Vaccine composition and method for controlling foot-and-mouth disease

Abstract

This disclosure provides a method for preventing foot lesions caused by FMD virus infection in ruminants through the use of a vaccine presented as an emulsion containing one or more adjuvants and viral antigens.

Description

[Technical Field]

[0001] Names of parties to the joint research agreement

[0001] The present invention is the result of activities conducted within the scope of a collaborative research agreement between Zoetis Pharmaceutical Research and ICAR-Indian Veterinary Research Institute, Hebbal, Bengaluru, Karnataka 560024, India.

[0002]

[0002] The present invention generally belongs to the field of vaccines against foot-and-mouth disease (FMD). [Background technology]

[0003]

[0003] Foot-and-mouth disease (FMD) is one of the most serious and contagious diseases affecting livestock. This disease is endemic in many countries around the world, particularly in Africa, Asia, and South America. Furthermore, outbreaks can occur periodically. When this disease occurs in a country, it can have very serious economic consequences, such as decreased productivity, weight loss and reduced milk production in infected groups, and trade restrictions imposed on the country. Measures against this disease consist of import restrictions, strict application of sanitation and quarantine, culling of diseased animals, and vaccination programs using inactivated vaccines, which are carried out at the national or regional level as preventive measures or regularly during outbreaks.

[0004]

[0004] FMD is characterized by a short incubation period, extremely high infectivity, the formation of ulcers in the mouth and feet, and sometimes death in young animals. FMD affects many animal species, especially cattle, pigs, sheep, and goats. The causative agent of this disease is a ribonucleic acid (RNA) virus belonging to the genus Aftovirus of the family Picornaviridae (Cooper et al., 1978, Intervirology 10, pp. 165-180). Currently, at least seven types of foot-and-mouth disease virus (FMDV) are known: European type (A, O, and C), African type (SAT1, SAT2, and SAT3), and Asian type (Asia 1). Numerous subtypes have also been distinguished (Kleid et al., 1981, Science 214, pp. 1125-1129).

[0005]

[0005] FMDV is a naked icosahedral virus with a diameter of approximately 25 nm, containing a single-stranded RNA molecule consisting of approximately 8500 nucleotides and possessing positive polarity. This RNA molecule contains a single open reading frame (ORF) and encodes a single polyprotein, in particular, a capsid precursor also known as protein P1. Protein P1 is myristylated at its amino terminus. During maturation, protein P1 is cleaved by protease 3C into three proteins known as VP0, VP1, and VP3 (or 1AB, 1D, and 1C, respectively) (Belsham GJ, Progress in Biophysics and Molecular Biology, 1993, 60, pp. 241-261). Within the viral particle, protein VP0 is further cleaved into two proteins, VP4 and VP2 (or 1A and 1B, respectively). The mechanism by which protein VP0 is converted to VP1 and VP3, as well as the mechanism of mature viral particle formation, is unknown. While proteins VP1, VP2, and VP3 have molecular weights of approximately 26,000 daltons, protein VP4 has a smaller molecular weight of approximately 8,000 daltons.

[0006]

[0006] Many proposals have been made as attempts to design effective vaccines against FMD. Cao et al. (Antiviral Research, 2013, 97:145-153; Veterinary Microbiology, 2014, 168:294-301) reported the design of a specific epitope protein that is immunogenic to FMDV antigen administration. It has been demonstrated that a synthetic polypeptide fused with one T epitope and two B epitopes derived from Asian serotypes induces a protective response (Ren et al., Vaccine, 2011, 29:7960-7965). [Overview of the project] [Problems that the invention aims to solve]

[0007]

[0007] Most FMD vaccines require a vaccination regimen of two doses administered 28 days or 6 months apart, but low compliance rates among farmers have created some limitations in the effective control of the disease. No FMD vaccine provides immunity lasting longer than 6 months. The World Organisation for Animal Health (OIE) emphasizes the need for two doses (prime-booster regimen) and revaccination every six months. [Means for solving the problem]

[0008] Summary of the Invention

[0008] In one embodiment, the present invention provides a vaccine comprising an FMD antigen and an adjuvant comprising an oil, a polycationic carrier, and an immunostimulatory oligonucleotide, wherein the vaccine is a water-in-oil (W / O) emulsion and is intended for use in the prevention of foot lesions caused by FMD virus infection in ruminants, wherein the vaccine is administered as a single prime inoculation and one or more booster inoculations, the first of the one or more booster inoculations being administered 7 to about 12 months after the single prime inoculation. In a particular embodiment, the ruminant is FMD-negative.

[0009]

[0009] In a particular embodiment, each subsequent booster dose is administered 7 to about 12 months after the previous booster dose, for example, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months after a single prime dose.

[0010]

[0010] In certain embodiments, the cationic carrier is DEAE dextran, which may be present in an amount of 25 to 250 mg per inoculation.

[0011] In certain embodiments, the immunostimulatory oligonucleotide is present in an amount of 25 to 250 μg per inoculation, for example, 50 to 150 μg per inoculation.

[0011]

[0012] In certain embodiments, the vaccine comprises at least 40% oil (v / v), for example, at least 45% oil (v / v), at least 48% oil (v / v), at least 52% oil (v / v), or at least 60% oil (v / v). The oil may be a non-metabolized oil such as light mineral oil.

[0012]

[0013] In certain embodiments, the antigen is derived from the FMD virus and may belong to serotypes O, A, C, Asia 1, SAT1, SAT2, or SAT3. In certain embodiments, the vaccine is monovalent, and in other specific embodiments, the vaccine is polyvalent, having combinations of different serotypes, such as FMD virus serotypes A, O, and / or C. In other embodiments, the vaccine contains antigens derived from FMD virus serotypes A, O, and / or Asia 1, or other serotypes such as SAT1, SAT2, and / or SAT3.

[0013]

[0014] In certain embodiments, the FMD antigen is an inactivated FMD virus. In certain embodiments, the inactivated FMD virus is a recombinant FMD virus. In certain embodiments, the recombinant FMD virus lacks a functional leader protein and / or contains one or more DIVA markers in, for example, the 3B and / or 3D proteins. In some embodiments, the recombinant virus expresses a capsid derived from a heterologous FMD strain. In some embodiments, the heterologous FMD strain is derived from serotypes O, A, and / or C. In other embodiments, the heterologous FMD strain is derived from serotypes O, A, and / or Asia 1. In yet another embodiment, the heterologous FMD strain is derived from serotypes SAT1, SAT2, and / or SAT3.

[0014]

[0015] In certain embodiments, the FMD virus is present in an amount of at least 4 μg per inoculation per strain, for example, at least 8 μg per inoculation per strain, or about 10 μg per inoculation per strain. [Modes for carrying out the invention]

[0015]

[0016] To better understand the present invention, the following non-limiting definitions are provided.

[0017] When used in relation to measurable numerical variables, "approximately" or "about" refers to all values ​​of the variable that are within the indicated value and within the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean), whichever is greater, or within 10% of the indicated value. Regarding periods, the term "approximately" refers to the indicated value and a range within 10% of the indicated value (e.g., "approximately 8 months" includes 8 months and 8 months plus or minus 10%). However, the upper limit for "approximately 11 months" is 12 months, and the upper limit for "approximately 12 months" is 12.5 months.

[0016]

[0018] An "adjuvant" refers to any substance that increases the humoral or cellular immune response to an antigen. Adjuvants are generally used to achieve two purposes: controlled release of the antigen from the injection site and stimulation of the immune system.

[0017]

[0019] An "antibody" refers to an immunoglobulin molecule that can bind to a specific antigen as a result of an immune response to that antigen.

[0020] An "antigen" or "immunogen" refers to any substance recognized by an animal's immune system that generates an immune response. This term includes dead, inactivated, attenuated, or modified live fungi, viruses, or parasites. The term "antigen" also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, polysaccharides, or lipids, either alone or in any combination thereof. The term "antigen" also includes antibodies such as anti-idiotype antibodies or fragments thereof, and synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).

[0018]

[0021] A "buffer solution" refers to a chemical system that prevents changes in the concentration of other chemical substances. For example, proton donor and acceptor systems function as buffer solutions that prevent significant changes in hydrogen ion concentration (pH). Further examples of buffer solutions include solutions containing a weak acid and its salt (conjugate base) or a mixture of a weak base and its salt (conjugate acid).

[0019]

[0022] "Subsequent booster vaccinations" refer to the second booster vaccination, the third booster vaccination, etc. "Previous booster vaccination" refers to the vaccination that immediately precedes the "subsequent booster vaccination." Therefore, if the third booster vaccination is a subsequent booster vaccination, the booster vaccination preceding it is the second booster vaccination. Generally, if vaccination N is a "subsequent booster vaccination," vaccination N-1 is the previous booster vaccination for vaccination N.

[0020]

[0023] The term "essentially consisting of" as applied to adjuvant formulations means that the formulation does not contain additional adjuvants or immunomodulators not listed in amounts that exert a measurable adjuvant or immunomodulatory effect.

[0021]

[0024] "Vaccination" refers to a single dose of vaccine or immunogenic composition administered to a target. "First vaccination," "priming vaccine," or "single prime vaccination" refers to the administration of the composition on day 0. "Second vaccination" or "annual vaccination" refers to the amount of the composition administered at a predetermined interval following the first vaccination, which may or may not be the same vaccine or immunogenic composition as the first vaccination.

[0022]

[0025] The term “emulsifier” is used broadly in this disclosure. This includes substances generally recognized as emulsifiers, such as various products in the TWEEN® or SPAN® product lines (fatty acid esters of polyethoxylated sorbitol and fatty acid-substituted sorbitan surfactants, respectively), and various solubility enhancers such as PEG-40 castor oil or other PEGylated hydrogenated oils.

[0023]

[0026] The term "FMD-negative" refers to an animal that has not previously been vaccinated against FMD. Preferably, the term "FMD-negative" refers to an animal that has not previously been vaccinated against FMD and has not been infected with FMD in the past (or currently).

[0024]

[0027] "Humoral immune response" refers to a response mediated by antibodies.

[0028] In this context, "immune response" refers to the expression of humoral, cellular, or combined humoral and cellular immune responses to an antigen. Immune responses can typically be determined using standard immunoassays and neutralization assays known in the field.

[0025]

[0029] An “immunologically effective amount” or “effective amount to produce an immune response” of an antigen refers to an amount effective in inducing an immunogenic response in a vaccinated individual. This immunogenic response may be sufficient for diagnostic purposes or other testing, or sufficient to prevent signs or symptoms of disease caused by pathogen infection, including adverse health effects and their complications. Humoral immunity, cell-mediated immunity, or both may be induced. An animal’s immunogenic response to an immunogenic composition can be evaluated indirectly, for example, by measuring antibody titers, lymphocyte proliferation tests, or directly by monitoring signs and symptoms after antigen administration with wild-type strains. On the other hand, protective immunity from a vaccine can be evaluated by measuring reductions in clinical signs such as mortality, morbidity, body temperature, overall physical condition, and the overall health and function of the subject. The immune response may include, but is not limited to, the induction of cellular and / or humoral immunity.

[0026]

[0030] "Immunogenicity" means the ability to evoke an immune response or antigen response. Therefore, an immunogenic composition would be any composition that induces an immune response.

[0031] "Pharmacologically acceptable" means a substance that, within the bounds of sound medical judgment, is suitable for use in contact with the target tissue without causing excessive toxicity, irritation, or allergic reactions, has a reasonable efficacy-to-risk ratio, and is effective for its intended use.

[0027]

[0032] The inventors found that a vaccine according to any of the above embodiments can provide advantageous protection from foot lesions caused by FMD for approximately 12 months, even when administered as a single dose.

[0028]

[0033] Accordingly, the present invention provides a vaccine for the prevention of foot lesions caused by FMD in animals, the vaccine administered in a single prime dose and one or more booster doses, the first booster dose administered 7 to about 12 months after the single prime dose, the vaccine comprising an FMD antigen and an adjuvant comprising (or consisting of, or essentially consisting of) an oil, an immunostimulatory oligonucleotide containing CpG, a polycationic carrier, and optionally one or more emulsifiers, the vaccine being a water-in-oil emulsion. In certain embodiments, the animal is a ruminant such as a cattle. In certain embodiments, the animal is FMD-negative.

[0029]

[0034] When multiple booster doses are administered, each subsequent booster dose is administered 7 to approximately 12 months after the previous booster dose. Therefore, the first of the one or more booster doses is administered 7 to approximately 12 months after the prime single dose, the second of the one or more booster doses is administered 7 to approximately 12 months after the first, the third of the one or more booster doses is administered 7 to approximately 12 months after the second, and so on. In certain embodiments, the interval between the prime single dose and the first booster dose is 7 months, or approximately 8 months, or approximately 9 months, or approximately 10 months, or approximately 11 months, or approximately 12 months.

[0030]

[0035] The interval between the two vaccinations is chosen independently of each other and can be the same or different, between 7 months and approximately 12 months, as mentioned above.

[0036] Table 1 shows non-limiting examples regarding the intervals between the prime single dose (PS) and the first booster dose (B1), and between the first booster dose and the second booster dose (B2). The symbol "X" indicates that the proposed vaccination regimen is suitable for the practice of the present invention.

[0031] [Table 1]

[0032]

[0037] Similarly, if the interval between the third booster dose and the second booster dose is in the range of 7 to approximately 12 months, then both of these intervals are perfectly acceptable, even if they are specific, as long as the intervals between SP and B1 and between B1 and B2 are also in the range of 7 to approximately 12 months.

[0033]

[0038] The vaccines described herein are suitable for any of the dosing regimens disclosed above. These vaccines comprise an FMD antigen and an adjuvant comprising an immunostimulatory oligonucleotide including an oil, a polycationic carrier, and CpG, and optionally one or more emulsifiers, wherein the vaccine is a w / o emulsion.

[0034] antigen

[0039] Viruses derived from any FMD serotype are suitable for use with the vaccine of this invention. Currently, seven serotypes of FMD have been isolated. Of these seven serotypes, A, C, O, Asia 1, and SAT3 appear to be distinct lineages; SAT 1 and SAT 2 are unresolved lineages. Multiple strains exist within each serotype. For example, A24 Cruzeiro belongs to serotype A, and O1 Campos belongs to serotype O.

[0035]

[0040] It should be noted that antigens from different serotypes may provide only limited cross-protection against antigen administration with heterologous serotype viruses. U.S. Patent No. 10,010,605 discloses the results of vaccination of pigs with VLPs containing a peptide pool of various strains of FMD virus. The authors report that when Asia1 Shamir FMDV antigen was used, a specific IFN-γ response (cellular response) was detected in both Asia1 Shamir VLP groups, and when A22 Iraq FMDV antigen was used, a specific IFN-γ response (cellular response) was detected in both A22 Iraq VLP groups. When A22 Iraq FMDV antigen was used, cross-immunogenicity was shown in the Asia1 Shamir VLP group. Specific plasma cells (humoral response) were detected in both the Asia1 Shamir VLP group and the A22 Iraq VLP group with the Asia1 Shamir antigen and the A22 Iraq antigen, respectively. Cross-immunogenicity (plasma cells) was observed in the Asia1 Shamir VLP group with the A22 Iraq antigen. In the Asia1 Shamir VLP group and the A22 Iraq VLP group, specific memory B cells (humoral response) were detected for the Asia1 Shamir antigen and the A22 Iraq antigen, respectively. In the Asia1 Shamir VLP group, good cross-immunogenicity (B cells) was shown with the A22 Iraq antigen. In the A22 Iraq VLP group, some degree of cross-immunogenicity (B cells) was also shown with the Asia1 Shamir antigen. The authors concluded that VLPs can induce an immune response sufficient to protect against heterologous antigen administration.

[0036]

[0041] However, it is preferable that the vaccine contains antigens derived from serotypes for which protection is desired, and this may vary depending on the geographical region. Therefore, in Africa, where SAT-1, SAT-2, and SAT-3 are most prevalent, it is preferable that the vaccine contains antigens derived from strains belonging to serotypes SAT-1, SAT-2, and / or SAT-3. In Europe, where strains of serotypes A, O, and C are prevalent, it is preferable that the vaccine contains antigens from strains belonging to serotypes O, A, and C. In Asia, it is preferable that the vaccine contains antigens from strains belonging to serotype Asia.

[0037]

[0042] Any serotype of FMD virus may be used as needed, provided the virus is not pathogenic. Pathogenicity can be reduced by inactivating the virus, for example, by treatment with formaldehyde or BEI.

[0038]

[0043] In certain embodiments, the virus may be attenuated by culture passaging or recombinant means. For example, leader protein L pro It has been previously demonstrated that deletion of the coding region produces attenuated FMD viruses in cattle and pigs. See, for example, U.S. Patents 5,824,316 and 8,765,141, Virology 1997 227(1):96-102, and J.Virol 2012 86:11675-11685. Point mutations at positions 55 and 58 within the SAP domain of the L protein also resulted in viable viruses exhibiting a mild attenuated phenotype in cell culture and protective effects in a porcine FMD model. See, U.S. Patent 8,846,057.

[0039]

[0044] In certain embodiments, the virus also includes a negative antigen marker that enables a DIVA (differentiating infected from vaccinated animals) assay. In certain embodiments, the negative antigen marker is introduced into 3D and / or 3B proteins.

[0040]

[0045] Like other viruses, FMD viruses are constantly evolving and mutating, and one of the challenges in vaccination is the vast amount of variation between and within serotypes. There is no cross-protection between serotypes (a vaccine against one serotype does not necessarily protect against others), and furthermore, two strains within a given serotype can differ by up to 30% in nucleotide sequences in the same gene. This means that, although some cross-protection between strains has been reported, FMD vaccines must be specific to the strain in question.

[0041]

[0046] Therefore, in certain embodiments, an endonuclease restriction site is introduced into the viral genome, thereby enabling the introduction of proteins derived from heterologous FMD strains (e.g., proteins that form the outer capsid).

[0042]

[0047] In certain embodiments, the antigenic component comprises FMD strain A24 Cruzeiro, which may optionally be modified by inactivation of the leader protein (by substitution, insertion or deletion, or a combination thereof), negative marker mutations in the 3B and / or 3D proteins, and the introduction of restriction enzyme cleavage sites to facilitate the introduction of heterologous antigen sequences (e.g., capsid proteins). Suitable non-limiting examples of antigens are described in U.S. Patent No. 8,765,141. See also U.S. Patents No. 9,180,179 and No. 10,478,487.

[0043]

[0048] Therefore, the DNA sequence complementary to the reference DNA sequence is a template for the RNA genome of the FMDV virus (i.e., the RNA encoding FMDV), that is, complementary to or "encodes" it. In certain embodiments, the virus contains the capsid protein of a heterologous FMD strain (i.e., FMD strains other than A24 Cruzeiro, including, but not limited to, strains of lineages C, O, Asia 1, SAT3, SAT 1 and SAT 2, Turkey 06, and other strains of lineage A).

[0044]

[0049] In certain non-limiting embodiments, FMD may be the following strains: O1 Manisa, O1 BFS or Campos, A24 Cruzeiro, Asia 1 Shamir, A Iran '96, A22 Iraq, or SAT2 Saudi Arabia.

[0045]

[0050] As other strains, there are FMDV strains A10-61, A5, A12, A24 / Cruzeiro, C3 / Indaial, O1, C1-Santa Pau, C1-C5, A22 / 550 / Azerbaijan / 65, SAT1-SAT3, A, A / TNC / 71 / 94, A / IND / 2 / 68, A / IND / 3 / 77, A / IND / 5 / 68, A / IND / 7 / 82, A / IND / 16 / 82, A / IND / 17 / 77, A / IND / 17 / 82, A / IND / 19 / 76, A / IND / 20 / 82, A / IND / 22 / 82, A / IND / 25 / 81, A / IND / 26 / 82, A / IND / 54 / 79, A / IND / 57 / 79, A / IND / 73 / 79, A / IND / 85 / 79, A / IND / 86 / 79, A / APA / 25 / 84, A / APN / 41 / 84, A / APS / 44 / 05, A / APS / 50 / 05, A / APS / 55 / 05, A / APS / 66 / 05, A / APS / 68 / 05, A / BIM / 46 / 95, A / GUM / 33 / 84, A / ORS / 66 / 84, A / ORS / 75 / 88, A / TNAn / 60 / 947 / Asia / 1, A / IRN / 05, Asia / IRN / 05, 0 / HK / 2001, 0 / UKG / 3952 / 2001, O / UKG / 4141 / 2001, Asia 1 / HNK / CHA / 05 (GenBank accession number EF149010 incorporated herein by reference), Asia I / XJ (Li, ZhiYong et al., Chin Sci Bull, 2007), HK / 70 (Chin Sci Bull, 2006, 51(17): 2072-2078), O / UKG / 7039 / 2001, O / UKG / 9161 / 2001, O / UKG / 7299 / 2001, O / UKG / 4014 / 2001, O / UKG / 4998 / 2001, O / UKG / 9443 / 2001, O / UKG / 5470 / 2001, O / UKG / 5681 / 2001, O / ES / 2001, HKN / 2002, O5India, O / BKF / 2 / 92, K / 37 / 84 / A, KEN / 1 / 76 / A, GAM / 51 / 98 / A, A10 / Holland, O / KEN / 1 / 91, O / IND49 / 97, O / IND65 / 98, O / IND64 / 98, O / IND48 / 98, O / IND47 / 98, O / IND82 / 97, O / IND81 / 99, O / IND81 / 98, O / IND79 / 97,O / IND78 / 97, O / IND75 / 97, O / IND74 / 97, O / IND70 / 97, O / IND66 / 98, O / IND63 / 97, O / IND61 / 97, O / IND57 / 98, O / IND56 / 98, O / IND55 / 98, O / IND54 / 98, O / IND469 / 98, O / IND465 / 97, O / IND464 / 97, O / IND424 / 97, O / IND423 / 97, O / IND420 / 97, O / IND414 / 97, O / IND411 / 97, O / IND410 / 97, O / IND409 / 97, O / IND407 / 97, ​​O / IND399 / 97, O / IND39 / 97, O / IND391 / 97, O / IND38 / 97, O / IND384 / 97, O / IND380 / 97, O / IND37 / 97, ​​O / IND352 / 97, O / IND33 / 9 7, O / IND31 / 97, O / IND296 / 97, O / IND23 / 99, O / IND463 / 97, O / IND461 / 97, O / IND427 / 98, O / IND28 / 97, O / IND2 87 / 99, O / IND285 / 99, O / IND282 / 99, O / IND281 / 97, O / IND27 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, This may include O / IND210 / 99, O / IND208 / 99, O / IND207 / 99, O / IND205 / 99, O / IND185 / 99, O / IND175 / 99, O / IND170 / 97, O / IND164 / 99, O / IND160 / 99, O / IND153 / 99, O / IND148 / 99, O / IND146 / 99, O / SKR / 2000, and A22 / India / 17 / 77.

[0046]

[0051] Such antigen variants are also conceivable. These variants are at least 80% identical to the reference sequence (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) using one of the alignment programs described with standard parameters. Multiple alignment tools are available for determining sequence identity, including, but are not limited to, BLAST, CLUSTAL, or PHILIP.

[0047]

[0052] Those skilled in the art will recognize that by considering codon degeneracy, amino acid similarity, reading frame position, and the like, these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences.

[0048]

[0053] In certain embodiments, variants encompass beyond specific exemplary nucleotide or amino acid sequences and include their functional equivalents. Modifications of nucleic acid fragments that produce chemically equivalent amino acids at a given site but do not affect the functional properties of the encoded polypeptide are well known in the art. Thus, the codon of the hydrophobic amino acid alanine can be replaced with a codon encoding another less hydrophobic residue such as glycine, or a highly hydrophobic residue such as valine, leucine, or isoleucine. Similarly, it is expected that functionally equivalent products will be produced by changes such as replacing a negatively charged residue with another residue, such as replacing aspartic acid with glutamic acid, or replacing a positively charged residue with another residue, such as replacing lysine with arginine. Nucleotide changes resulting in mutations in the N-terminal and C-terminal portions of a polypeptide molecule are also expected not to alter the activity of the polypeptide. Each of the proposed modifications, as well as the determination of the preservation of the biological activity of the encoded product, is within the scope of the ordinary art of those skilled in the art.

[0049]

[0054] Furthermore, the polypeptides of the present invention can be modified in various ways, including amino acid substitution, deletion, cleavage, and insertion. By combining elements and fragments of the proteins of the present invention, as well as other proteins, novel proteins with desired properties can be generated. Such manipulation methods are known in the art. Therefore, the gene and nucleotide sequences of the present invention include both naturally occurring sequences and mutants. Similarly, the proteins of the present invention include naturally occurring proteins, as well as their mutants and modifications. Such mutants continue to retain the desired modification activity of the parental FMD virus. Mutations applied to the DNA encoding the mutant should not remove the sequence from the reading frame and preferably do not generate complementary regions that may give rise to secondary mRNA structures.

[0050]

[0055] Methods for amplifying and purifying antigens suitable for the present invention are well known in the art and include, without limitation, hollow fiber filtration and PEG precipitation. These methods produce somewhat different antigen compositions. For example, in PEG precipitation, the antigen composition is free of non-structural proteins. On the other hand, in other methods such as hollow fiber filtration, the antigen composition contains both structural and non-structural FMD proteins. Therefore, in some embodiments, the FMD antigen contains structural proteins. In other embodiments, such as when the FMD antigen is prepared by hollow fiber filtration, the FMD antigen contains both structural and non-structural proteins, particularly 3D proteins.

[0051]

[0056] In other embodiments, the antigen may include an empty FMD virus capsid or virus-like particle, or a VLP. Both are known in the art. For example, U.S. Published Patent Application 2004 / 0001864 teaches a vaccine against foot-and-mouth disease in which an empty capsid is produced by co-expressing P1 and protease 3C. U.S. Patent No. 8,409,588 discloses a method for producing foot-and-mouth disease virus-like particles comprising the following steps: providing a host cell comprising an expression system comprising a promoter operably linked to a nucleic acid molecule encoding a polyprotein, wherein the polyprotein comprises FMDV 1A protein-non-FMDV protease recognition sequence-FMDV 1B protein-non-FMDV protease recognition sequence-FMDV 1C protein-non-FMDV protease recognition sequence-FMDV 1D protein, and the host cell expresses a protease that recognizes the non-FMDV protease recognition sequence; culturing the host cell under conditions such that a polyprotein is produced and degraded by the protease into 1A, 1B, 1C, and 1D, and 1A, 1B, 1C, and 1D are assembled into virus-like particles; and recovering the virus-like particles. See also Subramanian et al., "Development of foot-and-mouth disease virus (FMDV) serotype O virus-like-particles (VLPs) vaccine and evaluation of its potency." Antiviral Res. December 2012; 96(3): pp. 288-2895, which reports on VLPs obtained by co-expressing FMDV structural protein and 3C protease in Sf9 cells.

[0052]

[0057] The vaccines described herein may be monovalent (using only one strain of FMD antigen) or polyvalent (using two or more strains of FMD antigen). Therefore, for example, the vaccine may use antigens derived from a single strain of serotype A, or a single strain of serotype C, or a single strain of serotype Asia. A polyvalent vaccine may include strains derived from serotype A, serotype O, serotype C, serotype Asia, or any two or any three strains from these. In other embodiments, a polyvalent vaccine may include several strains derived from the same serotype.

[0053]

[0058] The vaccines described herein allow for some flexibility regarding the amount of antigen per dose. In certain embodiments, the amount of antigen may include at least 4 μg of antigen per dose per strain (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 μg per dose per strain). In the polyvalent vaccines described herein, the amounts of different antigens per dose are selected independently of other antigens.

[0054]

[0059] Furthermore, the use of heterologous (non-FMD) viral vectors encoding FMD antigens has been reported to provide a protective effect against antigen administration. See, for example, U.S. Patent No. 10,188,721, which discloses a composition or vaccine comprising a recombinant viral vector, such as an adenovirus vector expressing an FMDV antigen that induces a protective response in animals.

[0055]

[0060] Therefore, in certain embodiments, the FMD antigen is provided in the form of an adenovirus vector expressing FMDV structural protein P1 (VP4-VP2-Vp3-VP1), non-structural protein P2 (2A, 2B, and 2C), or non-structural protein P3 (3A, 3B, 3C, and 3D), or active fragments or variants thereof.

[0056]

[0061] In other embodiments, the antigen may include at least one immunogenic peptide derived from the FMD virus. Suitable non-limiting examples of such peptides are disclosed, for example, in International Publication No. 03068169, 20110206718, incorporated herein by reference, and in Ren et al., Vaccine 29 (2011), pp. 7960–7965.

[0057]

[0062] If the immunogenicity of a particular antigen is determined by standard methods for inactivated viruses, those skilled in the art will have no difficulty selecting an appropriate antigen dose. In the polyvalent vaccines described herein, the amounts of different antigens per dose are selected independently of each other.

[0058] Adjuvant

[0063] The vaccine of the present invention is formulated as a water-in-oil (W / O) emulsion and comprises (or essentially comprises) an oil, an immunostimulatory oligonucleotide containing CpG, a polycationic carrier, and optionally one or more emulsifiers.

[0059]

[0064] The present invention is suitable for a variety of oils, including non-metabolized oils (e.g., light mineral oils), metabolized oils (e.g., vegetable oils, or fatty acids, polyols, or alcohol esters), or mixtures thereof. When mixtures of non-metabolized and metabolized oils are used, it is preferable that the non-metabolized oil constitutes more than 50% v / v of the mixture (e.g., more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99%). In certain embodiments, 100% of the oil in the emulsion is non-metabolized. In the most preferred embodiment, 100% of the oil in the W / O emulsion is mineral oil. As used herein, the term “mineral oil” refers to a mixture of liquid hydrocarbons obtained by distillation techniques from petrolatum. This term is synonymous with “liquefied paraffin,” “liquid petrolatum,” and “white mineral oil.” This term is also intended to include “light mineral oil,” i.e., oils similarly obtained by distillation of petrolatum but with a slightly lower specific gravity than white mineral oil. See, for example, Remington's Pharmaceutical Sciences, 18th edition (Easton, Pa.: Mack Publishing Company, 1990, pp. 788 and 1323). Mineral oil can be obtained from various commercial sources, such as JTBaker (Phillipsburg, Pennsylvania) or USB Corporation (Cleveland, Ohio). The preferred mineral oil is light mineral oil, marketed under the name DRAKEOL®.

[0060]

[0065] The volume fraction of oil in the vaccine should be sufficient for a water-in-oil (W / O) emulsion. The integrity of the water-in-oil emulsion can be maintained as long as the dispersed spherical water droplets are not present in a form more concentrated than the maximum random packing rate of monodisperse droplets, i.e., 0.64. See Tadros, Emulsion Formation, Stability and Rheology, 1st edition 2013, Wiley-VCH GmbH & Co KGaA. This is as long as the total volume fraction occupied by water droplets does not exceed 0.64, i.e., 64% v / v. Therefore, in different embodiments, oil may be present in amounts exceeding 36% of the vaccine v / v (e.g., 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 55%, 60%, 65%, 70%, 75%, 80% or more of the vaccine v / v).

[0061]

[0066] The emulsion may also contain one or more emulsifiers. In additional embodiments, the emulsifiers used herein either do not contain lecithin or contain an immunologically ineffective amount of lecithin.

[0062]

[0067] Suitable non-natural synthetic emulsifiers for use in the adjuvant formulations of the present invention include sorbitan-based nonionic surfactants, such as fatty acid-substituted sorbitan surfactants (commercially available under the names SPAN® or ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids derived from raw materials such as castor oil (EMULFOR®); polyethoxylated fatty acids (e.g., stearic acid available under the name SIMULSOL® M-53), polyethoxylated isooctylphenol / formaldehyde polymers (TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®); polyoxyethylene nonphenyl ethers (TRITON® N); and polyoxyethylene isooctylphenyl ethers (TRITON® X). Preferred synthetic surfactants are those available under the names SPAN® and TWEEN®, such as TWEEN®-80 (sorbitan polyoxyethylene (20) monooleate) and SPAN®-80 (sorbitan monooleate).

[0063]

[0068] Emulsifiers can generally be present in the emulsion at an amount of 0-20% v / v. In certain embodiments, TWEEN® is present at an amount of 1% to 10% v / v of the vaccine, and SPAN® is also present at an amount of 1% to 10% v / v of the vaccine. For example, TWEEN® is present at an amount of 1% to 6% v / v of the vaccine, and SPAN® is also present at an amount of 1% to 6% v / v of the vaccine. In other embodiments, TWEEN® is present at an amount of 1% to 2% v / v of the vaccine, and SPAN® is present at an amount of 5% to 8% v / v of the vaccine. In yet another embodiment, TWEEN® is present at an amount of 4% to 7% v / v of the vaccine, and SPAN® is present at an amount of 1% to 3% v / v of the vaccine.

[0064]

[0069] The adjuvant component of the vaccine further includes a polycationic carrier. Suitable non-limiting examples of polycationic carriers include, but are not limited to, PEG, guar gum, chitosan derivatives, polycellulose derivatives such as hydroxyethylcellulose (HEC) polyethyleneimine, polyaminos such as polylysine, and cationized dextran. In certain embodiments, the polycationic carrier is diethylaminoethyl (DEAE) dextran.

[0065]

[0070] Polycationic carriers such as DEAE dextran may be present in the vaccine in amounts of 5 to approximately 500 mg per dose (for example, 10 to 500 mg, or 10 to 300 mg, or 10 to 50 mg, or 50 to 200 mg, or approximately 100 mg per dose).

[0066]

[0071] The adjuvant component of the vaccine further includes an immunomodulatory oligonucleotide. The immunomodulatory oligonucleotide according to the present invention contains CpG (also called "CpG-containing immunomodulatory oligonucleotide," "CpG oligonucleotide," or simply "CpG"). The effects of CpG-containing oligonucleotides on the immune system have been known for more than 20 years.

[0067]

[0072] Generally, CpGs suitable for the present invention are 15 to 100 bases in length, for example, 15 to 50 bases, or 18 to 40 bases, or 20 to 30 bases, or 20 to 24 bases.

[0068]

[0073] Several classes of CpGs have been reported, including Class A CpGs, Class B CpGs, Class C CpGs, and Class P CpGs. In certain embodiments, the CpG-containing immunostimulatory oligonucleotide is a Class P CpG. Class P CpGs are characterized by the presence of one or more TLR-9 activating motifs(s) and two palindromes or two complementary regions. Preferably, one or more TLR-9 activating motifs are located at the 5' end of the oligonucleotide and may be fully or partially incorporated into the 5' palindrome or 5' complementary region. The TLR-9 activating motifs are known and include, but are not limited to, TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT, or TTTT. The 5' palindrome or 5' complementary region is at least 6 nucleotides long. The 3' palindrome or 3' complementary region is at least 8 nucleotides long and is generally rich in C and G. These structural features of P-class CpGs confer upon them the ability to spontaneously self-assemble into a constituent in vitro and / or in vivo.

[0069]

[0074] To enhance the lipophilicity of CpG oligonucleotides, at least one lipophilic substitution nucleotide analog may be preferably included at the 5' end of the oligonucleotide. P-class immunostimulatory oligonucleotides can be modified according to techniques known in the art. For example, J modification refers to iodine-modified nucleotides, and E modification refers to ethyl-modified nucleotides. Therefore, an E-modified P-class immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide in which at least one nucleotide (preferably the 5' nucleotide) is ethylated. Additional modifications include the addition of 6-nitrobenzimidazole, O-methylation, modification with propynyl-dU, inosine modification, and 2-bromovinyl addition (preferably to uridine).

[0070]

[0075] Oligonucleotides modified by the addition of lipophilic moieties are generally described in U.S. Patent Application Publication No. 20100166780.

[0076] In certain embodiments, the CpG according to the present invention includes a modified backbone that includes, without limitation, phosphorothioate modifications, halogenations, alkylations (e.g., ethyl or methyl modifications), and phosphodiester modifications.

[0071]

[0077] Suitable non-limiting examples of modified class P immunostimulatory oligonucleotides are provided below (where "*" refers to a phosphorothioate bond, "-" refers to a phosphodiester bond, "JU" refers to 5'-iodo-2'-deoxyuridine, and "EU" refers to 5-ethyl-2'-deoxyuridine).

[0072] SEQ ID NO:1 5’ T * C-G * T * C-G * A * C-G * A * T * C-G * G * C * G * C-G * C * G * C * C * G 3’ SEQ ID NO:2 5’ T * C-G * A * C * G * T * C * G * A * T * C * G * G * C * G * C * G * C * G * C * C * G 3’ SEQ ID NO:3 5’ T * C * G * A * C* G * T * C * G * A * T * C * G * G * C * G * C * G * C * G * C * C * G * T 3' Allocation number 4 5' JU * CG * A * C * G * T * C * G * A * T * C * G * G * C * G * C * G * C * G * C * C * G 3' Allocation number 5 5' JU * CG * A * C * G * T * C * G * A * T * C * G * G * C * G * C * G * C * G * C * C * G * T 3' Allocation number 6 5' JU * C * G * A* C * G * T * C * G * A * T * C * G * G * C * G * C * G * C * G * C * C * G * T 3' Allocation number 7 5' EU * CG * A * C * G * T * C * G * A * T * C * G * G * C * G * C * G * C * G * C * C * G 3' Allocation number 8 5' JU * CG * T * C * G * A * C * G * A * T * C * G * G * C * G * G * C * C * G * C * C * G * T 3' Allocation number 9 5' JU * C * G* T * C * G * A * C * G * A * T * C * G * G * C * G * G * C * C * G * C * C * G * T 3' Sequence ID 10 5' T * CG * T * CG * A * CG * A * T * CG * G * C * G * CG * C * G * C * C * G 3'

[0078] In certain embodiments, the CpG oligonucleotide according to the present invention comprises an oligonucleotide comprising any one of SEQ ID NOs: 1 to 10, or an oligonucleotide comprising at least 15 consecutive bases of any one of SEQ ID NOs: 1 to 10. In the most preferred embodiment, the vaccine comprises an oligonucleotide comprising at least 15 consecutive bases of SEQ ID NO: 8 (for example, at least 16 bases, at least 17 bases, at least 18 bases, at least 19 bases, at least 20 bases, at least 21 bases, at least 22 bases, or at least 23 bases).

[0073]

[0079] CpG oligonucleotides may be present in the vaccine in amounts of 10-400 μg per dose, or 25-300, 50-200, or 50-100 mg per dose.

[0074]

[0080] Other components of the composition may include pharmaceutically acceptable excipients, such as carriers, solvents, diluents, isotonic agents, buffers, stabilizers, preservatives, antimicrobial agents, and antifungal agents. Typical carriers, solvents, and diluents include water, saline, dextrose, ethanol, glycerol, and oil. Typical isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, and lactose. Useful stabilizers include gelatin and albumin. The composition may also contain antibiotics or preservatives, such as gentamicin, methylthiolate, or chlorocresol. Various classes of antibiotics or preservatives that can be selected are well known to those skilled in the art.

[0075]

[0081] In certain embodiments, the vaccine of the present invention is prepared by creating a first solution comprising oil and an optional oil-soluble emulsifier, and a second solution comprising an aqueous diluent, an antigen, a CpG oligonucleotide, a polycationic carrier, and an optional water-soluble emulsifier. The first solution is then mixed with the second solution, for example, by adding the second solution dropwise to the first solution, thereby preparing a W / O emulsion vaccine according to the present invention.

[0076]

[0082] This disclosure further provides the following:

[0083] Item 1. A vaccine comprising an FMD antigen and an adjuvant comprising (or consisting of, or essentially consisting of) an oil, a polycationic carrier, and an immunostimulatory oligonucleotide, wherein the vaccine is a water-in-oil (W / O) emulsion and is intended for use in the prevention of foot lesions caused by FMD infection in ruminants, wherein the vaccine is administered as a single prime dose and one or more booster doses, the first of which is administered 7 months to about 12 months after the single prime dose.

[0077]

[0084] Item 2. Each subsequent booster dose is administered 7 to approximately 12 months after the previous booster dose, using the vaccines listed in Item 1.

[0085] Item 3. The vaccine described in Item 1 or 2, wherein the first booster dose is administered approximately 8 months, or 9 months, or 10 months, or 11 months, or 12 months after the single prime dose.

[0078]

[0086] Item 4. A vaccine described in any one of items 1-3, wherein the cationic carrier is DEAE dextran.

[0087] Item 5. A vaccine described in Item 4 that contains DEAE dextran in an amount of 25-250 mg per dose.

[0079]

[0088] Item 6. A vaccine as described in Item 5, containing at least 25 μg of immunostimulatory oligonucleotides per dose.

[0089] Item 7. A vaccine as described in Item 6, containing immunostimulatory oligonucleotides in an amount of 50-150 μg per dose.

[0080]

[0090] Item 8. A vaccine described in any one of items 1-6, wherein the immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide.

[0091] Item 9. The vaccine described in Item 8, wherein the P-class immunostimulatory oligonucleotide is a modified P-class immunostimulatory oligonucleotide.

[0081]

[0092] Item 10. The vaccine described in Item 9, comprising a modified P-class immunostimulatory oligonucleotide, which contains 5'-iodo-2'-deoxyuridine or 5-ethyl-2'-deoxyuridine.

[0082]

[0093] Item 11. The vaccine described in Item 10, wherein the P-class immunostimulatory oligonucleotide comprises 15 consecutive oligonucleotides of Sequence ID No. 8.

[0094] Item 12. The vaccine described in Item 11, wherein the P-class immunostimulatory oligonucleotide comprises 20 consecutive oligonucleotides of Sequence ID No. 8.

[0083]

[0095] Item 13. The vaccine described in Item 12, comprising a P-class immunostimulatory oligonucleotide, SEQ ID NO: 8.

[0096] Item 14. A vaccine described in any one of items 1-13, containing at least 40% oil (v / v).

[0084]

[0097] Item 15. A vaccine as described in Item 14, containing at least 45% oil (v / v).

[0098] Item 16. A vaccine as described in Item 15, containing at least 48% oil (v / v).

[0099] Item 17. A vaccine as described in Item 16, containing at least 52% oil (v / v).

[0085]

[0100] Item 18. A vaccine as described in Item 17, containing at least 60% oil (v / v).

[0101] Item 19. A vaccine described in any one of items 1-18, wherein the oil is a non-metabolized oil.

[0086]

[0102] Item 20. A vaccine as described in Item 19, in which the non-metabolized oil is light mineral oil.

[0103] Item 21. A vaccine described in any one of items 1-20, wherein the FMD antigen is derived from type O, type A, and / or Asia-1 FMD virus.

[0087]

[0104] Item 22. A vaccine described in any one of items 1-20, wherein the FMD antigen is derived from FMD virus of serotype O, A, and / or C.

[0105] Item 23. A vaccine described in any one of items 1-20, wherein the FMD antigen is derived from the FMD virus of serotypes SAT-1, SAT-2, and / or SAT-3.

[0088]

[0106] No. 24. FMD licensed O1 Manisa, O1 BFS, Campos, Asia 1 Shamir, A Iran '96, A22 Iraq, SAT2 Saudi Arabia, A10-61, A5, A12, A24 / Cruzeiro, C3 / Indaial, O1, C1-Santa Pau、C1-C5、A22 / 550 / Azerbaijan / 65、SAT1-SAT3、A、A / TNC / 71 / 94、A / IND / 2 / 68、A / IND / 3 / 77、A / IND / 5 / 68、A / IND / 7 / 82、A / IND / 16 / 82、A / IND / 17 / 7 7、A / IND / 17 / 82、A / IND / 19 / 76、A / IND / 20 / 82、A / IND / 22 / 82、A / IND / 25 / 81 A / IND / 26 / 82, A / IND / 27 / 11, A / IND / 54 / 79, A / IND / 57 / 79, A / IND / 73 / 79 A / IND / 85 / 79, A / IND / 86 / 79, A / APA / 25 / 84, A / APN / 41 / 84, A / APS / 44 / 05 / APS / 50 / 05、A / APS / 55 / 05、A / APS / 66 / 05、A / APS / 68 / 05、A / BIM / 46 / 95、A / GUM / 33 / 84, A / ORS / 66 / 84, A / ORS / 75 / 88, A / TNAn / 60 / 947 / Asia / 1, A / IRN / 05、Asia / IRN / 05、O / HK / 2001、0 / UKG / 3952 / 2001、O / UKG / 4141 / 2001、Asia 1 / HNK / CHA / 05、Asia I / XJ、HK / 70、O / UKG / 7039 / 2001、O / UKG / 9161 / 2001、O / UKG / 7299 / 2001、O / UKG / 4014 / 2001、O / UKG / 4998 / 20 01、O / UKG / 9443 / 2001、O / UKG / 5470 / 2001、O / UKG / 5681 / 2001、O / ES / 2001、HKN / 2002、O5India、O / BKF / 2 / 92、 K / 37 / 84 / A、KEN / 1 / 76 / A、GAM / 51 / 98 / A、A10 / Netherlands、O / KEN / 1 / 91、O / IND49 / 97、O / IND65 / 98、O / IND64 / 98 O / IND48 / 98, O / IND47 / 98, O / IND82 / 97, O / IND81 / 99, O / IND81 / 98, O / IND79 / 97, O / IND78 / 97, O / IND75 / 97O / IND74 / 97, O / IND70 / 97, O / IND66 / 98, O / IND63 / 97, O / IND61 / 97, O / IND57 / 98, O / IND56 / 98, O / IND55 / 98, O / IN D54 / 98, O / IND469 / 98, O / IND465 / 97, O / IND464 / 97, O / IND424 / 97, O / IND423 / 97, O / IND420 / 97, O / IND414 / 97, O / IND411 / 97, O / IND410 / 97, O / IND409 / 97, O / IND407 / 97, ​​O / IND399 / 97, O / IND39 / 97, O / IND391 / 97, O / IND38 / 97, O / IND384 / 97, O / IND380 / 97, O / IND37 / 97, ​​O / IND352 / 97, O / IND33 / 97, O / IND31 / 97, O / IND296 / 97, O / IND23 / 99, O / IND463 / 97, O / IND461 / 97, O / IND427 / 98, O / IND28 / 97, O / IND287 / 99, O / IND285 / 99, O / IND282 / 99, O / IND281 / 9 7, O / IND27 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, O / IND210 / 99, O / IND208 / 99, O / IND207 / 99, O / IND205 The vaccine described in item 24 is derived from a strain selected from the group consisting of O / IND / 99, O / IND185 / 99, O / IND175 / 99, O / IND170 / 97, O / IND164 / 99, O / IND160 / 99, O / IND153 / 99, O / IND148 / 99, O / IND146 / 99, O / IND / R2 / 75, O / SKR / 2000, A22 / India / 17 / 77, and any combination thereof.

[0089]

[0107] Item 25. A vaccine described in any one of items 1-24, wherein the antigen is an inactivated FMD virus.

[0108] Item 26. The vaccine described in Item 24, in which the inactivated FMD virus is a recombinant virus.

[0090]

[0109] Item 27. Recombinant FMD virus lacking a functional leader protein, as described in Item 26.

[0110] Item 28. The vaccine described in item 26 or 27, wherein the recombinant FMD virus further comprises one or more DIVA markers.

[0091]

[0111] Item 29. The vaccine described in Item 26, wherein recombinant FMD virus further comprises one or more deletion sequences as negative markers.

[0112] Item 30. The vaccine described in Item 26, wherein the recombinant FMD virus further comprises one or more stability-inducing mutations in the capsid-coding sequence.

[0092]

[0113] Item 31. A vaccine described in any one of items 26-30, wherein a recombinant FMD virus expresses a capsid derived from a heterologous FMD strain.

[0114] Item 32. The vaccine described in Item 31, wherein the heterologous FMD strain is a strain of serotype A, serotype O, serotype Asia, serotype C, serotype SAT1, serotype SAT2 and / or serotype SAT3.

[0093]

[0115] Item 33. The vaccine described in Item 31, wherein the heterologous FMD strain is a strain of serotype A, or serotype O, and / or serotype Asia 1.

[0116] Item 34. The vaccine described in Item 31, wherein the heterologous FMD strain is a strain of serotype A, or serotype O, and / or serotype C.

[0094]

[0117] Item 35. The vaccine described in Item 31, wherein the heterologous FMD strain is a strain of serotype SAT1, or serotype SAT2 and / or serotype SAT3.

[0118] Material 36, O1 Manisa, O1 BFS, Campos, Asia 1 Shamir, A Iran '96, A22 Iraq, SAT2 Saudi Arabia, A24 / Cruzeiro, A10-61, A5, A12, C3 / Indaial, O1, C1-Santa Pau、C1-C5、A22 / 550 / Azerbaijan / 65、SAT1-SAT3、A、A / TNC / 71 / 94、A / I ND / 2 / 68、A / IND / 3 / 77、A / IND / 5 / 68、A / IND / 7 / 82、A / IND / 16 / 82、A / IND / 1 7 / 77、A / IND / 17 / 82、A / IND / 19 / 76、A / IND / 20 / 82、A / IND / 22 / 82、A / IND / 25 / 81、A / IND / 26 / 82、A / IND / 54 / 79、A / IND / 57 / 79、A / IND / 73 / 79、A / IND / 85 / 79, A / IND / 86 / 79, A / APA / 25 / 84, A / APN / 41 / 84, A / APS / 44 / 05, A / APS / 50 / 05、A / APS / 55 / 05、A / APS / 66 / 05、A / APS / 68 / 05、A / BIM / 46 / 95、A / GUM / 33 / 84、A / ORS / 66 / 84、A / ORS / 75 / 88、A / TNAn / 60 / 947 / Asia / 1、A / IRN / 0 5、Asia / IRN / 05、O / HK / 2001、0 / UKG / 3952 / 2001、O / UKG / 4141 / 2001、Asia 1 / HNK / CHA / 05、Asia I / XJ、HK / 70、O / UKG / 7039 / 2001、O / UKG / 9161 / 2001、O / UKG / 7299 / 2001、O / UKG / 4014 / 2001、O / UKG / 4998 / 2001. O / UKG / 9443 / 2001、O / UKG / 5470 / 2001、O / UKG / 5681 / 2001、O / ES / 2001、HKN / 2002、O5India、O / BKF / 2 / 92、K / 37 / 2001. 84 / A、KEN / 1 / 76 / A、GAM / 51 / 98 / A、A10 / Holland.O / KEN / 1 / 91.O / IND49 / 97.O / IND65 / 98.O / IND64 / 98.O / IND48 / 98、O / IND47 / 98、O / IND82 / 97、O / IND81 / 99、O / IND81 / 98、O / IND79 / 97、O / IND78 / 97、O / IND75 / 97、O / IND74 / 97、O / IND70 / 97, O / IND66 / 98, O / IND63 / 97, O / IND61 / 97, O / IND57 / 98, O / IND56 / 98, O / IND55 / 98, O / IND54 / 98, O / IND469 / 98, O / IND465 / 97, O / IND464 / 97, O / IND424 / 97, O / IND423 / 97, O / IND420 / 97, O / IND414 / 97, O / IND411 / 97, O / IND410 / 97, O / IND409 / 97, O / IND407 / 97, ​​O / IND399 / 97, O / IND39 / 97, O / IND391 / 97, O / IND38 / 97, O / IND 384 / 97, O / IND380 / 97, O / IND37 / 97, ​​O / IND352 / 97, O / IND33 / 97, O / IND31 / 97, O / IND296 / 97, O / IND23 / 99, O / IN D463 / 97, O / IND461 / 97, O / IND427 / 98, O / IND28 / 97, O / IND287 / 99, O / IND285 / 99, O / IND282 / 99, O / IND281 / 97 , O / IND27 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, O / IND210 / 99, O / IND208 / 99, O / IND207 / 99, O / IND20 The vaccines described in item 31, selected from the group consisting of 5 / 99, O / IND185 / 99, O / IND175 / 99, O / IND170 / 97, O / IND164 / 99, O / IND160 / 99, O / IND153 / 99, O / IND148 / 99, O / IND146 / 99, O / SKR / 2000, A22 / India / 17 / 77, O / IND / R2 / 75, and any combination thereof.

[0095]

[0119] Item 37. A vaccine described in any one of items 24-36, containing at least 4 μg of FMD virus per dose per strain.

[0120] Item 38. A vaccine described in any one of items 24-37, containing at least 8 μg of FMD virus per dose per strain.

[0096]

[0121] Item 39. A vaccine described in any one of items 24-38, in which the FMD virus is present in an amount of approximately 10 μg per dose per strain.

[0122] Item 40. A vaccine described in any one of items 1-39, in which the ruminant is a cow.

[0097]

[0123] Item 41. Use of a vaccine, which is a water-in-oil (W / O) emulsion comprising an FMD antigen and an adjuvant comprising (or consisting of, or essentially consisting of) an oil, a polycationic carrier, and an immunostimulatory oligonucleotide, for the prevention of foot lesions caused by FMD infection in ruminants, wherein the vaccine is administered as a single prime dose and one or more booster doses, the first of which is administered 7 months to about 12 months after the single prime dose.

[0098]

[0124] Item 42. Each subsequent booster dose is administered 7 to approximately 12 months after the previous booster dose, as described in Item 41.

[0125] Item 43. The use described in Item 41 or 42, wherein the first booster dose is administered about 8, or about 9, or about 10, or about 11, or about 12 months after the single prime dose.

[0099]

[0126] Item 44. Use as described in any one of items 41-43, wherein the cationic carrier is DEAE dextran.

[0127] Item 45. Use as described in Item 44, where DEAE dextran is present in amounts of 25-250 mg per dose.

[0100]

[0128] Item 46. Use as described in Item 45, where the immunostimulatory oligonucleotide is present in an amount of at least 25 μg per dose.

[0129] Item 47. Use as described in Item 46, where immunostimulatory oligonucleotides are present in amounts of 50-150 μg per dose.

[0101]

[0130] Item 48. Uses described in any one of items 41-46, wherein the immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide.

[0131] Item 49. Use as described in Item 48, where a P-class immunostimulatory oligonucleotide is a modified P-class immunostimulatory oligonucleotide.

[0102]

[0132] Item 50. Use as described in Item 49, wherein the modified P-class immunostimulatory oligonucleotide comprises 5'-iodo-2'-deoxyuridine or 5-ethyl-2'-deoxyuridine.

[0103]

[0133] Item 51. Use as described in Item 50, wherein the P-class immunostimulatory oligonucleotide comprises 15 consecutive oligonucleotides of SEQ ID NO: 8.

[0134] Item 52. Use as described in Item 51, wherein the P-class immunostimulatory oligonucleotide comprises 20 consecutive oligonucleotides of SEQ ID NO: 8.

[0104]

[0135] Item 53. Uses of a P-class immunostimulatory oligonucleotide, including SEQ ID NO: 8, as described in Item 52.

[0136] Item 54. Use as described in any one of items 41-53, where the vaccine contains at least 40% oil (v / v).

[0105]

[0137] Item 55. Use as described in Item 54, where the vaccine contains at least 45% oil (v / v).

[0138] Item 56. Use as described in Item 55, where the vaccine contains at least 48% oil (v / v).

[0106]

[0139] Item 57. Use as described in Item 56, where the vaccine contains at least 52% oil (v / v).

[0140] Item 58. Use as described in Item 57, where the vaccine contains at least 60% oil (v / v).

[0107]

[0141] Item 59. Uses described in any one of items 41-58, where the oil is a non-metabolized oil.

[0142] Item 60. Use as described in Item 59, where the non-metabolized oil is light mineral oil.

[0108]

[0143] Item 61. Use as described in any one of items 41-60, where the FMD antigen is derived from type O, type A, and / or Asian type 1 FMD virus.

[0144] Item 62. Use as described in any one of items 41-60, where the FMD antigen is derived from FMD virus of serotype O, A, and / or C.

[0109]

[0145] Item 63. Use as described in any one of items 41-60, where the FMD antigen is derived from the FMD virus of serotypes SAT-1, SAT / 2, and / or SAT3.

[0146] No. 64. FMD licensed O1 Manisa, O1 BFS, Campos, Asia 1 Shamir, A Iran '96, A22 Iraq, SAT2 Saudi Arabia, A10-61, A5, A12, A24 / Cruzeiro, C3 / Indaial, O1, C1-Santa Pau、C1-C5、A22 / 550 / Azerbaijan / 65、SAT1-SAT3、A、A / TNC / 71 / 94、A / I ND / 2 / 68、A / IND / 3 / 77、A / IND / 5 / 68、A / IND / 7 / 82、A / IND / 16 / 82、A / IND / 1 7 / 77、A / IND / 17 / 82、A / IND / 19 / 76、A / IND / 20 / 82、A / IND / 22 / 82、A / IND / 25 / 81、A / IND / 26 / 82、A / IND / 54 / 79、A / IND / 57 / 79、A / IND / 73 / 79、A / IND / 85 / 79, A / IND / 86 / 79, A / APA / 25 / 84, A / APN / 41 / 84, A / APS / 44 / 05, A / APS / 50 / 05、A / APS / 55 / 05、A / APS / 66 / 05、A / APS / 68 / 05、A / BIM / 46 / 95、A / GUM / 33 / 84、A / ORS / 66 / 84、A / ORS / 75 / 88、A / TNAn / 60 / 947 / Asia / 1、A / IRN / 0 5、Asia / IRN / 05、O / HK / 2001、0 / UKG / 3952 / 2001、O / UKG / 4141 / 2001、Asia 1 / HNK / CHA / 05、Asia I / XJ、HK / 70、O / UKG / 7039 / 2001、O / UKG / 9161 / 2001、O / UKG / 7299 / 2001、O / UKG / 4014 / 2001、O / UKG / 4998 / 2001. O / UKG / 9443 / 2001、O / UKG / 5470 / 2001、O / UKG / 5681 / 2001、O / ES / 2001、HKN / 2002、O5India、O / BKF / 2 / 92、K / 37 / 2001. 84 / A、KEN / 1 / 76 / A、GAM / 51 / 98 / A、A10 / Holland.O / KEN / 1 / 91.O / IND49 / 97.O / IND65 / 98.O / IND64 / 98.O / IND48 / 98、O / IND47 / 98、O / IND82 / 97、O / IND81 / 99、O / IND81 / 98、O / IND79 / 97、O / IND78 / 97、O / IND75 / 97、O / IND74 / 97、O / IND70 / 97, O / IND66 / 98, O / IND63 / 97, O / IND61 / 97, O / IND57 / 98, O / IND56 / 98, O / IND55 / 98, O / IND54 / 98, O / IND469 / 98, O / IND465 / 97, O / IND464 / 97, O / IND424 / 97, O / IND423 / 97, O / IND420 / 97, O / IND414 / 97, O / IND411 / 97, O / IND410 / 97, O / IND409 / 97, O / IND407 / 97, ​​O / IND399 / 97, O / IND39 / 97, O / IND391 / 97, O / IND38 / 97, O / IND 384 / 97, O / IND380 / 97, O / IND37 / 97, ​​O / IND352 / 97, O / IND33 / 97, O / IND31 / 97, O / IND296 / 97, O / IND23 / 99, O / IN D463 / 97, O / IND461 / 97, O / IND427 / 98, O / IND28 / 97, O / IND287 / 99, O / IND285 / 99, O / IND282 / 99, O / IND281 / 97 , O / IND27 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, O / IND210 / 99, O / IND208 / 99, O / IND207 / 99, O / IND20 Use as described in any one of items 41-63, derived from strains selected from the group consisting of 5 / 99, O / IND185 / 99, O / IND175 / 99, O / IND170 / 97, O / IND164 / 99, O / IND160 / 99, O / IND153 / 99, O / IND148 / 99, O / IND146 / 99, O / SKR / 2000, A22 / India / 17 / 77 and any combination thereof.

[0110]

[0147] Item 65. Use as described in any one of items 41-64, where the antigen is an inactivated FMD virus.

[0148] Item 66. Use as described in Item 64, where the inactivated FMD virus is a recombinant virus.

[0111]

[0149] Item 67. Recombinant FMD virus lacks a functional leader protein; use as described in Item 66.

[0150] Item 68. Uses of item 66 or 67, wherein the recombinant FMD virus further comprises one or more DIVA markers.

[0112]

[0151] Item 69. Use as described in any one of items 66-68, wherein a recombinant FMD virus expresses a capsid derived from a heterologous FMD strain.

[0152] Item 70. Use as described in Item 69, where the heterologous FMD strain is a strain of serotype A, or serotype O, or serotype Asia, or serotype C, or serotype SAT1, or serotype SAT2 and / or serotype SAT3.

[0113]

[0153] Item 71. Use as described in Item 69, where the heterologous FMD strain is a strain of serotype A, or serotype O, and / or serotype Asia 1.

[0154] Item 72. Use as described in Item 69, where the heterologous FMD strain is a strain of serotype A, or serotype O, and / or serotype C.

[0114]

[0155] Item 73. Use as described in Item 69, where the heterologous FMD strain is a strain of serotype SAT1, or serotype SAT2 and / or serotype SAT3.

[0156] Material 74: O1 Manisa, O1 BFS, Campos, Asia 1 Shamir, A Iran '96, A22 Iraq, SAT2 Saudi Arabia, A24 / Cruzeiro, A10-61, A5, A12, C3 / Indaial, O1, C1-Santa Pau、C1-C5、A22 / 550 / Azerbaijan / 65、SAT1-SAT3、A、A / TNC / 71 / 94、A / I ND / 2 / 68、A / IND / 3 / 77、A / IND / 5 / 68、A / IND / 7 / 82、A / IND / 16 / 82、A / IND / 1 7 / 77、A / IND / 17 / 82、A / IND / 19 / 76、A / IND / 20 / 82、A / IND / 22 / 82、A / IND / 25 / 81、A / IND / 26 / 82、A / IND / 54 / 79、A / IND / 57 / 79、A / IND / 73 / 79、A / IND / 85 / 79, A / IND / 86 / 79, A / APA / 25 / 84, A / APN / 41 / 84, A / APS / 44 / 05, A / APS / 50 / 05、A / APS / 55 / 05、A / APS / 66 / 05、A / APS / 68 / 05、A / BIM / 46 / 95、A / GUM / 33 / 84、A / ORS / 66 / 84、A / ORS / 75 / 88、A / TNAn / 60 / 947 / Asia / 1、A / IRN / 0 5、Asia / IRN / 05、O / HK / 2001、0 / UKG / 3952 / 2001、O / UKG / 4141 / 2001、Asia 1 / HNK / CHA / 05、Asia I / XJ、HK / 70、O / UKG / 7039 / 2001、O / UKG / 9161 / 2001、O / UKG / 7299 / 2001、O / UKG / 4014 / 2001、O / UKG / 4998 / 2001. O / UKG / 9443 / 2001、O / UKG / 5470 / 2001、O / UKG / 5681 / 2001、O / ES / 2001、HKN / 2002、O5India、O / BKF / 2 / 92、K / 37 / 2001. 84 / A、KEN / 1 / 76 / A、GAM / 51 / 98 / A、A10 / Holland.O / KEN / 1 / 91.O / IND49 / 97.O / IND65 / 98.O / IND64 / 98.O / IND48 / 98、O / IND47 / 98、O / IND82 / 97、O / IND81 / 99、O / IND81 / 98、O / IND79 / 97、O / IND78 / 97、O / IND75 / 97、O / IND74 / 97、O / IND70 / 97, O / IND66 / 98, O / IND63 / 97, O / IND61 / 97, O / IND57 / 98, O / IND56 / 98, O / IND55 / 98, O / IND54 / 98, O / IND469 / 98, O / IND465 / 97, O / IND464 / 97, O / IND424 / 97, O / IND423 / 97, O / IND420 / 97, O / IND414 / 97, O / IND 411 / 97, O / IND410 / 97, O / IND409 / 97, O / IND407 / 97, ​​O / IND399 / 97, O / IND39 / 97, O / IND391 / 97, O / IND38 / 97, O / IND384 / 97, O / IND380 / 97, O / IND37 / 97, ​​O / IND352 / 97, O / IND33 / 97, O / IND31 / 97, O / IND296 / 97, O / IND23 / 99, O / IND463 / 97, O / IND461 / 97, O / IND427 / 98, O / IND28 / 97, O / IND287 / 99, O / IND285 / 99, O / IND282 / 99, O / IND281 / 97, O / IND27 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, O / IND210 / 99, O / IND208 / 99, O / IND207 Use as described in item 69, selected from the group consisting of / 99, O / IND205 / 99, O / IND185 / 99, O / IND175 / 99, O / IND170 / 97, O / IND164 / 99, O / IND160 / 99, O / IND153 / 99, O / IND148 / 99, O / IND146 / 99, O / SKR / 2000, A22 / India / 17 / 77, and any combination thereof.

[0115]

[0157] Item 75. Use as described in any one of items 64-74, where the FMD virus is present in an amount of at least 4 μg per inoculation per strain.

[0158] Item 76. Use as described in any one of items 64-75, where the FMD virus is present in an amount of at least 8 μg per inoculation per strain.

[0116]

[0159] Item 77. Use as described in any one of items 64-76, where the FMD virus is present in an amount of approximately 10 μg per inoculation per strain.

[0160] Item 78. Uses described in any one of items 41-77, where the ruminant is a cattle.

[0117]

[0161] Item 79. Use as described in any one of items 41-78, where the ruminant is FMD-negative.

[0162] Item 80. A vaccine described in any one of items 1-40, in which the ruminant animal is FMD-negative.

[0118]

[0163] The following embodiments are presented as illustrative examples and should not be construed as limiting the scope of the invention. Many variations, modifications, alterations, and other uses and applications of the invention will be apparent to those skilled in the art. [Examples]

[0119] Example 1: A single-dose vaccine provides immunity for one year.

[0164] The purpose of this study is to evaluate the one-year duration of immunity (DOI) of a trivalent inactivated FMDV vaccine adjuvanted with an optimized Zoetis adjuvant formulation, with the aim of improving protective immunity against antigen administration with allogeneic type O FMD virus in Indian cattle.

[0120]

[0165] In this study, FMD seronegative (SN titer < 1:8) 6-12 month old, 100-200 kg male cattle (Bos indicus) were used. All animals were healthy at the time of vaccination. During the vaccination phase, the animals were housed in a non-containment animal laboratory in accordance with the regulations of the local authority. The animals were housed in one barn. During the antigen administration phase, the vaccinated and unvaccinated calves were housed in a secure containment animal laboratory in accordance with the regulations of the local authority. The animals were housed in five identical rooms. Each room contained eight animals, with two animals per treatment group in each room. The animals were transferred to the antigen administration facility 358 days prior to antigen administration. Water and other feed components were supplied in accordance with the requirements set by the local authority.

[0121]

[0166] The animals were acclimatized for at least two weeks from the date of arrival at the testing facility. The animals were dewormed by administering Panacur Vet (fenbendazole 2.5% suspension, M / s Intervet) at a dose of 5 mg / kg body weight per animal.

[0122]

[0167] Table 2 lists the treatments.

[0168] The animals were administered a 2 ml dose of vaccine appropriate for their group by intramuscular injection on day 0.

[0123]

[0169] Blood collection for serology: To detect antibody responses to three antigens, blood samples were collected from all calves in all treatment groups on days 14, 0, 29, 60, 90, 120, 149, 180, 210, 240, 270, 300, 330, 365, and 379 of the study using sterile syringes and needles. Blood was collected from all calves in all groups via the jugular vein, and 8-10 ml of blood was collected using a vacuum container.

[0124] [Table 2]

[0125]

[0170] The adjuvants used in T03 and T04 were identical and contained the following components (per 2ml inoculation): light mineral oil 45% v / v, SPAN® 80 6.3% v / v, DEAE dextran 100mg, CpG 50μg, TWEEN® 80 1.45% v / v.

[0126]

[0171] Heparinized whole blood samples for testing cell-mediated immune responses (IFNγELISPOT assay) were also collected from all calves in all treatment groups using sterile syringes and needles on days 0 (pre-vaccination), 9, 29, 180, and 330.

[0127]

[0172] Antigen administration: Animals received antigen administration on day 365 of the study. Animals were transferred to the antigen administration facility one week prior to the day of antigen administration. Antigen administration dose: 10,000 BID 50 Sublingual intradermal inoculation was performed with bovine adaptive allogeneic type O FMD virus (median bovine infectivity in a 0.1 mL volume).

[0128]

[0173] Post-antigen administration observations were conducted and recorded during a 10-day period following antigen administration.

[0174] The serum neutralization test (SNT) is based on the neutralization of viral infection of target cells BHK21 using animal-derived antibodies. The SNT was performed in the same manner as described by Crowther et al. (1984). In short, serum dilutions were prepared in 100 TCID. 50 Mix with either A / IND / 40 / 00, O / IND / R2 / 75, or Asia-1 / IND / 63 / 72 in a microtiter plate and incubate at 37°C for 1 hour. Then, 3 × 10⁻¹⁶ 6 Equal volumes of BHK21 cells at a concentration of cells / ml were added to the plates, and the plates were further incubated at 37°C. After 48 hours, the decrease in virus-specific cytopathic effect (CPE) was read, and the serum viral neutralization titer was calculated using the Reid-Münch method.

[0129]

[0175] The final test results were based on the protective effect against the development of foot lesions from FMD virus serotype "O".

[0176] After antigen administration, the development of foot lesions was monitored and recorded. The presence or absence of foot lesions was considered a significant indicator (primary variable) and used to define the effectiveness of various vaccines. Since foot lesions are located far from the antigen administration site, their presence indicated that the virus had spread from the tongue to the foot. Tongue lesions (secondary variable) were scored on day 367, and foot lesions were scored on day 375. Therefore, the primary variable (foot lesions) is a secondary lesion, and the secondary variable (tongue lesions) is a primary lesion.

[0130]

[0177] While 100% of the control T01 animals developed tongue lesions (primary lesions) and foot lesions (secondary lesions), 100% of the T02 (positive control) animals developed tongue lesions and 60% developed foot lesions. In contrast, 60% of the T03 (standard-dose vaccine) and T04 (high-dose vaccine) animals developed tongue lesions, but no foot lesions were observed in the T03 group (0%), and only 10% (1 / 10) of the T04 group developed foot lesions (Table 3).

[0131] [Table 3]

[0132]

[0178] In the T03 and T04 groups, significantly fewer animals developed foot lesions compared to the T01 control group (p ≤ 0.0001; Table 4). In contrast, no significant difference was observed in the T02 group compared to the T01 control group (p = 0.0867; Table 4). Furthermore, the proportion of animals developing foot lesions in the T03 group was significantly lower than in the T02 group (p = 0.0108), and there was no significant difference in foot lesions between the T04 and T02 groups (p = 0.0573; Table 4). However, numerically, 1 out of 10 animals in the T04 group and 6 out of 10 animals in the T02 group tested positive.

[0133] [Table 4]

[0134]

[0179] No significant pairwise differences were observed between any group regarding the proportion of animals with tongue lesions (p>0.05) or the proportion of animals with either lesion (foot or tongue) (p>0.05). However, groups T03 and T04 were numerically different from groups T01 and / or T02 (p=0.0867).

[0135]

[0180] Tables 5-7 summarize the geometric mean values ​​of the SN titer for the O antigen for T02 through T04.

[0181] Before antigen administration, the signal-to-noise ratio (SN) was less than 8 in all animals in the T01 group, but it increased to 256 (95% confidence interval 183.9–356.3) by day 379. Animals with a titer of 8 or higher were considered seropositive. Animals with a titer of less than 8 were considered seronegative.

[0136]

[0182] All but one individual in group T02 responded to the vaccine. Of the nine calves that responded, five became seronegative between days 180 and 210, and four maintained their titer until day 365 (three of these four calves were negative for foot lesions). In contrast, all animals in groups T03 and T04 underwent seroneversion and developed titers that persisted until day 365.

[0137]

[0183] Table 8 summarizes the LSM analysis results (logarithmically transformed) of SN titers for the O antigen. This summary shows that animals in groups T03 and T04 developed significantly higher geometric mean titers (p<0.05) compared to the positive control group T02 at multiple days of the analysis. There was no significant difference in geometric mean titers between T03 and T04 at the time before antigen administration. At 14 and 379 days after antigen administration, the SN titers of groups T02 to T04 were significantly higher than those of the control group T01 (p<0.05). There was no difference in SN titers between T02 to T04 at 379 days.

[0138] [Table 5]

[0139] [Table 6]

[0140] [Table 7]

[0141] [Table 8-1]

[0142] [Table 8-2]

[0143]

[0184] CMI responses were measured before and after vaccination using the IFNγELISPOT assay. The analysis of these responses is summarized in Table 9. Significant differences in CMI responses were observed between T01 and T03 at days 0, 9, and 180 (p<0.05), between T04 and T04 at days 9, 180, and 330, and between T02 and T02 at day 180 (p<0.05). Significant differences in CMI responses were observed between T02 and T03 at day 9, and between T03 and T04 at day 180 (p<0.05).

[0144] [Table 9-1]

[0145] [Table 9-2]

[0146]

[0185] The single-dose trivalent FMD experimental vaccine administered to the T03 group animals showed significantly higher efficacy than the commercially available vaccine sold in India, which served as a positive control (p<0.05). The T04 vaccine did not induce a statistically significant protective effect compared to the T02 vaccine, but numerically, only 1 out of 10 calves in the T04 group tested positive for foot lesions, compared to 6 out of 10 calves in the T02 group (p=0.0573).

[0147]

[0186] Based on the primary variable, foot lesions, the T03 and T04 vaccines provided a one-year DOI for antigen administration to the O strain.

[0187] Similarly, the antibody response to type A antigen was low in the T02 group, with 7 out of 10 animals becoming seronegative by day 365, while titers were high in the T03 and T04 groups and persisted until day 365. Tables 10-12 show the results for the T02-T04 groups. Data for T01 are not shown because all animals in T01 had an SN value of less than 8 by day 365.

[0148] [Table 10]

[0149] [Table 11]

[0150] [Table 12]

[0151]

[0188] Table 13 shows a statistical analysis of the response to antigen A.

[0152] [Table 13-1]

[0153] [Table 13-2]

[0154]

[0189] SN titers for the Asia 1 antigen remained significantly higher in the T03 and T04 groups, but titers were more consistently induced in the T02 group and persisted until day 365. Only one animal in the T02 group was seronegative at day 365. See Tables 14-17. All animals in the T01 group remained seronegative throughout the study period.

[0155]

[0190] In all animals in groups T03 and T04, the SN titers for type A and Asia 1 were seropositive in all animals at 365 days post-vaccination. The lowest SN titers for type A in groups T03 and T04 were 45 and 45, respectively, at 365 days post-vaccination. The lowest titers for Asia 1 in groups T03 and T04 were 45 and 64, respectively, at 365 days post-vaccination.

[0156]

[0191] These titers are high enough to conclude that the animals are protected against antigen administration to serotype A or Asia 1 virus 365 days after vaccination.

[0157] [Table 14]

[0158] [Table 15]

[0159] [Table 16]

[0160] [Table 17-1]

[0161] [Table 17-2]

[0162]

[0192] All publications cited in this specification, including patent and non-patent publications, represent the state of the art relevant to the present invention. All of these publications are incorporated herein in full by reference to the same extent that each individual publication is specifically and individually incorporated by reference.

[0163]

[0193] Although the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Therefore, it should be understood that numerous modifications can be made to the exemplary embodiments and other configurations can be devised without departing from the spirit and scope of the invention as defined by the following claims.

Claims

1. A vaccine for use in the prevention of foot lesions caused by FMD infection in ruminants, comprising an FMD antigen and an adjuvant comprising an oil, a polycationic carrier, and an immunostimulatory oligonucleotide, wherein the vaccine is a water-in-oil (W / O) emulsion, and is administered as a single prime dose and one or more booster doses, the first of which is administered 7 months to about 12 months after the single prime dose.

2. The vaccine according to claim 1, wherein each subsequent booster dose is administered 7 to approximately 12 months after the previous booster dose.

3. The vaccine according to claim 1, wherein the first booster dose is administered approximately 8 months, 9 months, 10 months, 11 months, or 12 months after the single prime dose.

4. The vaccine according to claim 1, wherein the cationic carrier is DEAE dextran.

5. The vaccine according to claim 4, wherein DEAE dextran is present in an amount of 25 to 250 mg per dose.

6. The vaccine according to claim 5, wherein the immunostimulatory oligonucleotide is present in an amount of 25 to 250 μg per dose.

7. The vaccine according to claim 6, wherein the immunostimulatory oligonucleotide is present in an amount of 50 to 150 μg per dose.

8. The vaccine according to claim 1, comprising at least 40% oil (v / v).

9. The vaccine according to claim 8, comprising at least 45% oil (v / v).

10. The vaccine according to claim 9, comprising at least 48% oil (v / v).

11. The vaccine according to claim 10, comprising at least 52% oil (v / v).

12. The vaccine according to claim 11, comprising at least 60% oil (v / v).

13. The vaccine according to claim 1, wherein the oil is a light mineral oil.

14. The vaccine according to any one of claims 1 to 13, wherein the antigen is derived from serotype O, A and / or Asia 1 FMD virus.

15. The vaccine according to any one of claims 1 to 13, wherein the antigen is derived from FMD virus of serotype O, A and / or C.

16. The vaccine according to any one of claims 1 to 13, wherein the antigen is derived from FMD virus of serotype SAT1, SAT2, and / or SAT3.

17. The antigen is O1 Manisa, O1 BFS or Campos, Asia 1 Shamir, A Iran ’96, A22 Iraq, SAT2 Saudi Arabia, A10-61, A5, A12, A24 / Cruzeiro, C3 / Indaiál, O1, C1-Santa Pau, C1-C5, A22 / 550 / Azerbaijan / 65, SAT1-SAT3, A, A / TNC / 71 / 94, A / IND / 2 / 68, A / IND / 3 / 77, A / IND / 5 / 68, A / IND / 7 / 82, A / IND / 16 / 82, A / IND / 17 / 77, A / IND / 17 / 82, A / IND / 19 / 76, A / IND / 20 / 82, A / IND / 22 / 82, A / IND / 25 / 81, A / IND / 26 / 82, A / IND / 54 / 79, A / IND / 57 / 79, A / IND / 73 / 79, A / IND / 85 / 79, A / IND / 86 / 79, A / APA / 25 / 84, A / APN / 41 / 84, A / APS / 44 / 05, A / APS / 50 / 05, A / APS / 55 / 05, A / APS / 66 / 05, A / APS / 68 / 05, A / BIM / 46 / 95, A / GUM / 33 / 84, A / ORS / 66 / 84, A / ORS / 75 / 88, A / TNA n / 60 / 947 / Asia / 1, A / IRN / 05, Asia / IRN / 05, O / HK / 2001, 0 / UKG / 3952 / 2001, 0 / UKG / 4141 / 2001, Asia 1 / HNK / CHA / 05, Asia I / XJ, HK / 70, O / UKG / 7039 / 2001, O / UKG / 9161 / 2001, O / UKG / 7299 / 2001, O / UKG / 4014 / 2001, O / UKG / 4998 / 2001, O / UKG / 9443 / 2001, O / UKG / 5470 / 2001, O / UKG / 5681 / 2001, O / ES / 2001, HKN / 2002, O5India, O / BKF / 2 / 92, K / 37 / 84 / A, KEN / 1 / 76 / A, GAM / 51 / 98 / A, A10 / Holland, O / KEN / 1 / 91, O / IND49 / 97, O / IND65 / 98, O / IND64 / 98, O / IND48 / 98, O / IND47 / 98, O / IND82 / 97, O / IND81 / 99, O / IND81 / 98, O / IND79 / 97, O / IND78 / 97, O / IND75 / 97, O / IND74 / 97,O / IND70 / 97, O / IND66 / 98, O / IND63 / 97, O / IND61 / 97, O / IND57 / 98, O / IND56 / 98, O / IND55 / 98, O / IND54 / 98, O / IND IND469 / 98, O / IND465 / 97, O / IND464 / 97, O / IND424 / 97, O / IND423 / 97, O / IND420 / 97, O / IND414 / 97, O / IND411 / 97, O / IND410 / 97, O / IND409 / 97, O / IND407 / 97, ​​O / IND399 / 97, O / IND39 / 97, O / IND391 / 97, O / IND38 / 97, O / IND3 84 / 97, O / IND380 / 97, O / IND37 / 97, ​​O / IND352 / 97, O / IND33 / 97, O / IND31 / 97, O / IND296 / 97, O / IND23 / 99, O / IND 463 / 97, O / IND461 / 97, O / IND427 / 98, O / IND28 / 97, O / IND287 / 99, O / IND285 / 99, O / IND282 / 99, O / IND281 / 97, O / IND27 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, O / IND210 / 99, O / IND208 / 99, O / IND207 / 99, O / IND205 The vaccine according to claim 1, selected from the group consisting of O / IND185 / 99, O / IND175 / 99, O / IND170 / 97, O / IND164 / 99, O / IND160 / 99, O / IND153 / 99, O / IND148 / 99, O / IND146 / 99, O / SKR / 2000, A22 / India / 17 / 77, O / IND / R2 / 1975, and any combination thereof.

18. The vaccine according to claim 1, wherein the antigen is an inactivated FMD virus.

19. The vaccine according to claim 17, wherein the inactivated FMD virus is a recombinant virus.

20. The vaccine according to claim 18, wherein the recombinant FMD virus lacks a functional leader protein.

21. The vaccine according to claim 18, wherein the recombinant FMD virus further comprises a DIVA marker.

22. The vaccine according to claim 19, further comprising one or more deletion sequences as a negative marker for recombinant FMD virus.

23. The vaccine according to claim 19, wherein the recombinant FMD virus further comprises one or more mutations in a capsid-coding sequence that induce thermal stability.

24. The vaccine according to any one of claims 19 to 23, wherein the recombinant FMD virus expresses a capsid derived from a heterologous FMD strain.

25. The vaccine according to claim 24, wherein the heterologous FMD strain is a strain of serotype A, or serotype O, or serotype Asia, or serotype C, or serotype SAT1, or serotype SAT2, or serotype SAT3.

26. The vaccine according to claim 25, wherein the heterologous FMD strain is a strain of serotype A, or serotype O, and / or serotype Asia 1.

27. The vaccine according to claim 25, wherein the heterologous FMD strain is a strain of serotype A, or serotype O, and / or serotype C.

28. The vaccine according to claim 25, wherein the heterologous FMD strain is a strain of serotype SAT1, serotype SAT2 and / or serotype SAT3.

29. The heterologous strains are O1 Manisa, O1 BFS or Campos, Asia 1 Shamir, A Iran ’96, A22 Iraq, SAT2 Saudi Arabia, A10-61, A5, A12, A24 / Cruzeiro, C3 / Indaiál, O1, C1-Santa Pau, C1-C5, A22 / 550 / Azerbaijan / 65, SAT1-SAT3, A, A / TNC / 71 / 94, A / IND / 2 / 68, A / IND / 3 / 77, A / IND / 5 / 68, A / IND / 7 / 82, A / IND / 16 / 82, A / IND / 17 / 77, A / IND / 17 / 82, A / IND / 19 / 76, A / IND / 20 / 82, A / IND / 22 / 82, A / IND / 25 / 81, A / IND / 26 / 82, A / IND / 54 / 79, A / IND / 57 / 79, A / IND / 73 / 79, A / IND / 85 / 79, A / IND / 86 / 79, A / APA / 25 / 84, A / APN / 41 / 84, A / APS / 44 / 05, A / APS / 50 / 05, A / APS / 55 / 05, A / APS / 66 / 05, A / APS / 68 / 05, A / BIM / 46 / 95, A / GUM / 33 / 84, A / ORS / 66 / 84, A / ORS / 75 / 88, A / TNA n / 60 / 947 / Asia / 1, A / IRN / 05, Asia / IRN / 05, O / HK / 2001, 0 / UKG / 3952 / 2001, 0 / UKG / 4141 / 2001, Asia 1 / HNK / CHA / 05, Asia I / XJ, HK / 70, O / UKG / 7039 / 2001, O / UKG / 9161 / 2001, O / UKG / 7299 / 2001, O / UKG / 4014 / 2001, O / UKG / 4998 / 2001, O / UKG / 9443 / 2001, O / UKG / 5470 / 2001, O / UKG / 5681 / 2001, O / ES / 2001, HKN / 2002, O5India, O / BKF / 2 / 92, K / 37 / 84 / A, KEN / 1 / 76 / A, GAM / 51 / 98 / A, A10 / Holland, O / KEN / 1 / 91, O / IND49 / 97, O / IND65 / 98, O / IND64 / 98, O / IND48 / 98, O / IND47 / 98, O / IND82 / 97, O / IND81 / 99, O / IND81 / 98, O / IND79 / 97, O / IND78 / 97, O / IND75 / 97, O / IND74 / 97,O / IND70 / 97, O / IND66 / 98, O / IND63 / 97, O / IND61 / 97, O / IND57 / 98, O / IND56 / 98, O / IND55 / 98, O / IND54 / 98, O / IND IND469 / 98, O / IND465 / 97, O / IND464 / 97, O / IND424 / 97, O / IND423 / 97, O / IND420 / 97, O / IND414 / 97, O / IND411 / 97, O / IND410 / 97, O / IND409 / 97, O / IND407 / 97, ​​O / IND399 / 97, O / IND39 / 97, O / IND391 / 97, O / IND38 / 97, O / IND 384 / 97, O / IND380 / 97, O / IND37 / 97, ​​O / IND352 / 97, O / IND33 / 97, O / IND31 / 97, O / IND296 / 97, O / IND23 / 99, O / IN D463 / 97, O / IND461 / 97, O / IND427 / 98, O / IND28 / 97, O / IND287 / 99, O / IND285 / 99, O / IND282 / 99, O / IND281 / 97 , O / IND27 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, O / IND210 / 99, O / IND208 / 99, O / IND207 / 99, O / IND20 The vaccine according to claim 24, selected from the group consisting of 5 / 99, O / IND185 / 99, O / IND175 / 99, O / IND170 / 97, O / IND164 / 99, O / IND160 / 99, O / IND153 / 99, O / IND148 / 99, O / IND146 / 99, O / SKR / 2000, A22 / India / 17 / 77, O / IND / R2 / 75, and any combination thereof.

30. The vaccine according to claim 17, wherein the FMD virus is present in an amount of at least 4 μg per dose per strain.

31. The vaccine according to claim 17, wherein the FMD virus is present in an amount of at least 8 μg per strain per dose.

32. The vaccine according to claim 17, wherein the FMD virus is present in an amount of approximately 10 μg per strain per dose.

33. The vaccine according to claim 1, wherein the ruminant animal is a cow.

34. The vaccine according to claim 1, wherein the ruminant animals are FMD-negative.