Vaccines with replicon particles and oil adjuvants

Abstract

The present invention relates to vaccination against animal pathogens using alpha virus-replicon RNA particles and oil adjuvants. To vaccines and kits comprising said replicon particles and said oil adjuvant. The invention also relates to methods and uses for preparing and using the vaccine and the components of the kit.

Description

[0001] This application is a divisional application of patent application no. 201880078366.7, filed on December 3, 2018, entitled “Vaccine with replicon particles and oil adjuvant”. TECHNICAL FIELD

[0002] The present invention relates to vaccination against animal pathogens using alphavirus-replicon RNA particles and oil adjuvants. It relates to vaccines and kits of parts comprising said replicon particles and said oil adjuvants. It also relates to methods and uses of preparing and using the components of said vaccines and said kits. BACKGROUND

[0003] Over the years, various vector strategies have been used in vaccines in an effort to generate protection against animal pathogens. One such vector strategy includes the use of alphavirus-derived replicon RNA particles (RPs) [Van der 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 [Liljestrom and Garoff, Biotechnology (NY) 9: 1356-361 (1991)]. Encoded pathogen antigens are expressed from the replicon particles upon their infection of human or animal target cells. The result is the induction of protective antibodies against the expressed antigens. RPs have attractive safety and efficacy properties when compared to some traditional vaccine formulations [Van der Veen et al., Anim Health Res Rev. 13(1): 1-9 (2012)]. The RP platform is the basis for 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 Prescription Product, RNA Particles (Product Code 9PP0.00).

[0004] Alpha virus-derived replicon RNA particles lack the alpha virus structural protein genes, but retain the replication elements necessary for cytoplasmic RNA self-amplification and express an inserted heterologous nucleic acid under the drive of a highly active 26S alpha virus subgenomic promoter. Thus, RPs are single-cycle infectious particles that are 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 RPs [see Vajdy et al., Immunol. and Cell Biol. 82:617-627 (2004)]. Structural proteins are usually provided by transient co-transfection of replicon RNA and one or more "helper" RNAs encoding structural proteins. Alternatively, RPs 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, as structural proteins are not included in the resulting RP genome [Polo et al., Dev. Biol., 104:181-185 (2000)]. These replication-defective alpha virus RNA replicon particles induce protective immune responses in vivo when used to immunize a target human or animal. For example, VEE-based alpha virus vectors elicit strong mucosal and systemic immune responses following systemic immunization of mice and large animals [Davis et al., IUBMB Life 53:209-211 (2002)].

[0005] Adjuvants are known compounds that are able to provide non-specific stimulation of the immune system of a target human or animal. Standard use of adjuvants is in vaccines based on inactivated or subunit antigens. There are multiple adjuvant types and components, for example: aluminum salts (aluminum hydroxide or aluminum phosphate), liposomes, dextran, alginate, bacterial components (such as cell wall components), mineral or non-mineral oils, synthetic adjuvants (such as: non-ionic block polymers), polyamines (such as dextran sulfate), Carbopol TM , pyran and saponins (such as: Quil A TM or Q-vac TM ). Saponins can be combined with vaccine components in ISCOM TM s.

[0006] In addition, peptides such as muramyl dipeptide, dimethylglycine, tuftsin are often used as adjuvants. Similarly, combination products such as ISA TM compositions (Seppic, France).

[0007] A handbook on adjuvants and their use and effects is: "Vaccine adjuvants" (Methods in molecular medicine, vol. 42, D. O'Hagan ed., 2000, Humana press, NJ, ISBN: 0896037355).

[0008] Any reference cited herein should not be considered as an admission that the reference is "prior art" to this application. SUMMARY

[0009] The present application provides a vaccine comprising alphavirus RNA replicon particles encoding an antigen derived from an animal pathogen, wherein the vaccine further comprises an oil adjuvant.

[0010] In one embodiment of the vaccine of the present application, the oil adjuvant comprises at least one oil selected from the group consisting of mineral oil and non-mineral oil.

[0011] In one embodiment of the vaccine of the present application, the oil adjuvant comprises mineral oil; preferably, the mineral oil is liquid paraffin oil.

[0012] In one embodiment of the vaccine of the present application, the oil adjuvant comprises non-mineral oil; preferably, the non-mineral oil is selected from the group consisting of synthetic oil, semi-synthetic oil, animal oil and plant oil. More preferably, the non-mineral oil is selected from the group consisting of squalane, squalene, tocopherol and plant oil. In one embodiment, the tocopherol is alpha tocopherol; more preferably, the alpha tocopherol is selected from the group consisting of vitamin E and vitamin E-acetate. In one embodiment, the plant oil is an oleate, more preferably, the oleate is ethyl oleate.

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

[0014] In one preferred embodiment of the vaccine of the present application, the oil adjuvant comprises more than one oil.

[0015] In one embodiment of the oil adjuvant comprising more than one oil, the adjuvant comprises mineral oil and one or more non-mineral oil. More preferably, the oil adjuvant comprises liquid paraffin oil as the mineral oil and one or more non-mineral oil 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 .

[0016] In an alternative embodiment of the oil adjuvant comprising more than one oil, the adjuvant comprises more than one non-mineral oil. Preferably, the oil adjuvant comprises more than one non-mineral oil 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 .

[0017] In an embodiment of the vaccine of the application, the amount of mineral oil in the oil adjuvant is 1-70% v / v of the oil adjuvant. Preferably, the oil adjuvant comprises mineral oil in an amount of 5-60% v / v of the oil adjuvant.

[0018] In an embodiment of the vaccine of the application, the total amount of non-mineral oil is 0.1-30% w / v of the oil adjuvant. Preferably, the oil adjuvant comprises non-mineral oil in a total amount of 0.5-25% v / v of the oil adjuvant.

[0019] In an embodiment, when the non-mineral oil comprises squalane, the oil adjuvant comprises squalane in an amount of 0.5-30% w / v of the oil adjuvant; more preferably, the oil adjuvant comprises 1-25% w / v, 2-15% w / v squalane, or even comprises squalane in an amount of 3-10% w / v of the oil adjuvant.

[0020] Alternatively or additionally, in an embodiment, when the non-mineral oil comprises vitamin E-acetate, the oil adjuvant comprises vitamin E-acetate in an amount of 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 comprises vitamin E-acetate in an amount of 2-10% w / v of the oil adjuvant.

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

[0022] In an embodiment, the aqueous phase comprises water in a pharmaceutically acceptable amount.

[0023] In an embodiment, the emulsion of the oil adjuvant is formulated as a microemulsion, wherein the droplets of the internal phase are smaller 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.

[0024] In an embodiment of the vaccine of the application, the emulsion of the oil adjuvant comprises an emulsifying agent, preferably the emulsifying agent comprises a polysorbate, more preferably the emulsifying agent comprises polysorbate 80.

[0025] 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.

[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 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In a preferred embodiment, the nucleotide sequence encoding the antigen gene derived from the animal pathogen of the animal of the application is optimized taking into account codon usage and secondary structure of the RNA transcript. Preferably, the optimization of the nucleotide sequence is performed 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.

[0032] In a particularly preferred embodiment of the vaccine of the application, the oil adjuvant comprises a mineral oil and a 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.

[0033] In a preferred embodiment of the vaccine of the application, the antigen-encoding gene derived from the animal pathogen is a hemagglutinin (HA)- or neuraminidase (NA) protein of an influenza virus, or an antigenic fragment of such HA or NA protein. The HA and / or NA protein is preferably derived from an influenza A virus, more preferably from a swine influenza A virus or from PEDV.

[0034] The present application also provides a multivalent vaccine comprising the alphavirus RNA replicon particles of the application, wherein the vaccine comprises more than one RP encoding an antigen, or the vaccine comprises one or more RPs each encoding one or more than one antigen of the application.

[0035] The vaccine of the application comprises an immunologically effective amount of the alphavirus RNA replicon particles of the application. In one embodiment, the vaccine comprises about 1 x 10^3 to about 1 x 10^11 RPs. In a more particular embodiment, the vaccine comprises about 1 x 10^4 to about 1 x 10^10 RPs. In an even more particular embodiment, the vaccine comprises about 1 x 10^5 to about 1 x 10^9 RPs.

[0036] The vaccine of the application comprises an immunologically effective amount of the oil adjuvant of the application. In one embodiment, the vaccine comprises the oil adjuvant in an amount of about 10% - 90% v / v of the vaccine. More preferably, the vaccine comprises the oil adjuvant in an amount of between about 20% - 80% v / v, 30 - 70% v / v or even 40 - 60% v / v of the vaccine. Most preferably, the vaccine comprises the oil adjuvant in an amount of about 50% v / v of the vaccine.

[0037] In a particular embodiment, the vaccine of the application is administered in a volume of 0.05 mL to 5 mL per animal dose. In a more particular embodiment, the administration dose per animal is 0.1 mL to 2 mL. In an even more particular embodiment, the administration dose is 0.2 mL to 1.5 mL. In an even more particular embodiment, the administration dose is 0.3 to 1.0 mL. In an even more particular embodiment, the administration dose per animal is 0.4 mL to 0.8 mL.

[0038] In one embodiment of the vaccine of the application, the vaccine comprises a further adjuvant. Preferably, the further adjuvant is selected from the group consisting of bacterial cell wall components, cytokines and immunostimulatory nucleic acids comprising unmethylated CpG. In one embodiment, the immunostimulatory nucleic acid is one or more selected from the group consisting of WO 2012 / 089.800 (X4 family), WO 2012 / 160.183 (X43 family) and WO 2012 / 160.184 (X23 family).

[0039] In one embodiment of the vaccine of the application, the vaccine comprises a further antigen of an animal pathogen. In a preferred embodiment, the further antigen is selected from the group consisting of live attenuated microorganisms, inactivated microorganisms and subunits of microorganisms.

[0040] In a further aspect, the application provides a kit comprising at least two containers, wherein at least one container comprises alphavirus RNA replicon particles encoding an antigen derived from an animal pathogen and at least one container comprises an oil adjuvant. Each of the at least two containers comprises an immunologically effective amount of the alphavirus RNA replicon particles or the oil adjuvant.

[0041] In a preferred embodiment of the kit of the application, the alphavirus RNA replicon particles, the antigen-encoding, the animal pathogen and the oil adjuvant are one or more or all as defined in any one or more of the embodiments described herein.

[0042] In a preferred embodiment, the at least one container comprising the RP comprises the RP in the form of a lyophilisate.

[0043] In an alternative embodiment, the at least one container comprising the RP comprises the RP in an aqueous solution; the aqueous solution preferably comprises a buffer; the aqueous solution is preferably kept cool or frozen. In one embodiment, the aqueous solution is a reconstituted RP solution resulting from mixing a RP lyophilisate with a suitable aqueous diluent.

[0044] In embodiments wherein at least one of the containers comprises an RP in the form of a lyophilisate, the kit of the application can comprise further containers containing a suitable diluent for reconstituting the lyophilised RP. In a preferred embodiment, the diluent is an aqueous solution, preferably comprising a pharmaceutically acceptable quality of a buffer and / or a stabilizer and water.

[0045] In a preferred embodiment, the container comprising the oil adjuvant comprises an oil adjuvant formulated in the form of an emulsion of oil and aqueous phase; preferably the emulsion is an oil-in-water emulsion.

[0046] In embodiments of the kit, the kit comprises instructions for use of the kit and / or its components. In preferred embodiments, the instructions for use are provided on or with one or more of the components of the kit; or in the form of a reference to an electronic version of the instructions, such as information viewable or downloadable from an internet website of the manufacturer or distributor of the kit.

[0047] In one embodiment, the kit comprises a box of at least two containers, and the instructions for use are shown on the box or on an information carrier (e.g. a card or a leaflet) inside the box.

[0048] In one embodiment of the kit, the kit can also provide its components on an internet website, e.g. relating to the use of the immunization method of the application (in connection with commercial marketing).

[0049] In one embodiment of the kit, one or more of the containers can comprise further adjuvants as described herein; likewise or alternatively, one or more of the containers can comprise further antigens of animal pathogens as described herein.

[0050] Both the alphavirus RNA replicon encoding an antigen derived from an animal pathogen and the oil adjuvant, as defined herein, can be administered to a target animal. Such administration will induce effective immunoprotection in said animal against infection or disease caused by said animal pathogen. For example, administration can be performed in accordance with the EMA-CVMP guidelines for related uses of immunological veterinary medicinal products.

[0051] Thus, in a further aspect, the application provides a method of immunizing an animal, comprising administering to the animal an immunologically effective amount of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen and an oil adjuvant.

[0052] In a preferred embodiment of the method of immunizing an animal of the application, the method comprises administering to the animal a vaccine of the application.

[0053] In a preferred embodiment of the method of immunizing an animal of the present application, the alphavirus RNA replicon particles, the antigen-encoding, the animal pathogen and the oil adjuvant are as defined in any one or more of the embodiments described herein.

[0054] In a preferred embodiment of the method of immunizing an animal of the present application, the antigen-encoding derived from an animal pathogen is an antigen derived from a pathogen from the group consisting of fish, a member of the Cichlidae family, Tilapia, a mammal, an avian and a chicken.

[0055] In an embodiment of the method of immunizing an animal of the present application, the alphavirus RNA replicon particles and the oil adjuvant are administered in or on the target animal body in a simultaneous use or a parallel use.

[0056] In a preferred embodiment of the method of immunizing an animal of the present application, the alphavirus RNA replicon particles and the oil adjuvant are administered in or on the target animal body in a simultaneous use (i.e. as a single composition).

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

[0058] In a preferred embodiment, the single composition is prepared immediately prior to administration to the target animal by mixing a composition comprising the RP as described herein with a composition comprising the oil adjuvant; more preferably by mixing the contents of the containers of the kit of the present application; even more preferably by mixing an aqueous solution comprising the RP with a composition comprising an O / W emulsion of the oil adjuvant. In an alternatively even more preferred embodiment, the single composition is prepared by reconstitution of a RP lyophilisate as defined herein with an O / W emulsion of the oil adjuvant. Effectively, the preparation of the single composition results in a vaccine of the present application.

[0059] Preferably, "immediately prior to administration to the target animal" is within 24 hours prior to administration to the target animal, more preferably within 16 hours, within 12 hours, within 8 hours, within 4 hours or even within 2 hours prior to administration to the target animal, in this order of preference.

[0060] In an alternatively preferred embodiment of the method of immunizing an animal of the present application, the alphavirus RNA replicon particles and the oil adjuvant are administered in or on the target animal body in a parallel use, i.e. comprised in separate compositions which are administered separately in terms of location and / or time.

[0061] In a preferred embodiment, the parallel use comprises administration of the alphavirus RNA replicon particles and the oil adjuvant in or on the target animal body comprised in the kit of the present application, but separately in terms of location and / or time.

[0062] In a preferred embodiment of the concurrent use of the present application, separate compositions are administered to separate sites in or on the target animal body by the same or by different routes of administration within a limited amount of time of each other; preferably, the "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 this order of preference. Most preferably, the administration of the concurrent use is essentially simultaneous.

[0063] In a preferred embodiment of the concurrent use of the present application, separate compositions are administered to separate sites in or on the target animal body by the same or by different routes of administration within a limited amount of time of each other. For the present application, the separate sites of administration are at least 1 cm apart from each other on or in the animal body; preferably at least 2 cm, at least 5 cm, at least 10 cm or even at least 25 cm apart from each other, in this order of preference.

[0064] In a preferred embodiment of the concurrent use of the present application, separate compositions are administered to substantially the same site in or on the target animal body by the same or by different routes of administration within a limited amount of time of each other, but sufficiently separated in time from each other to prevent mixing of the compositions at the site of administration. For the present application, the time sufficiently separated to prevent mixing is not within 2 hours of each other, preferably not within 6 hours, not within 12 hours, not within 1 day, not within 2 days or even not within 1 week of each other, in this order of preference.

[0065] In an embodiment of the method of immunizing an animal of the present application, the administration in or on the target animal body is by parenteral administration. In an alternative embodiment, the administration is by a method of mucosal administration. In yet another alternative embodiment, the vaccine is administered by a method of topical administration.

[0066] Preferred methods of administration are selected from the group consisting of intradermal, intramuscular, intraperitoneal, subcutaneous, immersion and spray. The method of intradermal administration is preferably administered by needleless means, more preferably by using a device (intradermal application of liquids).

[0067] ​In one embodiment of administering a vaccine of the application, the vaccine is administered as a prime vaccine and / or as a boost vaccine. In a particular embodiment, the vaccine of the application is administered in one (one injection) vaccination and no subsequent boost administration is required. In certain embodiments, where both a prime vaccine and a boost vaccine are administered, the prime vaccine and the boost vaccine can be administered in the same route. In an alternative embodiment, where both a prime vaccine and a boost vaccine are administered, the administration of the prime vaccine can be by one route and the boost vaccine can be administered by another route.

[0068] In certain embodiments of administering a vaccine of the application, the vaccine is administered to a pig, and both the prime vaccine and the boost vaccine are administered by intradermal injection. In an alternative embodiment, the prime vaccine is administered by intradermal injection and the boost vaccine is administered by another route.

[0069] In a further embodiment, the application provides a method of producing a vaccine of the application, the method comprising the step of mixing an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen with an oil adjuvant. The alphavirus RNA replicon particle is mixed with the oil adjuvant in an immunologically effective amount.

[0070] In a preferred embodiment of the method for producing a vaccine of the application, one or more or all of the vaccine, the alphavirus RNA replicon particle, the antigen-encoding, the animal pathogen and the oil adjuvant are as defined in any one or more embodiments described herein.

[0071] In one embodiment of the method for producing a vaccine of the application, the alphavirus RNA replicon particle is comprised in an aqueous solution.

[0072] In a preferred embodiment of the method for producing a vaccine of the application, the mixing is performed such that the alphavirus RNA replicon particle, respectively the aqueous solution comprising the alphavirus RNA replicon particle, is mixed with the oil adjuvant in a volume ratio of 1 : 10 to 10: 1; more preferably in a volume ratio of between 1 : 5 to 5: 1, between 1 : 4 to 4: 1, between 1 : 3 to 3: 1 or even between 1 : 2 to 2: 1, in this order of preference. Most preferably, the alphavirus RNA replicon particle, respectively the aqueous solution comprising the alphavirus RNA replicon particle, is mixed with the oil adjuvant in a volume ratio of about 1 : 1.

[0073] In one embodiment of the method for producing a vaccine of the application, the mixing comprises mixing the contents of the containers of the kit of the application.

[0074] In one embodiment of the method for producing the vaccine of the application, the alphavirus RNA replicon particles, respectively, are mixed with an oil adjuvant as defined according to the application, the oil adjuvant being comprised in another O / W emulsion containing a pathogen antigen. Preferably, the other O / W emulsion is a vaccine comprising inactivated viral and / or bacterial pathogen. In a more preferred embodiment, the RP encoding an antigen from SIV or from PEDV is mixed with an O / W emulsion vaccine comprising porcine circovirus (PCV) and / or Mycoplasma hyopneumoniae, such as PCVM.

[0075] In a further embodiment, the application provides an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for use in protecting an animal against an infection or a disease caused by the animal pathogen, wherein the alphavirus RNA replicon particle and the oil adjuvant are administered in or on the target animal in a simultaneous use or a concurrent use. Both the immunologically effective amount of the alphavirus RNA replicon particle and the oil adjuvant are comprised in the use for protecting the animal.

[0076] In a preferred embodiment of the use of the alphavirus RNA replicon for the protective use of the application, the alphavirus RNA replicon particle, the antigen encoding, the animal pathogen and the oil adjuvant are one or more or all as defined in any one or more of the embodiments described herein.

[0077] In a preferred embodiment of the use of the alphavirus RNA replicon for the protective use of the application, the use comprises the use of the vaccine of the application.

[0078] In one embodiment of the use of the alphavirus RNA replicon for the protective use of the application, the protection is effective in different age and type of target animals.

[0079] In one embodiment, the use is for protecting young animals. Preferably, the young animals are pigs up to 3 weeks of age, or chickens up to 1 week of age, or salmon up to 14 months of age.

[0080] In a further embodiment, the use is for protecting juvenile animals. Preferably, the juvenile animals are pigs from 3 weeks of age to 8 months of age, or chickens from 1 week of age to 22 weeks of age, or salmon from 14 months of age to 24 months of age.

[0081] In a further embodiment, the use is for protecting adult animals. Preferably, the adult animals are pigs older than 8 months of age, or chickens older than 22 weeks of age, or salmon older than 24 months of age.

[0082] For tilapia, the preferred period for use of the application for the purposes of protection is generally not expressed in terms of age, but in terms of overall weight: when the tilapia weighs between 0.5 g and 5 g, immunization is preferably performed by bath treatment. When the tilapia weighs between 10 g and 100 g; more preferably when the tilapia weighs between 20 g and 25 g, immunization is preferably performed by parenteral injection.

[0083] In one embodiment of use of the alphavirus RNA replicon for the purposes of protection of the application, the target animal can be seropositive or seronegative for antibodies against the animal pathogen, or for the animal pathogen, respectively, for the antigen from the animal pathogen.

[0084] In one embodiment of use of the alphavirus RNA replicon for the purposes of protection of the application, the target animal is an MDA (maternally derived antibody) positive animal, whereby the MDA can react with the animal pathogen for which protection is intended. More preferably, the MDA positive animal is an avian, a ruminant or a pig. Still more preferably, the MDA positive animal is a pig.

[0085] In one embodiment of use of the alphavirus RNA replicon for the purposes of protection of the application, 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.

[0086] In one embodiment of use of the alphavirus RNA replicon for the purposes of protection of the application, the protection is protection of a production animal. Preferably, the production animal is a pig raised for fattening, or a broiler or a layer chicken, or a ruminant raised for milk or meat production, or a salmon, or a tilapia.

[0087] In a further embodiment, the protection is protection of an animal for restocking. Preferably, the animal for restocking is a parental or grand-parental line of the animal of production.

[0088] In a further embodiment, the application provides the use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for the preparation of a vaccine for protection of an animal against infection or disease caused by the animal pathogen, which comprises the simultaneous or concurrent use of said alphavirus RNA replicon particle with an oil adjuvant. Both the alphavirus RNA replicon particle and the oil adjuvant are used in an immunologically effective amount.

[0089] In a preferred embodiment of the use for the preparation of a vaccine of the application, one or more or all of the alphavirus RNA replicon particle, the antigen-encoding, the animal pathogen and the oil adjuvant are as defined in any one or more of the embodiments described herein.

[0090] In a further embodiment, the present application provides the use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for the manufacture of a component of a kit as defined by the present application, wherein the kit is for protecting an animal against an infection or disease caused by the animal pathogen by simultaneous or concurrent use of the components of the kit. Both the alphavirus RNA replicon particle and the oil adjuvant are used in an immunologically effective amount.

[0091] In a preferred embodiment of the use for the manufacture of a component of a kit of the present application, the alphavirus RNA replicon particle, the antigen-encoding, the animal pathogen, the oil adjuvant and one or more or all of the components of the kit are as defined in any one or more of the embodiments described herein.

[0092] In a further embodiment, the present application provides the use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for protecting an animal against an infection or disease caused by the animal pathogen, wherein the use comprises the simultaneous or concurrent use of the alphavirus RNA replicon particle with an oil adjuvant. Both the alphavirus RNA replicon particle and the oil adjuvant are used in an immunologically effective amount.

[0093] In a preferred embodiment of the use for protecting an animal of the present application, the alphavirus RNA replicon particle, the antigen-encoding, the animal pathogen and the oil adjuvant are as defined in any one or more of the embodiments described herein.

[0094] These and other aspects of the present application will be better appreciated by reference to the following drawings and detailed description.

[0095] The present application also includes the following embodiments.

[0096] 1. A vaccine comprising an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen, wherein the vaccine comprises an oil adjuvant.

[0097] 2. The vaccine according to embodiment 1, wherein the oil adjuvant comprises a mineral oil.

[0098] 3. The vaccine according to embodiment 1, wherein the oil adjuvant comprises a non-mineral oil.

[0099] 4. The vaccine according to embodiment 1, 2 or 3, wherein the oil adjuvant comprises a mineral oil and one or more non-mineral oils.

[0100] 5. The vaccine according to embodiment 4, wherein the oil adjuvant comprises a liquid paraffin oil and vitamin E-acetate.

[0101] 6. The vaccine according to embodiment 1 or 3, wherein the oil adjuvant comprises more than one non-mineral oil.

[0102] 7. The vaccine of claim 6, wherein the oil adjuvant comprises squalane and vitamin E-acetate.

[0103] 8. The vaccine of embodiment 1, 2, 4 or 5, wherein the amount of the mineral oil in the oil adjuvant is 1-70% v / v of the oil adjuvant.

[0104] 9. The vaccine of embodiment 1, 3, 4, 5, 6 or 7, wherein the total amount of the non-mineral oil in the oil adjuvant is 0.1-30% w / v of the oil adjuvant.

[0105] 10. The vaccine of embodiment 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein the oil adjuvant is formulated as an oil-in-water emulsion.

[0106] 11. The vaccine of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein the alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle.

[0107] 12. The vaccine of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, wherein the antigen-encoding antigen derived from an animal pathogen is an antigen derived from a virus or a bacterium.

[0108] 13. The vaccine of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the antigen-encoding antigen derived from an animal pathogen is an antigen derived from a pathogen of an animal selected from the group consisting of a fish, a bird and a mammal.

[0109] 14. The vaccine of embodiment 13, wherein the mammal is a pig.

[0110] 15. The vaccine of embodiment 13, wherein the bird is a chicken or a turkey.

[0111] 16. The vaccine of embodiment 13, wherein the fish is a member of the Cichlidae family.

[0112] 17. The vaccine of embodiment 16, wherein the member of the Cichlidae family is a tilapia.

[0113] 18. 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.

[0114] 19. 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.

[0115] 20. The method of embodiment 19, comprising administering to the animal the vaccine of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

[0116] 21. The method of embodiment 19 or 20, wherein the animal is a fish, and the antigen encoded derived from an animal pathogen is an antigen derived from a fish pathogen.

[0117] 22. The method of embodiment 21, wherein the fish is a member of the Cichlidae family, and the antigen encoded derived from a fish pathogen is an antigen derived from a fish pathogen of a member of the Cichlidae family.

[0118] 23. The method of embodiment 22, wherein the member of the Cichlidae family is a tilapia, and the antigen encoded derived from a fish pathogen of the Cichlidae family is an antigen derived from a tilapia pathogen.

[0119] 24. The method of embodiment 19 or 20, wherein the animal is a mammal, and the antigen encoded derived from an animal pathogen is an antigen derived from a mammal pathogen.

[0120] 25. The method of embodiment 24, wherein the mammal is a pig, and the antigen derived from a mammal pathogen is an antigen derived from a pig pathogen.

[0121] 26. The method of embodiment 19 or 20, wherein the animal is an avian, and the antigen encoded derived from an animal pathogen is an antigen derived from an avian pathogen.

[0122] 27. The method of embodiment 26, wherein the avian is a chicken, and the antigen encoded derived from an avian pathogen is an antigen derived from a chicken pathogen.

[0123] 28. The method of embodiment 19, 20, 21, 22, 23, 24, 25, 26, or 27, wherein the alphavirus RNA replicon particles and the oil adjuvant are administered in the target animal or on the target animal in a simultaneous use or a parallel use.

[0124] 29. A method for producing a vaccine according to embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, said method comprising the step of mixing the alphavirus RNA replicon particles encoding an antigen derived from an animal pathogen with the oil adjuvant.

[0125] 30. An alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for use in protecting an animal from an infection or disease caused by the animal pathogen, wherein the alphavirus RNA replicon particle and the oil adjuvant are administered in the target animal or on the target animal in a simultaneous use or a parallel use.

[0126] 31. Use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for the manufacture of a vaccine protecting an animal from an infection or disease caused by the animal pathogen, which comprises the simultaneous use or the parallel use of the alphavirus RNA replicon particle and the oil adjuvant.

[0127] 32. Use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for the manufacture of a component of a kit as defined in embodiment 18, wherein the kit is for protecting an animal from an infection or disease caused by the animal pathogen by the simultaneous use or the parallel use of the components of the kit.

[0128] 33. Use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for protecting an animal from an infection or disease caused by the animal pathogen, which use comprises the simultaneous use or the parallel use of the alphavirus RNA replicon particle and the oil adjuvant. BRIEF DESCRIPTION OF DRAWINGS

[0129] Figure 1 and Figure 2 Results in Example 2:

[0130] Figures 1A-1B Pulmonary lesions

[0131] Figures 1C-1D Nasal shedding

[0132] Figures 1E-1F NI titers

[0133] Figure 2 HI titers results (Figures numbered from left to right, top to bottom, respectively 2A, 2B, 2C, 2D, 2E, 2F)

[0134] Figure 3 Serum antibody responses against N1-Classic antigen in pigs vaccinated with multivalent NA-RP in Example 3:

[0135] Graph showing serum neuraminidase inhibition (NI) antibody responses specific for N1 -classic strain of the vaccine composition as described in Example 3. Serum samples were collected prior to the first immunization (3 weeks of age), prior to the second immunization (7 weeks of age), and prior to challenge (10 weeks of age).

[0136] Figure 4 Efficacy of the 4-way NA-RP vaccine against challenge infection as determined in Example 3, involving macroscopic lung lesion scores 5 days post-infection with H1N1 virus:

[0137] Graph showing lung lesion scores of pigs administered the immunization composition described in Example 3 and challenged with H1 -gamma-N1 -classic virus.

[0138] Figure 5 and 6 NI titer results in Example 8:

[0139] Figure 5 Graph showing NI titers against N1 -classic NA antigen measured at three time points over time, including standard deviation. This represents the NI titer profile measured for the other three NA types.

[0140] Figure 6 Graph showing group mean NI titers for the combined 4 NA types at 7 days post-2nd immunization. DETAILED DESCRIPTION

[0141] 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 an animal pathogen and an oil adjuvant. The alphavirus RNA replicon particles are in effect similar to live viruses in that they are capable of infecting host cells of the target human or animal and expressing the genes they contain. This is also evidenced by the fact that RPs are typically quantified by cell infectivity titration. Thus, similar to live (attenuated) virus vaccines, such RPs in a pharmaceutically acceptable carrier are typically the only component of an effective vaccine. Several unadjuvanted RP-based vaccines have been developed and commercialized.

[0142] Adjuvants are primarily used in combination with inactivated or subunit vaccine antigens only. Also, oil adjuvants can be very aggressive to other vaccine components, and thus oil adjuvants are typically not used in combination with live vaccines. Furthermore, alphavirus RNA RP vaccines, particularly those based on VEE alphavirus, are known to induce a strong anti-viral response from the target's innate immune system itself following the acquired immune response. Thereby, any need for additional immune stimulation is effectively prevented.

[0143] However, it was surprisingly discovered that oil 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. Enhanced efficacy can be obtained when the RP and oil adjuvant are combined into a single composition (i.e., used simultaneously), or when they are administered as separate compositions (i.e., used concurrently). The degree of enhanced efficacy by oil adjuvants is also unexpected, as immunization with the RP and oil adjuvant can reduce the minimum effective dose of the RP by several orders of magnitude compared to the vaccine without (oil) adjuvant. In addition, the use of oil adjuvants can increase the duration of the immune response to RP immunization. Moreover, the combination of oil adjuvant with RP can provide superior immune responses when the RP component alone does not elicit immunity at all.

[0144] To more fully understand the application, the following definitions are provided.

[0145] A "vaccine" is a well-known composition with a medical effect, comprising an immunologically active component and a pharmaceutically acceptable carrier. An aqueous solution and / or an oil adjuvant can serve as a "carrier" for the vaccine. The "immunologically active component" of the vaccine of the present application is an antigen-encoding derived from an animal pathogen, which is delivered and expressed by the RP. The vaccine stimulates the immune system of the target animal to which it is administered, inducing a protective immune response. The response can be derived from the animal's innate and / or acquired immune system, and can be cellular and / or humoral.

[0146] A vaccine can provide "protection" against an infection or disease by reducing the severity of a subsequent infection or infestation of the vaccinated animal, for example by reducing the number of pathogens, or shortening the duration of replication of the pathogen in or on the animal, and reducing the number, intensity or severity of lesions caused by the infection or infestation. Also, or correspondingly, the vaccine is effective in reducing or alleviating the (clinical) symptoms of a disease that can be caused by such an infection, infestation or replication, or by the target's response to the infection, infestation or replication. A reference book for such diseases and clinical signs is "The Merck Veterinary Manual" (10th edition, 2010, C.M. Kahn, editor, ISBN: 091191093X). Such a vaccine is colloquially referred to as a "vaccine against" a particular pathogen.

[0147] As used herein, the term "comprising" (as well as variations such as "comprise", "comprises", "comprised" and "comprised of") refers to all possible combinations of the elements and is intended to encompass both the textually stated elements and any possible additional elements not expressly recited in the text portion, paragraph, claim, etc. in which the term is used, even if such elements or combinations are not explicitly stated; and is not intended to exclude any such elements or combinations. Thus, any such text portion, paragraph, claim, etc. can also refer to one or more embodiments in which the term "comprising" (or variations thereof) is replaced by terms such as "consist of", or "consisting essentially of".

[0148] As used herein, the term "replicon" refers to a modified RNA virus genome that lacks one or more elements (e.g., a coding sequence for a structural protein) that, if present, would enable the parental 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.

[0149] 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) that are also derived from an alphavirus, e.g., as described in Pushko et al. [Virology 239(2): 389-401 (1997)]. The RP infects a suitable target cell and then expresses one or more inserted heterologous genes, but is unable to propagate in cell culture or an animal host (without a helper plasmid or similar component) because the replicon does not encode alphavirus structural components (e.g., capsid and viral glycoproteins).

[0150] For ease of description, the use of singular terminology shall never be intended to be limiting. Thus, for example, a reference to an "alphavirus RNA replicon particle" includes a reference to a plurality of such alphavirus RNA replicon particles, unless otherwise specified.

[0151] That a RP "encodes" an antigen means the transcription and / or translation of a nucleic acid against that protein antigen included in the RP, resulting in the expression of the protein antigen. Typically, such a nucleic acid encoding a protein is an open reading frame (ORF), indicating the absence of an undesirable stop codon that would prematurely terminate translation of the protein. The nucleic acid can be a full gene encoding the entire protein, or can be a gene fragment encoding a portion of the 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 can be of natural or synthetic origin.

[0152] Heterologous nucleic acid sequences expressing antigens of the application 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 "PCR protocols" by J. Bartlett and D. Stirling (Humana press, ISBN: 0896036421).

[0153] For the purposes of the present application, a "protein" is a molecular chain of amino acids. A protein can be a natural or mature protein, a pre- or pro-protein, or a portion of a protein. Among these: peptides, oligopeptides and polypeptides are included in the definition of a protein.

[0154] For the purposes of the present application, an "antigen" refers to a protein capable of inducing a protective immune response in a target animal under the appropriate circumstances.

[0155] The terms "originate from", "originates from" and "originating from" can be used interchangeably with respect to a given protein antigen and the pathogen or strain of the pathogen that naturally encodes it. As used herein, these terms indicate 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 a nucleic acid construct of the application for a protein antigen originating from a pathogen can have been genetically manipulated to result in an amino acid sequence modification, truncation and / or extension of the expressed protein antigen relative to the corresponding coding sequence of the protein antigen in the pathogen or strain of the pathogen (including naturally attenuated strains) from which it originates.

[0156] "Animal pathogen" refers to any biological entity capable of causing an infection and / or disease in a veterinary relevant animal, such as a wild animal, a farm animal or a companion animal.

[0157] For the present application, the animal pathogen can or can not be a natural pathogen of the target animal receiving the vaccine of the present application.

[0158] "Oil" is used herein in its usual meaning and refers to a non-polar chemical with a relatively high hydrocarbon content, which is usually a relatively viscous liquid, has a density lighter than water, and is hydrophobic and lipophilic. The oil can be of mineral origin, or "non-mineral" origin, such as synthetic, semi-synthetic, animal or plant origin. Some oils are metabolizable.

[0159] The term "mineral" indicates that the corresponding oil is of mineral origin, usually from petroleum.

[0160] "Semisynthetic oil" is an oil of non-mineral origin, such as animal or plant oil, but which has been modified in its structure and / or composition by chemical or physical means.

[0161] The term "adjuvant" is used herein in its usual meaning, the composition being able to stimulate the immune response in the target animal in a non-specific manner.

[0162] "Light 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 usually available in pharmaceutical grade quality. Examples are: 6VR (Penreco), 52 (Exxon Mobile) and (Sonneborn).

[0163] "Vitamin E acetate" refers to the chemical compound with 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 material, such as seeds, nuts, fruits or leaves, or from animal fat, but can also be produced synthetically. The definition of vitamin E acetate thus includes natural, synthetic or semi-synthetic forms, or mixtures thereof. Vitamin E acetate is commercially available in different purities.

[0164] "Squalane" refers to the chemical compound with CAS number 111-01-3. Some alternative names are: hydrogenated shark liver oil, hexamethyltetracosane or perhydro squalene. It is not to be confused with squalene (CAS number 111-02-4), which is a polyunsaturated C30 oil, which can be metabolized as a compound of the cholesterol pathway.

[0165] The precursor of squalane was originally obtained from shark livers, but due to environmental concerns, other natural sources such as olive oil or chemical synthesis have been turned to. Therefore, the definition of squalane includes natural, synthetic or semi-synthetic forms, or mixtures thereof. Squalane is commercially available in various purities, e.g. from vegetable sources, from Worlee (Squalane, Vegetable) or Croda (Pripure Squalane) or synthetic, e.g. from Kuraray (Squalane-PE). For the present application, high purity squalane is preferred: preferably a purity higher than 75%, more preferably a purity higher than 80, 90 or even higher than 95%, in this order of preference.

[0166] An "emulsion" is a mixture of at least two immiscible liquids, in which one is dispersed in the other. Typically, the dispersed phase droplets are very small, in the micrometer or smaller range. For the present application, the emulsion comprises an oil phase and an aqueous phase.

[0167] The procedures and equipment to prepare emulsions at any scale are well known in the art and described, e.g. 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).

[0168] When the oil adjuvant used in the present application 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.

[0169] By selecting the appropriate kind and concentration of emulsifying agent(s), such emulsions can be formed and stably maintained.

[0170] Emulsifying agents are located at the interface between water and oil and stabilize the droplets of the internal dispersed phase. Many different emulsifying agents are well known and suitable for pharmaceutical use, e.g. in vaccines. A preferred emulsifying agent for the oil adjuvant of the present application is polysorbate 80, also known as polyoxyethylene sorbitan monooleate, and is commercially available as Tween® 80. Tween 80 is used in the oil adjuvant of the present application in an amount of 0.1-10% w / v of the oil adjuvant.

[0171] For the oil adjuvant of the present application, when in the form of an O / W emulsion, it consists of an external aqueous phase and a dispersed internal oil phase. This facilitates mixing of the oil adjuvant in the form of an O / W emulsion with the RP encoding antigens derived from animal pathogens in the present application. For example, by mixing an aqueous composition comprising the RP with the oil adjuvant O / W emulsion. A simple manual shaking for about 1 minute is sufficient to properly mix the two aqueous compositions. ​

[0172] Alternatively, and very advantageously, the oil adjuvant in the form of an O / W emulsion can be used directly for reconstitution of the RP in lyophilized form. This means that the RP can be provided in a highly stable form such as a lyophilisate and the vaccine of the application can be prepared on site by mixing at a convenient time before administration to the target animal.

[0173] As used herein, the term "about" is used interchangeably with "approximately," to mean within 50 percent of the indicated value, i.e., within 50 percent of the value per milliliter contains "about" 1 x 10 8 A composition of alpha virus RNA replicon particles contains 5 x 10 7 to 1.5 x 10 8 alpha virus RNA replicon particles per milliliter.

[0174] Examples of O / W emulsion oil adjuvants for use in the vaccine of the application 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 a liquid paraffin oil (see WO 2009 / 144.088).

[0175] In these emulsions, the volume average size of the mineral oil and non-mineral oil droplets can be different. Preferably, the mineral oil droplets have a sub-micron size.

[0176] Conveniently, the oil adjuvant emulsion is prepared separately from the RP of the application. Thus, methods and equipment for emulsifying the oil adjuvant can be used which are not compatible with the quality of the RP which is maintained in the oil adjuvant. One example is the high shear emulsification method for obtaining sub-micron emulsions by high pressure homogenization, such as using a Microfluidiser processor (Microfluidics, MA, USA).

[0177] A further example of an O / W emulsion oil adjuvant for use in the vaccine of the application is: SVEA TM which comprises squalane and vitamin E acetate, and as described in WO 2018 / 115.435.

[0178] For 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 respective systematic classification of those microorganisms as currently applied. However, these names can change over time as new insights can lead to reclassification into a new or different taxon. However, since this does not change the microorganism itself nor its antigen pool, only its scientific name or classification, such reclassified microorganisms are still within the scope of the present invention.

[0179] Reference to a taxonomic family includes any microorganism of a species, subspecies, variant, biotype, serotype or genotype within that family.

[0180] For the present invention, "pigs" refer to swine animals, preferably to Sus scrofa animals, such as, for example, wild or domestic pigs, wild boars, peccaries or warthogs. Also included are pigs denoted by any name referring to sex, age or size, such as, for example, sows, boars, barrows, gilts, weaners or piglets.

[0181] As used herein, the term "poultry" refers to agriculturally relevant birds, such as, for example, chickens, turkeys, ducks, geese, stone pheasants, peafowls, quails, pigeons, pheasants, guineafowls or ostriches. Preferably, the poultry is a chicken, a turkey, a duck or a goose. More preferably, the poultry is a chicken or a turkey. Most preferably, the poultry is a chicken.

[0182] The poultry can be of any type, such as, for example, layers, breeders, broilers, a combi breed or a parent line of any such breed. Preferably, the type of poultry is a broiler.

[0183] As used herein, the term "tilapia" can include the nearly one hundred species of osteichthids from the family Cichlidae. Tilapia are primarily freshwater fish, inhabiting creeks, ponds, rivers and lakes, and rarely living in brackish water.

[0184] A "kit" for use in the present invention is generally a packaged combination of containers with predetermined amounts of specific ingredients, which can include or refer to instructions for performing the preparation and immunization of the present invention.

[0185] The vaccines, immunization methods, as well as the compounds and uses for protecting animals of the present invention are directed to animals in need of immunization, in order to protect the animals from infection or disease caused by the specific pathogen that is the source of the encoded antigen. The age, weight, sex, immunological status and other parameters of the target to be vaccinated / protected / immunized are not critical, but it is obviously advantageous to vaccinate healthy, uninfected targets and as early as possible.

[0186] As used herein, a "phylogenetic cluster" is a group of influenza virus neuraminidases that have been grouped together (on the same branch) in a phylogenetic tree or evolutionary tree rooted in a similar (homologous) ancestor. For IAV-S neuraminidases (NA) found in the United States, there are two major N1 phylogenetic clusters, N1-Classic and N1-Pandemic, and two major N2 phylogenetic clusters, N2-1998 and N2-2002. The N1-Classic phylogenetic cluster contains NAs grouped together with the NA of H1N1 classic swine influenza viruses. The N1-Pandemic phylogenetic cluster contains NAs grouped together with the NA from H1N1 pandemic influenza viruses. The N2-1998 phylogenetic cluster contains NAs grouped together with the NA of human H3N2 influenza viruses that jumped into swine in 1998, while the N2-2002 phylogenetic cluster contains NAs grouped together with the NA of human H3N2 influenza viruses that jumped into swine in 2002. [See, Anderson et al., Influenza and other Respiratory Viruses 7 (Suppl. 4): 42-51 (2013)].

[0187] The term "non-IAV-S" is used to modify terms such as pathogen and / or antigen (or immunogen) to indicate that the corresponding pathogen and / or antigen (or immunogen) is neither an IAV-S pathogen nor an IAV-S antigen (or immunogen), and that a non-IAV-S protein antigen (or immunogen) is not derived from an IAV-S.

[0188] As used herein, a multivalent vaccine is a vaccine that contains two or more different antigens, where the difference can be any difference at many biological levels, such as genus, species, serotype, and the like. In one particular embodiment of this type, a multivalent vaccine stimulates the immune system of a target animal against two or more different animal pathogens, or against immunologically different variants of the same pathogen.

[0189] As used herein, the term "pharmaceutically acceptable" as an adjective to modify a noun means that the modified noun is suitable for use in a pharmaceutical product. For example, when used to describe an excipient in a pharmaceutical vaccine, it characterizes the excipient as being compatible with the other ingredients in the composition, and as having no adversely deleterious effects on the target recipient animal (e.g., a pig).

[0190] Any feasible method and route can be used to "administer" the vaccine (respectively the components of the kit of the application) to the animal target. Generally, the optimal mode of administration will be determined by the type of vaccine / compound applied, the characteristics of the target and the disease to be prevented. Depending on the method of formulation of the vaccine / compound, different administration techniques can be employed. For example, the vaccine / compound of the application in the form of an O / W emulsion can be administered by enteral or mucosal routes, i.e. by eye drops, nose drops, oral, enteral, mouth-nose drops, spray. Other possible ways are by mass administration methods, such as by drinking water, coarse spraying, atomization, feeding, etc.

[0191] "Parenteral administration" includes subcutaneous injection, submucosal injection, intravenous injection, intramuscular injection, intradermal injection and infusion.

[0192] "Mucosal administration" includes ocular, nasal, oral, ocular-nasal, intratracheal, intestinal, anal and vaginal routes of administration.

[0193] "Topical administration" includes dermal and transdermal routes of administration.

[0194] Preferably, the method, timing and volume of administration of the vaccine (respectively the components of the kit of the application) are integrated into the existing immunization schedule of the target animal, possibly in need of other immunizations, in order to reduce stress on the target animal and reduce labor costs. These other immunizations can themselves be administered by associated methods of use in a manner compatible with their registered applications.

[0195] As used herein, the term "antigenic fragment" with respect to a particular protein (e.g., a protein antigen) refers to a fragment of that protein that is antigenic (including large fragments missing only one amino acid from the full-length protein), i.e., capable of specifically interacting 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 fragments of the application are immunodominant for antibody and / or T cell receptor recognition. In particular embodiments, an antigenic fragment with respect to a given protein antigen is a fragment of that protein that retains at least 25% of the antigenicity of the full-length protein antigen. In preferred embodiments, the antigenic fragment retains at least 50% of the antigenicity of the full-length protein. In more preferred embodiments, the antigenic fragment retains at least 75% of the antigenicity of the full-length protein. Antigenic fragments can be as small as 12 amino acids, or at the other extreme, large fragments missing only one amino acid from the full-length protein. In particular embodiments, the antigenic fragment comprises 25 to 150 amino acid residues. In other embodiments, the antigenic fragment comprises 50 to 250 amino acid residues.

[0196] As used herein, one amino acid sequence is 100% "identical" or has 100% "identity" to another amino acid sequence when the amino acid residues of the two sequences are the same at each corresponding amino acid residue position. Thus, one amino acid sequence is 50% "identical" to another amino acid sequence when 50% of the amino acid residues of the two sequences are the same. Sequence comparisons are made over a contiguous block of amino acid residues encompassed by a given protein (e.g., a portion of the protein or polypeptide being compared). In a particular embodiment, selected deletions or insertions that can alter correspondence between two amino acid sequences are taken into account.

[0197] As used herein, the percent identity of nucleotide and amino acid sequences can be determined using C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996), and the Clustal W algorithm, using default alignment parameters and default parameters for identity. These commercially available programs can also be used to determine sequence similarity, using the same or similar default parameters. Alternatively, high- level Blast searches under default filtering conditions can be used, such as the GCG (Genetics Computer Group, GCG software package program manual, 7th edition, Madison, Wisconsin) Accumulator program using default parameters.

[0198] When the alphavirus RNA replicon particles are stored separately but 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.

[0199] The vaccines of the present application can be readily administered by any standard "method of immunizing an animal". The skilled artisan will recognize that the route of administration is selected with consideration of the target animal and the characteristics of the vaccine to be administered. Preferably, the vaccine composition is appropriately formulated for each type of target animal and route of administration.

[0200] For the present application, a "subunit of a microorganism" can be a biological or synthetic molecule, such as a protein, carbohydrate, lipopolysaccharide, lipid, or nucleic acid molecule.

[0201] Further optimization of the vaccines, kits, methods, or uses of the present application is well within the capabilities of the skilled artisan. Typically, this involves fine-tuning of the inoculation / immunization efficacy to further improve the immune protection it provides. This can be accomplished by adjusting the dose, volume, adjuvant, or antigen content of the material administered, or by administering it using a different route, method, or regimen. All of these are within the scope of the present application.

[0202] It is also to be understood that the application is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials can vary. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting as the scope of the present application will only be limited by the appended claims and equivalents thereof.

[0203] SEQUENCE LISTING

[0204]

[0205] The following non-limiting examples are provided to give further appreciation of the application, but are in no way meant to limit the effective scope of the application.

[0206] Example

[0207] Example 1

[0208] Oil adjuvants improve the extent and duration of antibody responses in pigs to alphavirus RNA replicon particles encoding swine influenza virus hemagglutinin

[0209] Materials and Methods

[0210] VEE replicon vectors designed to express hemagglutinin (HA) genes were constructed according to previous descriptions [see US 9,441,247 B2; the content of which is incorporated herein by reference] with the following modification: sequence optimization of the HA gene insert (ATUM, CA, USA). The replicon vector “pVEK” [disclosed and described in U.S. 9,441,247 B2] derived from TC-83 was digested using the restriction enzymes Ascl and PacI. The DNA plasmid containing the codon-optimized open reading frame sequence of the N1 or N2 gene with 5’ flanking sequence (5’-GGCGCGCCGCACC-3’) [SEQ ID NO: 1] and 3’ flanking sequence (5’-TTAATTAA-3’) [SEQ ID NO: 2] was likewise digested using the restriction enzymes Ascl and PacI. The synthetic gene cassette was then ligated to 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 refers to replicon vectors derived from pVEK containing a transgene expression cassette cloned through the Ascl and PacI sites in the multiple cloning site of pVEK.

[0211] TC-83 RNA replicon particles were prepared according to previously described methods [US9,441,247 B2 and US8,460,913 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.

[0212] 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.

[0213] Table 1: Geometric Mean Hemagglutination Inhibition (HI) Titers

[0214]

[0215] At the relatively low dose of 5 x 10Λ5 RPs, the unadjuvanted vaccine induced lower intensity, short-lived and transient HI titers. In contrast, the adjuvanted vaccine induced higher HI titers after the boost and these HI titers remained elevated until the end of the trial at 84 days.

[0216] This finding clearly demonstrates for the first time that XSolve is able to have such a dramatic impact on immunization with porcine RPs at very low doses (5 x 10Λ5 RPs). Previous studies using unadjuvanted RP vaccines had to use much higher amounts of the RP component per dose, for example: FMD RPs at 1 x 10Λ9 and SIV RPs at 5 x 10Λ7.

[0217] Example 2

[0218] Oil adjuvant improves the extent and duration of antibody responses in pigs to multivalent alphavirus RNA replicon particles encoding swine influenza virus antigens

[0219] As shown in Table 2, weaned piglets from a swine influenza negative herd were randomized into treatment groups. Eight individual RNA particle antigens expressing HA or NA from different swine influenza virus strains were mixed for a multivalent alphavirus RNA replicon particle vaccine for swine influenza, each antigen mixed to approximately 1 x 10Λ7 RPs / dose. Two H3 antigens, four H1 antigens, one N1 antigen, and one N2 antigen were included and paired serology assays (HI or NI) were performed to assess antibody responses to each antigen. The study was conducted in a replicate design with half of the animals challenged with H1N1 virus and the other half with H1N2 virus.

[0220] Table 2: Experimental protocol for Example 2

[0221]

[0222] We found that the use of XSolve oil adjuvant significantly increased the extent of serological responses to all eight vaccine components. Although both vaccine formulations conferred protection from lung lesions Figures 1A-1B ), we found that the addition of XSolve improved the efficacy of the vaccine Figures 1C-1D when measured by nasal shedding of H1N1 and N1N2 influenza viruses. Figures 1E-1F The corresponding NI titer scores are shown. In Figure 2 A-2F shows the effect of adjuvant on HI titers. Figure 2 A-2F shows the effect of adjuvant on HI titers.

[0223] This study shows that unadjuvanted multivalent SIV RP induced antibodies against all fractions, but the addition of oil adjuvant significantly enhanced the immune response to all fractions. Reduced nasal shedding and tighter clustering of lung scores represent important clinical advantages, for example involving limitation of the level of infection spread in a herd or human population.

[0224] Example 3

[0225] Efficacy of adjuvanted 4-way NA-RP vaccine in weaned piglets with N1 antibodies at first vaccination against H1N1 infection

[0226] An immunization-challenge study was conducted to determine the efficacy and immunogenicity of two dose levels of adjuvanted 4-way NA-RP vaccine. The adjuvanted vaccine comprised four RP constructs, each of which individually encoded a single, different NA gene of contemporary U.S. IAV-S isolates. These NA genes collectively represent two N1 phylogenetic clusters (N1-Classic and N1-Pandemic), and two N2 clusters (N2-1998 and N2-2002) (see Table 3). The adjuvanted vaccine was administered at 1 mL / dose by two intramuscular (IM) inoculations at first vaccination in N1-Classic antibody-positive weaned piglets. The efficacy of the adjuvanted 4-way NA-RP vaccine against heterologous N1 (H1N1 virus) challenge infection was examined.

[0227] Materials and Methods

[0228] Construction of NA-RP antigens:

[0229] Replication-defective alphavirus RNA replicon particles (RP) encoding neuraminidase (NA) genes were prepared essentially as described in Example 1 herein.

[0230] Table 3: N1 and N2 genes encoded in the NA-RP constructs of Example 3

[0231]

[0232] n = 10 pigs per group

[0233] Virus

[0234] Challenge virus was obtained from the USDA National Veterinary Service Laboratories. A / Swine / Illinois / A01554351 / 2015 (H1N1) has an HA gene of the H1-gamma cluster and an NA gene of the N1-classic cluster. Virus was propagated in MDCK cell culture. Infection of confluent cells was approximately 48 hours until prominent cytopathic effect was observed in more than 70% of the cell monolayer. At harvest, supernatant was removed from the vessel and clarified by centrifugation before virus was stored frozen.

[0235] Animals

[0236] Weanling piglets were selected from a high health pig population based on serological screening to confirm the absence of pre-existing HI or NI (neuraminidase inhibition) antibodies against the vaccine and challenge strains. Animals were mixed gender and approximately 3 weeks of age at the time of the first vaccination.

[0237] Vaccination and challenge

[0238] Treatment groups are outlined in Table 4. The 4-way NA-RP vaccine was formulated at 10^6 copies of each RP / dose, quantifying functional RPs according to an immunofluorescence-based titer assay. NA-RP antigens were formulated in stabilizer consisting of 1% porcine serum and 5% sucrose. The placebo vaccine consisted of the same stabilizer without antigens. Vaccines were mixed with XSolve oil adjuvant (1 : 1 v / v, 1 mL dose) and administered immediately by the IM route to pigs at 3 and 7 weeks of age. Dose levels were confirmed by back-titrating residual vaccine material by IFA detection 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.

[0239] All pigs were weighed 1 day prior to the first vaccination and pigs in the N1-classic antibody positive group were injected subcutaneously with a 2 mL / kg dose of N1-classic hyperimmune serum (anti-NI antibody titer of 1 :2560 against N1-classic antigen).

[0240] Table 4: Treatment groups using H1N1 challenge strain as in Example 3

[0241]

[0242] (n = 10 pigs per group)

[0243] Challenge infection was administered to pigs 3 weeks after the second immunization. Challenge material H1N1 (H1-gamma-N1-classic) was formulated to a target dose of 10^6.5 TCID50 / pig (volume of 6 mL). Challenge material was administered by the intratracheal route. Challenge virus dose was confirmed by back-titration of residual challenge material. Nasal swab samples were taken from all pigs on days -1, 1, 3, and 5 post-challenge.

[0244] Necropsy was performed 5 days post challenge. Pigs were euthanized under the supervision of a licensed veterinarian with an overdose of barbiturates at 5 DPC. Lungs were collected and observed to record the surface area of each lung lobe affected by macroscopic lesions to derive a comprehensive lung lesion score percentage. 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.

[0245] Immune response analysis:

[0246] IAV-S specific antibodies in pig serum samples were determined by NI test. Serum was heat inactivated at 56°C for 30-60 minutes. The NI test was performed using a slightly modified Sandbulte & Eichelberger method (Methods Mol Biol 1161:337-45, 2014). Briefly, 2-fold serial dilutions of serum were mixed in equal volume with expressed protein antigen on fetuin-coated 96-well plates and incubated overnight at 37°C. Peanut agglutinin-horseradish peroxidase conjugate was added for 2 hours at room temperature to bind the fetuin molecules stripped of sialic acid. Signal was obtained by TMB chromogenic substrate and results were read at 650 nm. The mean optical density (OD) of negative controls containing no NA antigen was subtracted from all wells. OD values of test samples were then normalized in the 0-100% range, with the mean OD of positive control wells containing NA antigen but no serum defined as 100%. NI antibody titers were defined as the highest dilution of samples that inhibited > 50% of neuraminidase activity.

[0247] Lung pathology

[0248] Gross lesions (well-demarcated purple to plum-colored consolidations) observed on the exterior of all lung lobes were recorded on a grid map of the anterior and posterior lobes of the lung. A composite score (lung lesion percentage) was calculated for each pig based on the number of grids affected by lesions.

[0249] Virus shedding

[0250] Nasal swab samples and BAL fluid were serially diluted 10-fold using infection medium [Dulbeco Minimal Essential Medium (DMEM) supplemented with 0.3% bovine serum albumin, component V; 2 mM L-glutamine; 25 pg / mL gentamicin; 2 pg / mL trypsin IX] and 100 pL of each dilution was added to confluent MDCK cells in 96-well plates (quadruplicate). Plates were incubated at 37°C, 5% CO2 and 72 hours later, presence of infectious virus was observed by hemagglutinin assay on supernatant of each well. Presence of virus was determined by the presence of hemagglutination in the supernatant of at least one well of each dilution. The highest dilution of each sample that contained virus was defined as the virus shedding titer. IAV-S titers were calculated and expressed as Log10 TCID50 / mL.

[0251] Results

[0252] The immune response of pigs vaccinated with the multivalent NA-RP is shown in Figure 3 It is noteworthy that: - pigs that were passively transferred with N1 -classical hyperimmune serum the day before their first vaccination had a N1 -classical antibody titer of 40-80 at the time of vaccination.

[0253] - both seronegative and N1 -antibody seropositive pigs showed a significant increase in their N1 -classical antibody titers after 2 vaccinations with the 4-way NA-RP vaccine with XSolve adjuvant. This demonstrates that the use of an oil adjuvant for the RP vaccination is effective even in target animals that are positive for antibodies against the RP encoded antigens.

[0254] - in the N1 classical negative / placebo vaccinated group, the pigs remained seronegative for NI.

[0255] Figure 4 The efficacy of the 4-way NA-RP vaccine against the challenge infection in terms of lung lesions is shown in Table 3. It is noteworthy that the 4-way NA-RP vaccination was very effective in reducing lung lesions regardless of whether the pigs were vaccinated with or without N1 -classical antibodies at their first vaccination. The percentage of lung lesions was significantly reduced in both of these groups compared to the two placebo vaccinated groups.

[0256] Example 4

[0257] The extent to which an oil adjuvant improves the antibody response of pigs to alphavirus RNA replicon particles encoding porcine epidemic diarrhea virus antigens relative to aluminum adjuvant or water

[0258] Nine piglets of approximately 3 weeks of age were randomly assigned to groups of 3 animals. Alphavirus RNA replicon particle vaccines expressing the porcine epidemic diarrhea virus (PEDV) spike glycoprotein were prepared and lyophilized in 20 dose vials. On study day 0, pigs were injected intramuscularly with 1.0 ml of the alphavirus RNA replicon particle vaccine that had been rehydrated with water, aluminum hydroxide adjuvant, or XSolve adjuvant. On study day 21, this was repeated using a new vial of vaccine. The final titer of each dose of the alphavirus RNA replicon particle after rehydration was determined by immunofluorescence assay and was approximately 7 x 10^6 RPs / dose for all groups. Sera collected during the trial were assayed for PEDV neutralizing antibodies, see Table 5.

[0259] Table 5: Effect of different adjuvants on the generation of neutralizing antibodies

[0260]

[0261] Pigs given the unadjuvanted vaccine had little or no detectable neutralizing antibodies after two doses of vaccine. All pigs given the aluminum hydroxide adjuvanted vaccine produced neutralizing antibodies just above the limit of detection. In contrast, the XSolve adjuvanted vaccine induced higher levels of neutralizing antibodies after two doses of vaccine, with all three pigs producing a minimum antibody titer higher than the peak titer observed in the other test groups. Thus, the use of aluminum hydroxide as an adjuvant was ineffective, and the oil adjuvant was greatly superior to the aluminum hydroxide adjuvant.

[0262] Example 5

[0263] Oil adjuvant improves the efficacy of alphavirus RNA replicon particle vaccines expressing influenza virus antigens in chickens

[0264] One-day-old chickens were randomly assigned to groups of 10. Alphavirus RNA replicon particle vaccines expressing the hemagglutinin antigen of the H3N2 swine influenza virus strain were prepared at a titer of 1 x 108RP / dose. Vaccines for the RP only group were diluted 1 : 1 (v / v) with sterile PBS diluent, while vaccines for the alphavirus RNA replicon particle plus adjuvant group were diluted 1 : 1 (v / v) with XSolve adjuvant, and then immediately inoculated. Hemagglutinin inhibition (HI) titers of chicken sera were determined on days 0, 7, and 14 post-inoculation study days.

[0265] Table 6: Effect of oil adjuvant on HI titers

[0266]

[0267] The addition of XSolve adjuvant to the alphavirus RNA replicon particle vaccine resulted in a significant increase in HI titers in birds after a single inoculation compared to the unadjuvanted and placebo vaccines.

[0268] Example 6

[0269] Oil adjuvant improves the efficacy of alphavirus RNA replicon particle vaccines expressing IBDV antigens in chickens

[0270] This vaccination-challenge experiment tested the effect of oil adjuvants on RP vaccination-efficacy against a severe challenge with the infectious bursal disease virus (IBDV) Faragher 52 / 70 strain. The test animals were SPF (specific pathogen free) level chickens that were negative for antibodies against IBDV. Group size was 5 or 10 chickens. Vaccination was performed at day of birth (hatch day) by the subcutaneous route; one group (n=5) was sham-vaccinated with phosphate buffered saline (PBS). One vaccination group received the RP vaccine in aqueous buffer, the other vaccine group received the RP vaccine mixed 1 : 1 with XSolve adjuvant. At 28 days post-vaccination, all groups were challenged with the virulent IBDV strain CS89 by the ocular route. Vaccination efficacy was monitored by necropsy at 10 days post-challenge, gross lesions and histopathological changes in bursal tissue were scored according to well-known standards for IBDV infection.

[0271] Table 7: Effect of oil adjuvants on RP vaccination-efficacy against a severe IBDV challenge

[0272]

[0273] Clearly, the addition of oil adjuvants to the RP vaccine had a significant positive effect on vaccination-efficacy against a severe challenge infection: the immune response of the chickens was raised from zero to full protection.

[0274] Example 7

[0275] Oil adjuvants also improve the efficacy of alphavirus RNA replicon particle vaccines in fish

[0276] An alphavirus RNA replicon particle vaccine expressing the major capsid protein of red sea bream iridovirus (RSIV) was prepared by standard methods as described herein. Tilapia fish were vaccinated intramuscularly with 0.05 ml of the vaccine, and then challenged at different times post-vaccination. In the study, 30 fish were used per treatment and per challenge time. The alphavirus RNA replicon particle vaccine was mixed 1 : 1 (v / v) with PBS or with SVEA (a double non-mineral oil adjuvant) immediately prior to vaccination. The RP titer for each treatment was 1 x 10Λ7 RPs / dose. Control fish were vaccinated with a placebo vaccine.

[0277] Table 8A: Effect of oil adjuvants on the percentage survival of tilapia fish

[0278]

[0279] Table 8B: Effect of oil adjuvants on the relative percentage survival of tilapia fish

[0280]

[0281] The adjuvant greatly improved the relative survival rate of fish vaccinated with the RP vaccine in both the "3 weeks low dose" and "6 weeks high dose" challenges. The same results were produced for the other two challenge treatments. Thus, the alphavirus RNA RP vaccine with adjuvant was significantly more effective than the RP vaccine without adjuvant, and provided effective vaccination even with only a single vaccination.

[0282] Example 8

[0283] Efficacy of simultaneous and concurrent use of RP and oil adjuvant

[0284] An experiment was performed in pigs using a 4-way swine influenza virus NA vaccine with alphavirus RNA replicon particles as described in Example 3. This was used to illustrate the efficacy of different types of use of the vaccine components (RP and oil adjuvant).

[0285] Materials and Methods

[0286] Pigs were vaccinated at approximately 4 and 7 weeks of age. The 4-way NA vaccine was a mixture of dual-gene N1 and dual-gene N2 RP constructs each administered at approximately 2 x 10Λ6 RPs / dose as described in Example 3. Each group contained 4 pigs. The schedule of the test regimen is listed in Table 9. Group 2 received a mixture of NA RPs with XSolve adjuvant used simultaneously.

[0287] Groups 3 and 4 tested the effect of concurrent use of RPs and oil adjuvant: both were administered within approximately 10 minutes and on the same side of the neck (separated by >5 cm) or on opposite sides of the neck.

[0288] A control group (Group 5) was included using RPs encoding swine influenza HA H1 antigen. This was administered at approximately 1 x 10Λ7 RPs / dose.

[0289] Sera were collected on the day of the first vaccination, on 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 4 NA types.

[0290] The resulting NI titers measured are shown in Figure 5 and Figure 6 where Figure 5 The NI titers measured against the N1 classic NA antigen over time are shown in Figure 6 represent the group mean NI titers for the combination of the 4 NA types at 7 days after the second vaccination.

[0291] Table 9: Schedule of experiment for Example 8

[0292] Results and Conclusions

[0293] From the results measured in Example 8, the following effects can be observed:

[0294] - The NI titer pattern for the 4 NA types is almost identical at the 3 time points measured, thus the NA NI classical Figure 5 The results shown in Table 2 demonstrate the titer pattern for the other 3 NA types.

[0295] - Moreover, as shown in Table 2, at 7 dpv2, the NI titer response pattern for the 4 NA types is essentially identical. Figure 6

[0296] - As expected, the HA H1 RP control (Group 5) did not induce NI titers; this sets the titer threshold for a specific response.

[0297] - Except for some experimental variations, the majority of the NI titers in Groups 2-4, which received the RP and the oil adjuvant, were significantly higher than the group that received only the RP (Group 1). This demonstrates that the oil adjuvant strongly boosts the RP immunization.

[0298] - The group that received the RP and the oil adjuvant simultaneously (Group 2) confirmed the strong effect of the oil adjuvant that has been observed in previous examples.

[0299] - The immunization effect of the concurrent use of the RP and the oil adjuvant (Groups 3-4) is very close to the simultaneous use:

[0300] - The strong stimulation of the RP immune response. The difference in the site of administration does not have a significant effect.

[0301] The present application is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the application, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0302] 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 provided for description.​

Claims

1. A vaccine comprising alphavirus RNA replicon particles encoding an antigen derived from an animal pathogen, wherein the vaccine further comprises an oil adjuvant, wherein the oil adjuvant comprises one or more non-mineral oils, and wherein the animal is a fish, and wherein the encoded antigen derived from the animal pathogen is an antigen of a pathogen derived from fish.

2. The vaccine of claim 1, wherein the oil adjuvant comprises squalane and vitamin E-acetate.

3. The vaccine according to claim 1 or 2, wherein the total amount of the non-mineral oil in the oil adjuvant is 0.1-30% w / v of the oil adjuvant.

4. The vaccine of claim 1, 2 or 3, wherein the oil adjuvant is formulated as an oil-in-water emulsion.

5. The vaccine of claim 1, 2, 3 or 4, wherein the alphavirus RNA replicon particle is a Venezuelan equine encephalitis (VEE) alphavirus RNA replicon particle.

6. The vaccine of claim 1, 2, 3, 4 or 5, wherein the fish is a member of the family Cichlidae.

7. The vaccine of claim 6, wherein the member of the Cichlidae family is tilapia.

8. 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.

9. A method for 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.

10. The method of claim 9 comprising administering to the animal the vaccine of claim 1, 2, 3, 4, 5, 6, 7 or 8.

11. The method of claim 9 or 10, wherein the animal is a fish, and the encoded antigen derived from an animal pathogen is an antigen derived from a fish pathogen.

12. The method of claim 11, wherein the fish is a member of the family Cichlidae, and the encoded antigen derived from a fish pathogen is an antigen of a fish pathogen derived from a member of the family Cichlidae.

13. The method of claim 12, wherein the member of the Cichlidae family is tilapia, and the encoded antigen derived from a pathogen of a fish of the Cichlidae family is an antigen derived from a pathogen of tilapia.

14. The method of claim 9, 10, 11, 12 or 13, wherein the alphavirus RNA replicon particles and the oil adjuvant are administered simultaneously or concurrently in or on the target animal.

15. A method for producing the vaccine of claim 1, 2, 3, 4, 5, 6 or 7, comprising the step of mixing the alphavirus RNA replicon particles encoding antigens derived from animal pathogens with the oil adjuvant.

16. 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 in or on the target animal.

17. Use of an alphavirus RNA replicon particle encoding an antigen derived from an animal pathogen for preparing a vaccine for protecting animals from infection or disease caused by the animal pathogen, comprising using the alphavirus RNA replicon particle and an oil adjuvant simultaneously or in parallel.

18. Use of alphavirus RNA replicon particles encoding antigens derived from animal pathogens for the preparation of components of a kit as defined in claim 8, wherein the kit is for protecting animals from infection or disease caused by said animal pathogen by simultaneous or parallel use of said components of the kit.

19. 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 the simultaneous or concurrent use of the alphavirus RNA replicon particle and an oil adjuvant.

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