COMPOSIÇÕES DE VACINAS E MÉTODOS PARA CONTROLE DA FEBRE AFTOSA

Abstract

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

Description

1 / 60 Vaccine compositions and methods for controlling foot-and-mouth disease. NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

[0001] The claimed invention was made as a result of activities carried out within the scope of a joint research agreement between Zoetis Pharmaceutical Research Ltd and ICAR - Indian Veterinary Research Institute, Hebbal, Bengaluru, Karnataka 560024, India. FIELD OF THE INVENTION

[0002] The present invention, in general, is in the field of vaccines against foot and mouth disease (FMD). BACKGROUND OF THE INVENTION

[0003] Foot-and-mouth disease (FMD) is one of the most serious and contagious diseases affecting livestock. This disease is endemic in several countries around the world, especially in Africa, Asia, and South America. Furthermore, epidemic outbreaks can occur periodically. The presence of this disease in a country can have very serious economic consequences, resulting from loss of productivity, weight loss, and reduced milk production in infected herds, as well as trade embargoes imposed on those countries. Measures taken against this disease consist of the strict application of import restrictions, hygiene and quarantine controls, culling of diseased animals, and vaccination programs with inactivated vaccines, either as a preventive measure at the national or regional level, or periodically when an epidemic outbreak occurs.

[0004] Foot-and-mouth disease is characterized by its short incubation period, its highly contagious nature, the formation of ulcers in the mouth and feet, and sometimes the death of young animals. Foot-and-mouth disease affects several animal species, especially cattle, pigs, sheep, and goats. The agent responsible for this disease is a ribonucleic acid (RNA) virus belonging to the genus Aphthovirus of the family Petition 870250084645, dated 09 / 19 / 2025, page 13 / 85 2 / 60 Picornaviridae (Cooper et al., 1978, Intervirology 10, 165-180). Currently, at least seven types of foot-and-mouth disease virus (FMDV) are known: the European types (A, O, and C), the African types (SAT1, SAT2, and SAT3), and an Asian type (Asia 1). Several subtypes have also been distinguished (Kleid et al., 1981, Science 214, 1125-1129).

[0005] FMDV is a non-enveloped icosahedral virus of approximately 25 nm in diameter, containing a single-stranded RNA molecule composed of approximately 8500 nucleotides, with positive polarity. This RNA molecule comprises a single open reading frame (ORF), which encodes a single polyprotein containing, among other things, the capsid precursor, also known as the P1 protein. The P1 protein is myristylated at its amino-terminal end. During the maturation process, the P1 protein 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, 241-261). In the virion, the VP0 protein is then cleaved into two proteins, VP4 and VP2 (or 1A and 1B, respectively). The mechanism for the conversion of VP0 proteins into VP1 and VP3, and for the formation of mature virions, is not known. VP1, VP2, and VP3 proteins have a molecular weight of approximately 26.000 Da, while the VP4 protein is smaller, around 8,000 Da.

[0006] Many proposals have been developed in an attempt to create effective vaccines against foot-and-mouth disease. Cao et al. (Antiviral Research, 2013, 97: 145-153; Veterinary Microbiology, 2014, 168: 294-301) reported the design of proteins for specific epitopes with immunogenicity against FMDV challenge. A synthetic polypeptide corresponding to the fusion of one T epitope and two B epitopes of the Asia serotype has been shown to induce a protective response (Ren et al., Vaccine, 2011, 29:7960-7965). Petition 870250084645, dated 09 / 19 / 2025, p. 14 / 85 3 / 60

[0007] Most FMD vaccines require a vaccination regimen involving two doses administered 28 days or 6 months apart, resulting in low adherence by farmers and causing some limitations in providing effective disease control. There are no FMD vaccines that provide immunity lasting longer than six months. The Office of World Organisation for Animal Health (OIE) emphasizes the need for a two-dose regimen (primary booster) and semi-annual revaccinations. SUMMARY OF THE INVENTION

[0008] In one aspect, the invention provides a vaccine comprising an FMD antigen and an adjuvant comprising oil, a polycationic carrier and an immunostimulatory oligonucleotide, wherein the vaccine is a water-in-oil (W / O) emulsion, for use in preventing foot lesions caused by FMD virus infection in a ruminant, wherein the vaccine is administered as a primary vaccination dose and one or more booster doses, wherein the first of the one or more booster doses is administered 7 to about 12 months after the initial single dose. In certain embodiments, the ruminant is FMD negative.

[0009] In certain modalities, each subsequent booster dose is administered 7 to approximately 12 months after the previous booster dose, for example, approximately 8, or approximately 9, or approximately 10, or approximately 11, or approximately 12 months after the primary vaccination dose.

[00010] In certain embodiments, the cationic vehicle is DEAE Dextran, which may be present in amounts of 25 to 250 mg per dose.

[00011] In certain embodiments, the immunostimulatory oligonucleotide is present in amounts of 25 to 250 pg per dose, for example, in amounts of 50 to 150 pg per dose.

[00012] In certain forms, the vaccine comprises at least 40% v / v oil, for example, at least 45% v / v oil, by Petition 870250084645, dated 09 / 19 / 2025, p. 15 / 85 4 / 60 less 48% oil v / v, at least 52% oil v / v, or at least 60% oil v / v. The oil may be a non-metabolizable oil, such as light mineral oil.

[00013] In certain embodiments, the antigen is from an FMD virus that may belong to serotype O, A, C, Asia 1, SAT1, SAT2, or SAT3. In certain embodiments, the vaccine is monovalent, and in certain other embodiments, the vaccine is multivalent, having a combination of different serotypes, such as FMD virus of serotypes A, O, and / or C. In other embodiments, the vaccine comprises the antigen(s) of FMD virus of serotypes A, O, and / or Asia 1 or other serotypes, such as SAT1, SAT2, and / or SAT3.

[00014] In certain embodiments, the FMD antigen is a virus of Inactivated FMD. 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, for example, in the 3B and / or 3D protein. In some embodiments, the recombinant virus expresses a capsid from a heterologous FMD strain. In some embodiments, the heterologous FMD strain is serotype O, A, and / or C. In other embodiments, the heterologous FMD strain is serotype O, A, and / or Asia 1. In other embodiments, the heterologous FMD strain is serotype SAT1, SAT2, and / or SAT3.

[00015] In certain embodiments, the FMD virus is present in the amount of at least 4 pg per dose per strain, for example, at least 8 pg per dose per strain, or about 10 pg per dose per strain. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[00016] For a better understanding of the invention, the following non-limiting definitions are provided.

[00017] “About” or “approximately”, when used in connection with a measurable numerical variable, refers to the stated value of the variable and to all values ​​of the variable that are within the error. Petition 870250084645, dated 09 / 19 / 2025, p. 16 / 85 5 / 60 of the stated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the stated value, whichever is greater. Regarding the time period, the term “approximately” refers to the stated value and an interval within 10% of the stated value (e.g., “approximately 8 months” includes 8 months as well as 8 months plus or minus 10%), except for the upper limit of “approximately 11 months” which is 12 months and the upper limit of “approximately 12 months” which is 12.5 months.

[00018] “Adjuvant” means any substance that enhances the humoral or cellular immune response to an antigen. Adjuvants are generally used to achieve two goals: controlled release of antigens from the injection site and stimulation of the immune system.

[00019] “Antibody” refers to an immunoglobulin molecule that can bind to a specific antigen as a result of an immune response to that antigen.

[00020] “Antigen” or “immunogen” refers to any substance that is recognized by the animal’s immune system and generates an immune response. The term includes live, dead, inactivated, attenuated, or modified bacteria, viruses, or parasites. The term “antigen” also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, polysaccharides or lipids, or fragments thereof, individually 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).

[00021] “Buffer” means a chemical system that prevents changes in the concentration of another chemical substance, for example, proton donor and acceptor systems serve as buffers, preventing Petition 870250084645, dated 09 / 19 / 2025, p. 17 / 85 6 / 60 significant changes in hydrogen ion concentration (pH). Another example of a buffer is a solution containing a mixture of a weak acid and its salt (conjugate base) or a weak base and its salt (conjugate acid).

[00022] “Subsequent booster dose” refers to the second booster dose, the third booster dose, and so on. “Previous booster dose” is a dose that immediately precedes the “subsequent booster dose”. Thus, if a third booster dose is the consequent booster dose, then the previous booster dose will be a second booster dose. In general, if dose N is a “consequent booster dose”, then dose N-1 will be the previous booster dose for dose N.

[00023] “Essentially consisting”, as applied to adjuvant formulations, refers to a formulation that does not contain additional adjuvant or immunomodulatory agents not specified in the amounts in which said agent exerts measurable adjuvant or immunomodulatory effects.

[00024] “Dose” refers to a vaccine or immunogenic composition administered to an individual in a single administration. A “first dose” or “induction vaccine” or “primary vaccination dose” refers to the dose of such composition administered on Day 0. A “second dose” or an “annual dose” refers to an amount of this composition administered after the first dose at the indicated intervals, which may or may not be the same vaccine or immunogenic composition as the first dose.

[00025] The term “emulsifier” is used extensively in the present invention. It includes substances generally accepted as emulsifiers, for example, different products from the TWEEN® or SPAN® product lines (polyethoxylated sorbitol fatty acid esters and surfactants). Petition 870250084645, dated 09 / 19 / 2025, p. 18 / 85 7 / 60 of sorbitan replaced by fatty acids, respectively) and different solubility enhancers, such as PEG-40 castor oil or other PEGylated hydrogenated oil.

[00026] The term “negative for FMD” refers to an animal that has not been previously vaccinated against foot-and-mouth disease. Preferably, the term “negative for foot-and-mouth disease” refers to an animal that has not been previously vaccinated against foot-and-mouth disease or previously (or currently) infected with foot-and-mouth disease.

[00027] “Humoral immune response” refers to that mediated by antibodies.

[00028] “Immune response” in an individual refers to the development of a humoral immune response, a cellular immune response, or a humoral and cellular immune response to an antigen. Immune responses can generally be determined using standard immunoassays and neutralization assays, which are known in the art.

[00029] “Immunologically effective quantity” or “effective quantity to produce an immune response” of an antigen is an amount effective in inducing an immunogenic response in the recipient. The immunogenic response may be sufficient for diagnostic purposes or other testing, or it may be adequate to prevent signs or symptoms of disease, including adverse health effects or complications arising from infection by a disease agent. Both humoral immunity and cell-mediated immunity, or both, may be induced.An animal's immunogenic response to an immunogenic composition can be assessed, for example, indirectly by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs and symptoms after challenge with a wild strain, while the protective immunity conferred by a vaccine can be assessed by measuring, for example, a reduction in clinical signs such as mortality, morbidity, and number of... Petition 870250084645, dated 09 / 19 / 2025, page 19 / 85 8 / 60 temperature, general physical condition, and overall health and performance of the individual. The immune response may include, but is not limited to, induction of cellular and / or humoral immunity.

[00030] “Immunogenic” means to evoke an immune or antigenic response. Therefore, an immunogenic composition would be any composition that induces an immune response.

[00031] “Pharmaceutically acceptable” refers to substances that are within the scope of medical common sense, suitable for use in contact with the tissues of individuals without undue toxicity, irritation, allergic response and the like, compatible with a reasonable risk-benefit ratio and effective for the intended use.

[00032] It has been found that vaccines according to any of the modalities described above can provide advantageous protection against foot lesions caused by foot-and-mouth disease for about twelve months when administered even as a single-dose vaccine.

[00033] Therefore, the invention provides a vaccine for the prevention of foot lesions caused by foot-and-mouth disease in an animal, wherein the vaccine is administered in a primary vaccination dose and in one or more booster doses, wherein the first booster dose is administered 7 to about 12 months after the single primary dose, wherein the vaccine comprises a foot-and-mouth disease 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, wherein the vaccine is a water-in-oil emulsion. In certain embodiments, the animal is a ruminant, such as a bovine. In certain embodiments, the animal is negative for FMD.

[00034] If more than one booster dose is administered, each subsequent booster dose will be given 7 to approximately 12 months after the previous booster dose. In this way, the first of said booster doses will be given 7 to approximately 12 months after the previous booster dose. Petition 870250084645, dated 09 / 19 / 2025, p. 20 / 85 9 / 60 One or more booster doses are administered 7 to approximately 12 months after the primary vaccination dose; the second of said one or more booster doses is administered 7 to approximately 12 months after the first of said one or more booster doses; the third of said one or more booster doses is administered 7 to approximately 12 months after the second of said one or more booster doses, and so on. In certain embodiments, the interval between the primary vaccination 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.

[00035] The intervals between the two doses are selected independently of each other and are, as noted above, between 7 and approximately 12 months and may be the same or different.

[00036] Table 1 provides a non-limiting illustration relating to the intervals between the primary vaccination dose (PS) and the first booster dose (B1) and between the first booster dose and the second booster dose (B2). The “X” mark indicates that the proposed regimen is suitable for the practice of the invention. TABLE 1 Interval between PS and B1, months Interval between B1 and B2, months 7 ~8 ~9 ~10 ~11 ~12 7 XXXXXX ~8 XXXXXX ~9 XXXXXX ~10 XXXXXX ~11 XXXXXX ~12 XXXXXX

[00037] Similarly, the time interval between the third booster dose and the second booster dose, which is between 7 months and approximately 12 months, is adequate, regardless of the specific interval. Petition 870250084645, dated 09 / 19 / 2025, p. 21 / 85 10 / 60 between PS and B1 and between B1 and B2, provided that both intervals are between 7 and approximately 12 months.

[00038] The vaccines described in this application are suitable for any of the administration regimens disclosed above. The vaccines comprise an FMD antigen and an adjuvant comprising an oil, a polycationic carrier, an immunostimulatory oligonucleotide containing CpG and, optionally, one or more emulsifiers, wherein the vaccine is an O / W emulsion. Antigens

[00039] Foot-and-mouth disease viruses of any serotype are suitable for use with the vaccine of the invention. Currently, seven foot-and-mouth disease serotypes have been isolated. Of the seven serotypes of this virus, A, C, O, Asia 1, and SAT3 appear to be distinct lineages; SAT1 and SAT2 are unresolved clades. Within each serotype, there are multiple strains. For example, A24 Cruzeiro belongs to serotype A, and O1 Campos belongs to serotype O.

[00040] It should be noted that antigens from different serotypes may provide limited cross-protection against challenge with a heterologous serotype virus. US Patent 10,010,605 disclosed the results of vaccinating pigs with VLPs containing peptide sets of foot-and-mouth disease viruses from different strains. The authors reported that a specific IFN-gamma response (cellular response) was detected in both Asia 1 Shamir VLP groups when FMDV Asia 1 Shamir antigens were used, and a specific IFN-gamma response (cellular response) was detected in both A22 Iraq VLP groups when FMDV A22 Iraq antigens were used. The Asia 1 Shamir VLP group showed cross-immunogenicity when FMDV A22 Iraq antigens were used. Specific plasma cells (humoral response) were detected in both Asia 1 Shamir VLP groups and both Iraqi A22 VLP groups with Asia 1 antigens. Petition 870250084645, dated 09 / 19 / 2025, page 22 / 85 11 / 60 Shamir and A22 Iraq, respectively. Cross-immunogenicity (plasma cells) in Asia 1 Shamir VLP groups was observed with A22 Iraq antigens. Specific memory B cells (humoral response) were detected in Asia 1 Shamir VLP groups and A22 Iraq VLP groups with Asia 1 Shamir and A22 Iraq antigens, respectively. Good cross-immunogenicity (B cells) was observed in Asia 1 Shamir VLP groups with A22 Iraq antigens. Some cross-immunogenicity (B cells) was also observed in A22 Iraq VLP groups with Asia 1 Shamir antigens. The authors concluded that VLPs can elicit a sufficient immune response to protect against heterologous challenge.

[00041] However, it is preferable that the vaccine contain the antigens of the serotype against which protection is desired, which may vary according to the geographical region. Thus, for the African region, where SAT-1, SAT-2 and SAT-3 are most prevalent, it is preferable that the vaccine contain the antigens of the strain(s) belonging to serotypes SAT-1, SAT-2 and / or SAT-3. For Europe, where strains of serotypes A, O and C are prevalent, it is preferable that the vaccine contain antigens of the strain(s) belonging to serotypes O, A and C. For Asia, it is preferable that the vaccine contain the antigens of the strain(s) belonging to the Asia serotype.

[00042] Foot-and-mouth disease virus of any serotype may be used as needed, provided the virus is not pathogenic. Pathogenicity may be reduced by inactivating the virus, for example, by treatment with formaldehyde or BEI.

[00043] In certain embodiments, the virus can be attenuated by culture passage or by recombinant means. It has been previously demonstrated, for example, that the elimination of the coding region of the Lpro leader protein results in a foot-and-mouth disease virus that is attenuated in cattle and swine. See, for example, US Patent No. 5,824,316, Petition 870250084645, dated 09 / 19 / 2025, page 23 / 85 12 / 60 8,765,141, Virology 1997 227(1): 96-102, J. Virol 2012 86:11675-11685. Point mutations at positions 55 and 58 within the SAP domain of the L protein also resulted in a viable virus that exhibited a mildly attenuated phenotype in cell culture and was protective in the swine foot-and-mouth disease model. See US Patent No. 8,846,057.

[00044] In certain embodiments, the virus also contains negative antigenic markers that allow DIVA assays (differentiation between infected and vaccinated animals). In certain embodiments, the negative antigenic markers are introduced into 3D and / or 3B proteins.

[00045] Like other viruses, the FMD virus evolves and mutates continuously, therefore, one of the difficulties in vaccination against it is the enormous variation between serotypes, and even within them. There is no cross-protection between serotypes (a vaccine for one serotype will not necessarily protect against others) and, in addition, two strains within a given serotype may have nucleotide sequences that differ by up to 30% for a given gene. This means that FMD vaccines must be specific to the strain involved, although some cross-protection between strains has been reported.

[00046] Thus, in certain embodiments, endonuclease restriction sites are introduced into the virus genome, thereby allowing the introduction of proteins (e.g., proteins that form the outer capsids) from heterologous FMD strains.

[00047] In certain embodiments, the antigen component comprises the FMD A24 Cruzeiro strain, which may optionally be modified by inactivation of the leader protein (by substitutions or insertions, or deletions, or combinations thereof), mutations of negative markers in proteins 3B and / or 3D, and by the introduction of restriction endonuclease sites for easier introduction of sequences. Petition 870250084645, dated 09 / 19 / 2025, page 24 / 85 13 / 60 for antigens (e.g., capsid proteins) of heterologous strains. Suitable, non-limiting examples of antigens are described in U.S. Patent No. 8,765,141. See also U.S. Patents 9,180,179 and 10,478,487.

[00048] Thus, a DNA sequence complementary to the reference DNA sequence is a template for, i.e., it is complementary to or “encodes” the RNA genome of the FMDV virus (i.e., RNA that encodes FMDV). In certain embodiments, the virus comprises capsid protein(s) from heterologous FMD strains (i.e., FMD strains other than A24 Cruzeiro, including, without limitation, strains from lineages C, O, Asia 1, SAT3, SAT 1 and SAT 2, Turkey 06 and other lineage A strains).

[00049] In certain non-limiting modalities, FMD may be of the following strains: 01 Manisa, 01 BFS or Campos, A24 Cruzeiro, Asia 1 Shamir, A Iran '96, A22 Iraq, SAT2 Saudi Arabia.

[00050] Other strains may also include FMDV strains A1061, A5, A12, A24 / Cruzeiro, C3 / Indaial, O1, C1-Santa Pau, C1-C5, A22 / 550 / Azerbaijan / 65, SAT1-SAT3, 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, O / HK / 2001, O / UKG / 3952 / 2001, O / UKG / 4141 / 2001, Asia 1 / HNK / CHA / 05 (GenBank accession number EF149010, incorporated herein by reference), Asia 1 / 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, Petition 870250084645, dated 09 / 19 / 2025, p. 25 / 85 14 / 60 HKN / 2002, OSIndia, 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 / IND397 / 97, ​​O / IND391 / 97, O / IND388 / 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 / IND277 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, 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, A22 / India / 17 / 77.

[00051] Variants of these antigens are also predicted. Variants are at least 80% identical (e.g., 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical) to a reference sequence using one of the alignment programs described using standard parameters. Several alignment tools are available to determine sequence identity, including, but not limited to, BLAST, CLUSTAL, or PHILIP.

[00052] One skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[00053] In certain modalities, the variants encompass more than Petition 870250084645, dated 09 / 19 / 2025, page 26 / 85 15 / 60 that specific nucleotide or amino acid sequences exemplify and include functional equivalents thereof. Alterations in a nucleic acid fragment that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, can be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, it can also be expected that alterations resulting in the substitution of a negatively charged residue for another, such as aspartic acid for glutamic acid, or a positively charged residue for another, such as lysine for arginine, will produce a functionally equivalent product.It is also not expected that nucleotide changes that result in alterations to the N-terminal and C-terminal portions of the polypeptide molecule will alter the polypeptide's activity. Each of the proposed modifications is within the routine capabilities of the technique, as is the determination of the retention of the biological activity of the encoded products.

[00054] The polypeptides of the invention can also be altered in various ways, including substitutions, deletions, truncations, and insertions of amino acids. New proteins with properties of interest can be created by combining elements and fragments of proteins of the present invention, as well as with other proteins. Methods for such manipulations are generally known in the art. Thus, the genes and nucleotide sequences of the invention include both natural sequences and mutant forms. Similarly, the proteins of the invention encompass natural proteins as well as variations and modified forms thereof. These variants will continue to possess the desired modified activities of the virus. Petition 870250084645, dated 09 / 19 / 2025, page 27 / 85 16 / 60 original foot-and-mouth disease. The mutations that will be made to the DNA encoding the variant should not place the sequence out of the reading frame and, preferably, should not create complementary regions that could produce secondary mRNA structure.

[00055] Methods for culturing 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 antigenic compositions. For example, in PEG precipitation, the antigenic composition is devoid of non-structural proteins. In other methods, such as hollow fiber filtration, the antigenic composition contains structural and non-structural FMD proteins. Consequently, in some embodiments, the FMD antigen comprises structural proteins. In other embodiments, such as where the FMD antigen is prepared by hollow fiber filtration, the FMD antigen comprises structural and non-structural proteins, particularly the 3D protein.

[00056] In other embodiments, the antigen may include empty FMD virus capsids or virus-like particles, or VLPs. Both are known in the art. For example, US Published Patent Application 2004 / 0001864 teaches a foot-and-mouth disease vaccine in which empty capsids are produced by co-expression of P1 and protease 3C. US Patent No. 8,409,588 discloses a method for producing a foot-and-mouth disease virus-like particle, comprising: providing a host cell containing an expression system comprising a promoter functionally linked to a nucleic acid molecule encoding a polyprotein, said polyprotein comprising FMDV protein 1A - non-FMDV protease recognition sequence, FMDV protein 1B - non-FMDV protease recognition sequence, FMDV protein 1C - non-FMDV protease recognition sequence, and FMDV protein 1D, the host cell being Petition 870250084645, dated 09 / 19 / 2025, page 28 / 85 17 / 60 capable of expressing a protease that recognizes said non-FMDV protease recognition sequence, cultivate the host cell under conditions such that the polyprotein is produced, cleaved into 1A, 1B, 1C, and 1D by the protease, and 1A, 1B, 1C, and 1D self-assemble into virus-like particles, and recover 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. Dec. 2012;96(3):288-95 reporting VLPs resulting from the expression of FMDV structural proteins along with protease 3C in Sf9 cells.

[00057] The vaccines described in this application may be monovalent (using the foot-and-mouth disease antigen of only one strain) or multivalent (using foot-and-mouth disease antigens of two or more strains). Thus, for example, the vaccine may use the antigen of a single strain of serotype A, or a single strain of serotype C, or a single strain of serotype C, or a single strain of serotype Asia, and so on. Multivalent vaccines may contain a strain of serotype A, a strain of serotype O, a strain of serotype C, a strain of serotype Asia, or any two or three of them. In other embodiments, the multivalent vaccine may contain several strains of the same serotype.

[00058] The vaccines described in this application allow some flexibility in the amount of antigen per dose.In certain embodiments, the quantity of antigen comprises at least 4 pg 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 pg or more per dose per strain). In the multivalent vaccines described in this application, the quantities of different antigens per dose are selected independently of other antigens.

[00059] In addition, it has been reported that the use of heterologous (non-FMD) viral vectors encoding FMD antigens provides protection. Petition 870250084645, dated 09 / 19 / 2025, page 29 / 85 18 / 60 against a challenge. See, for example, US patent 10,188,721 which discloses compositions or vaccines comprising recombinant viral vectors, such as adenoviral vectors expressing FMDV antigens that elicit a protective response in animals.

[00060] Thus, in certain embodiments, the FMD antigen is provided in the form of an adenovirus vector that expresses the FMDV structural protein P1 (VP4-VP2-VP3-VP1), the non-structural protein P2 (2A, 2B and 2C) or the non-structural protein P3 (3A, 3B, 3C and 3D) or an active fragment or variant thereof.

[00061] In other embodiments, the antigen may comprise at least one immunogenic peptide of the foot-and-mouth disease virus. Suitable non-limiting examples of such peptides are disclosed, for example, in documents W003068169, 20110206718 and Ren et al., Vaccine 29 (2011) 7960-7965, incorporated herein by reference.

[00062] Since the immunogenicity of a given antigen is determined in relation to the inactivated virus by standard methods, one skilled in the art would have no difficulty in selecting the appropriate dose of the antigen. In the multivalent vaccines described in this application, the quantities of different antigens per dose are selected independently of other antigens. Adjuvants

[00063] The vaccine according to the invention is formulated as a water-in-oil emulsion (W / O emulsion) and comprises (or essentially consists of or consists of) an oil, an immunostimulatory oligonucleotide comprising CpG, a polycationic carrier and, optionally, one or more emulsifiers.

[00064] Different oils are suitable for the invention, including non-metabolizable oils (e.g., a light mineral oil), metabolizable oils (e.g., vegetable oil or fatty acid esters, Petition 870250084645, dated 09 / 19 / 2025, page 30 / 85 19 / 60 polyols or alcohol) or a mixture thereof. If a mixture of non-metabolizable and metabolizable oils is used, it is preferred that the non-metabolizable oils comprise more than 50% v / v (e.g., more than 60%, or more than 70%, or more than 80%, or more than 90%, or more than 95%, or more than 99%) of the mixture. In certain embodiments, 100% of the oil in the emulsion is non-metabolizable. In the most preferred embodiments, 100% of the oil in the W / O emulsion is a mineral oil. As used in this application, the term “mineral oil” refers to a mixture of liquid hydrocarbons obtained from petrolatum by means of a distillation technique. The term is synonymous with “liquefied paraffin,” “liquid petrolatum,” and “white mineral oil.” The term is also intended to include "light mineral oil," that is, oil obtained similarly by distillation of petrolatum, but which has 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, for example, JT Baker (Phillipsburg, Pennsylvania, USA) or USB Corporation (Cleveland, Ohio, USA). The preferred mineral oil is commercially available light mineral oil under the name DRAKEOL®.

[00065] The volume fraction of oil in the vaccine must be sufficient for the 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 compaction fraction for the random compaction of monodisperse droplets, i.e.: 0.64. See Tadros, Emulsion Formation, Stability and Rheology, 1st ed. 2013, Wiley-VCH GmbH & Co KGaA. Provided that the total volume fraction occupied by aqueous droplets does not exceed 0.64, i.e.: 64% v / v. Thus, in different embodiments, the oil may be present in an amount greater than 36%. Petition 870250084645, dated 09 / 19 / 2025, p. 31 / 85 20 / 60 of the v / v vaccine (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 v / v vaccine).

[00066] The emulsion may also comprise one or more emulsifiers. In other embodiments, the emulsifiers used in this application do not include lecithin or use lecithin in an amount that is not immunologically effective.

[00067] Suitable non-natural synthetic emulsifiers for use in the adjuvant formulations of the present invention include nonionic sorbitan-based surfactants, for example, fatty acid-substituted sorbitan surfactants (commercially available under the names SPAN® or ARLACEL®), polyethoxylated sorbitol fatty acid esters (TWEEN®), polyethylene glycol fatty acid esters from sources such as castor oil (EMULFOR®); polyethoxylated fatty acid (for example, stearic acid available under the name SIMULSOL®M-53), polyethoxylated iso-octylphenol / formaldehyde polymer (TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRU®); Non-phenyl polyoxyethylene ethers (TRITON® N), iso-octylphenyl polyoxyethylene ethers (TRITON® X). Preferred synthetic surfactants are surfactants available under the names SPAN® and TWEEN®, such as TWEEN®-80 (polyoxyethylene (20) sorbitan monooleate) and SPAN®-80 (sorbitan monooleate).

[00068] Emulsifiers are generally present in the emulsion in amounts of 0 to 20% v / v. In certain embodiments, TWEEN® is present in amounts of 1% to 10% v / v of the vaccine and SPAN® is also present in amounts of 1% to 10% v / v of the vaccine. Thus, for example, TWEEN® is present in amounts of 1% to 6% v / v of the vaccine and SPAN® is also present in amounts of 1% to 6% v / v of the vaccine. In other embodiments, TWEEN® is Petition 870250084645, dated 09 / 19 / 2025, page 32 / 85 21 / 60 is present in a quantity of 1% to 2% v / v of the vaccine, and SPAN® is present in a quantity of 5% to 8% v / v of the vaccine. In other dosage forms, TWEEN® is present in a quantity of 4% to 7% v / v of the vaccine, and SPAN® is present in a quantity of 1% to 3% v / v of the vaccine.

[00069] The vaccine adjuvant component further comprises a polycationic carrier. Suitable but not limiting examples of polycationic carriers include, without limitation, PEGs, guar gum, chitosan derivatives, polycellulose derivatives such as hydroxyethylcellulose (HEC), polyethyleneimene, polyaminos such as polylysine, cationized dextran and the like. In certain embodiments, the polycationic carrier is Diethylaminoethyl (DEAE) Dextran.

[00070] The polycationic vehicle, such as DEAE Dextran, may be present in the vaccine in amounts between 5 and 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).

[00071] The adjuvant component of the vaccine also comprises an immunostimulatory oligonucleotide. The immunomodulatory oligonucleotides according to the invention comprise CpG (and are also referred to as “CpG-containing immunostimulatory oligonucleotides”, “CpG oligonucleotides” or simply “CpGs”). The effect of CpG-containing oligonucleotides on the immune system has been known for more than 20 years.

[00072] Generally, the CpGs suitable for the invention have between 15 and 100 bases in length, for example, between 15 and 50 bases in length, or between 18 and 40 bases in length, or between 20 and 30 bases in length, or 20 to 24 bases in length.

[00073] Several classes of CpGs have been described, including class A CpGs, class B CpGs, class C CpGs, and class P CpGs. In certain embodiments, the immunostimulatory oligonucleotide containing CpG is a class P CpG. Class P CpGs are characterized Petition 870250084645, dated 09 / 19 / 2025, page 33 / 85 22 / 60 by the presence of one or more TLR-9 activating motifs and two palindromes or two complementarity areas. Preferably, one or more TLR-9 activating motifs are in the 5' of the oligonucleotide and may be completely or partially incorporated into the 5' palindrome or the 5' complementarity area. TLR-9 activating motifs are known and include, without limitation, TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT, or TTTT. The 5' palindrome or the 5' complementary area is at least 6 bases long. The 3' palindrome or the 3' complementary area is at least 8 bases long and is generally rich in C and G. These structural features of class P CpGs confer the ability to spontaneously self-assemble into concatemers in vitro and / or in vivo.

[00074] To increase the lipophilicity of CpG oligonucleotides, at least one lipophilic substituted nucleotide analog may be included, preferably at the 5' end of the oligonucleotide. Class P immunostimulatory oligonucleotides may be modified according to techniques known in the art. For example, the J modification refers to iodine-modified nucleotides. The E modification refers to ethyl-modified nucleotide(s). Thus, E-modified class P immunostimulatory oligonucleotides are class P immunostimulatory oligonucleotides in which at least one nucleotide (preferably the 5' nucleotide) is ethylated. Additional modifications include 6-nitrobenzimidazole linkage, O-methylation, proynyl-dU modification, inosine modification, 2-bromovinyl linkage (preferably to uridine).

[00075] Oligonucleotides modified by the addition of a lipophilic moiety are generically described in document US 20100166780.

[00076] In certain embodiments, CpGs according to the invention Petition 870250084645, dated 09 / 19 / 2025, page 34 / 85 23 / 60 comprise the modified structure including, without limitation, phosphorothioate modifications, halogenations, alkylation (e.g., ethyl or methyl modifications) and phosphodiester modifications.

[00077] Suitable non-limiting examples of modified class P immunostimulatory oligonucleotides are provided below (“*” refers to a phosphorothioate linkage, “JU” refers to a phosphodiester linkage, and “EU” refers to 5-ethyl-2'-deoxyuridine). 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*T3' SEQ ID NO: 4 5' JU*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: 5 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’ SEQ ID NO: 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' SEQ ID NO: 7 5' EU*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: 8 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' SEQ ID NO: 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' SEQ ID NO: 10 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'

[00078] In certain embodiments, the CpG oligonucleotide according to the invention comprises any of the SEQ ID NOs 1 to 10 or an oligonucleotide comprising at least 15 consecutive bases of any of the 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, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 or at least 23).

[00079] The CpG oligonucleotide may be present in the vaccine in amounts of 10 to 400 pg per vaccine dose, or 25 to 300, 50 to 200, or 50 to 100 mg per dose.

[00080] Other components of the compositions may include exci Petition 870250084645, dated 09 / 19 / 2025, p. 35 / 85 24 / 60 pharmaceutically acceptable components, such as vehicles, solvents and diluents, isotonic agents, buffering agents, stabilizers, preservatives, antibacterial agents, antifungal agents and the like. Typical vehicles, solvents and diluents include water, saline solution, dextrose, ethanol, glycerol, oil and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose and the like. Useful stabilizers include gelatin, albumin and the like. The compositions may also contain antibiotics or preservatives, including, for example, gentamicin, merthiolate or chlorocresol. The various classes of antibiotics or preservatives to be selected are well known to qualified practitioners.

[00081] In certain embodiments, the vaccines of the invention are prepared by producing a first solution containing the oil and the optional oil-soluble emulsifier, and a second solution comprising the aqueous diluent, the antigen, the CpG oligonucleotide, the polycationic carrier and the optional water-soluble emulsifier. The first solution is then combined with the second solution, for example, by adding the second solution drop by drop to the first solution, thus preparing the W / O emulsion vaccine according to the invention.

[00082] The invention also provides the following items:

[00083] Item 1. A vaccine comprising an FMD antigen and an adjuvant comprising (or consisting of or consisting essentially of) oil, a polycationic carrier and an immunostimulatory oligonucleotide, wherein the vaccine is a water-in-oil (W / O) emulsion, for use in the prevention of foot lesions caused by FMD infection in a ruminant, wherein the vaccine is administered as a primary vaccination dose and one or more booster doses, wherein the first of the one or more booster doses is administered 7 to about 12 months after the initial single dose.

[00084] Item 2. The vaccine according to item 1, in which each dose Petition 870250084645, dated 09 / 19 / 2025, page 36 / 85 The subsequent 25 / 60 booster dose is administered 7 to approximately 12 months after the previous booster dose.

[00085] Item 3. The vaccine according to item 1 or 2, wherein the first booster dose is administered approximately 8, or approximately 9, or approximately 10, or approximately 11, or approximately 12 months after the initial single dose.

[00086] Item 4. The vaccine according to any of items 1 to where the cationic vehicle is DEAE Dextran.

[00087] Item 5. The vaccine according to item 4, in which the DEAE Dextran is present in amounts ranging from 25 to 250 mg per dose.

[00088] Item 6. The vaccine according to item 5, in which the immunostimulatory oligonucleotide is present in an amount of at least 25 pg per dose.

[00089] Item 7. The vaccine according to item 6, in which the immunostimulatory oligonucleotide is present in the amount of 50 to 150 pg per dose.

[00090] Item 8. The vaccine according to any of items 1 to 6, where the immunostimulatory oligonucleotide is a class P immunostimulatory oligonucleotide.

[00091] Item 9. The vaccine according to item 8, wherein the class P immunostimulatory oligonucleotide is a modified class P immunostimulatory oligonucleotide.

[00092] Item 10. The vaccine according to item 9, wherein the modified class P immunostimulatory oligonucleotide comprises 5'iodo-2'-deoxyuridine or 5-ethyl-2'-deoxyuridine.

[00093] Item 11. The vaccine according to item 10, wherein the class P immunostimulatory oligonucleotide comprises 15 consecutive oligonucleotides of SEQ ID NO: 8.

[00094] Item 12. The vaccine according to item 11, in which the oligo Petition 870250084645, dated 09 / 19 / 2025, page 37 / 85 26 / 60 class P immunostimulatory nucleotide comprises 20 consecutive oligonucleotides of SEQ ID NO: 8.

[00095] Item 13. The vaccine according to item 12, wherein the class P immunostimulatory oligonucleotide comprises SEQ ID NO: 8.

[00096] Item 14. The vaccine according to any of items 1 to 13, wherein the vaccine comprises at least 40% v / v oil.

[00097] Item 15. The vaccine according to item 14, wherein the vaccine comprises at least 45% v / v oil.

[00098] Item 16. The vaccine according to item 15, wherein the vaccine comprises at least 48% v / v oil.

[00099] Item 17. The vaccine according to item 16, wherein the vaccine comprises at least 52% v / v oil. [000100] Item 18. The vaccine according to item 17, wherein the vaccine comprises at least 60% v / v oil. [000101] Item 19. The vaccine according to any of items 1 to 18, wherein the oil is a non-metabolizable oil. [000102] Item 20. The vaccine according to item 19, wherein the non-metabolizable oil is a light mineral oil. [000103] Item 21. The vaccine according to any of items 1 to 20, wherein the foot-and-mouth disease antigen is from a foot-and-mouth disease virus of Type O, A and / or Asia-1. [000104] Item 22. The vaccine according to any of items 1 to 20, wherein the foot-and-mouth disease antigen is of a foot-and-mouth disease virus of serotype Type O, A and / or C. [000105] Item 23. The vaccine according to any of items 1 to 20, wherein the foot-and-mouth disease antigen is from a foot-and-mouth disease virus of serotypes SAT-1, SAT-2 and / or SAT-3. [000106] Item 24. The vaccine according to item 24, wherein the foot-and-mouth disease antigen is of a strain selected from the group consisting Petition 870250084645, dated 09 / 19 / 2025, page 38 / 85 27 / 60 in O1 Manisa, O1 BFS or 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 / 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 / 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, 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, O / HK / 2001, O / UKG / 3952 / 2001, O / UKG / 4141 / 2001, Asia 1 / HNK / CHA / 05, Asia 1 / 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, O5 India, 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 / 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 / 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 / IND277 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, 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 / IND / R2 / 75, O / SKR / 2000, A22 / India / 17 / 77 and any combination thereof. Petition 870250084645, dated 09 / 19 / 2025, page 39 / 85 28 / 60 [000107] Item 25. The vaccine according to any of items 1 to 24, wherein the antigen is an inactivated FMD virus. [000108] Item 26. The vaccine according to item 24, in which the inactivated FMD virus is a recombinant virus. [000109] Item 27. The vaccine according to item 26, in which the recombinant FMD virus does not have a functional leader protein. [000110] Item 28. The vaccine according to item 26 or 27, wherein the recombinant FMD virus further comprises one or more DIVA markers. [000111] Item 29. The vaccine according to item 26, wherein the recombinant FMD virus further comprises one or more deletion sequences as negative markers. [000112] Item 30. The vaccine according to item 26, wherein the recombinant FMD virus further comprises one or more mutations in the capsid coding sequences that induce stability. [000113] Item 31. The vaccine according to any of items 26 to 30, wherein the recombinant FMD virus expresses a capsid of a heterologous strain of FMD. [000114] Item 32. The vaccine according to item 31, wherein the heterologous strain of FMD 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. [000115] Item 33. The vaccine according to item 31, wherein the heterologous strain of FMD is a strain of serotype A, or serotype O, and / or serotype Asia 1. [000116] Item 34. The vaccine according to item 31, wherein the heterologous strain of FMD is a strain of serotype A, or serotype O, and / or serotype C. [000117] Item 35. The vaccine according to item 31, wherein the heterologous strain of FMD is a strain of serotype SAT1, or serotype SAT2 Petition 870250084645, dated 09 / 19 / 2025, p. 40 / 85 29 / 60 and / or serotype SAT 3. [000118] Item 36. The vaccine according to item 31, wherein the heterologous strain is selected from the group consisting of O1 Manisa, O1 BFS or 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 / 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, O / HK / 2001, O / UKG / 3952 / 2001, O / UKG / 4141 / 2001, Asia 1 / HNK / CHA / 05, Asia 1 / 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, OSIndia, 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 / 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 / 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 / IND277 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, 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, Petition 870250084645, dated 09 / 19 / 2025, page 41 / 85 30 / 60 O / IND153 / 99, O / IND148 / 99, O / IND146 / 99, O / SKR / 2000, A22 / India / 17 / 77, O / IND / R2 / 75 and any combination thereof. [000119] Item 37. The vaccine according to any of items 24 to 36, in which the foot-and-mouth disease virus is present in the amount of at least 4 pg per dose per strain. [000120] Item 38. The vaccine according to any of items 24 to 37, in which the foot-and-mouth disease virus is present in the amount of at least 8 pg per dose per strain. [000121] Item 39. The vaccine according to any of items 24 to 38, in which the foot-and-mouth disease virus is present in the amount of approximately 10 pg per dose per strain. [000122] Item 40. The vaccine according to any of items 1 to 39 where the ruminant is a bovine. [000123] Item 41. Use of a vaccine comprising an FMD antigen and an adjuvant comprising (or consisting of or consisting essentially of) oil, a polycationic carrier and an immunostimulatory oligonucleotide, wherein the vaccine is a water-in-oil (W / O) emulsion, for use in the prevention of foot lesions caused by FMD infection in a ruminant, wherein the vaccine is administered as a primary vaccination dose and one or more booster doses, wherein the first of the one or more booster doses is administered 7 to about 12 months after the initial single dose. [000124] Item 42. Use in accordance with item 41, where each subsequent booster dose is administered 7 to approximately 12 months after the previous booster dose. [000125] Item 43. Use in accordance with item 41 or 42, where the first booster dose is administered approximately 8, or approximately 9, or approximately 10, or approximately 11, or approximately 12 months after the primary vaccination dose. [000126] Item 44. Use in accordance with any of items 41 to Petition 870250084645, dated 09 / 19 / 2025, page 42 / 85 31 / 60 where the cationic vehicle is DEAE Dextran. [000127] Item 45. Use in accordance with item 44, where DEAE Dextran is present in the amount of 25 to 250 mg per dose. [000128] Item 46. Use in accordance with item 45, where the immunostimulatory oligonucleotide is present in an amount of at least 25 pg per dose. [000129] Item 47. Use in accordance with item 46, where the immunostimulatory oligonucleotide is present in the amount of 50 to 150 pg per dose. [000130] Item 48. Use in accordance with any of items 41 to 46, wherein the immunostimulatory oligonucleotide is a class P immunostimulatory oligonucleotide. [000131] Item 49. The use in accordance with item 48, wherein the class P immunostimulatory oligonucleotide is a modified class P immunostimulatory oligonucleotide. [000132] Item 50. The use in accordance with item 49, wherein the modified class P immunostimulatory oligonucleotide comprises 5'iodo-2'-deoxyuridine or 5-ethyl-2'-deoxyuridine. [000133] Item 51. The use in accordance with item 50, wherein the class P immunostimulatory oligonucleotide comprises 15 consecutive oligonucleotides of SEQ ID NO: 8. [000134] Item 52. The use in accordance with item 51, wherein the class P immunostimulatory oligonucleotide comprises 20 consecutive oligonucleotides of SEQ ID NO: 8. [000135] Item 53. Use in accordance with item 52, wherein the class P immunostimulatory oligonucleotide comprises SEQ ID NO: 8. [000136] Item 54. Use in accordance with any of items 41 to 53, wherein the vaccine comprises at least 40% v / v oil. [000137] Item 55. Use in accordance with item 54, wherein the vaccine comprises at least 45% v / v oil. Petition 870250084645, dated 09 / 19 / 2025, page 43 / 85 32 / 60 [000138] Item 56. Use in accordance with item 55, wherein the vaccine comprises at least 48% v / v oil. [000139] Item 57. Use in accordance with item 56, wherein the vaccine comprises at least 52% v / v oil. [000140] Item 58. Use in accordance with item 57, wherein the vaccine comprises at least 60% v / v oil. [000141] Item 59. Use in accordance with any of items 41 to 58, wherein the oil is a non-metabolizable oil. [000142] Item 60. Use in accordance with item 59, where the non-metabolizable oil is a light mineral oil. [000143] Item 61. Use in accordance with any of items 41 to 60, wherein the FMD antigen is from an FMD virus of Type O, A and / or Type Asia 1. [000144] Item 62. Use in accordance with any of items 41 to 60, where the FMD antigen is from an FMD virus of serotype Type O, A and / or C. [000145] Item 63. Use in accordance with any of items 41 to 60, wherein the FMD antigen is from an FMD virus of serotype SAT1, SAT / 2 and / or SAT3. [000146] Item 64. Use in accordance with items 41 to 63, wherein the FMD antigen is from a strain selected from the group consisting of O1 Manisa, O1 BFS or 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 / 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, Petition 870250084645, dated 09 / 19 / 2025, p. 44 / 85 33 / 60 A / IRN / 05, Asia / IRN / 05, O / HK / 2001, O / UKG / 3952 / 2001, O / UKG / 4141 / 2001, Asia 1 / HNK / CHA / 05, Asia 1 / 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, O5 India, 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 / 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 / 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 / IND277 / 97, O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, 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, A22 / India / 17 / 77 and any combination thereof. [000147] Item 65. Use in accordance with any of items 41 to 64, wherein the antigen is an inactivated FMD virus. [000148] Item 66. The use in accordance with item 64, where the inactivated FMD virus is a recombinant virus. [000149] Item 67. Use in accordance with item 66, wherein the recombinant FMD virus does not possess a functional leader protein. [000150] Item 68. Use in accordance with item 66 or 67, wherein the recombinant FMD virus further comprises one or more DIVA markers. [000151] Item 69. Use in accordance with any of items 66 to Petition 870250084645, dated 09 / 19 / 2025, page 45 / 85 34 / 60 68, in which the recombinant FMD virus expresses a capsid from a heterologous strain of FMD. [000152] Item 70. Use in accordance with item 69, where the heterologous strain of FMD 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. [000153] Item 71. Use in accordance with item 69, where the heterologous strain of FMD is a strain of serotype A, or serotype O and / or serotype Asia 1. [000154] Item 72. Use in accordance with item 69, where the heterologous strain of FMD is a strain of serotype A, or serotype O and / or serotype C. [000155] Item 73. Use in accordance with item 69, where the heterologous strain of FMD is a strain of serotype SAT1, or serotype SAT2 and / or serotype SAT3. [000156] Item 74. The use in accordance with item 69, wherein the heterologous strain is selected from the group consisting of O1 Manisa, O1 BFS or 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 / 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, O / HK / 2001, O / UKG / 3952 / 2001, O / UKG / 4141 / 2001, Asia 1 / HNK / CHA / 05, Asia 1 / 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 / Petition 870250084645, dated 09 / 19 / 2025, page 46 / 85 35 / 60 2002, O5 India, 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 / 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 / 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 / IND277 / 97, ​​O / IND278 / 97, O / IND256 / 99, O / IND249 / 99, 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, A22 / India / 17 / 77 and any combination thereof. [000157] Item 75. Use in accordance with any of items 64 to 74 where FMD virus is present in an amount of at least 4 pg per dose per strain. [000158] Item 76. Use in accordance with any of items 64 to 75 where FMD virus is present in an amount of at least 8 pg per dose per strain. [000159] Item 77. Use in accordance with any of items 64 to 76, where FMD virus is present in the amount of approximately 10 pg per dose per strain. [000160] Item 78. Use in accordance with any of items 41 to 77, wherein the ruminant is a bovine. [000161] Item 79. Use in accordance with any of items 41 to 78, where the ruminant is negative for FMD. Petition 870250084645, dated 09 / 19 / 2025, page 47 / 85 36 / 60 [000162] Item 80. Vaccine, according to any one of claims 1 to 40, wherein the ruminant is negative for foot-and-mouth disease. [000163] The following examples are presented as illustrative embodiments, but should not be considered as limiting the scope of the invention. Many changes, variations, modifications, and other uses and applications of this invention will be apparent to those skilled in the art. EXAMPLES Example 1: A single-dose vaccine provides immunity lasting for one year. [000164] The aim of the study was to evaluate the 1-year duration of immunity (DOI) of an inactivated trivalent FMDV vaccine with adjuvants, with a Zoetis adjuvant formulation optimized to enhance protective immunity in Indian cattle against challenge with homologous type O FMD virus. [000165] This study used male cattle (Bos indicus) seronegative for foot-and-mouth disease (SN titer <1:8), aged six to twelve months, weighing between 100 and 200 kg. All animals were healthy at the time of vaccination. During the vaccination phase, the animals were kept in an animal experimentation unit without confinement, as determined by local government authorities. The animals were housed in a barn. During the challenge phase, vaccinated and unvaccinated calves were housed in secure containment facilities for animal experimentation, as determined by local government authorities. The animals were housed in five identical rooms. Each room housed 8 animals, with 2 animals per treatment group per room. The animals were transported to the challenge unit on day 358 before the challenge. Water and other feed ingredients were provided as required by local authorities. Petition 870250084645, dated 09 / 19 / 2025, page 48 / 85 37 / 60 [000166] The animals were acclimated for at least two weeks from the date of arrival at the test facilities. The animals were dewormed with Panacur Vet (2.5% Fenbendazole suspension, M / s Intervet) 5 mg / kg of body weight for each animal. [000167] The treatments are provided in Table 2. [000168] The animals were vaccinated on day zero by intramuscular injection of 2 ml of the vaccine appropriate for the group. [000169] Blood collection for serology: Blood samples were collected for detection of antibody responses to the three antigens using a sterile syringe and needle on days -14, 0, 29, 60, 90, 120, 149, 180, 210, 240, 270, 300, 330, 365, and 379 of the study from all calves in all treatment groups. Calves from all groups underwent blood collection via the jugular vein, and 8 to 10 ml of blood were collected using vacutainers. Group N Vaccine Composition T01 10 None Saline solution T02 10 Commercial Property of the manufacturer. Contains FMD-A, FMD-O, FMD-Asia 1 T03 10 Experimental vaccine, Normal dose of AG FMD-A (5 pg / dose), FMD-O (8 pg / dose), FMD-Asia 1 (5 pg / dose) + Adjuvant T04 10 Experimental vaccine, High dose of AG FMD-A (10 pg / dose), FMD-O (16 pg / dose), FMD-Asia 1 (8 pg / dose) + Adjuvant Table 2 [000170] The adjuvant used in T03 and T04 was identical and contained (per 2 ml dose): Light mineral oil 45% v / v, SPAN®80 6.3% v / v; DEAE Dextran 100 mg; CpG 50 pg; TWEEN® 80 1.45% v / v. [000171] Heparinized whole blood samples for testing cell-mediated immune responses (ELISPOT gamma IFN assay) were also collected using a sterile syringe and needle on days 0 (pre-vaccination), 9, 29, 180, and 330 from all calves in all groups. Petition 870250084645, dated 09 / 19 / 2025, page 49 / 85 38 / 60 treatment. [000172] Challenge: On the day of the study, 365 animals were challenged. The animals were transported to the challenge center one week before the day of the challenge. Challenge dose: 10000 BID50 (average bovine infectivity dose in a volume of 0.1 mL) of type O homologous foot-and-mouth disease virus adapted to cattle by intradermal lingual inoculation. [000173] Post-challenge observations were observed and documented during the 10-day post-challenge period. [000174] The serum neutralization test (SNT) is based on the neutralization of BHK21 target cell virus infection using animal antibodies. The SNT was performed similarly to the test described by Crowther et al. (1984). Briefly, serum dilutions were mixed with 100 TCID50 of A / IND / 40 / 00, O / IND / R2 / 75 or Asia1 / IND / 63 / 72 in microtiter plates and incubated for 1 hour at 37 °C. Then, an equal volume of BHK21 cells at 3 x 10⁶ cells / ml was added and the plates were again incubated at 37 °C. The reduction in virus-specific cytopathic effect (cpe) was assessed after 48 hours, and the serum viral neutralization titer was calculated using the Reed and Muench method. [000175] The final result of the study was based on protection against the development of lesions on the paws, against the FMD virus serotype “O”. [000176] The development of paw lesions after the challenge was monitored and recorded. The presence or absence of paw lesions was considered significant (primary variable) and used to define the effectiveness of the various vaccines. As the paw lesions were distant from the site where the challenge was administered, their presence meant that the virus was able to spread from the tongue to the paws. Tongue lesions (secondary variable) were scored on day 367 and lesions Petition 870250084645, dated 09 / 19 / 2025, page 50 / 85 39 / 60 on the paws on day 375. Thus, the primary variable (paw lesions) are the secondary lesions and the secondary variable (tongue lesions) are the primary lesions. [000177] While 100% of the T01 control animals developed lesions on the tongue (primary lesions) and paws (secondary lesions), 100% of the T02 animals (positive control) developed lesions on the tongue and 60% developed lesions on the paws. In contrast, 60% of the T03 (normal dose vaccine) and T04 (high dose vaccine) animals developed lesions on the tongue, but no lesions on the paws (0%) were observed in the T03 group animals and only 10% of the T04 animals (1 / 10) developed lesions on the paws (Table 3). Petition 870250084645, dated 09 / 19 / 2025, page 51 / 85 Table 3: Summary of Injuries TREATMENT GROUP T01: Negative Control (Saline) T02: Single dose of positive control (commercial FMD vaccine) T03: Trivalent FMD vaccine - Normal dose T04: Trivalent FMD vaccine - High Dose N % N % n % n % TYPE OF LESION LESION PRESENT 0 0.0 0 0.0 4 40.0 4 40.0 PRIMARY (LESSIONS ON THE TONGUE) No Yes 10 100.0 10 100.0 6 60.0 6 60.0 SECONDARY (LESSIONS ON THE PAW) No 0 0.0 4 40.0 10 100.0 9 90.0 Yes 10 100.0 6 60.0 0 0.0 1 10.0 ANY (LESSIONS ON THE (TONGUE OR ON THE PAW) No 0 0.0 0 0.0 4 40.0 4 40.0 Yes 10 100.0 10 100.0 6 60.0 6 60.0 40 / 60 [000178] Significantly fewer animals in groups T03 and T04 developed paw lesions compared to the control group T01 (p<0.0001; Table 4). In contrast, group T02 was not significantly different from the control group T01 (p=0.0867; Table 4). Furthermore, the proportion of animals that developed paw lesions in group T03 was significantly lower than in group T02 (p=0.0108), and there was no significant difference between groups T04 and T02 for paw lesions (p=0.0573; Table 4), although numerically group T04 had 1 out of 10 positive animals and group T02 had 6 out of 10 positive animals. Petition 870250084645, dated 09 / 19 / 2025, page 52 / 85 41 / 60 Table 4: Analysis of Paw Injuries: Primary Outcome Variable Comparison of treatments. Overall p-value <0.0001 T01 vs. T02 0.0867 T01 vs. T03 <0.0001 T01 vs. T04 0.0001 T02 vs. T03 0.0108 T02 vs. T04 0.0573 T03 vs. T04 1.0000 [000179] There were no significant pairwise differences between any of the groups in terms of the proportion of animals with tongue lesions (p>0.05) or the proportion of animals with any lesion (paw or tongue, p>0.05). However, groups T03 and T04 were numerically different from groups T01 and / or T02 (p = 0.0867). [000180] The geometric means of SN titers against antigen O for T02 to T04 are summarized in Tables 5 to 7. [000181] The SN was below 8 in all animals in the T01 pre-challenge group and rose to 256 on day 379 (95% confidence interval, 183.9 to 356.3). Animals with a titer of 8 and above were considered seropositive. Animals with a titer below 8 were considered seronegative. [000182] All T02 animals, except one, responded to vaccination. Five of the nine calves that responded became seronegative between days 180 and 210, four showed persistent titers until day 365 (three of these four were negative for foot lesions). In contrast, all T03 and T04 animals seroconverted and developed titers that persisted until day 365. [000183] The LSM analysis of SN titers for the O antigen (log-transformed) is summarized in Table 8. The summary shows Petition 870250084645, dated 09 / 19 / 2025, p. 53 / 85 42 / 60 that animals in groups T03 and T04 developed significantly higher geometric mean titers (p<0.05) on several days of analysis compared to the positive control group T02. There were no significant differences between the geometric mean titers of T03 and T04 at any time before the challenge. The SN titers of groups T02 to T04 on day 379, 14 days after the challenge, were significantly higher than the SN titers of control animals T01 (p<0.05). There were no differences between the SN titers on day 379 of T02 to T04. Table 5. Geometric means of SN titers in animals from the T02 positive control group for the O antigen. Study day, number of animals, geometric mean (conf.) less than 95%, limit for mean (conf.) plus 45 120 10 21.1 12.3 36.2 8 45 149 10 16.5 9.7 28.0 4 45 180 10 6.7 4.1 11.1 4 23 210 10 6.7 4.4 10.1 4 23 240 10 6.1 3.9 9.5 4 16 270 10 7.7 4.5 13.3 4 23 300 10 8.0 5.0 12.9 4 23 330 10 7.0 4.1 11.9 4 16 365 10 7.2 4.4 11.8 4 23 379 10 954.4 860.5 1060.7 724 1024 Table 6. Geometric means of SN titers in animals from the normal dose AG group of the experimental vaccine T03 in relation to antigen O. Petition 870250084645, dated 09 / 19 / 2025, p. 54 / 85 43 / 60 Study day, number of animals, geometric mean (conf.) less than 95%, limit for mean (conf.) plus 132.7 23 181 120 10 73.6 43.0 125.9 16 181 149 10 61.6 36.3 104.6 16 181 180 10 73.3 44.4 121.0 32 181 210 10 59.6 39.3 90.2 23 128 240 10 61.6 39.3 96.7 32 181 270 10 73.3 42.6 126.0 32 256 300 10 66.0 40.9 106.3 45 181 330 10 73.3 43.1 125.0 32 181 365 10 70.9 43.2 116.2 32 181 379 10 831.7 607.8 1138.1 256 1024 Table 7. Geometric means of SN titers in animals from the high-dose AG group of the experimental T04 vaccine in relation to the O antigen. Study day, number of animals, geometric mean (conf. less than 95%), limit for mean (conf.). upper limit of 95%, average limit minimum maximum 14 10 187.3 108.8 322.3 64 362 29 10 107.5 68.0 169.9 64 181 50 10 127.7 77.5 210.5 45 512 30 10 103.8 61.7 174.8 23 362 120 10 78.7 46.0 134.7 16 362 149 10 73.7 43.4 125.1 23 362 180 10 59.7 36.2 98.6 8 256 210 10 55.5 36.7 84.1 8 128 240 10 57.7 36.8 90.5 8 181 270 10 70.7 41.1 121.6 11 256 300 10 55.6 34.5 89.6 11 256 330 10 61.8 36.3 105.4 8 256 365 10 59.5 36.3 97.6 11 256 379 10 955.4 860.5 1060.7 724 1024 Petition 870250084645, dated 09 / 19 / 2025, page 55 / 85 Table 8. Significant least squares differences between treatment groups. AND 95% CONFIDENCE INTERVALS FOR O ANTIGEN TITRATES (Transformed into Log) Comparison of treatments, study day, difference of means, least squares standard error, confidence level less than 95%, limit for mean difference, confidence level. greater than 95%, limit for mean difference p-value T02 - T03 14 -0.694 0.374 -1.462 0.074 0.0747 T02 - T04 14 -0.972 0.374 -1.740 -0.204 0.015 T03 - T04 14 -0.278 0.374 -1.046 0.489 0.4634 T02 - T03 29 -0.860 0.315 -1.507 -0.214 0.011 T02 - T04 29 -1.070 0.315 -1.717 -0.423 0.0021 T03 - T04 29 -0.210 0.315 -0.856 0.437 0.5117 T02 - T03 60 -1.278 0.345 -1.985 -0.571 0.001 T02 - T04 60 -1.763 0.345 -2.469 -1.056 <0.0001 T03 - T04 60 -0.485 0.345 -1.192 0.222 0.1709 T02 - T03 90 -1.422 0.359 -2.158 -0.685 0.0005 T02 - T04 90 -1.698 0.359 -2.435 -0.961 <0.0001 T03 - T04 90 -0.276 0.359 -1.013 0.461 0.4486 T02 - T03 120 -1.247 0.371 -2.008 -0.487 0.0023 T02 - T04 120 -1.315 0.371 -2.075 -0.554 0.0014 T03 - T04 120 -0.068 0.371 -0.828 0.693 0.8565 T02 - T03 149 -1.317 0.365 -2.065 -0.568 0.0012 44 / 60 Petition 870250084645, dated 09 / 19 / 2025, p. 56 / 85 Comparison of treatments, study day, difference of means, least squares standard error, confidence level less than 95%, limit for mean difference, confidence level.greater than 95%, limit for mean difference. p-value T02 - T04 149 -1.496 0.365 -2.244 -0.747 0.0003 T03 - T04 149 -0.179 0.365 -0.928 0.569 0.627 T02 - T03 180 -2.387 0.346 -3.096 -1.678 <0.0001 T02 - T04 180 -2.182 0.346 -2.891 -1.473 <0.0001 T03 - T04 180 0.205 0.346 -0.504 0.914 0.5586 T02 - T03 210 -2.185 0.286 -2.772 -1.598 <0.0001 T02 - T04 210 -2.115 0.286 -2.702 -1.528 <0.0001 T03 - T04 210 0.070 0.286 -0.517 0.657 0.8075 T02 - T03 240 -2.319 0.310 -2.955 -1.682 <0.0001 T02 - T04 240 -2.253 0.310 -2.889 -1.616 <0.0001 T03 - T04 240 0.066 0.310 -0.571 0.703 0.8332 T02 - T03 270 -2,249 0.374 -3.016 -1.483 <0.0001 T02 - T04 270 -2.214 0.374 -2.980 -1.447 <0.0001 T03 - T04 270 0.036 0.374 -0.731 0.802 0.9244 T02 - T03 300 -2.111 0.329 -2.786 -1.436 <0.0001 T02 - T04 300 -1.940 0.329 -2.615 -1.265 <0.0001 T03 - T04 300 0.171 0.329 -0.504 0.846 0.6072 T02 - T03 330 -2.354 0.367 -3.108 -1.601 <0.0001. 45 / 60 Petition 870250084645, dated 09 / 19 / 2025, page 57 / 85 Comparison of treatments, study day, difference of means, least squares standard error, confidence level less than 95%, limit for mean difference, confidence level. greater than 95%, limit for mean difference. p-value T02 - T04 330 -2.184 0.367 -2.937 -1.430 <0.0001 T03 - T04 330 0.171 0.367 -0.583 0.924 0.6461 T02 - T03 365 -2.283 0.341 -2.983 -1.584 <0.0001 T02 - T04 365 -2.109 0.341 -2.809 -1.409 <0.0001 T03 - T04 365 0.174 0.341 -0.525 0.874 0.6130 T01-T02 379 -1.317 -0.153 -1.655 0.979 <0.0001 T01 - T03 379 -1.178 -0.201 -1.602 0.755 <0.0001 T01-T04 379 -1.317 -0.153 -1.655 0.979 <0.0001 T02-T03 379 0.139 0.146 -0.183 0.460 0.3632 T02-T04 379 0.000 0.065 -0.137 0.137 1.0000 T03 - T04 379 -0.139 0.146 -0.460 0.183 0.3632 46 / 60 [000184] MIC responses were measured before and after vaccination using the IFN gamma ELISPOT assay. The analysis of these responses is summarized in Table 9. There were significant differences (p<0.05) between MIC responses of T01 versus T03 on days 0, 9, and 180 (p<0.05), versus T04 on days 9, 180, and 330, and versus T02 on day 180. There were significant differences (p<0.05) between MIC responses of T02 versus T03 on day 9 and between groups T03 and T04 on day 180. Petition 870250084645, dated 09 / 19 / 2025, page 58 / 85 Table 9. Differences in least squares means between treatment groups and 95% confidence intervals - CMI responses (log-transformed) Comparison of treatments, study day, difference of means, least squares standard error, confidence level less than 95%, limit for mean difference, confidence level. upper 95%, limit for mean difference p-value T01 - T02 0 -0.056 0.110 -0.278 0.166 0.6119 T01 - T03 0 -0.236 0.110 -0.458 -0.014 0.0381 T01 - T04 0 -0.069 0.110 -0.292 0.153 0.5313 T02 - T03 0 -0.180 0.110 -0.402 0.042 0.1094 T02 - T04 0 -0.013 0.110 -0.236 0.209 0.9048 T03 - T04 0 0.167 0.110 -0.056 0.389 0.1369 T01 - T02 9 -0.302 0.217 -0.742 0.138 0.1723 T01 - T03 9 -0.747 0.217 -1.187 -0.306 0.0015 T01 - T04 9 -0.523 0.217 -0.963 -0.082 0.0213 T02 - T03 9 -0.445 0.217 -0.885 -0.004 0.0479 T02 - T04 9 -0.220 0.217 -0.661 0.220 0.3164 T03 - T04 9 0.224 0.217 -0.216 0.664 0.3087 T01 - T02 29 -0.094 0.229 -0.559 0.370 0.6828 T01 - T03 29 -0.166 0.229 -0.630 0.299 0.4746 T01 - T04 29 -0.270 0.229 -0.734 0.195 0.2468 T02 - T03 29 -0.071 0.229 -0.536 0.393 0.758 09 / ZÍ7 Petition 870250084645, dated 09 / 19 / 2025, page 59 / 85 Comparison of treatments, study day, difference of means, least squares standard error, confidence level less than 95%, limit for mean difference, confidence level. upper 95%, limit for mean difference p-value T02 - T04 29 -0.175 0.229 -0.640 0.289 0.4491 T03 - T04 29 -0.104 0.229 -0.569 0.360 0.652 T01 - T02 180 -0.361 0.143 -0.652 -0.071 0.0162 T01 - T03 180 -0.594 0.143 -0.885 -0.304 0.0002 T01 - T04 180 -0.300 0.143 -0.590 -0.009 0.0437 T02 - T03 180 -0.233 0.143 -0.523 0.058 0.1131 T02 - T04 180 0.062 0.143 -0.229 0.352 0.6682 T03 - T04 180 0.295 0.143 0.004 0.585 0.0471 T01 - T02 330 -0.121 0.125 -0.375 0.132 0.3388 T01 - T03 330 -0.231 0.125 -0.485 0.023 0.0734 T01 - T04 330 -0.321 0.125 -0.575 -0.067 0.0147 T02 - T03 330 -0.109 0.125 -0.363 0.144 0.3881 T02 - T04 330 -0.199 0.125 -0.453 0.055 0.1204 T03 - T04 330 -0.090 0.125 -0.344 0.164 0.4782 48 / 60 Petition 870250084645, dated 09 / 19 / 2025, pp. 60 / 85 49 / 60 [000185] The experimental trivalent single-dose FMD vaccines administered to animals in group T03 were significantly more effective than the commercial positive control vaccine sold in India (p<0.05). Vaccine T04 did not induce statistically significant protection compared to vaccine T02, but numerically only 1 of 10 calves in group T04 tested positive for foot lesions, while 6 of 10 calves in group T02 tested positive (p=0.0573). [000186] Based on the primary variable, paw lesions, vaccines T03 and T04 provided a DOI of 1 year against challenge with the O strain. [000187] Similarly, antibody responses to type A antigen in group T02 were lower, and 7 out of 10 animals were seronegative on day 365, while titers were higher and persisted until day 365 in groups T03 and T04. Results for groups T02-T04 are provided in Tables 10 to 12. Data for T01 are not shown, as all animals in T01 had SN below 8 until day 365. Table 10: Geometric means of SN titers against antigen A of the T02 positive control vaccine. Study day Number of animals Geometric mean conf. less than 95%, limit for mean conf. more 120 10 25.1 18.4 34.2 11 64 149 10 20.4 14.3 29.0 8 45 180 10 10.5 6.6 16.9 4 23 210 10 11.3 7.0 18.2 4 32 Petition 870250084645, dated 09 / 19 / 2025, p. 61 / 85 50 / 60 Study day Number of animals Geometric mean Confidence below 95%, limit for mean Confidence above 95%, limit for mean Minimum Maximum 240 10 11.3 7.0 18.2 4 45 270 10 10.5 6.5 16.9 4 45 300 10 9.2 5.6 15.0 4 64 330 10 7.5 4.5 12.6 4 64 365 10 7.5 4.5 12.4 4 64 Table 11: Geometric means of SN titers against antigen A. Experimental vaccine T03, standard dose of AG. Study day, number of animals, geometric mean (conf. less than 95%), limit for mean (conf.). upper limit of 95%, average limit minimum maximum 14 10 93.5 62.6 139.6 32 256 29 10 57.5 33.5 98.7 8 181 60 10 100.0 52.4 190.7 11 256 90 10 152.1 105.5 219.3 64 256 120 10 187.4 137.4 255.5 64 362 149 10 163.1 114.5 232.5 64 256 180 10 90.6 56.7 145.0 23 256 210 10 93.7 58.3 150.8 23 256 240 10 107.4 66.4 173.6 45 256 270 10 123.5 76.7 198.9 45 256 300 10 111.2 68.2 181.4 32 256 330 10 111.4 66.4 186.9 32 256 365 10 119.2 71.9 197.6 45 256 Table 12: Geometric means of SN titers against antigen A. The experimental vaccine T04, high dose of AG. Study day Number of animals Geometric mean Confidence less than 95%, limit for mean Confidence greater than 95%, limit for mean Minimum Maximum 14 10 132.2 88.6 197.4 45 362 29 10 78.6 45.8 134.8 16 256 Petition 870250084645, dated 09 / 19 / 2025, p. 62 / 85 51 / 60 Study day, number of animals, geometric mean (conf.) less than 95%, limit for mean (conf.) upper limit of 95%, average limit minimum maximum 60 10 93.5 49.0 178.4 8 362 90 10 215.1 149.2 310.2 90 362 120 10 230.7 169.2 314.6 128 362 149 10 215.0 150.9 306.5 90 362 180 10 146.9 91.8 234.8 45 256 210 10 168.8 104.9 271.5 45 256 240 10 146.9 90.9 237.6 32 256 10 157.4 97.7 253.5 45 256 300 10 157.3 96.5 256.5 45 256 330 10 146.9 87.5 246.5 64 256 365 10 141.8 85.6 235.0 45 256 [000188] The statistical analysis of the responses to antigen A is provided in Table 13. Petition 870250084645, dated 09 / 19 / 2025, pp. 63 / 85 Table 13: Statistical analysis of antigen A responses Comparison of treatments, study day, difference of means, least squares standard error, confidence level less than 95%, limit for mean difference, confidence level.greater than 95%, limit for mean difference. p-value T02 - T03 14 -1.142 0.276 -1.708 -0.575 0.0003 T02 - T04 14 -1.488 0.276 -2.055 -0.922 <0.0001 T03 - T04 14 -0.347 0.276 -0.913 0.220 0.2202 T02 - T03 29 -1.109 0.372 -1.872 -0.345 0.006 T02 - T04 29 -1.420 0.372 -2.183 -0.657 0.0007 T03 - T04 29 -0.311 0.372 -1.075 0.452 0.4099 T02 - T03 60 -2.004 0.445 -2.917 -1.091 0.0001 T02 - T04 60 -1.937 0.445 -2.851 -1.024 0.0002 T03 - T04 60 0.066 0.445 -0.847 0.980 0.8824 T02 - T03 90 -1.983 0.252 -2.500 -1.465 <0.0001 T02 - T04 90 -2.329 0.252 -2.847 -1.812 <0.0001 T03 - T04 90 -0.347 0.252 -0.864 0.171 0.1807 T02 - T03 120 -2.011 0.214 -2.449 -1.572 <0.0001 T02 - T04 120 -2.219 0.214 -2.657 -1.780 <0.0001 T03 - T04 120 -0.208 0.214 -0.646 0.231 0.3392 T02 - T03 149 -2.080 0.244 -2.581 -1.579 <0.0001 T02 - T04 149 -2.356 0.244 -2.857 -1.855 <0.0001 T03 - T04 149 -0.276 0.244 -0.777 0.225 0.2682 T02 - T03 180 -2.151 0.324 -2.815 -1.488 <0.0001. 52 / 60 Petition 870250084645, dated 09 / 19 / 2025, pp. 64 / 85 Comparison of treatments, study day, difference of means, least squares standard error, confidence level less than 95%, limit for mean difference, confidence level. greater than 95%, limit for mean difference p-value T02 - T04 180 -2.634 0.324 -3.298 -1.970 <0.0001 T03 - T04 180 -0.483 0.324 -1.146 0.181 0.1474 T02 - T03 210 -2.117 0.328 -2.789 -1.445 <0.0001 T02 - T04 210 -2.705 0.328 -3.377 -2.033 <0.0001 T03 - T04 210 -0.588 0.328 -1.260 0.084 0.0838 T02 - T03 240 -2.255 0.331 -2.935 -1.575 <0.0001 T02 - T04 240 -2.569 0.331 -3.248 -1.889 <0.0001 T03 - T04 240 -0.314 0.331 -0.993 0.366 0.352 T02 - T03 270 -2.464 0.328 -3.138 -1.790 <0.0001 T02 - T04 270 -2.707 0.328 -3.380 -2.033 <0.0001 T03 - T04 270 -0.243 0.328 -0.916 0.431 0.4665 T02 - T03 300 -2,495 0.337 -3.186 -1.803 <0.0001 T02 - T04 300 -2.841 0.337 -3.533 -2.150 <0.0001 T03 - T04 300 -0.347 0.337 -1.038 0.345 0.313 T02 - T03 330 -2.699 0.357 -3.432 -1.967 <0.0001 T02 - T04 330 -2.976 0.357 -3.708 -2.244 <0.0001 T03 - T04 330 -0.277 0,357 -1.009 0.456 0.4449 T02 - T03 365 -2.768 0.348 -3.482 -2.053 <0.0001 T02 - T04 365 -2.941 0.348 -3.655 -2.227 <0.0001 T03 - T04 365 -0.173 0.348 -0.888 0.541 0.6227, 53 / 60 Petition 870250084645, dated 09 / 19 / 2025, pp. 65 / 85 54 / 60 [000189] For SN titers for Asia 1 antigen, titers were still significantly higher in groups T03 and T04, but titers in T02 were induced more consistently and persisted until day 365. Only one T02 animal was seronegative on day 365. See Tables 14 to 17. All animals in group T01 were seronegative throughout the study. [000190] In all animals from groups T03 and T04, SN titers for A and Asia 1 were seropositive in all animals 365 days after vaccination. The lowest SN titers for type A in groups T03 and T04 were 45 and 45, respectively, 365 days after vaccination. The lowest titers for type Asia 1 in groups T03 and T04 were 45 and 64, respectively, 365 days after vaccination. [000191] These titers were high enough to conclude that the animals would be protected against challenges with serotype A or Asia 1 virus 365 days after vaccination. Petition 870250084645, dated 09 / 19 / 2025, pp. 66 / 85 Table 14: Geometric means of SN titers against Asia Type 1 antigen - positive control vaccine T02. Study day, number of animals, geometric mean (conf.) less than 95%, limit for mean (conf.) plus 4 181 120 10 26.0 13.7 49.1 4 128 149 10 27.8 14.7 52.7 4 128 180 10 45.2 25.7 79.4 11 256 210 10 41.9 23.9 73.3 8 181 240 10 43.5 27.1 69.6 11 181 270 10 23.4 13.5 40.5 4 90 300 10 35.5 21.0 59.9 8 256 330 10 29.7 17.0 52.0 4 181 365 10 29.7 17.2 51.2 4 181 55 / 60 Petition 870250084645, dated 09 / 19 / 2025, p. 67 / 85 Table 15: Geometric means of SN titers against Asia Type 1 antigen - positive control vaccine T03. Study day, number of animals, geometric mean (conf.) less than 95%, limit for mean (conf.) upper limit of 95%, average limit minimum maximum 14 10 152.0 98.3 235.0 45 362 29 10 97.0 52.8 178.2 23 362 60 10 75.9 43.7 131.8 32 256 90 10 78.7 45.0 137.6 16 181 120 10 90.5 47.8 171.0 23 362 149 10 97.0 51.2 183.8 23 256 180 10 123.4 70.2 217.0 45 256 210 10 63.5 194.5 45 256 240 10 127.8 79.8 204.6 45 256 270 10 90.6 52.2 157.2 23 256 300 10 103.7 61.5 175.0 45 256 330 10 87.2 49.9 152.6 32 256 365 10 90.3 52.4 155.5 45 256 56 / 60 Petition 870250084645, dated 09 / 19 / 2025, pp. 68 / 85 Table 16: Geometric means of SN titers against Asia Type 1 antigen - positive control vaccine T04. Study day, number of animals, geometric mean (conf.) less than 95%, limit for mean (conf.) upper limit of 95%, average limit minimum maximum 14 10 187.2 121.0 289.5 45 362 29 10 115.3 62.8 211.8 32 362 60 10 103.8 59.8 180.3 16 256 90 10 115.2 65.8 201.4 32 256 120 10 107.5 56.8 203.1 32 256 149 10 115.3 60.8 218.5 32 256 180 10 141.9 80.7 249.6 64 256 210 10 137.0 78.3 239.8 64 256 240 10 141.9 88.6 227.2 64 256 270 10 119.2 68.7 206.9 45 256 300 10 132.2 78.4 223.0 64 256 330 10 141.8 81.0 248.1 64 256 365 10 115.2 66.8 198.5 64 256 57 / 60 Petition 870250084645, dated 09 / 19 / 2025, p. 69 / 85 Table 17: Statistical analysis of responses to Asia type 1 antigen Comparison of treatments on study day, difference of means, least squares standard error, confidence level less than 95%, limit for mean difference, confidence level. greater than 95%, limit for mean difference. p-value T02 - T03 14 -0.103 0.300 -0.720 0.513 0.7334 T02 - T04 14 -0.312 0.300 -0.928 0.305 0.3085 T03 - T04 14 -0.208 0.300 -0.825 0.408 0.4937 T02 - T03 29 -0.660 0.419 -1.520 0.200 0.1268 T02 - T04 29 -0.833 0.419 -1.693 0.027 0.0571 T03 - T04 29 -0.173 0.419 -1.033 0.687 0.6833 T02 - T03 60 -1.140 0.380 -1.920 -0.360 0.0058 T02 - T04 60 -1.453 0.380 -2.234 -0.673 0.0007 T03 - T04 60 -0.314 0.380 -1.094 0.467 0.4168 T02 - T03 90 -0.760 0.385 -1.551 0.030 0.0588 T02 - T04 90 -1.141 0.385 -1.932 -0.351 0.0063 T03 - T04 90 -0.381 0.385 -1.172 0.409 0.3312 T02 - T03 120 -1.247 0.439 -2.148 -0.346 0.0085 T02 - T04 120 -1.419 0.439 -2.320 -0.519 0.0032 T03 - T04 120 -0.172 0.439 -1.073 0.728 0.6979 T02 - T03 149 -1.250 0.441 -2.154 -0.346 0.0085 58 / 60 Petition 870250084645, dated 09 / 19 / 2025, pp. 70-85 Comparison of treatments on study day, difference of means, least squares standard error, confidence level less than 95%, limit for mean difference, confidence level.greater than 95%, limit for mean difference. p-value T02 - T04 149 -1.423 0.441 -2.327 -0.519 0.0032 T03 - T04 149 -0.173 0.441 -1.077 0.731 0.698 T02 - T03 180 -1.005 0.389 -1.803 -0.207 0.0155 T02 - T04 180 -1.145 0.389 -1.943 -0.347 0.0066 T03 - T04 180 -0.140 0.389 -0.938 0.658 0.7221 T02 - T03 210 -0.976 0.386 -1.767 -0.185 0.0175 T02 - T04 210 -1.185 0.386 -1.976 -0.394 0.0048 T03 - T04 210 -0.209 0.386 -1.000 0.582 0.5921 T02 - T03 240 -1.078 0.324 -1.744 -0.413 0.0026 T02 - T04 240 -1.183 0.324 -1.849 -0.517 0.0011 T03 - T04 240 -0.105 0.324 -0.770 0.561 0.7498 T02 - T03 270 -1,355 0.380 -2.135 -0.575 0.0014 T02 - T04 270 -1.630 0.380 -2.410 -0.850 0.0002 T03 - T04 270 -0.275 0.380 -1.055 0.505 0.4755 T02 - T03 300 -1.073 0.360 -1.812 -0.333 0.0061 T02 - T04 300 -1.315 0.360 -2.055 -0.576 0.0011 T03 - T04 300 -0.243 0.360 -0.982 0.497 0.5066. 59 / 60 Petition 870250084645, dated 09 / 19 / 2025, pp. 71 / 85 Comparison of treatments on study day, difference of means, least squares standard error, confidence level less than 95%, limit for mean difference, confidence level. greater than 95%, limit for mean difference. p-value T02 - T03 330 -1.077 0.386 -1.868 -0.286 0.0095 T02 - T04 330 -1.563 0.386 -2.354 -0.772 0.0004 T03 - T04 330 -0.486 0.386 -1.277 0.305 0.2185 T02 - T03 365 -1.111 0.375 -1.881 -0.341 0.0063 T02 - T04 365 -1.355 0.375 -2.125 -0.585 0.0012 T03 - T04 365 -0.244 0.375 -1.014 0.526 0.5214 [000192] All publications cited in the descriptive report, both patent publications and non-patent publications, are indicative of the skill level of those skilled in the art to which this invention relates. All such publications are fully incorporated into this application by reference, to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference. [000193] Although the invention described in this application has been made with reference to particular 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 illustrative embodiments and that other arrangements can be designed without departing from the spirit and scope of the present invention, as defined by the following claims. 60 / 60 Petition 870250084645, dated 09 / 19 / 2025, pp. 72-85

Claims

1 / 6 CLAIMS 1. A vaccine, characterized by comprising an FMD antigen and an adjuvant comprising oil, a polycationic carrier and an immunostimulatory oligonucleotide, wherein the vaccine is a water-in-oil (W / O) emulsion, for use in the prevention of foot lesions caused by FMD infection in a ruminant, wherein the vaccine is administered as a primary vaccination dose and one or more booster doses, wherein the first of the one or more booster doses is administered 7 to about 12 months after the primary vaccination dose.

2. Vaccine, according to claim 1, characterized in that each subsequent booster dose is administered 7 to about 12 months after the previous booster dose.

3. Vaccine, according to claim 1 or 2, characterized in that the first booster dose is administered approximately 8, or approximately 9, or approximately 10, or approximately 11, or approximately 12 months after the primary vaccination dose.

4. Vaccine, according to any one of claims 1 to 3, characterized in that the cationic carrier is DEAE Dextran.

5. Vaccine, according to claim 4, characterized in that DEAE Dextran is present in the amount of 25 to 250 mg per dose.

6. Vaccine, according to claim 5, characterized in that the immunostimulatory oligonucleotide is present in an amount of 25 to 250 pg per dose.

7. Vaccine, according to claim 6, characterized in that the immunostimulatory oligonucleotide is present in an amount of 50 to 150 pg per dose.

8. Vaccine, according to any one of claims 1 to 7, characterized in that the vaccine comprises at least 40% v / v oil. Petition 870250084645, dated 09 / 19 / 2025, p. 73 / 85 2 / 6 9. Vaccine, according to claim 8, characterized in that the vaccine comprises at least 45% v / v oil.

10. Vaccine, according to claim 9, characterized in that the vaccine comprises at least 48% v / v oil.

11. Vaccine, according to claim 10, characterized in that the vaccine comprises at least 52% v / v oil.

12. Vaccine, according to claim 11, characterized in that the vaccine comprises at least 60% v / v oil.

13. Vaccine, according to any one of claims 1 to 12, characterized in that the oil is a light mineral oil.

14. Vaccine, according to any one of claims 1 to 13, characterized in that the antigen is from an FMD virus of serotype O, A and / or Asia 1.

15. Vaccine, according to any one of claims 1 to 13, characterized in that the antigen is from an FMD virus of serotype O, A and / or C.

16. Vaccine, according to any one of claims 1 to 13, characterized in that the antigen is from an FMD virus of serotype SAT1, SAT2 and / or SAT3.

17. Vaccine, according to any one of claims 1 to 13, characterized in that the antigen is selected from the group consisting of O1 Manisa, O1 BFS or 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 / 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, Petition 870250084645, dated 19 / 09 / 2025, p. 74 / 85 3 / 6 A / TNAn / 60 / 947 / Asia / 1, A / IRN / 05, Asia / IRN / 05, O / HK / 2001, O / UKG / 3952 / 2001, O / UKG / 4141 / 2001, Asia 1 / HNK / CHA / 05, Asia 1 / 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, O5 India, 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 / 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 / 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 / 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,O / IND / R2 / 1975 and any combination thereof.

18. Vaccine, according to any one of claims 1 to 17, characterized in that the antigen is an inactivated FMD virus.

19. Vaccine, according to claim 17, characterized in that the inactivated FMD virus is a recombinant virus.

20. Vaccine, according to claim 18, characterized in that the recombinant FMD virus does not possess a functional leader protein.

21. Vaccine, according to claim 18 or 19, characterized in that the recombinant FMD virus further comprises DIVA markers. Petition 870250084645, dated 19 / 09 / 2025, pp. 75 / 85 4 / 6 22. Vaccine, according to claim 19, characterized in that the recombinant FMD virus further comprises one or more deletion sequences as negative markers.

23. Vaccine, according to claim 19, characterized in that the recombinant FMD virus further comprises one or more mutations in the capsid coding sequences that induce thermostability.

24. Vaccine, according to any one of claims 19 to 23, characterized in that the recombinant FMD virus expresses a capsid from a heterologous strain of FMD.

25. Vaccine, according to claim 24, characterized in that the heterologous strain of FMD 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. Vaccine, according to claim 25, characterized in that the heterologous strain of FMD is a strain of serotype A, or serotype O, and / or serotype Asia 1.

27. Vaccine, according to claim 25, characterized in that the heterologous strain of FMD is a strain of serotype A, or serotype O, and / or serotype C.

28. Vaccine, according to claim 25, characterized in that the heterologous strain of FMD is a strain of serotype SAT1, serotype SAT2 and / or serotype SAT3.

29. Vaccine, according to any one of claims 24 to 28, characterized in that the heterologous strain is selected from the group consisting of O1 Manisa, O1 BFS or 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, SAT1SAT3, 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, Petition 870250084645, of 09 / 19 / 2025, p. 76 / 85 5 / 6 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, O / HK / 2001, O / UKG / 3952 / 2001, O / UKG / 4141 / 2001, Asia 1 / HNK / CHA / 05, Asia 1 / 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, O5 India, 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 / 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 / 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 / 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, O / IND / R2 / 75 and any combination thereof.

30. Vaccine, according to any one of claims 17 to 28, characterized in that the FMD virus is present in an amount of at least 4 pg per dose per strain.

31. Vaccine, according to any of claims 17 to 29, characterized in that the FMD virus is present in the quantity of at least 8 pg per dose per strain. Petition 870250084645, dated 09 / 19 / 2025, page 77 / 85 6 / 6.

32. Vaccine, according to any one of claims 17 to 30, characterized in that the FMD virus is present in an amount of about 10 pg per dose per strain.

33. Vaccine, according to any one of claims 1 to 32, characterized in that the ruminant is a bovine.

34. Vaccine, according to any one of claims 1 to 33, characterized in the ruminant being negative for FMD. Petition 870250084645, dated 09 / 19 / 2025, pp. 78 / 85