutinin, swine influenza virus neuraminidase or porcine epidemic diarrhea virus spike glycoprotein.

5. Use of a VEE alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen and an oil adjuvant in the preparation of a medicament for protecting an animal from a disease caused by the animal pathogen, wherein the alphavirus RNA replicon particle and the oil adjuvant are administered simultaneously or concurrently in or on a target animal, wherein the animal pathogen is a mammalian pathogen, and wherein the mammal is a pig. wherein the oil adjuvant is Xsolve adjuvant, wherein the antigen derived from an animal pathogen is swine influenza virus hemagglutinin, swine influenza virus neuraminidase or porcine epidemic diarrhea virus spike glycoprotein, The disease caused by the animal pathogen is a disease caused by swine influenza virus or porcine epidemic diarrhea virus.

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