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 RAR-related orphan receptor gamma t, which is a T cell agonist, as an active ingredient.SELECTED DRAWING: Figure 8b

Description

[Technical field]

[0001] The present invention relates to a novel immune enhancing agent, more specifically to a novel immune enhancing agent 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, infectious disease that spreads very rapidly in cloven-hoofed animals, especially in cattle and pigs, causing severe animal productivity loss and economic losses. Most clinical studies on FMD have focused on improving vaccine efficacy in cattle rather than in 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 is highly dependent 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, posing a high risk of severe disease transmission.

[0003] In recent years, according to the vaccination trend in Korea, FMD vaccine effectively induces immune response in cattle, but in pigs, despite high virus neutralization (VN) titer in serum, it does not induce sufficient immune response for complete protection against FMD virus infection. Such differences can be explained by the susceptibility of each livestock species to FMD virus or the host immune response to vaccination.

[0004] Vaccination containing inactivated foot-and-mouth disease virus (FMDV), i.e., inactivated antigen, is mainly used for host defense and FMD control. To improve the immunogenicity and efficacy of the vaccine, adjuvants such as oil emulsions and immune enhancers such as saponins, gels, etc. 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, such studies have been conducted at a basic level, and little mechanistic research has been conducted to further deepen the understanding of the cellular and humoral immune responses mediated by FMDV antigens or FMD vaccines.

[0005] The objective of the present invention is to use unconventional T cells, including gamma delta T cells, invariant natural killer T cells, and mucosal-associated invariant T cells, which act as a linker between innate and humoral immune responses and have been proposed as "new guardians" in host defense, and agonists of conventional T cells, such as T cells, as vaccine adjuvants, to directly stimulate T cells without stimulating antigen presenting cells (APCs), such as dendritic cells (DCs), macrophages (MΦs), and monocytes, thereby leading to a more rapid and stronger cellular immune response, which plays an important role in the initial defense of the host upon vaccination, and to simultaneously stimulate cellular and humoral immune responses, thereby generating a strong memory response in animals, particularly in pigs. The present invention aims to develop an immune enhancing adjuvant capable of inducing a specific immune response and an antibody titer thereof, and a FMD 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 immune enhancing composition. [Means for solving the problem]

[0009] In order to achieve the above object, the present invention provides a novel immune enhancing 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] In the present invention, the term "immunostimulant" or "adjuvant" refers to a substance that enhances the immune response caused by an antigen. The immunostimulant can exert the same effect in a vaccine even with a small amount of antigen, so that a vaccine can be made with about half to one third of the amount of antigen used in the past.

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

[0013] The atypical T cell agonist in the present invention means a T cell agonist excluding the typical T cell agonists already known 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% by weight, preferably 0.1 to 0.5% by weight, more preferably 0.2 to 0.3% by weight. If the amount is less than the above range, the immunopotentiating effect may not be exhibited, and if the amount is more than the above 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, and the like, which are widely known in the art, in addition to the above-mentioned components.

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

[0017] The emulsifiers may include, but are not limited to, materials commonly 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, while an immune enhancing (or adjuvant) composition generally has excellent immune enhancing effects when in an oil formulation, the immune enhancing composition of the present invention has excellent immune enhancing effects even when in a non-oil formulation.

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

[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 can include, but is not limited to, immunity against infection with any one of the pathogens selected from the group consisting of 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), West Nile virus, and the like. Examples of viruses that may be used include, but are not limited to, WNV.

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

[0024] The immune enhancer composition may be included in an amount of 30-70% by weight, preferably 40-50% by weight, 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 be exhibited 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] Moreover, the vaccine composition can be prepared by any conventional manufacturing 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 contained in a FMD vaccine composition, the FMD vaccine composition can exert an immune effect against FMD viruses of serotype O, serotype A, serotype Asia1, serotype C, serotype SAT1, serotype SAT2, serotype SAT3, etc. Desirably, the composition can exert a stronger immune effect against FMD viruses of serotype O and serotype A.

[0029] The vaccine composition may be administered by sublingual, transdermal, rectal, transmucosal, topical, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intraspinal, or intraarticular administration, or the like. Effect 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 description of the drawings]

[0031] [Figure 1] FIG. 1 shows the results of PBMCs, lymphocytes, monocytes and T cell proliferation in cattle and pigs induced by inactivated FMDV O / TWN / 97 antigen. [Diagram 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. [Diagram 3] FIG. 3 shows the results of expression of inflammatory cytokines in porcine immune cells 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. [Diagram 5] 5a and 5b show that FMDV antigens are endocytosed by porcine DCs and MΦs through phagocytosis to initiate cellular immunity. [Figure 6] Figure 6a shows the experimental process to clarify the difference 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 an atypical T cell agonist and a typical T cell agonist. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 antigens were 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 the virus by incubating them at 37°C with 5% carbon dioxide for 1 h. Extracellular virus was then removed. 24 h after infection, the virus was inactivated by treating twice with 0.003 N binary ethyleneimine for 24 h in a shaking incubator, and then concentrated in polyethylene glycol (PEG) 6000 (Sigma-Aldrich, St. Louis, MO, USA). The obtained virus concentrate was layered on a 15-45% sucrose density gradient and centrifuged.

[0035] After ultracentrifugation, 1 mL fractions were collected by punching the bottom of the centrifuge tube. The presence of FMDV particles in each fraction sample 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 the field experiments, the absence of live virus was confirmed in the supernatant by passing the PEG-pretreated supernatant at least twice through ZZ-R127 and BHK-21 cells to ensure that no cytopathic effect (CPE) occurred.

[0036] 2. Isolation of PBMCs To examine antigen-mediated immune responses, whole blood from pigs and cows was donated by the Gyeonggi-do Animal Health Laboratory, Korea. Whole blood (15 mL) was collected in BD Vacutainer heparin tubes (BD Biosciences, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and transfected with Ficoll-Paque. TMThe PBMCs were then cultured in ammonium chloride·potassium chloride (ACK) lysis buffer (Gibco, Carlsbad, CA, USA) supplemented with 2% fetal bovine serum (FBS) (Gibco), and the remaining red blood cells were lysed by treatment with ACK lysis buffer (Gibco, Carlsbad, CA, USA). 2+ and Mg 2+ The PBMCs were then suspended in Dulbecco's PBS free of 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 allowed to adhere to tissue culture flasks (Eppendorf, Hamburg, Germany) and cultured in a 5% CO2, 37°C environment. After 3 h of culture, non-adherent 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 The purified cells were further separated 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 with FlowJo software version vX.0.7 (TreeStar Inc., Ashland, OR, USA). The purity of the isolated cells was higher 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 mentioned medium was added, and on day 7, non-adherent 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 more than 95%, and the cells were maintained in a 5% CO2 incubator at 37°C.

[0039] For differentiation of Mo-MΦs, isolated monocytes were cultured at 106 The cells were aliquoted at 12 cells / mL into 12-well plates 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 more than 95%. The cells were maintained in a 5% CO2 incubator at 37°C.

[0040] 5. Preparation of Mature DCs Mature DCs were generated by adding one of six treatments: no treatment (unstimulated), LPS (E. coli 055:B5, Sigma-Aldrich [100 ng / mL]) + 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 + rpIFNγ + antigen (LPS [100 ng / mL], rpIFN-γ [20 ng / mL], and antigen [1 μg / mL]), or rpTNFα + antigen (rpTNFα [20 ng / mL] and antigen [1 μg / mL]). At specific time points (0, 6, 12, 24, 48, 72, and 96 h) after treatment, cell culture supernatants were collected for ELISA.

[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] and 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 after treatment (0, 6, 12, 24, 48, 72, and 96 h), 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 was tested using the BrdU Cell Proliferation Assay Kit (Cell Signaling Technology, MA, USA), based on the incorporation of BrdU during DNA synthesis, 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-developing substrate tetramethylbenzidine was used for color development. The absorbance was measured at two wavelengths, 450 / 550 nm. Cell Titer-Blue TMCell viability was monitored using an assay kit (Promega, Madison, WI, USA). Compared to the medium alone 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 manufacturers' 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 min before antigen treatment. CytD-treated Mo-DCs and Mo-MΦs were then cultured with 1 μg / mL antigen. After 6 h, culture supernatants were collected for ELISA.

[0045] 10. Cows and pigs To understand the fundamental differences between immune responses in natural conditions between cattle and pigs, and the immune responses mediated by FMDV O antigen and related mechanisms, field experiments using cattle and pigs were conducted by the method described by Lee et al.. FMD antibody-negative animals were used (cattle aged 5 months and pigs aged 10–12 weeks). Cattle and pigs were divided into two groups (n = 5 / group). The animals were maintained in isolation during 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 was carried out in accordance with the institutional guidelines.

[0046] 11. Vaccination and Sampling O / TWN / 97-R Ag was used as FMD antigen. The vaccine composition for the positive control group was as follows: 1 mL of vaccine manufactured in a single dose containing 15 μg 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 of the naive control group (FMD antibody negative) for naive PBMCs isolation. Vaccination was performed twice with an interval of 28 days, and 1 mL of vaccine (single dose) was injected via intramuscular route in the neck of the positive control animals. Blood samples were collected from cattle and pigs at 0, 14, 28, 42, 56, 70 and 84 dpv for serological analysis, and blood samples were collected from cattle and pigs at 28 dpv (28 days after the first vaccination, before the second vaccination) for PBMCs isolation. The animals were monitored daily for 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 transferred to Ficoll-Paque Ficoll Immunosorbent Assay (FIMA) 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 a PLUS (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).

[0048] 13. Library Construction and Sequencing To obtain high-throughput bovine and porcine transcriptome data, Illumina next generation sequencing (NGS) was performed. Total RNA was extracted from bovine and porcine PBMCs using TRIzol Reagent (Invitrogen) and 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 by an RNA6000Nano analysis kit (Agilent Technologies, Santa Clara, USA) and Bioanalyzer2100 (Agilent). NGS sequencing libraries were generated from 1 μg of total RNA using a 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-coupled 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 addition of poly(A). The short fragments were then ligated with a sequencing adapter. Each library was isolated with flanking distinct MID tags. 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. Analysis of Gene Expression Low quality bases (PHERD score(Q)<20) and adapter contamination were removed by Trimmomatic v.0.36 using the 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 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 with statistical significance. Additionally, filters were applied to select differentially expressed transcripts, i.e., 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. Then, enrichment pathways, networks, and functions were analyzed using IPA (QIAGEN Inc., https: / / www.qiagenbioinformatics.com / products / ingenuity-pathway-analysis). Finally, a binary heatmap showing all genes involved in significant pathways was generated using in-house Rscript.

[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 performed in accordance with the institutional guidelines and approved by the Animal Experiment Ethics Committee of the Animal and Plant Quarantine Agency (approval numbers IACUC-2018-800 and IACUC-2019-185).

[0052] 2. Helper and host defense mediated by atypical and classical T cell agonists To evaluate the potential of atypical and typical T cell agonists as FMD vaccine adjuvants, as well as the simultaneous induction of potent cellular and humoral immune responses, and to examine the protective effect against FMDV infection, an experiment was carried out using the presented strategy (Fig. 7a) (n=10 per group). O / TWN / 97-R antigen was used as the inactivated FMDV antigen. The vaccine composition for the PC group was as follows: O / TWN / 97-R antigen (15μg / dose / mL, 1 / 10 dose for use in cattle and pigs), 10% Al(OH)3 and 15μg / Quil-A / mouse, with a total volume of 100μL. All mice in the experimental groups were provided with a vaccine of 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% by weight of the total weight of the immune enhancer composition and approximately 0.1% by weight 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 were administered the same volume of phosphate-buffered saline (PBS, pH 7.0) via the same route. Briefly, vaccination was performed twice with an interval of 35 days, and mice were vaccinated intramuscularly in the thigh muscle. After vaccination, mice were challenged with FMDV (100 LD50 of O / VET / 2013, ME-SA topotype) by intraperitoneal injection at 84 or 168 dpv. Survival rate and body weight of mice were monitored up to 7 days post challenge (dpc). Sera sampled from mice at 0, 7, 14, 28, 56, 84, and 168 dpv were also analyzed by structural protein A enzyme-linked immunosorbent assay (SPA ELISA) and virus neutralization (VN) titers to investigate the induced cellular and humoral immune responses.

[0055] 3. Serological analysis To detect SP antibodies from serum, the PrioCHECK FMDV type O ELISA kit (Prionics AG, Switzerland) was used 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 min. The cell density was adjusted to form a 70% monolayer, and two-fold serial dilutions (1:8 to 1:1024) of serum samples were prepared. The diluted serum samples were then transferred 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 porcine PBMCs isolation. Whole blood (15 mL / each donor) was collected independently into BD Vacutainer heparin tubes. Detailed protocols for PBMCs isolation have been previously described. All cells were freshly isolated immediately before use, and no cryopreserved cells were 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 100x the number of cells and incubated in a 5% CO2, 37°C environment. 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 together with various atypical T cell agonists and T cell agonists or PRR ligands.

[0058] 5. Integration Analysis of BrdU in Porcine PBMCs The detailed protocol for cell proliferation assay 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). The 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), Abcam), and MAIT cell agonist (6-formylpterin (F), Cayman). The cells were treated with atypical T cell agonists including T cell agonist (RORγT(R), Abcam) and 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, InvivoGen, San Diego, CA, USA). After 12 h 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 All quantitative data are presented as mean ± SEM unless otherwise stated. Comparison of values ​​for statistical significance between groups was performed using one-way ANOVA 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 assay was used to observe the proliferation of bovine and porcine PBMCs, lymphocytes, monocytes, and T cells mediated by O / TWN / 97-R antigen. In 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 O / TWN / 97-R antigen showed that in pig PBMCs, cytokine expression peaked at 12 to 48 hours and then declined rapidly, whereas in bovine PBMC, cytokine expression increased significantly within 24 hours and remained at this level for up to 240 hours (Figure 2a-d; Table 1).

[0063] Cytokine expression mediated by 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 cytokine expression mediated by O / TWN / 97-R antigen 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. It then gradually decreased in porcine T cells, but 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. The expression of IL-1β and IL-12 / 23p40 as proinflammatory cytokines was high, whereas the expression of IL-10 as an anti-inflammatory cytokine 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 response of APCs 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 confirm whether antigen-mediated cytokines were directly secreted. In Mo-DCs (Fig. 4a a-e, Table 2) and / or Mo-MΦs (Fig. 4a f and Fig. 4b g-j, Table 3), the expression of IL-1β, IL-6, IL-12 / 23p40 (48 h) and TNFα (24 h) decreased after reaching peak levels. O / TWN / 97-R antigen induced the expression of all these inflammatory cytokines at significantly higher levels, 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 pig DCs and MΦs via phagocytosis, initiating cellular immunity. To identify the pathway that initiates and amplifies the innate immune response by endocytosis of O / TWN / 97-R antigen in porcine Mo-DCs and Mo-MΦs, we treated cells with antigen before and after treatment with phagocytosis inhibitor cytochalasin D (CytD) and observed the expression of cytokines in the cell culture medium (Figure 5a and 5b). In Mo-DCs and Mo-MΦs, the expression of cytokines was increased 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 was almost the same as the level before treatment.

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

[0071] By comparing the immune responses of cattle and pigs according to the above procedure, fundamental differences in the immune responses induced by FMD vaccination in vivo between cattle and pigs were confirmed (Figures 6a-c). Under natural conditions, the innate immune response of cattle is well regulated and maintained, whereas in pigs it is abnormally overactivated (Figures 6a-c). As a result, 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 demonstrated that IL23A and IL23R expression was effectively induced by FMD vaccination in cattle. 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 a strong cellular immune response by directly activating T cells without stimulating APCs, the feasibility of using atypical T cell agonists, including γδT cells, iNKT cells, and MAIT cells, and typical T cell agonists as novel FMD vaccine adjuvants was evaluated in mice prior to experiments in pigs as the target animals. We also evaluated whether a vaccine without oil emulsion could efficiently induce early, mid, and long-term immune responses and have a host protective effect 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 agonist (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 typical 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 were significantly higher than those of the control group up to 168 dpv (Fig. 7b).

[0074] Virus neutralizing antibody (VN) titers showed a similar trend to those measured by SP-O ELISA. The γδT cell agonist (I), γδT cell agonist (H), and iNKT cell agonist (G) groups showed approximately 100-fold increases in neutralizing antibody titers at 7 dpv, and the MAIT cell agonist (F) group also showed high levels of VN titers at 14 dpv. At 28 dpv, VN titers were highest in the γδT cell agonist (I) and iNKT cell agonist (G) groups. VN titers reached a maximum at 56 dpv after boosting, and all atypical T cell agonist groups maintained significantly higher neutralizing antibody titers than the control group at 168 dpv (Figure 7b). FMDV (100LD) administered at 84 and 168 dpv were significantly higher than the control group at 168 dpv. 50In a challenge test using O / VET / 2013, all adjuvant-treated groups showed 100% survival rate (Figure 7c) and little change in body weight (Figure 7d). Thus, it was confirmed that the FMD vaccine containing an atypical T cell agonist has a strong effect on the induction of 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, cell proliferation mediated by atypical T cell agonists was observed in naive porcine PBMCs isolated from FMD antibody-negative producers at 12 and 36 hours (Fig. 8a). To provide conditions similar to those of the actual test vaccine, FMD serotype O (O / TWN / 97-R) antigen was administered together with atypical T cell agonists. Cell proliferation was highest 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 (Fig. 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 low immunogenicity of pigs compared to cattle by inducing a strong cellular immune response in pigs and provide a solution to 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 PRRs 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 + 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 + atypical T cell agonists (γδ T cell agonist, I; γδ T cell agonist, H; iNKT cell agonist, G; MAIT cell agonist, F) or antigen + 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. The RNA was extracted using a kit (QIAGEN) according to the method recommended by the manufacturer, and RNA-Seq was performed to confirm the immune enhancer-mediated cellular immune response and the expression profile of related genes (Figures 9a to 9e).

[0077] The expression profile of TLR / CDS signaling-related genes showed that PRR ligands induced the expression of TLR-7 / 8, cGAS, and RUNX3, and conventional atypical T cell agonists significantly affected the expression of TBK1, RUNX1, IL23A, and IL23R (Fig. 9a, a). The CLR signaling pathway was significantly induced by treatment with typical cell agonists compared with PRR ligand treatment. IL23A and IL23R were highly expressed in the iNKT cell agonist (G) and typical T cell agonist (R)-treated groups, especially among the atypical T cell agonists (Fig. 9a, b). The expression of genes related to the pathway of T cell exhaustion was improved by PRR ligand and atypical T cell agonist treatment (Fig. 9a, c). Gene expression in Th1, Th2, Th9, Th17, Th22, Tfh, pTreg and tTreg cells was significantly increased in the atypical T cell agonist and typical T cell agonist treated groups compared to the PRR ligand treated group. IL23A and IL23R expression was also enhanced, and LTA, STAT4, CCL17, CCL22, IL10, RORA, CCL20, IL17A, IL17F, IL1α and IL1β expression was also increased by T cell agonist treatment (Fig. 9a d, Fig. 9b e-h, Fig. 9c i-k). In particular, gene expression in M1, M2a, M2b, M2c, M2d and DCs was significantly improved by atypical T cell agonist and typical T cell agonist treatment compared to 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 by treatment with atypical T cell agonists and T cell agonists (Fig. 9e, r).

[0078] [Acknowledgements] Project unique number: 1545019609 Assignment number: B-1543386-2019-21-03 Name of issue management agency: 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 capable of inducing long-term immunity using a non-oil type

Claims

1. An immune enhancing composition comprising a T cell agonist, RORγt (RAR-related orphan receptor gamma t), as an active ingredient.

2. The immune enhancing composition according to claim 1, wherein the active ingredient is contained in an amount of 0.01 to 1% by weight of the immune enhancing composition.

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

4. The immune enhancing composition according to claim 1, wherein the immune enhancing composition is in an oil or non-oil formulation.

5. A vaccine composition comprising the immune enhancing composition according to any one of claims 1 to 4.

6. The vaccine composition according to claim 5, wherein the immune enhancer composition is contained in an amount of 30 to 70% by weight of the vaccine composition.

7. The vaccine composition according to claim 5, which is a foot and mouth disease vaccine composition.

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