Novel immune enhancing agent and vaccine composition containing the same

Abstract

To provide a novel immune enhancer composition which induces a strong cellular immune response by direct activation of unconventional T cells and conventional T cells in bovine and pig immune cells, and a vaccine composition including the immune enhancer composition.SOLUTION: An immune enhancer composition comprises α-Galactosyl ceramide, which is an iNKT cell agonist, as an active ingredient.SELECTED DRAWING: Figure 8b

Description

[Technical Field]

[0001] The present invention relates to a novel immunopotentiator, more specifically to a novel immunopotentiator comprising an atypical T cell agonist and a typical T cell agonist, and a vaccine composition comprising the same. [Background technology]

[0002] Foot-and-mouth disease (FMD) is an acute, rapidly transmitted, infectious disease in cloven-hoofed animals, particularly cattle and pigs, causing severe animal productivity losses and economic losses. Most clinical research on FMD has focused on improving vaccine efficacy in cattle rather than pigs. The OIE (Office International Epizooties) guidelines for FMD vaccine production only define efficacy testing procedures for cattle, not pigs. However, the severity of FMD depends heavily on both the virus strain and the affected host species. Acute clinical FMD is more severe in pigs than other ruminants, and infected pigs can shed large amounts of aerosol virus, increasing the risk of severe disease transmission.

[0003] Recent vaccination trends in Korea have shown that FMD vaccines effectively induce immune responses in cattle, but in pigs, despite high virus neutralization (VN) titers in serum, they do not induce sufficient immune responses for complete protection against FMD virus infection. Such differences can be explained by the susceptibility of different livestock species to the FMD virus or the host's immune response to vaccination.

[0004] Vaccination containing inactivated foot-and-mouth disease virus (FMDV), i.e., inactivated antigen, is primarily used for host defense and FMD control. To improve vaccine immunogenicity and efficacy, adjuvants (auxiliary agents) such as oil emulsions and immunostimulants such as saponins and gels are added as vaccine components. Recently, cytokines (e.g., IL-15, IL-18, and IFNα) and pattern recognition receptor (PRR) ligands, including poly(I:C) as a Toll-like receptor (TLR)-3 ​​agonist, CpG as a TLR-9 agonist, and R848 (Resquimod) as a TLR-7 / 8 agonist, have been proposed as new FMD vaccine adjuvants. However, these studies have been conducted at a basic level, and little mechanistic research has been conducted to further understand the cellular and humoral immune responses mediated by FMDV antigens or FMD vaccines.

[0005] The objective of the present invention is to use, as a vaccine adjuvant, unconventional T cells, including gamma delta (γδ) T cells, invariant natural killer (iNK) T cells, and mucosal-associated invariant T cells (MAIT), which have been proposed as "new guardians" in host defense by acting as a linker between innate and humoral immune responses, and agonists of conventional T cells, such as T cells, to directly stimulate T cells without stimulating antigen-presenting cells (APCs), such as dendritic cells (DCs), macrophages (MΦs), and monocytes, thereby inducing a faster and stronger cellular immune response, which plays an important role in the initial defense of the host upon vaccination, and simultaneously stimulates both cellular and humoral immune responses, thereby producing a strong memory response in animals, particularly pigs. The present invention aims to develop an immune enhancing adjuvant that induces a response and antibody titer, and a foot-and-mouth disease vaccine composition containing the same. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lee, MJ et al. Mincle and STING-stimulating adjuvants elicit robust cellular immunity and drive long-lasting memory responses in a foot-and-mouth disease vaccine. Front. Immunol. 10, 2509 (2019). Summary of the Invention [Problem to be solved by the invention]

[0007] In order to solve the above-mentioned problems, the objective of the present invention is to provide a novel immune enhancing composition that induces a strong cellular immune response by directly activating atypical T cells and typical T cells in immune cells of cattle and pigs.

[0008] Another object of the present invention is to provide a vaccine composition comprising the immunopotentiator composition. [Means for solving the problem]

[0009] To achieve the above object, the present invention provides a novel immunopotentiator composition that induces a strong cellular immune response in immune cells of cattle and pigs.

[0010] The novel immune enhancing agent contains an unconventional T cell agonist and a conventional T cell agonist as active ingredients.

[0011] The term "immunostimulant" or "adjuvant" as used herein refers to a substance that enhances the immune response induced by an antigen. Such an immunostimulant can achieve the same efficacy with a smaller amount of antigen in a vaccine, allowing vaccines to be made using only half to one-third of the amount of antigen required in conventional vaccines.

[0012] Exemplary T cell agonists in the present invention include RORγt (RAR-related orphan receptor gamma t).

[0013] The atypical T cell agonist of the present invention refers to a T cell agonist excluding the known typical T cell agonists as described above. The atypical T cell agonist may be, but is not limited to, a γδ T cell agonist, an iNKT cell agonist, or a MAIT cell agonist.

[0014] The atypical T cell agonist or typical T cell agonist may be contained in the immunopotentiator composition at 0.01 to 1 wt %, preferably 0.1 to 0.5 wt %, more preferably 0.2 to 0.3 wt %. If the amount is less than this range, the immunopotentiating effect may not be exhibited, and if the amount is more than this range, toxicity may be induced.

[0015] The immune enhancing agent or immune enhancing agent composition of the present invention may further contain oil (or oil emulsion), emulsifier, gel, etc., which are well known in the art, in addition to the above ingredients.

[0016] The oil (or oil emulsion) can be, but is not limited to, ISA201, ISA61, ISA50, ISA206, or ISA207.

[0017] The emulsifier may include, but is not limited to, materials generally recognized as emulsifiers, such as other products in the TWEEN® or SPAN® product lines (polyethoxylated sorbitol fatty acid esters and fatty acid-substituted sorbitan surfactants, respectively), and other solubility enhancers such as PEG-40 castor oil or other PEGylated hydrogenated oils.

[0018] Furthermore, although an immunopotentiator (or adjuvant) composition generally has excellent immunopotentiating effects when in an oil formulation, the immunopotentiator composition of the present invention has excellent immunopotentiating effects even when in a non-oil formulation.

[0019] The immune enhancing agent may further contain additives, excipients, carriers, etc. commonly used in the art for preparing the immune enhancing agent, and may be prepared by a conventional preparation method used in the art for preparing immune enhancing agents.

[0020] The immune enhancing effect observed after administration of the immune enhancing composition of the present invention may be due to immune mediators or cells, and specifically may include, but is not limited to, cellular immunity, mucosal immunity, and humoral immunity enhancing effects.

[0021] The immunity may include, but is not limited to, immunity against infection with any one of the pathogens selected from viruses, fungi, bacteria, and parasites, or immunity against cancer.

[0022] The viruses include foot-and-mouth disease virus (FMDV), Leishmania, human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis E virus (HEV), hepatitis A virus (HAV), hepatitis B virus (HBV), tuberculosis, herpes simplex virus (HSV), malaria causing parasites, human papilloma virus (HPV), influenza virus, measles virus, mumps virus, Ebola virus, respiratory syncytial virus (RSV), and West Nile virus. virus, WNV), etc.

[0023] The present invention also provides a vaccine composition comprising the immunopotentiator composition.

[0024] The immune enhancer composition may be present in an amount of 30 to 70 wt %, preferably 40 to 50 wt %, based on the total weight of the vaccine composition, but is not limited thereto. If the amount is less than this range, the vaccine effect may not be exhibited, and if the amount is more than this range, toxicity may occur upon administration.

[0025] The vaccine composition may further include additives, excipients, carriers, etc. that are commonly used in the art for producing vaccine compositions.

[0026] The vaccine composition can be prepared by a conventional method used in the art for preparing vaccine compositions.

[0027] In the present invention, the type of the vaccine is not limited, but is preferably a virus vaccine, more preferably a foot-and-mouth disease vaccine.

[0028] When the immune enhancer according to the present invention is included in a foot-and-mouth disease vaccine composition, the foot-and-mouth disease vaccine composition can exert an immune effect against foot-and-mouth disease viruses of serotype O, serotype A, serotype Asia1, serotype C, serotype SAT1, serotype SAT2, serotype SAT3, etc. Preferably, the composition can exert a stronger immune effect against foot-and-mouth disease viruses of serotype O and serotype A.

[0029] The vaccine composition can be administered by sublingual, transdermal, rectal, transmucosal, topical, oral, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intraspinal, or intraarticular administration. [Effects of the Invention]

[0030] The present invention provides a novel immune enhancer capable of enhancing the innate cellular immune response of cattle and pigs and more efficiently improving the humoral immune response, and a vaccine composition containing the same. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 shows the results of proliferation of PBMCs, lymphocytes, monocytes, and T cells in cattle and pigs induced by inactivated FMDV O / TWN / 97 antigen. [Figure 2] 2a to 2d show the results of expression of inflammatory cytokines in porcine immune cells and bovine immune cells mediated by FMDV (O / TWN / 97-R) antigen. [Figure 3] FIG. 3 shows the results of the expression of inflammatory cytokines in porcine and bovine immune cells mediated by FMDV (O / TWN / 97-R) antigen. [Figure 4]FIG. 4 shows that FMDV antigens directly stimulate cytokine expression in porcine Mo-DCs and Mo-MΦs. [Figure 5] 5a and 5b show that FMDV antigens are endocytosed by porcine DCs and MΦs through phagocytosis, initiating cellular immunity. [Figure 6] Figure 6a shows the experimental process for investigating the differences in immune responses between cows and pigs, and Figures 6b and 6c show the results of abnormally overexpressed innate immune responses in pigs and the induction of T cell exhaustion pathways by FMD vaccination. [Figure 7] Figure 7a shows the experimental process of Experimental Example 1, and Figures 7b to 7d show the results confirming that atypical T cell agonists and typical T cell agonists induce early, intermediate, and long-term immunity in mice. [Figure 8] FIG. 8a shows the experimental process of Experimental Example 2, and FIG. 8b shows the results of cell proliferation in T cell agonist-induced porcine PBMCs. [Figure 9] Figures 9a to 9e show the results of Experimental Example 3, showing the improvement of abnormal innate immune responses in pigs and the activation of immune cells, including T cells, by atypical T cell agonists and typical T cell agonists. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention will be described in detail below with reference to examples and experimental examples. However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

[0033] <Experimental Materials and Methods> 1. Antigen purification and inactivation Purified inactivated viral antigen was produced in BHK-21 cells infected with FMDV O / TWN / 97-R (GenBank AY593823; P1), which was constructed for phenotypic switching of P1 (reference sequence) by reverse genetic techniques.

[0034] For viral infection, the culture medium was switched to serum-free Dulbecco's modified Eagle's medium (DMEM; HyClone, Logan, UT, USA), and the cells were inoculated with virus by incubating at 37°C and 5% carbon dioxide for 1 hour. Extracellular virus was then removed. At 24 hours postinfection, the virus was inactivated by two treatments with 0.003 N binary ethyleneimine for 24 hours in a shaking incubator, followed by concentration in polyethylene glycol (PEG) 6000 (Sigma-Aldrich, St. Louis, MO, USA). The resulting virus concentrate was layered on a 15-45% sucrose density gradient and centrifuged.

[0035] After ultracentrifugation, 1 mL fractions were collected by puncturing the bottom of the centrifuge tube. The presence of FMDV particles in each fraction was confirmed based on optical density using a lateral flow device, UA-6 (BioSign FMDV Ag; Princeton BioMeditech, Princeton, NJ, USA). Prior to use in field experiments, the absence of live virus in the PEG-pretreated supernatant was confirmed by passing it at least twice through ZZ-R127 and BHK-21 cells to confirm the absence of cytopathic effect (CPE).

[0036] 2. Isolation of PBMCs To investigate antigen-mediated immune responses, whole blood from pigs and cattle was donated by the Gyeonggi-do Animal Health Research Institute, Korea. Whole blood (15 mL) was collected in BD Vacutainer heparin tubes (BD Biosciences, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and immobilized on Ficoll-Paque membrane. TMThe gradient was centrifuged with PLUS (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA), and residual red blood cells were lysed by treatment with ammonium chloride potassium (ACK) lysis buffer (Gibco, Carlsbad, CA, USA). PBMCs were then cultured in CaCl2+ medium supplemented with 2% fetal bovine serum (FBS) (Gibco). 2+ and Mg 2+ The purified PBMCs were suspended in Dulbecco's PBS without PBS (Gibco) and counted using a flow cytometer (MACSQuant® Analyzer, Miltenyi Biotec, Bergisch Gladbach, Germany). All cells were freshly isolated immediately before use, and cryopreserved cells were not used in any experiments. The purified PBMCs were resuspended in RPMI 1640 (Gibco) medium supplemented with 10% FBS (HyClone, Logan, Utah, USA), 3 mM L-glutamine (Sigma-Aldrich), and 100 U / mL penicillin-streptomycin (Sigma-Aldrich). The purified PBMCs were then transferred to a 25 cm 2 Freshly isolated monocytes were then plated into tissue culture flasks (Eppendorf, Hamburg, Germany) and cultured at 37°C in a 5% CO2 atmosphere to allow for attachment. After 3 hours of culture, nonadherent cells were collected for lymphocyte isolation. The remaining adherent cells were washed extensively with Dulbecco's phosphate-buffered saline (DPBS) (Gibco) before adding 4 mL of RPMI 1640 growth medium to each flask and then cultured at 37°C in a 5% CO2 incubator.

[0037] 3. Cell Separation by Magnetic Activated Cell Sorting (MACS) Monocytes, lymphocytes, and T cells were isolated from PBMCs. For the isolation of primary immune cells, monocytes were purified from adherent PBMCs and T cells from non-adherent cells by MACS. Adherent and non-adherent cells obtained from PBMCs were briefly resuspended in MACS buffer (1x PBS supplemented with 0.5% BSA and 2mM EDTA). Monocytes and T cells were isolated using a Monocyte Isolation Kit and a PanT Cell Isolation Kit (Miltenyi Biotec) with magnetic microbeads, respectively, according to the manufacturer's instructions, and further sorted using a fluorescence-activated cell sorter (FACS, MoFlo® Astrios). TM Further isolation was performed using a Cell Sorter (Beckman Coulter, Brea, CA, USA). The purity of the isolated cells was confirmed by a flow cytometer (MACSQuant Analyzer, Miltenyi Biotec) and analyzed using FlowJo software version vX.0.7 (TreeStar Inc., Ashland, OR, USA). The purity of the isolated cells was greater than 95%.

[0038] 4. Generation of Mo-DCs and Mo-MΦs For differentiation of Mo-DCs, isolated monocytes were cultured for 7 days in complete RPMI 1640 medium (Gibco) supplemented with 10% FBS (HyClone), 3 mM L-glutamine (Sigma-Aldrich), 100 U / mL penicillin-streptomycin (Sigma-Aldrich), 50 ng / mL GM-CSF, and IL-4 (Miltenyi Biotec) to generate immature DCs. On day 3, the same volume of the aforementioned medium was added, and on day 7, nonadherent contaminating cells were removed by vigorous washing before cell lysis. For isolation of pure DCs, anti-CD11c magnetic beads were used according to the manufacturer's instructions (Miltenyi Biotec). CD11c + / MHCII + The purity of the cells was over 95%, and the cells were maintained in a 5% CO incubator at 37°C.

[0039] For differentiation of Mo-MΦs, isolated monocytes were cultured at 106 The cells were aliquoted into 12-well plates at 100 cells / mL and cultured for 6 days in RPMI 1640 medium (Gibco) supplemented with 10% FBS (HyClone), 1x MEM non-essential amino acids, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol (Sigma-Aldrich), 100 U / mL penicillin-streptomycin (Sigma-Aldrich), and 50 ng / mL macrophage colony-stimulating factor (M-CSF) (Abcam, Cambridge, MA, USA). Additional M-CSF was added on day 2, and all medium was replaced fresh on day 4. + / F4 / 80 + The purity of the cells was over 95%. The cells were maintained in a 5% CO2 incubator at 37°C.

[0040] 5. Preparation of Mature DCs Mature DCs were generated with one of six treatments: no treatment (unstimulated), LPS (E. coli 055:B5, Sigma-Aldrich [100 ng / mL]) plus rpIFN-γ (Novus Biologicals, LLC., Littleton, CO, USA [20 ng / mL]), rpTNFα (R&D Systems, Minneapolis, MN, USA [20 ng / mL]), antigen alone (antigen [1 μg / mL]), LPS plus rpIFNγ plus antigen (LPS [100 ng / mL], rpIFN-γ [20 ng / mL], and antigen [1 μg / mL]), or rpTNFα plus antigen (rpTNFα [20 ng / mL] and antigen [1 μg / mL]). Cell culture supernatants were collected for ELISA at specific time points (0, 6, 12, 24, 48, 72, and 96 h) after treatment.

[0041] 6. Polarization of M1 / M2MΦs After 7 days of growth, Mo-MΦs were treated with one of six treatments: no treatment (unstimulated), M1MΦs (LPS [100 ng / mL] and rpIFN-γ [20 ng / mL]), M2MΦs (rpIL-4, R&D Systems [20 ng / mL]), antigen alone (1 μg / mL), M1MΦs + antigen (IFN-γ [20 ng / mL], LPS [100 ng / mL], and antigen [1 μg / mL]), or M2MΦs + antigen (IL-4 [20 ng / mL] + antigen [1 μg / mL]). At specific time points (0, 6, 12, 24, 48, 72, and 96 h) posttreatment, cell culture supernatants were collected for ELISA.

[0042] 7. Integrated Analysis of Cell Culture, Antigen Treatment, and BrdU in Bovine and Porcine PBMCs Dissociated or differentiated cells (1 × 10 6 Cells (cells / well) were cultured in complete medium consisting of RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (HyClone), 3 mM L-glutamine (Sigma-Aldrich), 10 mM HEPES, 100 U / mL penicillin-streptomycin (Sigma-Aldrich), and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich) in a 5% CO2, 37°C incubator (Sigma-Aldrich). For stimulation, cells were treated with 1 μg of each antigen. At specific time points after treatment (0, 6, 12, 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 h), cell proliferation, based on BrdU incorporation during DNA synthesis, was assayed using a BrdU Cell Proliferation Assay Kit (Cell Signaling Technology, MA, USA) according to the manufacturer's instructions. Briefly, 10 μM BrdU was added to the cell culture medium and incubated at 37°C for 4 hours. The cells were then fixed and incubated with anti-BrdU mouse monoclonal antibody, followed by treatment with horseradish peroxidase-conjugated goat anti-mouse antibody. The color development was performed using tetramethylbenzidine, a colorimetric substrate. The absorbance was measured at two wavelengths: 450 and 550 nm. Cell Titer-Blue TMCell viability was monitored using an assay kit (Promega, Madison, WI, USA). Compared to the medium-only control treatment, the experimental treatments did not affect cell viability.

[0043] 8.ELISA ELISAs for bovine and porcine IL-1β, IL-6, IL-10, IL-12 / 23p40, IL-23, and TNFα (DuoSet, R&D Systems, Minneapolis, MN, USA; Cloud-Clone Corporation, Houston, USA) were performed using cell culture supernatants according to the manufacturer's instructions.

[0044] 9. Inhibition of phagocytosis To inhibit phagocytosis, Mo-DCs and Mo-MΦs were incubated with 5 μg / mL cytochalasin D (CytD) (Sigma-Aldrich) for 45 minutes before antigen treatment. CytD-treated Mo-DCs and Mo-MΦs were then cultured with 1 μg / mL antigen. After 6 hours, culture supernatants were collected for ELISA.

[0045] 10. Cows and Pigs To understand the fundamental differences in immune responses between cattle and pigs under natural conditions, as well as the immune responses mediated by FMDV O antigen and related mechanisms, field experiments using cattle and pigs were conducted using the method described by Lee et al. FMD antibody-negative animals were used (cattle 5 months of age, pigs 10–12 weeks of age). Cattle and pigs were divided into two groups (n = 5 / group). Animals were maintained in isolation throughout the study. The study was approved by the Animal Experiment Ethics Committee of the Agriculture, Forestry and Fisheries Quarantine Agency (approval numbers IACUC-2018-800 and IACUC-2019-185) and conducted in accordance with the institution's guidelines.

[0046] 11. Vaccination and Sampling O / TWN / 97-R Ag was used as the FMD antigen. The vaccine composition for the positive control group was as follows: 1 mL of vaccine was prepared as a single dose containing 15 μg of O / TNW / 97-R antigen (single dose for use in cattle and pigs), ISA206 (50%, w / w), 10% Al(OH)3, and 150 μg Quil-A. Whole blood was collected from cattle and pigs in the naive control group (FMD antibody-negative) for naive PBMC isolation. Vaccinations were administered twice, 28 days apart, with 1 mL of vaccine (single dose) injected intramuscularly into the neck of the positive control group. Blood samples were collected from cattle and pigs at 0, 14, 28, 42, 56, 70, and 84 days post-vaccination (dpv) for serological analysis, and at 28 days post-vaccination (dpv) for PBMC isolation. The animals were monitored daily for body temperature, symptoms at the vaccination site and appetite. Serum samples were stored at -80°C until testing was performed.

[0047] 12. RNA sequence analysis (RNA-Seq) For RNA-Seq analysis, PBMCs from whole blood of cattle and pigs from the naive control group (n=3 / group) and the positive control group (n=3 / group) (28 dpv) were collected using Ficoll-Paque membranes according to the method described in Lee, MJ et al. Mincle and STING-Stimulating Adjuvants Elicit Robust Cellular Immunity and Drive Long-Lasting Memory Responses in a Foot-and-Mouth Disease Vaccine. Front Immunol, 2509 (2019) (hereafter referred to as Lee et al.). TM The mixture was centrifuged by density gradient centrifugation using PLUS (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).

[0048] 13. Library Construction and Sequencing To obtain high-throughput transcriptome data from cattle and pigs, Illumina next-generation sequencing (NGS) was performed. Total RNA was extracted from cattle and pig PBMCs using TRIzol Reagent (Invitrogen) and the RNeasy Mini Kit (QIAGEN), respectively, according to the manufacturer's protocol. Total RNA was then quantified using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, USA), and its quality was assessed using an RNA6000 Nano Analysis Kit (Agilent Technologies, Santa Clara, USA) and a Bioanalyzer 2100 (Agilent). NGS sequencing libraries were generated from 1 μg of total RNA using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer's protocol. Poly(A)-containing RNA molecules were purified using poly(T) oligo-conjugated magnetic beads. After purification, total poly(A)+ RNA was fragmented into small pieces using divalent cations at high temperature. The cleaved mRNA fragments were reverse transcribed into single-stranded cDNA using random primers. The short fragments were purified with a QiaQuick PCR extraction kit and digested into elution buffer for final recovery and poly(A) addition. The short fragments were then ligated with a sequencing adapter. Each library was separated with a distinct MID tag flanking it. The resulting cDNA libraries were then sequenced using NovaSeq. TM Samples were subjected to paired-end sequencing (2 × 101 bp) using a 6000 system (Illumina).

[0049] 14. Gene Expression Analysis Low-quality bases (PHERD score (Q) < 20) and adapter contamination were removed using Trimmomatic v.0.36 with the following parameters: ILLUMINACLIP: TruSeq3-SE: 2:30:10 LEADING: 3 SLIDINGWINDOW: 4:15 MINLEN: 36. After quality score confirmation and read length confirmation, RNA-Seq reads were mapped to the reference Bostaurus genome (published April 2018; ARS-UCD1.2; GCA_002263795.2) using expectation maximization (RSEM, RNA-Seq) with STAR as the base parameter. Expression was estimated by the expectation maximization method to obtain expression values ​​for each gene / transcript from the genome. Read counts estimated by RSEM were applied to edgeR v3.22.5 to obtain differential expression scores along with statistical significance. Additionally, filters were applied to select differentially expressed transcripts: TPM (transcripts per million) ≥ 0.3, number of reads ≥ 5, and log2 fold change ≥ 1. Finally, expressed transcripts (i.e., TPM ≥ 0.3 and number of reads ≥ 5) were analyzed to show their expression patterns in each condition and gene family (immune genes, T cell markers, and TLR, CDS, and CLR signaling pathway genes).

[0050] 15. Ingenuity Pathway Analysis (IPA) The expression profiles were then classified into clusters of similar expression patterns. We then analyzed enriched pathways, networks, and functions using IPA (QIAGEN Inc., https: / / www.qiagenbioinformatics.com / products / ingenuity-pathway-analysis). Finally, we used in-house Rscript to generate a binary heatmap showing all genes involved in significant pathways.

[0051] <Evaluation of the effect of atypical T cell agonists as potent vaccine adjuvants and experimental methods> 1. Mouse Age- and sex-matched wild-type C57BL / 6 mice (6–7 weeks old, female) were purchased from KOSA BIO Inc. (Gyeonggi-do, Korea). All mice were housed in microisolated cages in the Animal Biosafety Level 3 (ABSL3) specific pathogen-free (SPF) animal facility at the Animal and Plant Quarantine Agency. The study was conducted in accordance with institutional guidelines and with approval from the Animal Experiment Ethics Committee of the Animal and Plant Quarantine Agency (approval numbers IACUC-2018-800 and IACUC-2019-185).

[0052] 2. Adjunctive and host defense mediated by atypical and typical T cell agonists To evaluate the potential of atypical and typical T cell agonists as FMD vaccine adjuvants to simultaneously induce potent cellular and humoral immune responses and to examine their protective effects against FMDV infection, we conducted experiments using the proposed strategy (Fig. 7a) (n = 10 per group). O / TWN / 97-R antigen was used as an inactivated FMDV antigen. The vaccine composition for the PC group was as follows: O / TWN / 97-R antigen (15 μg / dose / mL, 1 / 10 the dose for use in cattle and pigs), 10% Al(OH)3, and 15 μg / mouse of Quil-A in a total volume of 100 μL. All mice in the experimental group were given a vaccine with the same composition as the PC group, with an atypical T cell agonist or a typical T cell agonist added as an adjuvant (immune enhancer) at approximately 0.2 wt% of the total weight of the immune enhancer composition and approximately 0.1 wt% of the total weight of the vaccine.

[0053] The atypical and typical T cell agonists used in these experiments were purchased from Sigma-Aldrich (γδ T cell agonist; isopentenyl pyrophosphate trilithium salt, IPP (I), (E)-1-Hydroxy-2-methyl-2-butenyl 4-pyrophosphate lithium salt, HMP (H)), Abcam (iNKT cell agonist; α-Galactosyl ceramide, α-Galcer (G), T cell agonist (RORγT (R))), and Cayman (MAIT cell agonist; 6-Formylpterin (F), Cayman Chemical, Ann Arbor, MI, USA), respectively.

[0054] Mice in the negative control group received the same volume of phosphate-buffered saline (PBS, pH 7.0) via the same route. Briefly, vaccinations were administered twice, 35 days apart, via intramuscular injection into the thigh muscle of mice. After vaccination, mice were challenged intraperitoneally with FMDV (100 LD50 of O / VET / 2013, ME-SA topotype) at 84 or 168 dpv. Survival and body weight of mice were monitored up to 7 days postchallenge (dpc). Sera collected from mice at 0, 7, 14, 28, 56, 84, and 168 dpv were analyzed by structural protein A enzyme-linked immunosorbent assay (SPA) and virus neutralization (VN) titers to assess the induced cellular and humoral immune responses.

[0055] 3. Serological analysis To detect SP antibodies in serum, we used the PrioCHECK FMDV type O ELISA kit (Prionics AG, Switzerland) as described by Lee et al. The absorbance on the ELISA plate was converted into a percent inhibition (PI) value. Animals were considered antibody positive when the PI value was 50% or higher.

[0056] The VN test was performed according to the World Organization for Animal Health (OIE) manual, as described by Lee et al. Serum was inactivated by heat treatment in a 56°C water bath for 30 minutes. The cell density was adjusted to form a 70% monolayer, and two-fold serial dilutions of serum samples (1:8 to 1:1024) were prepared. The diluted serum samples were then diluted to 100 tissue culture infectious doses (TCID) 50 Each well was incubated with 0.5 mL of the same virus at 37°C for 1 hour. After 1 hour, LF-BK (bovine kidney) cell suspension was added to all wells. After 2-3 days, CPE was confirmed and the titer was determined. The titer was 100 TCID 50 Log of antibody replicate dilutions required to neutralize virus 10 was calculated as:

[0057] 4. Isolation of PBMCs FMD antibody-negative animals were used as donors (n = 3 / group) for the isolation of porcine PBMCs. Whole blood (15 mL / each donor) was collected independently into BD Vacutainer heparin tubes. The detailed protocol for PBMC isolation has been previously described. All cells were freshly isolated immediately before use; cryopreserved cells were not used in any experiments. Purified PBMCs were then resuspended in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (HyClone), 3 mM L-glutamine (Sigma-Aldrich), and 100 U / mL penicillin-streptomycin (Sigma-Aldrich). 1 × 10 cells were plated per well in a 96-well plate. 5The cells were plated at 1000 x 1000 cells / well and incubated in a 5% CO2 environment at 37°C. After 3 hours of incubation, the culture medium was replaced with serum-free medium, and the cells were stimulated with FMDV O(O / TWN / 97-R) antigen alone or in combination with various atypical T cell agonists and T cell agonists or PRR ligands.

[0058] 5. Integrative Analysis of BrdU in Pig PBMCs The detailed protocol for cell proliferation analysis is as described above. After treatment with atypical and typical T cell agonists, cell proliferation was tested 12 and 36 hours later according to the manufacturer's instructions.

[0059] 6.RNA-Seq For RNA-Seq analysis, pig PBMCs were isolated from whole blood of serum FMD antibody-negative pigs (n = 3 / group). Isolated PBMCs were incubated with FMDV O(O / TWN / 97-R) antigen along with γδT cell agonists (isopentyl pyrophosphate trilithium salt, IPP(I)), (E)-1-hydroxy-2-methyl-2-butenyl 4-pyrophosphate lithium salt, HMP(H)), iNKT cell agonist (α-galactosylceramide, α-Galcer(G) from Abcam), and MAIT cell agonist (6-formylpterin(F) from Cayman). The mice were treated with atypical T cell agonists, including a T cell agonist (RORγT®, Abcam) and PRR ligands (resiquimod, R848, a TLR-7 / 8 agonist) and trehalose-6,6-dibehenate (TDB, a Mincle agonist); TDB and cyclic bis(3'-5')diguanylic acid (c-di-GMP, a STING agonist, InvivoGen, San Diego, CA, USA). After 12 hours of incubation, PBMCs were harvested and RNA was extracted for qRT-PCR. Library construction and sequence analysis, gene expression analysis and IPA were performed as described above.

[0060] 7. Statistics Unless otherwise stated, all quantitative data are presented as mean ± SEM. Comparison of values ​​for statistical significance between groups was performed using one-way analysis of variance with Tukey's multiple comparison test or Student's t-test for comparing two data points. Statistical analysis was performed using GraphPad Prism 8.3.1 software (GraphPad Software, San Diego, USA).

[0061] <Preliminary Experiment Example> Evaluation of immune differences between cows and pigs 1. FMDV antigens induce stronger proliferation in PBMCs, lymphocytes, monocytes, and T cells from cattle than from pigs. BrdU cell proliferation assays were used to examine the O / TWN / 97-R antigen-mediated proliferation of bovine and porcine PBMCs, lymphocytes, monocytes, and T cells. For all cell types, proliferation of bovine cells was significantly higher than that of porcine cells (p<0.001) (Figure 1a-d).

[0062] 2. FMDV antigen significantly induced the expression of inflammatory cytokines in bovine immune cells compared to porcine immune cells. Analysis of cytokine expression mediated by the O / TWN / 97-R antigen showed that cytokine expression in pig PBMCs peaked between 12 and 48 hours and then declined rapidly, whereas in bovine PBMCs, cytokine expression increased significantly within 24 hours and remained at this level for up to 240 hours (Fig. 2a-d, Table 1).

[0063] Cytokine expression mediated by the O / TWN / 97-R antigen in lymphocytes was significantly higher in bovine cells than in porcine cells (Fig. 2b, e–h; Table 1). Regarding O / TWN / 97-R antigen-mediated cytokine expression in porcine monocytes (Fig. 2c, i–l; Table 1), the expression of IL-2, IL-6, TNFα, and IFNγ was significantly higher than that in bovine monocytes. The time course of O / TWN / 97-R antigen-mediated cytokine expression was evaluated in bovine and porcine T cells (Fig. 2d, m–p; Table 1). Cytokine expression rapidly increased in both bovine and porcine T cells up to 24 h. Thereafter, it gradually decreased in porcine T cells, whereas it remained almost constant in bovine cells up to 240 h.

[0064] The kinetics of IL-1β, IL-12 / 23p40, and IL-10 expression was confirmed in pig PBMCs, lymphocytes, monocytes, and T cells. Expression of the pro-inflammatory cytokines IL-1β and IL-12 / 23p40 was high, whereas expression of the anti-inflammatory cytokine IL-10 was significantly low (Figure 3).

[0065] [Table 1]

[0066] 3. FMDV antigens directly stimulate cytokine expression in porcine Mo-DCs and Mo-MΦs. To investigate the APC response in pigs, which have a lower immune response than cattle, porcine Mo-DCs and Mo-MΦs were polarized from monocytes and stimulated with O / TWN / 97-R antigen to determine whether antigen-mediated cytokines were directly secreted. In Mo-DCs (Figure 4a, a–e, Table 2) and / or Mo-MΦs (Figure 4a, f and Figure 4b, g–j, Table 3), the expression of IL-1β, IL-6, IL-12 / 23p40 (at 48 h), and TNFα (at 24 h) peaked and then declined. O / TWN / 97-R antigen induced significantly higher levels of all these pro-inflammatory cytokines, whereas IL-10 (an anti-inflammatory cytokine) was expressed at lower levels in Mo-DCs and Mo-MΦs. In particular, LPS- and IFNγ-stimulated M1MΦs responded more significantly than IL-4-stimulated M2MΦs.

[0067] [Table 2]

[0068] [Table 3]

[0069] 4. FMDV antigens are endocytosed by porcine DCs and MΦs through phagocytosis, initiating cellular immunity. To identify the pathway by which endocytosis of O / TWN / 97-R antigen into porcine Mo-DCs and Mo-MΦs initiates and amplifies the innate immune response, we examined cytokine expression in the cell culture medium by treating cells with antigen before and after treatment with the phagocytosis inhibitor cytochalasin D (CytD) and co-culture (Figures 5a and 5b). Cytokine expression in Mo-DCs and Mo-MΦs was elevated 24 and 48 hours after co-culture with antigen before CytD treatment, whereas it was significantly suppressed when co-cultured with antigen after CytD treatment. IL-10 expression in Mo-MΦs was slightly suppressed after CytD treatment but remained almost unchanged from pre-treatment levels.

[0070] 5. Abnormally overexpressed innate immune responses and induction of T cell exhaustion pathways by FMD vaccination in pigs To clarify the reason for the lower immune response in pigs compared to cattle, despite antigen-mediated stimulation of pig APCs and endocytosis of antigens by pig DCs and MΦs, we isolated naive bovine and porcine PBMCs from FMD antibody-negative animals and performed RNA-Seq.

[0071] Following the above procedure, we compared the immune responses of cattle and pigs and confirmed fundamental differences between the immune responses induced by FMD vaccination in vivo in cattle and pigs (Figures 6a–c). Under natural conditions, the innate immune response in cattle is well-regulated and maintained, whereas in pigs it is abnormally overactivated (Figures 6a–c). Consequently, while FMD vaccination induced normal immune responses in livestock, the expression of genes involved in the T cell exhaustion pathway (TBX21, NEAT1, NEAT3, NEAT5, EOMES, PRDM1, BCL6, and PDCD1) was significantly increased in pigs (Figure 6b). In particular, expression analysis of genes involved in the TLR / CDS and CLR signaling pathways confirmed that IL23A and IL23R expression was effectively induced in cattle by FMD vaccination. IL23A showed an overexpression pattern in pigs, but IL23R expression was not observed (Figure 6c).

[0072] <Experimental Example 1> Atypical T cell agonists as FMD vaccine adjuvants induce early, mid-term, and long-term immunity in mice. To induce potent cellular immune responses by directly activating T cells without stimulating APCs, we evaluated the feasibility of using atypical T cell agonists, including γδ T cells, iNKT cells, and MAIT cells, as well as typical T cell agonists as novel FMD vaccine adjuvants in mice prior to experiments in pigs as target animals. We also evaluated whether a vaccine without oil emulsion could efficiently induce early, mid, and long-term immune responses and provide host protection against FMDV infection (Figure 7a).

[0073] Compared with the control group, antibody titers by SP-O ELISA were significantly higher at 7 days post-vaccination (dpv) after administration of γδ T cell agonists (isopentyl pyrophosphate trilithium salt, IPP(I)), (E)-1-hydroxy-2-methyl-2-butenyl 4-pyrophosphate lithium salt, HMP(H)), and iNKT cell agonist (α-galactosylceramide, α-Galcer(G)). Titers were also increased in the MAIT cell agonist (6-formylpterin(F))-treated group at 14 dpv. For the classical T cell agonist (RORγT(R)), antibody titers were similar to those of the atypical T cell agonist at 28 dpv. In all experimental groups, antibody titers remained significantly higher than those of the control group up to 168 dpv (Figure 7b).

[0074] Virus-neutralizing antibody (VN) titers showed a similar trend to those measured by SP-O ELISA. The γδ T cell agonist (I), γδ T cell agonist (H), and iNKT cell agonist (G) treatment groups showed approximately a 100-fold increase in neutralizing antibody titers at 7 dpv. The MAIT cell agonist (F) treatment group also showed high levels of VN titers at 14 dpv. At 28 dpv, VN titers were highest in the γδ T cell agonist (I) and iNKT cell agonist (G) treatment groups. VN titers peaked at 56 dpv after boosting, and all atypical T cell agonist treatment groups maintained significantly higher neutralizing antibody titers than the control group at 168 dpv (Figure 7b). In vivo FMDV (100LD) treatments at 84 and 168 dpv were performed. 50In a challenge test using the O / VET / 2013 (Fig. 7c), all adjuvant-treated groups showed a 100% survival rate (Fig. 7d), with little change in body weight (Fig. 7d). Therefore, it was confirmed that the FMD vaccine containing an atypical T cell agonist has a strong effect on inducing early, intermediate, and long-term immunity in mice.

[0075] <Experimental Example 2> Atypical T cell agonists induce potent cell proliferation in porcine PBMCs. After incubation, atypical T cell agonist-mediated cell proliferation was observed in naive pig PBMCs isolated from FMD antibody-negative animals at 12 and 36 hours (Figure 8a). To provide conditions similar to those observed with the actual test vaccine, FMD serotype O (O / TWN / 97-R) antigen was administered together with the atypical T cell agonist. Cell proliferation was observed in the following order: antigen + γδ T cell agonist (H) > antigen + iNKT cell agonist (G) > antigen + MAIT cell agonist (F) > antigen + T cell agonist (R) > antigen + γδ T cell agonist (I) > antigen alone (Figure 8b).

[0076] <Experimental Example 3> Atypical T cell agonists directly activate T cells, which improve abnormal innate immune responses in pigs and induce strong immune responses without APC stimulation. In order to overcome the lower immunogenicity in pigs compared to cattle by inducing a strong cellular immune response in pigs and provide a solution to the various problems that have arisen in pigs, we compared a system that induces an immune response by indirectly activating T cells by stimulating APCs such as DCs and MΦs with PRR ligands, and a system that induces an immune response by directly stimulating T cells with atypical T cell agonists and T cell agonists. Naive PBMCs were isolated from FMD antibody-negative (seronegative) pigs and treated with antigen plus PRR ligands (resiquimod (R848, TLR-7 / 8 agonist) and trehalose-6,6-dibehenate (TDB, Mincle agonist); TDB and cyclic bis(3´-5´)diguanylic acid (c-di-GMP, STING agonist) or antigen plus atypical T cell agonists (γδ T cell agonist, I; γδ T cell agonist, H; iNKT cell agonist, G; MAIT cell agonist, F), or antigen plus T cell agonist, R, or antigen alone. PBMCs were collected 12 hours later, and total RNA was extracted using TRIzol Reagent (Invitrogen) and RNeasy Mini. RNA was extracted using a kit (QIAGEN) according to the manufacturer's recommended method, and RNA-Seq was performed to confirm the immunopotentiator-mediated cellular immune response and the expression profile of related genes (Figures 9a to 9e).

[0077] Expression profiles of TLR / CDS signaling-related genes showed that PRR ligands induced the expression of TLR-7 / 8, cGAS, and RUNX3, while conventional and atypical T cell agonists significantly affected the expression of TBK1, RUNX1, IL23A, and IL23R (Figure 9a, a). The CLR signaling pathway was significantly induced by treatment with conventional cell agonists compared with PRR ligand treatment. IL23A and IL23R were particularly highly expressed in the iNKT cell agonist (G) and conventional T cell agonist (R)-treated groups among the atypical T cell agonists (Figure 9a, b). The expression of genes related to the T cell exhaustion pathway was improved by treatment with PRR ligands and atypical T cell agonists (Figure 9a, c). Gene expression in Th1, Th2, Th9, Th17, Th22, Tfh, pTreg, and tTreg cells was significantly increased in the atypical and typical T cell agonist-treated groups compared with the PRR ligand-treated group. Expression of IL23A and IL23R was also enhanced, and expression of LTA, STAT4, CCL17, CCL22, IL10, RORA, CCL20, IL17A, IL17F, IL1α, and IL1β was also increased by T cell agonist treatment (Figure 9a-d, Figure 9b-e-h, Figure 9c-i-k). In particular, gene expression in M1, M2a, M2b, M2c, M2d, and DCs was significantly enhanced by atypical and typical T cell agonist treatment compared with APC-stimulated PRR ligand treatment. Furthermore, the expression of CD80, CD86, CCL1, CCL2, CCL3, CCL17, CCL22, IL1β, IL23A, TNFα, IL1R2, TGM2, CXCL10, CXCL16, and CD14 was significantly increased (Fig. 9c, l, Fig. 9d, m, n, Fig. 9e, o-q). In NK cells, increased expression of ITGB2 and IFNAR2 genes was observed after treatment with atypical T cell agonists and T cell agonists (Fig. 9e, r).

[0078] [Acknowledgments] Project unique number: 1545019609 Project number: B-1543386-2019-21-03 Name of issue management organization: Agriculture, Forestry and Fisheries Quarantine Agency Research project name: Development of agricultural, forestry and livestock quarantine inspection technology Research title: Construction of a next-generation swine foot-and-mouth disease vaccine platform that can induce long-term immunity using a non-oil type

Claims

1. A vaccine composition comprising an immune enhancing composition containing α-galactosyl ceramide, an iNKT cell agonist, as an active ingredient, The vaccine composition is a foot-and-mouth disease vaccine composition, The active ingredient is contained in an amount of 0.01 to 1% by weight of the immune enhancing composition, The vaccine composition is characterized in that the immune enhancing agent composition is contained in an amount of 30 to 70% by weight of the vaccine composition.

2. The vaccine composition according to claim 1, further comprising an additive, excipient or carrier in addition to the active ingredient.

3. The vaccine composition according to claim 1, wherein the immunopotentiator composition is in an oil or non-oil formulation.

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