阅读说明：本技术 可与油乳剂组合施用的免疫增强佐剂以及包含该佐剂的口蹄疫疫苗组合物 (Immunopotentiating adjuvant capable of being administered in combination with oil emulsion and foot and mouth disease vaccine composition comprising the same ) 是由 M·李 J－H·朴 S-M·金 H·赵 B·金 于 2019-05-02 设计创作，主要内容包括：本发明涉及用于口蹄疫疫苗的佐剂组合物。本发明可通过同时诱导牛、猪等偶蹄动物的细胞免疫和体液免疫来诱导强免疫应答,用于预防和治疗口蹄疫爆发,安全和优化的佐剂组合物以及包含该佐剂组合物的疫苗组合物。此外,当口蹄疫爆发时,本发明可作为现场应急疫苗用于有效应对疫情,并可为偶蹄动物提供稳定的系统性疫苗。(The present invention relates to adjuvant compositions for foot and mouth disease vaccines. The adjuvant composition can induce strong immune response by simultaneously inducing cellular immunity and humoral immunity of artiodactyls such as cattle, pigs and the like, is used for preventing and treating foot-and-mouth disease outbreak, and is safe and optimized, and the vaccine composition containing the adjuvant composition. In addition, when the foot-and-mouth disease outbreak occurs, the vaccine can be used as a field emergency vaccine for effectively coping with epidemic situations, and can provide a stable systemic vaccine for artiodactyls.)

1. An adjuvant composition comprising a Pattern Recognition Receptor (PRR) ligand or cytokine as an active ingredient.

2. An adjuvant composition comprising a Pattern Recognition Receptor (PRR) ligand and a cytokine as active ingredients.

3. An adjuvant composition according to claim 1 or 2, wherein the PRR ligand comprises any one or more of R848(TLR-7/8 agonist), TDB (Mincle agonist), Curdlan (Dectin-1 agonist), furfurfurmann (Dectin-2 agonist), MDP (NOD-2 ligand), MPLA-SM (TLR-4), zymosan (Dectin-2/TLR-2), chitosan (NLRP inflammasome inducer, MR), C-di-GMP, C-di-AMP, ccGAMP (STING ligand), poly (I: C) (TLR-3) and poly (dA: dT) (RIG-1/CDS agonist, AIM-2 inflammasome inducer).

4. An adjuvant composition according to claim 1 or 2, wherein the cytokine comprises any one or more of IL-1 β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-12/23p40, IL-15/15R, IL-17A, IL-21, IL-22, IL-23, IL-33, TNF- α and IFN- γ.

5. An adjuvant composition according to claim 1 or 2, wherein the PRR ligand is comprised in an amount of 1 to 100 μ g/ml.

6. An adjuvant composition according to claim 3, wherein two PRR ligands are included and either of the PRR ligands the remaining PRR ligand is included in a weight ratio of 1:1 to 3.

7. An adjuvant composition according to claim 6, wherein the PRR ligand is comprised in an amount of 1 to 30 μ g/ml.

8. An adjuvant composition according to claim 1 or 2, wherein the cytokine is comprised in an amount of 1 to 100 μ g/ml.

9. An adjuvant composition according to claim 1 or 2 wherein the composition is for foot and mouth disease.

10. A vaccine composition comprising the adjuvant composition of claim 1 or 2.

Technical Field

The present invention relates to an immunopotentiating adjuvant for a foot-and-mouth disease vaccine and a vaccine composition comprising the same. More particularly, the present invention relates to an adjuvant for enhancing immunity of a foot-and-mouth disease vaccine, which can be administered in combination with an oil emulsion, and a vaccine composition comprising the same.

Background

Foot-and-mouth disease (FMD) is a highly contagious viral disease that mainly affects artiodactyl livestock (i.e., artiodactyl), causing significant economic losses to the animal husbandry due to its rapid spread, high mortality, and reduced productivity. Susceptible species include about 70 or more wild animals, including ruminants (ruminants), such as cattle, pigs, buffalos, camels, sheep and goats. This disease is associated with high fever, which is known to cause blistering of the mouth, tongue, oronasal, nose, nipple, hooves and other glabrous skin areas.

Vaccination is used as a means of controlling the pathological conditions in countries with foot and mouth disease and plays an important role in emergency planning in countries without foot and mouth disease. The foot and mouth disease vaccines worldwide use killed (inactivated) vaccines, most of which are enhanced in effectiveness by vaccination with an oil adjuvant (double oil emulsion DOE or single oil emulsion SOE). However, a delay in the induction period of antibodies to defense levels, low antibody titers, short antibody durations and low immunogenicity in swine compared to cattle are indicated as disadvantages.

Foot-and-mouth disease vaccines currently focus primarily on inducing a humoral immune response rather than a cellular immune response, but their protective properties are not ideal. The induction period of the humoral immune response (mainly neutralizing antibody IgG) of the foot-and-mouth disease vaccine is 4 to 7 days, while the induction period of the cellular immune response is several hours to 3 days. Thus, induction of a cellular immune response appears to protect the host more effectively than a humoral immune response in the early stages of foot and mouth disease infection. In addition, current foot-and-mouth disease vaccines require periodic vaccination every 4 to 6 months because of the short antibody maintenance period after vaccination. In particular, when the breeding pigs are inoculated by intramuscular injection, there are disadvantages of low safety and local side effects such as formation of lesions at the inoculation site, such as formation of fibrosis and granuloma at the inoculation site. On the other hand, studies related to foot-and-mouth disease vaccines have been conducted so far in a number of evaluation studies to determine the effectiveness of the vaccine in livestock breeds on cattle rather than pigs. However, swine are reported to be less immunogenic than cattle upon vaccination. Therefore, an ideal vaccine design that overcomes the limitations of commercially available vaccines should meet requirements including: for example, both cellular and humoral immunity are induced, high antibody titer is maintained by inducing memory response, safety is ensured to reduce local side effects, and an adjuvant optimized for livestock species is developed according to a new strategy or the like.

In foreign countries, Marcol 52, ISA206 and ISA50 are mainly used as adjuvants for commercial foot-and-mouth disease vaccines. As adjuvant-related studies effective against foot-and-mouth disease, various studies have been conducted such as evaluating the effectiveness of foot-and-mouth disease vaccines in pigs and goats using, for example, Pattern Recognition Receptor (PRR) ligands such as R848, poly (I: C), MDP, MPL, β -glucan, etc., immunopotentiators such as rapeseed oil, ginseng root saponin, etc., or typical commercial adjuvants (ISA201, ISA206, Emulsigen-D, Carbigen, etc.), but these adjuvants have not yet induced complete immunity.

In addition to the foot-and-mouth disease vaccine, there have been efforts to improve immunogenicity by simultaneously inducing cellular and humoral immunity mainly using human vaccines, specifically: 1) vaccine delivery systems, such as oil emulsions, surfactants, liposomes, virosomes, ISCOMs, and the like; 2) immunopotentiators such as saponin, aluminum hydroxide, potassium phosphate, etc.; 3) toll-like receptors (TLRs), RIG-I-like receptors (RLRs), nucleotide binding oligomerization domain (NOD) -like receptors (NLRs), and receptor-specific immunostimulants, such as C-type lectin receptor (CLR) ligands; 4) various cytokines such as IL-1, IL-2, IL-6, IL-18, TNF α, IFN γ, and GM-CSF, etc., have been studied and used as adjuvants. Some of these materials are currently used as vaccine adjuvants for the prevention and treatment of various human diseases such as cancer, tuberculosis, hepatitis b, malaria, influenza, HIV and HSV, or in clinical trials, but are not used as vaccine compositions for foot-and-mouth disease. Furthermore, since multiple adjuvants have different modes of action, understanding the immunological mechanisms of the adjuvant is very important for FMD vaccine development, according to new strategies that can induce strong cellular and humoral immune responses.

Therefore, the present invention aims to propose the possibility of developing FMD vaccines according to a new strategy by developing various PPRR ligands and cytokines with adjuvant efficacy in mice, inducing memory immune responses by inducing cellular and humoral immune responses, as well as optimized adjuvants for different species (artiodactyla, such as cattle and pigs) and vaccine compositions comprising the same for foot and mouth disease.

Meanwhile, the prior art related to the above-mentioned technologies includes korean patent laid-open publication No. 10-2017-0097116 entitled "foot-and-mouth disease vaccine", korean patent laid-open publication No. 10-2018-002430 entitled "oily adjuvant", and the like, but the effects of the above-mentioned materials as an immunopotentiation aid or adjuvant that can be used in the foot-and-mouth disease vaccine and a vaccine composition comprising the same have not been specifically described.

Documents of the prior art

Patent document

(patent document 1) Korean patent laid-open No. 10-2017-0097116

(patent document 2) Korean patent laid-open No. 10-2018-0024030

Summary of The Invention

Problems to be solved by the invention

In order to solve the aforementioned problems, the present invention aims to provide an adjuvant that is safe and optimized for protective antibody induction in artiodactyl (i.e., artiodactyl) subjects such as cattle and pigs and mice to prevent and treat foot-and-mouth disease, and a vaccine composition comprising the same.

Means for solving the problems

In order to achieve the above objects, the present invention provides an adjuvant composition comprising a Pattern Recognition Receptor (PRR) ligand as an active ingredient.

In addition, the invention also provides a foot-and-mouth disease vaccine composition containing the composition.

The adjuvant composition comprises a cytokine as an active ingredient, and a Pattern Recognition Receptor (PRR) ligand.

In the present invention, artiodactyla generally refers to a group of animals with two hooves, such as cattle, pigs, sheep, goats, reindeer, buffalo and camels, and preferably cattle and pigs.

The foot-and-mouth disease virus antigen in the invention can comprise a foot-and-mouth disease virus O serotype, a foot-and-mouth disease virus A serotype, a foot-and-mouth disease virus Asia 1 serotype, a foot-and-mouth disease virus C serotype, a foot-and-mouth disease virus SAT1 serotype, a foot-and-mouth disease virus SAT2 serotype, a foot-and-mouth disease virus SAT3 and the like.

Cytokines are the general term for bioactive factors secreted by cells, and are involved in intercellular signal transduction, regulation of cell behavior, regulation of immune response, etc., and may refer to low molecular proteins such as interleukins, lymphokines, interferons, cell proliferation and differentiation factors, etc.

The cytokine of the present invention may include IL-1 β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-12/23p40, IL-15/15R, IL-17A, IL-21, IL-22, IL-23(IL-12p40/IL-23p19), IL-33, TNF- α, IFN- β, IFN- γ, etc., but they are not limited thereto.

The above cytokines are commonly used cytokines, and may include recombinant cytokines expressed in escherichia coli (e.coli), baculovirus, HEK 293 cells, and the like.

Among the Pattern Recognition Receptor (PRR) ligands of the present invention, the pattern recognition receptor can be used to recognize a conserved molecular pattern that distinguishes viruses, bacteria, fungi and parasites, and the ligand refers to an agonist of the receptor, i.e., a substance that binds to and activates the receptor.

PRR ligands may include, for example, R848(TLR-7/8 agonist), TDB (Mincle agonist), Curdlan (Dectin-1 agonist), Furfurman (Dectin-2 agonist), MDP (NOD-2 ligand), MPLA-SM (TLR-4), zymosan (Dectin-2/TLR-2), chitosan (NLRP inflammasome inducer, MR), poly (I: C) (TLR-3), poly (dA: dT) (RIG-1/CDS agonist, AIM-2 inflammasome inducer), or STING-based ligands, but are not limited thereto.

STING-based ligands can include DNA, RNA, proteins, peptide fragments, compounds, and the like that activate STING signaling, and preferably includes c-di-GMP (cyclic diguanidine ester), cGAMP, 3'3' -cGAMP, c-di-GAMP, c-di-AMP, 2'3' -cGAMP, 10- (carboxymethyl) 9(10H) acridone (CMA) (10- (carboxymethyl) 9(10H) acridone (CMA)), 5, 6-dimethylxanthone-4-acetic acid-DMXAA, methoxyflavone (methoxyvone), 6,4' -dimethoxyflavone, 4' -methoxyflavone, 3',6' -dihydroxyflavone, 7,2' -dihydroxyflavone, daidzein, formononetin, rituxin 7-methyl ether, xanthone, etc., but is not limited thereto.

Poly (I: C) refers not only to the naturally occurring polyinosinic acid, a polymer of polycytidylic acid, but also to its synthetic form.

The compositions of the present invention may comprise one or more components optionally selected from the PRRs described above.

When two components of the PRR ligands described above are included, any PRR ligand to the remaining PRR ligand may comprise a weight ratio of 1:1 to 3, and preferably 1:1 to 2.

With respect to the administration amount, i.e., the dose in the present invention, the PRR ligand or cytokine is not limited, but preferably contains a dose of 1 to 100 μ g/ml.

When two or more PRR ligands are included, each ligand may comprise 1/2 or less, preferably 1 to 30 μ g/ml, more preferably 5 to 15 μ g/ml, and most preferably 7 to 10 μ g/ml, based on the dosage included alone.

Since the above dose is in the range of being nontoxic in vivo, if it is outside the above range, there is a risk of toxicity in vivo upon administration to animals.

In addition to the above components, oils (or oil emulsions) and emulsifiers, gels, etc. known in the art may be included.

The oil (or oil emulsion) may be ISA201, ISA 61, ISA50, or ISA206, but is not limited thereto.

The emulsifier may include materials recognized as emulsifiers, such as tweensOrOther products of the product line (fatty acid esters of polyethoxylated sorbitol and fatty acid-substituted sorbitan surfactants, respectively), and other solubility-improving agents, such as PEG-40 castor oil or other pegylated hydrogenated oils, but are not limited thereto.

Advantageous effects

The present invention can provide an adjuvant composition that is safe and optimized for inducing protective antibodies of artiodactyla (e.g., cattle, swine, etc.) to prevent and treat picornaviruses including foot-and-mouth disease, and a vaccine composition comprising the adjuvant composition. In addition, the present invention can be used to effectively cope with emergency vaccines on the spot when foot-and-mouth disease occurs, and can provide effective and stable vaccines as conventional vaccines for artiodactyl purposes.

Brief Description of Drawings

Figure 1 shows the results of the induction of an initial cellular immune response in vivo based on serotype specific antigens.

Fig. 2A shows the results of inducing innate and cellular immune responses of DCs according to serotype-specific antigens.

Fig. 2B shows the results of inducing an innate immune response and a cellular immune response of M Φ according to serotype specific antigens.

FIG. 3 shows the cytokine expression profile induced by the foot and mouth disease virus (O TWN 97-R) antigen in mice and the strategy for time kinetic studies in vivo.

FIG. 4 shows the cytokine expression profile and in vivo time kinetic results (time points: 0, 6, 12, 24, 48, 72, 96h) induced by the foot and mouth disease virus (O TWN 97-R) antigen in mice.

FIG. 5 shows a strategy for identifying in vitro characteristics of cytokine expression in DC, M Φ, iNK T cells, γ δ T cells and T cells isolated from mouse Peritoneal Effusion Cells (PEC) and splenocytes mediated by foot and mouth disease virus (O TWN 97-R) antigen.

FIGS. 6A to 6C show the results of time-dependent cytokine expression in DC, M.phi., iNK T cells, γ.delta.T cells and T cells isolated from Peritoneal Effusion Cells (PEC) and splenocytes of mice mediated by foot-and-mouth disease virus (O TWN 97-R) antigen (time points: 0, 6, 12, 24, 48, 72, 96 h).

FIG. 7 shows strategies for identifying in vitro characteristics of foot and mouth disease virus (O TWN 97-R) antigen-mediated cytokine expression when co-culturing DCs, iNK T cells, γ δ T cells and T cells isolated from mouse Peritoneal Effusion Cells (PEC) and splenocytes, where (A) shows the strategy of in vitro studies and (B) is a schematic of the co-cultured cells.

Figure 8 shows the results of antigen-mediated cytokine expression in co-cultured cells, DCs, inkt cells, γ δ T cells and T cells in mice.

FIG. 9 shows a strategy to confirm the in vivo defense effect mediated by the antigen O TWN97-R early in FMDV infection.

FIG. 10 shows the effect on antigen O TWN97-R mediated in vivo defense effects (mouse survival, body weight change) early in FMDV infection.

Figure 11 shows the strategy of cell depletion, cytokine neutralization, and antigen-mediated defense effects in vivo.

Figures 12A and 12B show the in vivo defense effect (survival, weight change) of antigen against FMDV infection in hosts at the expense of DC and M Φ and neutralizing cytokines.

FIG. 13 shows the results of antigen-mediated expression of the stimulatory molecules CD40, CD80 and CD86, and cell proliferation in O TWN97-R antigen and stimuli (R848, chitosan, zymosan, ODN 2395, TDB, Furfuman and c-di-GMP) treated DCs.

FIG. 14 shows IL-23 activity produced by simultaneous activation of Dectin-1 and Mincle in DC and M.phi.

Figure 15 shows strategies for evaluating the potential as vaccine adjuvants and short-term immunological memory effects on FMDV infection when PRR ligands and cytokines are administered alone or in combination.

Figure 16 shows short-term immunological memory effects and in vivo defense effects (survival, weight change) when PRR ligands and cytokines are administered alone or in combination as adjuvants to host resistance to FMDV infection.

Figure 17 shows a strategy for evaluating long-term memory responses to FMDV infection (long-term immunity) mediated by cytokines, HSP70 protein and PRR ligand and their protective effects in mice.

Figures 18A to 18D show the results of long-term memory immune responses mediated by cytokines, HSP70 protein and PRR ligand and protection against FMDV infection in mice.

Fig. 19A to 19G show the results of cytokine, HSP70 protein and PRR ligand mediated immune cell expansion in mice.

Figure 20 shows a strategy to confirm IFN α and IL-23 mediated protection against infection by multiple FMDV serotypes (O, a and asia type 1).

Figure 21 shows IFN alpha and IL-23 mediated memory response and multiple FMDV serotype (O type, A type and Asia 1 type) infection widely protective effect results.

Figure 22 shows LDH release (cytotoxicity, after 96 hours) in PRR ligand-treated bovine PBMC.

Figure 23 shows the results of cell proliferation mediated by PRR ligands (after 96 hours) in bovine PBMC.

Figure 24 shows a strategy for evaluating PRR ligand-mediated adjuvant effects and memory responses in cattle.

Figure 25 shows the adjuvant effect and memory response results mediated by PRR ligands in cattle.

Figure 26 shows the results of LDH release (cytotoxicity, after 96 hours) in PRR ligand-treated porcine PBMCs.

Figure 27 shows the results of PRR ligand mediated cell proliferation (after 96 hours) in porcine PBMC.

Figure 28 shows a strategy for evaluating PRR ligand-mediated adjuvant effects and memory responses in pigs.

Figure 29 shows the results of PRR ligand-mediated adjuvant effect and memory response in pigs.

Modes for carrying out the invention

Hereinafter, the present invention will be described in detail by way of examples and experimental examples.

However, the following examples and experimental examples are only illustrative examples of the present invention, and the contents of the present invention are not limited to the following examples and experimental examples.

The experimental materials used in the examples and experimental examples of the present invention are as follows:

antigens

The antigen was purified and inactivated as follows.

The antigen was prepared from FMDV O TWN97-R (P1 GenBank AY593823) and BHK-21 cells. For viral infection, the medium was replaced with serum-free Dulbecco's modified Eagle Medium (DMEM, Cellgro, Manassas, Va., USA) and 5% CO at 37 deg.C₂Incubated in air for 1 hour to inoculate the virus. And (4) removing the extracellular viruses. After 24 hours of infection, the virus was inactivated by treatment with 0.003N binary ethylamine twice for 24 hours in a shaking incubator and concentrated with polyethylene glycol (PEG)6000(Sigma-Aldrich, St. Louis, Mo., USA). The virus concentrate was fractionated by a 15-45% sucrose density gradient and then centrifuged. After ultracentrifugation, the bottom of the centrifuge tube was pierced and 1ml fractions were collected. The presence of FMDV particles in each fraction sample was confirmed by optical density using a lateral flow device (BioSign FMDV Ag, Princeton biomedetch, Princeton, NJ, USA). Before use in the experiment, the PEG-pretreated supernatant was passed through ZZ-R and BHK-21 cells at least twice to confirm whether no cytopathic effect (CPE) occurred and, therefore, that no live virus was present in the supernatant.

Pattern recognition receptor (PPR) ligands and cytokines

As PRR ligand, Invivogen (san diego, CA, USA) was used, and a product of mitenyi biotec (miltenyi biotec, bergisch gladbach, germany) was used as a cytokine. Oil emulsion ISA206 is available from Seppic Corporation (Seppic Inc., Paris, France), while aluminum hydroxide gelAnd Quil-A from Invivogen.

Mouse

Age and gender matched wild type C57BL/6 mice (7 week old females) were purchased (KOSA BIO Inc, Gyeonggi-do, korea). All mice were housed in mini-quarantine cages of specific Sterile Pathogen Free (SPF) animal facilities with a biosafety rating of 3 (ABSL3) at the korean agricultural, forestry and livestock quarantine bureau.

Examples of the Main experiments

Assay to confirm host immunological mechanisms of foot-and-mouth disease antigens

1. Induction of an intrinsic immune response based on serotype-specific antigen confirmation

1-1. to confirm whether 8 antigens induce an intrinsic immune response in vivo, 2. mu.g of each antigen was injected into the abdominal cavity of mice and the abdominal cavity was washed with 2ml of cold PBS, and the collected intra-abdominal wash was centrifuged, and then the supernatant was analyzed for cytokine expression pattern and time kinetics.

Since the pattern and transient changes in cytokine expression caused by serotype-specific antigens were examined, it was highly demonstrated that the expression of pro-and anti-inflammatory cytokines IL-23 and IL-10 could competitively induce a "cytokine storm" by most antigens. Expression of the two cytokines was similarly observed from 48 hours to 72 hours and 96 hours in the SAT1-BOT-R, SAT2-ZIM-R and SAT3-ZIM-R injection groups, while the O PA-2-R, O TWN97-R, A22-R, C3-Resend-R and Asia 1-MOG-R groups showed significantly higher IL-23 expression than IL-10. IL-23 expression is characterized by a similar expression pattern in serotypes O, O PA-2-R and O TWN97-R, i.e., expression increases rapidly at 6 to 12 hours and then shows slightly lower levels at 24 hours. Expression then increased again at 48 to 72 hours, approaching levels at 12 hours, then decreased significantly at 96 hours (figure 1).

1-2 to study the innate and cellular immune responses of DCs and M.phi. to serotype specific FMDV antigens, DCs and M.phi. were isolated from naive mice and treated with the respective antigens, and the cell culture supernatants were then analyzed for inflammatory cytokines such as IL-1 alpha, IL-6, IL-10, IL-12p70, IL-12/23p40, IL-23 and TNF. alpha. the anti-inflammatory cytokine expression pattern and the temporal kinetics of cytokine expression after 0, 12, 24, 48, 72 and 96 hours. Thus, the expression in DC is as follows.

With respect to the expression levels of serotypes IL-23 and IL-10, the cytokine expression levels of SAT1-BOT-R, SAT2-ZIM-R, SAT3-ZIM-R, C3-Resend-R and Asian 1-MOG-R treated groups were substantially similar. On the other hand, in the case of A22-R, O PA-2-R and O TWN97-R, the expression level of IL-23 was significantly higher than that of IL-10. In addition, the maximum expression level of IL-6 is high, such as 593 to 753pg/ml, with the remaining cytokines being expressed at levels of about 50 to 200 pg/ml. In most antigen-treated groups, the expression levels of IL-23, IL-10 and IL-6 crossed each other at 12 hours, with the initial increased expression of IL-6 showing a tendency to be down-regulated by the anti-inflammatory cytokine IL-10, while IL-23 shows a tendency to be continuously up-regulated (FIG. 2A).

Overall cytokine expression in M Φ was similar to that in DC, as shown in fig. 2A. Furthermore, with respect to the change in serotype cytokine expression, it was observed that the level of IL-23 expression in most antigen treated cells, except Asia 1-MOG-R, was significantly higher than IL-10 after 48 hours. In the SAT1-BOT-R, SAT2-ZIM-R, SAT3-ZIM-R and C3-Resende-R treated groups, the expression levels were IL-10> IL-6> IL-23 in that order up to 12 hours. However, after the expression level of the above-mentioned cytokine was crossed at 12 to 48 hours, the expression pattern was changed after 48 hours in the order of IL-23> IL-10> IL-6. On the other hand, in the case of A22-R, O PA-2-R and O TWN97-R, the cytokine expression levels were higher up to 12 hours in the order IL-6> IL-23 and IL-10, and after crossing the expression levels of these cytokines between 12 and 24 hours, the expression pattern was changed after 24 hours in the order IL-23> IL-6 and IL-10 (FIG. 2B).

From the above results, it was confirmed that DCs and M Φ were efficiently stimulated in the early stage of serotype-specific antigen treatment. In addition, inflammatory cytokines (i.e., IL-23 and IL-6), as well as anti-inflammatory cytokines (i.e., IL-10), have been shown to be expressed primarily by stimulated cells.

2. Measurement of in vivo antigen-mediated cytokine changes

This assay was the same as that shown in FIG. 3, and attempted to confirm stimulation of immune cells by measuring changes in cytokines.

As a result of the experiment, IL-1 β (IL-1 beta) and IL-6 initially reacted and most were stimulated over time. IL-12/23p40, IL-17A and TNF α (TNF alpha) were stimulated between 12 and 24 hours, whereas IL-1 β (IL-1 β), IL-2, IL-10, IL-15/15R and IL-33 were fully stimulated within 72 hours. Furthermore, in the case of IL-4, IL-9, IL-12p70, IL-21, IL-22 and IFN gamma (IFN gamma), the response increased up to 96 hours. Since the cytokines stimulated in the cells were different, it was possible to predict which cells acted (FIG. 4).

3. Measurement of antigen-mediated changes in vitro cytokine expression profiles of immune cells isolated from mouse Peritoneal Effusion Cells (PEC) and splenocytes

This test method is the same as that shown in FIG. 5. Immune cells (DC, M Φ, inkt cells, γ δ T cells and T cells) were isolated from Peritoneal Effusion Cells (PEC) and splenocytes in mice, and then changes in cytokine expression were measured to confirm stimulation of specific immune cells. Specifically, cells were treated with O TWN97-R antigen (2. mu.g), and cells (DC, M.phi., iNK T cells, γ. delta. T cells and T cells) were isolated from PEC and spleen, and then changes over time were measured (0, 6, 12, 24, 48, 72, 96 h).

As a result of the experiment, the cytokine reactivities of cytokines IL-12p70, IL-12/23 total p40, IL-10, IL-5 and TNF α reacting with DC tended to be higher in the first 6 to 12 hours. In addition, IL-12/23p40 and IL-23 were confirmed to have a tendency to increase in expression at 24 to 48 hours (FIGS. 6A to 6C).

4. Measurement of in vitro changes in O TWN97-R antigen mediated cytokine expression profiles by simultaneous culture in sorted cells

For sorting cells, this assay method was the same as in the main experimental example 2, immune cells (DC, M Φ, inkt cells, γ δ T cells and T cells) were separated and mixed, and then changes in cytokines mediated by O TWN97-R antigen were measured to confirm stimulation of immune cells (fig. 7). Specifically, cells were treated with O TWN97-R antigen (2. mu.g), and the results were measured 48 hours after antigen stimulation.

As a result of the experiment, IL-2, IL-4, IL-5, IL-12/23p40, IL-15/15R, IL-21, IL-23, IFN gamma, etc. were confirmed to show reactivity only when DC was added. On the other hand, it was confirmed that IL- β, IL-6, IL-9, IL-10, IL-12p70, IL-17A, IL-22, IL-33, TNF α, etc., showed reactivity even without addition of DC (FIG. 8).

5. Analysis of defense mechanisms by antigen stimulation of innate immune response

5-1. FIG. 9 shows a study strategy to confirm whether the O TWN97-R antigen that activates both the innate and cellular immune responses indeed shows a defense effect when infected with FMD virus. First, to determine the vaccination period of the antigen, mice were intraperitoneally injected with 2 μ g of antigen, infected with virus at1, 3, 5 and 7dpi, respectively, and then monitored for survival and weight change until 7 dpc. Thereafter, to confirm the dose-specific response of the antigen, 2, 5 and 10 μ g of antigen were injected into the same route, mice were infected with virus at 5dpi, and survival and body weight changes were monitored up to 7 dpc.

As a result, the 5dpi group showed a 40% defense rate, while the group with an increased dose to 5, 10 μ g showed 100% survival without weight loss (fig. 10).

5-2 to protect the host from foot and mouth disease, attempts were made to identify which cells in DC and M.phi.are important, or which cytokines show important roles (FIG. 11).

Cells were consumed or cytokines were neutralized with antibodies, antigens were injected 24 hours later, and viruses were infected 5 days later (5dpi) to confirm defense performance. As a result, IL-23p19, TNF α and IL-10 were confirmed to play important roles. All mice in the group administered with the antibody against the inflammatory cytokine IL-23p19 died at day 5 (5dpc), all mice in the group administered with the TNF α antibody died at day 6 (6dpc), whereas the survival rate was 100% in the group administered with the antibody against the anti-inflammatory cytokine IL-10, and no weight loss was observed. In the antigen-induced cytokine neutralization experiments by specific antibodies, with regard to IL-23, TNF α, IL-1 β, IL-6 and IL-10, these cytokines were identified experimentally as playing an important role in protecting the host from FMDV attack, and the defense roles of these cytokines were individually identified. First, each cytokine was intraperitoneally injected into mice in an amount of 5. mu.g/100. mu.L of PBS, and the mice were infected with O-VET (ME-SA) 6 hours later, and then the survival rate and the change in body weight were monitored until 7 dpc. On the other hand, the negative control group was compared by administering 100. mu.l of PBS by the same route. As a result, after injection of IL-23, the survival rate of FMDV-infected group mice was confirmed to be 100%, demonstrating complete protection. Furthermore, no change in body weight was observed compared to before infection.

In the case of IL-1. alpha. and TNF. alpha. treated groups, survival was low. In addition, although body weight is reduced by about 30%, death is often delayed. On the other hand, in the case of IL-6 and IL-10 treated groups, mice died early in infection, demonstrating no defense.

From the above results, it is considered that the cytokine IL-23 plays the most important role in host defense against FMDV attack. IL-1 α and TNF α are also weaker than IL-23, but they were identified as contributing in part to host defense at the onset of viral infection.

On the other hand, in the case of IL-10 as an anti-inflammatory cytokine, 5. mu.g of IL-10 and antigen were injected simultaneously, and IL-10 alone was injected to compare the survival rate and the body weight change between the two.

To inhibit high levels of IL-23, IL-1 α, IL-6 and TNF α expression following antigen stimulation, each of these cytokines was neutralized with a specific mouse monoclonal antibody, injected simultaneously with IL-10 and antigen, and then subjected to viral infection at 24 hours. Isotype control antibodies for each antibody were injected and compared as controls. As a result, similar to the IL-10 treated group, the survival rate at day 4 (4dpc) after FMDV infection decreased to 20%, all animals died at day 6 (6dpc) and the body weight change decreased by about 30%. Thus, in the early stages of FMDC infection, IL-10 appears to suppress the immune response in the host, so as to increase susceptibility to the virus, thereby worsening host defense.

From the above results, it is considered that if the expression of IL-23, which is an inflammatory cytokine, is increased at an early stage of FMDV infection while inhibiting the production of IL-10, the cellular immune response in vivo can be activated, thereby more effectively protecting the host.

In addition, in order to confirm IL-23 as adjuvant (vaccine adjuvant) showed immune stimulation activity, 2.5 u g IL-23 and equivalent antigen combination administration.

As a result, the mice in the group administered with only 2.5. mu.g of antigen showed a survival rate of 40%, while the mice in the group administered with both IL-23 and antigen showed a survival rate of 80% or more, and the weight loss was not significant on days 2 to 3 (2dpc), and then showed a tendency of recovery. Thus, it was confirmed that the adjuvanting effect of IL-23 improves the defense effect against FMDV infection.

On the other hand, to confirm whether antigen-stimulated DCs and M Φ do indeed play an important role in host defense at the initial stage of FMDV infection, activated DCs and M Φ were isolated and transplanted into naive mice 6 hours after antigen administration, and virus infection was performed 1 hour later. Then, survival and body weight changes were observed up to 7 dpc.

As a result, it was confirmed that all mice survived 100% in the activated DC and M Φ transplantation groups, and no weight change was observed (fig. 12A and 12B).

From the above results, antigen stimulation of DCs and M Φ is expected to promote the secretion of inflammatory cytokines, chemokines, and co-stimulatory molecules by these cells at the initial stage of FMDV infection, and play an important role in protecting the host by presenting antigen to T cells.

The results of examining the expression of antigen-mediated costimulatory molecules in DCs are as follows (fig. 13). After treatment with the antigen alone or with PRR ligand added to the antigen as an adjuvant, high expression was observed in the treatment groups to which R848, zymosan, TDB and c-di-GMP were added, respectively, by detecting the expression of CD40, CD80 and CD 86. Thus, although DCs are stimulated with antigen alone to increase expression of co-stimulatory molecules, it is expected that treatment with PRR ligands as adjuvants may stimulate DCs more strongly, thereby effectively presenting antigen to T cells.

Previously, changes in O TWN97-R Ag derived cytokines were observed in DC and M Φ. To investigate which PRR ligand signals initiate expression of these cytokines, DC and M Φ were isolated from PECs of naive mice and treated with O TWN 97-rag. Then, after 6 hours of treatment, a cell sample was collected and RNA was prepared, and then the gene expression level of PRR ligand was confirmed by qRT-PCR.

Thus, as shown in FIG. 14, the expression of dectin1, dectin2, tlr7, tlr8, etc. was high in DC, while the expression of minicle and sting was high in M Φ, and the expression of tlr2, tlr4, nlrp3, etc. was also relatively high. From the overall gene expression level of DC and M phi, the PRR ligand gene expression level is higher, and the sequence is dectin1> mincle > dectin2> tlr7> sting > tlr 8.

As can be understood from the above results, O TWN 97-rsag simultaneously stimulates both intracellular PRR ligands such as tlr7, tlr8, sting, etc., and extracellular PRR ligands such as dectin1, dectin2, minicle, etc., thereby enhancing innate immune responses as well as cellular immune responses in APCs such as DCs and M Φ. In addition, the process of differentiation of Th cells into Th1, Th2 or Th17 cells can be promoted, thereby finally promoting the activation of T cells. Therefore, the above substances are expected to induce cellular immune responses and to play a crucial role in host defense in the early stage of FMDV infection.

< example 1> screening of candidate adjuvants by efficacy evaluation in mice

Analysis of the potency and memory Effect of PRR ligands and cytokines as vaccine adjuvants

To assess the potential of PRR ligands and recombinant cytokines as vaccine adjuvants and memory effects on FMDV infection, the materials of table 1 were tested as shown in fig. 15.

TABLE 1

Experimental design for evaluation of PRR ligand and cytokine-mediated adjuvant effect in mice

When PRR ligand and cytokine were administered alone, the survival rate was higher in the order of IFN α > IL-23> c-di-GMP administration group as shown in FIG. 16 (A). In addition, in the case of IFN α with a change in body weight in fig. 16(B), no change in body weight was substantially observed. In addition, fig. 16(C) shows survival when administered in combination with PRR ligands.

The survival rate was high, in the order of R848+ TDB > TDB + c-di-GMP > Furfurman + TDB, while only weight loss was observed in the range of less than about 20% change in body weight (FIG. 16 (D)).

As can be seen from the above results, mice were more effectively protected from FMDV infection when administered in combination with cytokines than when PRR ligand was administered alone. In particular, it was determined that combinations of TLR-7/8, Mincle, Dectin-2, Sting, etc. have higher vaccine adjuvant potential.

PRR ligand and cytokine mediated mouse memory immune response

To assess the potential of PRR ligands and recombinant cytokines as vaccine adjuvants and memory effects on FMDV infection, experiments were performed according to the strategy shown in fig. 17. The foot-and-mouth disease antigen used herein was O TWN97-R, and the Positive Control (PC) group of vaccines consisted of: o TNW97-R Ag (15. mu.g/dose/ml, 1/160 dose), ISA206 (50%, w/w), 10% Al (OH)₃15. mu.g/mouse Quil-A.

Mice were vaccinated 1 st at 0dpv (days post vaccination) I.M (intramuscular vaccination) and then vaccinated 2 nd (boost) 28 days later (day 28 post vaccination, dpv). Thereafter, on day 56 (56 days post inoculation, dpv), FMDV (100 LD of O VET 2013) was administered intraperitoneally to mice₅₀ME-SA topology), then survival and weight changes were monitored until day 7 (days post challenge, dpc). In addition, to confirm the ability to induce cellular and humoral immune responses, mouse serum and Peritoneal Effusion Cells (PECs) were sampled at 0, 28 and 56dpv and then used for analysis, e.g., SP ELISA and virus neutralization titers (VN titers, neutralizing antibodies).

As a result of the experiment, as shown in FIG. 18A, at 28dpv, IFN α, IFN γ + IL-2+ TNF α, IL-15+ IL-18, TDB + c-di-GMP and R848+ c-di-GMP administered groups showed a significant increase in antibody titer as compared to the control group. Furthermore, at 56dpv, antibody titers were significantly higher for all cytokine and PRR ligand adjuvant administered groups. Particularly in the case of IFN α, antibody titers remained high up to 56 dpv. In the case of the IL-23 administration group, the cellular immune response was significantly increased in the previous experiment, and the antibody titer was also observed to be high, thereby indicating that the humoral immune response can be efficiently induced.

Meanwhile, even in the case of IFN γ + IL-2+ TNF α administration group involving T cell activity and T cell-mediated cellular and humoral immune responses, the antibody titer remained stably high after 28dpv, while the IL-15+ IL-18 administration group involved in mucosal immunity as an adjuvant showed high antibody titer. Furthermore, when HSP70, which is known to be involved in long-term immunity, was administered, a relative increase in antibody titer was observed to a higher level, although it was slightly lower than that of the other adjuvant administration group. In addition, in the case of the PRR ligand administration group, all groups showed high antibody titers.

To observe the body weight changes caused by the vaccination itself, body weight changes were monitored weekly over 8 weeks (56dpv) after vaccination (fig. 18B). Thus, the IFN γ + IL-2+ TNF α combination group lost slightly, but not significantly, body weight at 7 dpv. Furthermore, since the dose of the recombinant cytokine (total dose 15. mu.g) was slightly higher than that of the other groups (total dose 5 to 10. mu.g), this result was presumed.

On day 56 after vaccination (56dpv), the negative control mice died 100% on day 4, while the positive control mice showed a trend of 40% survival by observing survival (fig. 18C) and Body Weight (BW) changes (fig. 18D) after infection with O VET 2013. On the other hand, the survival rate of mice given PRR ligand and cytokine adjuvant group was 100%, but no weight loss was observed.

Therefore, when these cytokines and PRR ligands are used as adjuvants, it is expected that both the cellular immune response and the humoral immune response induced by the vaccine can be effectively induced, thereby significantly promoting host defense.

To observe the induction of cellular and humoral immune responses by cytokines and PRR ligands, the expansion of cells involved in these immune responses was analyzed by FACS. In FIG. 19A, CD3 was confirmed⁺CD4⁺T cells, and cell expansion was observed in the group to which IFN α, HSP70, R848+ TDB, TDB + c-di-GMP, etc. were added. FIG. 19B shows CD3⁺CD8⁺A population of T cells. And CD4⁺The total number of cells appeared to be lower compared to T cells, and IFN α treated groups showed significance at 28 dpv.

In FIG. 19C, CD4 was confirmed⁺CD8⁺Expansion of T cells. At 28dpv, the cell numbers of all cytokine and PRR ligand addition groups, except HSP70, increased significantly, whereas at 56dpv, there was a tendency for the cell numbers to decrease significantly. Due to the observation of CD44^{Height of}CD62^{Is low in}Expansion of T cells (termed markers for memory T cells) (fig. 19D), the population increased rapidly at 56dpv compared to 28 dpv. Furthermore, at 28dpv, PRR ligands induced memory T cell expansion more significantly than cytokines, while at 56dpv, the number of cells increased for all adjuvant-added groups. Thus, these adjuvants are expected to be effective in inducing memory T cells, thereby facilitating stimulation of cellular and humoral immune responses.

In FIG. 19E, CD44 was observed^{Height of}CD27^{Is low in}Expression of γ δ T cells (memory γ δ T cells). It is known that about 5% of γ δ T cells are present in mice, but the number of these cells increases by about 8% to 10% due to the addition of adjuvant, while no significant difference is observed between 28 and 56 dpv. Thus, it can be seen that the addition of an adjuvant is effective not only for inducing an initial cellular immune response, but also for inducing intermediate and long-term cellular immune responses.

FIG. 19F shows memory B cells (CD 3)^-CD44⁺CD27⁺B cells). IL-23, R848+ TDB, TDB + c-di-GMP showed a significant increase in cell number at 28dpv, and the cell number was generally increased at 56dpv as compared with 28dpv, and significant expansion was confirmed in all cytokine and PRR ligand-added group memory B cells. In particular, IL-23, IFN γ + IL-2+ TNF α, IL-15+ IL-18, R848+ TDB and R848+ c-di-GMP addition groups showed significance p<0.001 or less.

FIG. 19G shows confirmation of CD3 called memory-like NK cells^-CD335(NKp46)⁺CD27⁺Results of cell expansion. In particular, the IFN α administration group showed a significantly high cell number, and as a result, IFN α appeared to effectively stimulate NK cells involved in the innate immune response, and couldAchieves excellent effect in inducing memory response.

1-3. cytokine potential as an adjuvant and protection against FMDV infection of multiple serotypes in mice

To observe the potential of cytokines, particularly IFN α and IL-23, as vaccine adjuvants, memory responses, and defense against FMDV infection of various serotypes, animal experiments were performed as shown in figure 20.

To confirm the above effects, O VET (O VET 2013), O JC (O Jincheon 2014), AMalay (AMalay 97), asian Shamir-R (whole genome recombinant clone of asian 1 Shamir) and PC groups were injected with ISA206 oil immunopotentiator only, and NC was a negative control group.

As a result of the experiment, administration groups of IFN α and IL-23 as adjuvants showed almost complete defense against challenge with O VET2013, OJC, a Malay 97 and asia 1Shamir, while the weight change was hardly reduced compared to before virus challenge (fig. 21).

Therefore, the memory function of IFN alpha and IL-23 in vivo is confirmed, and it is expected that the above substances can effectively protect the host from FMDV attack of various serotypes regardless of the specificity between antigen viruses contained in the vaccine.

< example 2> evaluation of livestock breed reactivity to candidate adjuvant

Based on the above experimental results of mice, experiments were performed using the adjuvant compositions of table 2 below to observe PRR ligand, HSP70 and cytokine-mediated cell proliferation and cytotoxicity in bovine and porcine immune cells.

TABLE 2

Isolation of PBMC

Whole blood from pig and cattle is prepared by Animal diagnosis Laboratory in Gyeonggi PrDonation of ovince. 15ml of whole blood was collected using a BD-Vaccutaier heparin tube (BD, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and passed through Ficoll-Paque^TMPeripheral Blood Mononuclear Cells (PBMC) were collected by PLUS (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) density gradient centrifugation. The lysed residual erythrocytes were treated with ACK (ammonium potassium chloride) lysis buffer (Gibco, Carlsbad, CA, USA).

The solution was suspended in Ca-free Ca supplemented 2% FBS (Gibco, Carlsbad, Calif., USA)²⁺And Mg²⁺(Gibco, Carlsbad, Calif., USA) in Dulbecco's PBS and the number of cells counted by a volume flow meter (MACSQuant Analyzer, Miltenyi Biotec, Bergisch Gladbach, Germany). All cells were freshly isolated immediately prior to use, without the use of cryopreserved cells.

Purified PBMCs were resuspended in RPMI-1640(Gibco, Carlsbad, CA, USA; Louis, Missouri) supplemented with 10% Fetal Bovine Serum (FBS) (HyClone, Logan, Utah, USA), 3mM L-glutamine (Sigma-Aldrich, USA) and 100U/ml penicillin-streptomycin (Sigma-Aldrich, St. Louis, Mo., USA), then at 37 deg.C, 5% CO₂Culturing, then culturing at 1X 10 per well⁴Individual cells were attached to 96-well plates. After 3 hours of culture, the medium was replaced with serum-free medium and then stimulated with various PRR ligands and cytokines.

PRR ligand and cytokine treatment

Porcine and bovine PBMCs were treated with PRR ligands and cytokines as shown in table 2. After 96 hours, the cell culture medium (supernatant) was collected, and cell proliferation caused by secretion of Lactate Dehydrogenase (LDH) and incorporation of 5-bromo-2' -deoxyuridine (BrdU) was observed.

Measurement of PRR ligand and cytokine mediated LDH secretion in porcine and bovine PBMCs

The cytotoxicity levels in the treated porcine and bovine PBMC supernatants were as described above. LDH secretion assays were performed according to the manufacturer's protocol using the CytoTox 96 nonradioactive cytotoxicity assay (Promega).

The percentage LDH secretion was calculated as follows: percentage LDH secretion 100 × (absorbance reading of untreated control)/(absorbance of maximum LDH emission control-absorbance of no absorbance control)

Cells were lysed using lysis buffer in the kit and supernatants of cells were treated with lysis buffer to determine the maximum LDH release control.

BrdU incorporation assay in porcine and bovine PBMCs

The effect of PRR ligands and cytokines on porcine and bovine PBMC proliferation was evaluated using the BrdU Cell proliferation assay kit (Cell Signaling Technology, Beverly, MA, USA), which is based on the incorporation of BrdU during DNA synthesis.

mu.M BrdU was added to the cell culture broth and incubated at 37 ℃ for 4 hours. Cells were then fixed and incubated with anti-BrdU mouse monoclonal antibody and goat anti-mouse antibody conjugated to horseradish peroxidase (HRP). Color development was performed with the chromogenic substrate tetramethylbenzidine. The absorbance was measured at a dual wavelength of 450/550 nm.

Vaccination and sampling

This experiment was performed to observe the potential of PRR ligands as adjuvants, the induction of cellular and humoral immune responses, and long-term immune effects in pigs and cattle. The positive control group vaccine used O TWN97-R as the foot-and-mouth disease antigen consisted of: o TNW97-R Ag (15. mu.g/dose/ml), ISA206 (50%, w/w), 10% Al (OH)₃150 μ g/dose/ml Quil-A.

1ml of vaccine was administered twice every 28 days (0dpv, 28dpv) by the deep intramuscular route in the animal neck. Blood samples were taken from pigs at 0, 14, 28, 42, 56, 70, 84dpv and cattle at 0, 14, 28, 56, 84, 112, 140, 168dpv, respectively.

The temperature and appetite of the animals were monitored daily. Serum samples were stored at-80 ℃ until the test was performed.

Serological test

1) ELISA (enzyme-linked immunosorbent assay) for detecting structural protein antibody

To detect Structural Protein (SP) antibodies in serum, PrioCHECK FMDV type O (Prionics, switzerland) was used. The absorbance of the ELISA plate was converted to Percent Inhibition (PI) values. If the PI value is 50% or higher, the animal is considered antibody positive.

2) Virus neutralization assay

Virus neutralization assays were performed according to the OIE manual. The virus was activated by soaking in serum at 56 ℃ for 30 minutes. Serum samples were prepared at 2-fold dilutions from 1:4 to 1:512 by adjusting cell density to form a 70% monolayer. Diluted serum samples were incubated with 100-fold Tissue Culture Infectious Dose (TCID)50/0.5ml of homologous virus for 1 hour at 37 ℃. After 1 hour, LF-BK (bovine kidney) cell suspension was added to all wells. After 2 to 3 days, CPE was measured to neutralize 100TCID by log 10 of titer antibody dilution₅₀The titer of the virus of (a).

2-1. evaluation of reactivity of bovine candidate adjuvant

As a result of confirming secretion of LDH (lactate dehydrogenase) to observe cytotoxicity of PRR ligand, cytokine, gel, saponin, and the like to bovine-derived PBMC, respectively, it was found that the cytotoxicity of the treated group itself was low. Therefore, there was no toxicity at the adjuvant concentration used in this experiment (fig. 22 (a)). Figure 22(B) shows the results of observing cytotoxicity after treatment of bovine PBMCs with PRR ligand and cytokine (mixed with vaccine adjuvant oil + gel + saponin), respectively. Even at this time, cytotoxicity caused by the adjuvant mixture was not observed as compared with the control group.

On the other hand, cytotoxicity of the adjuvant was not observed even after combined treatment with PRR ligand (fig. 22 (C)). Furthermore, even if the PRR ligand was combined, mixed with vaccine adjuvant oil + gel + saponin, and then used for treatment, no cytotoxicity was observed (fig. 22 (D)).

Figure 23(a) shows the results of observing cell proliferation by BrdU incorporation after 96 hours of treatment of bovine PBMC with PRR ligand and cytokines, oil, gel, saponin, and oil + gel + saponin, respectively. Cell proliferation was increased in all PRR ligand treated groups compared to the control group, and in particular the effects of Curdlan, TDB, c-di-GMP, R848 and Furfarnann were found to be large.

On the other hand, the effect of gel and saponin was not significant at 96 hours. In addition, after each PRR ligand and cytokine was mixed with oil + gel + saponin and treated with it, cell proliferation was observed. As a result of the experiment, cell proliferation was increased in all PRR ligands mixed with oil + gel + saponin compared to the control group, but the level of cell proliferation was lower compared to the case where oil + gel + saponin (W/O) was not added (fig. 23 (B)).

Fig. 23(C) shows the results of observing cell proliferation by combining PRR ligands. Cell proliferation was increased in all treated groups compared to the control group, especially the R848+ TDB, Furfurman + TDB and TDB + c-di-GMP treated groups showed higher values. From this result, these PRR ligand combinations appear to be effective in stimulating bovine PBMCs, thereby enhancing cell proliferation and stimulating immune responses. Furthermore, these adjuvants elicit significantly higher immune responses in bovine PBMC than in porcine PBMC, and thus, bovine immune cells appear to respond more sensitively than porcine immune cells.

Meanwhile, as a result of observing cell proliferation 96 hours after treating bovine-derived PBMC with a mixture of PRR ligand and oil + gel + saponin combination, the cell proliferation levels of all PRR ligand and oil + gel + saponin mixture-treated groups were increased compared to the control group. However, the above level was lower than without oil + gel + saponin (W/O) (fig. 23 (D)). From the above results, which are substantially similar to those of the swine immune cells, it is considered that when bovine immune cells are directly treated with PRR ligands, the cells are stimulated more rapidly than when PRR ligands and oil + saponin + gel treatment are used, thereby promoting cell proliferation and initiating an immune response.

For candidate substances that showed significant results in PRR ligand screening experiments using bovine PBMC, animal experiments were performed using the strategy shown in fig. 24 to observe PRR ligand-mediated adjuvant effects and memory responses in actual field cattle. In field trials, 5 month old cattle (FMD antibody seronegative) were used and were divided into 3 groups (n-5/group). Unvaccinated animals were used as negative controls and cattle were isolated in a closed space during the experiment.

As a result of confirmation of the Ab titer (antibody titer) by SP O ELISA, the antibody titer of the PRR ligand-added group at 28 days after inoculation (28dpv) was significantly higher than that of the control group. In addition, the level remained high for 140 days (140dpv) after the enhancement (fig. 25 (a)). Furthermore, as a result of confirming VN titer (virus neutralization titer), the PRR ligand-added group showed a significantly higher value than the 14dpv control group (p <0.01), and maintained a very high level (p <0.001) to 140dpv from then on (fig. 25 (B)).

From the above results, when R848+ TDB, Curdlan + c-di-GMP, etc. are added as vaccine adjuvants, the natural and cellular immune responses are effectively stimulated, thereby continuing to stimulate the humoral immune response, and thus being expected to contribute to host defense against FMDV infection.

2-2 evaluation of candidate adjuvants in pigs for reactivity and safety

Secretion of LDH (lactate dehydrogenase) was confirmed in order to observe the cytotoxicity of PRR ligand and cytokines, oil, gel, saponin and oil + gel + saponin, respectively, on porcine PBMC. Therefore, the treatment group itself was found to be less cytotoxic and not cytotoxic at the adjuvant concentration used in this experiment (fig. 26 (a)).

Figure 26(B) shows the results of observing cytotoxicity after treatment of porcine PBMCs with PRR ligands and a mixture of cytokines and oil + gel + saponin as vaccine adjuvants. Even at this time, no cytotoxicity of the adjuvant mixture was observed compared to the control group.

On the other hand, even though cytotoxicity was confirmed after treatment of porcine PBMCs with the combined PRR ligand, cytotoxicity of these adjuvants was not observed (fig. 26 (C)). Furthermore, a similar trend was shown even after treatment of porcine PBMCs with a mixture combining PRR ligand and vaccine adjuvants (i.e. oil + gel + saponin) (fig. 26 (D)).

FIG. 27(A) shows that as a result of observing cell proliferation by incorporation of BrdU in porcine PBMC 96 hours after treating porcine PBMC with PRR ligand and cytokine, oil, gel, saponin and oil + gel + saponin, respectively, cell proliferation was increased in all PRR ligand-treated groups compared to the control group, especially the effects of TDB and c-di-GMP were maximized. On the other hand, the effect of gel and saponin was not significant at 96 hours.

As a result of observing cell proliferation 96 hours after treating porcine PBMC with a mixture of PRR ligand and cytokine and oil + gel + saponin, cell proliferation increased in all PRR ligand and oil + gel + saponin mixture treated groups compared to the control group, however, at a level lower than that without adding oil + gel + saponin (W/O) (fig. 27 (B)). Fig. 27(C) shows the results of observing cell proliferation by PRR ligand combination. Cell proliferation was increased in all treated groups compared to the control group, in particular the R848+ TDB, Furfurman + TDB and TDB + c-di-GMP treated groups showed higher values. From these results, it is believed that the combination of these PRR ligands is effective in stimulating porcine PBMCs, thereby enhancing cell proliferation and stimulating immune responses. On the other hand, as a result of observing cell proliferation 96 hours after treating porcine PBMC with a mixture combining PRR ligand and oil + gel + saponin, the cell proliferation levels were increased in all PRR ligand and oil + gel + saponin mixture-treated groups compared to the control group, but were lower than those in the case where oil + gel + saponin (W/O) was not added (fig. 27 (D)). From the above results, when the porcine immune cells were treated with PRR ligand alone without any oil adjuvant (oil emulsion), the cells could be stimulated more rapidly than when treated with PRR ligand and oil + saponin + gel, thereby promoting cell spreading and initiating immune response.

For candidate substances that showed significant results in PRR ligand screening experiments using porcine PBMCs, the following animal experiments were performed to observe PRR ligand-mediated adjuvant effects and memory responses in live pigs (fig. 28). For the field trial, 10-week-old pigs (FMD antibody seronegative) were used and divided into 4 groups (n-5/group). Unvaccinated animals were used as negative controls and pigs were isolated in a closed space during the study.

To confirm the antibody titers generated by vaccination, SP OELISA was performed using pig sera. Therefore, as shown in FIG. 29(A), the TDB + c-di-GMP-administered group showed a significant increase in antibody titer at 14dpv (p <0.001) compared to the control group. Furthermore, at 28dpv, antibody titers were significantly increased for all PRR ligand-administered groups compared to the control group (p < 0.001). In particular, in the case of the TDB + c-di-GMP-added group, the antibody titer was found to be significantly higher than that of the control group, and was continuously maintained up to 84 dpv. The high antibody titers of the primary vaccination of pigs are explained by the efficient activation of innate and cellular immune responses by PRR ligands and by the strong induction of humoral immune responses.

In addition, it is believed that a second vaccination (boost) of 28dpv may effectively induce "recall stimulation" of immune cells stimulated by the first vaccination, while maintaining long-term immunity. In addition, as a result of confirming the VN titer, the VN titer at 14dpv was significantly higher in the TDB + c-di-GMP (p <0.001), R848+ TDB (p <0.01) added group than in the control group. Furthermore, VN titres at 28dpv were significantly higher for all PRR added groups than for the control group (p < 0.001). In particular, the TDB + c-di-GMP-added group maintained high VN titers up to 84dpv, while the R848+ TDB-added group showed good immunopotentiation effects from early (14dpv) to mid (42 dpv). However, the above-described effect shows a slightly decreasing tendency thereafter.

Furthermore, in the case of the furfurfurfurrman + TDB-added group, VN titres increased slowly until the initial 14dpv, but VN titres were significantly higher from 28 to 84dpv than the control group.

The PRR ligand + additive group showed significantly higher levels than the control group starting at 14dpv, even up to 84dpv, and remained at significantly higher levels compared to the control group (fig. 29 (B)). From the above results, it is expected that when R848+ TDB, furfurfurfurmann + TDB and TDB + c-di-GMP are added as adjuvants to a vaccine, the vaccine can effectively stimulate innate and cellular immune responses and continue to stimulate humoral immune responses, thereby significantly promoting host defense against FMDV infection. Furthermore, it is contemplated that the present invention may be used in the development of FMDV vaccines according to new strategies.