Novel immunostimulants and vaccine compositions comprising the same

Abstract

The inventors found that the overexpression of innate immune response and T cell exhaustion pathways is much more pronounced in pigs than in cattle, making pigs less likely than cattle to mount adaptive immune responses and humoral immune responses. This paper presents an innovative strategy to improve the abnormal immune responses in pigs by simultaneously inducing effective cellular and humoral immune responses and applying T cell agonists as new vaccine adjuvants. This result can provide important clues for understanding the differences in immune responses between cattle and pigs, while presenting a method for maximizing immune responses and vaccine efficacy, which are less expressed in pigs than in cattle.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel immune immune enhancer ("adjuvant"), and particularly to a novel immune adjuvant comprising an unconventional T cell agonist and a conventional T cell agonist and a vaccine composition comprising the same. BACKGROUND

[0002] Foot-and-mouth disease (FMD) is an acute infectious disease that spreads very rapidly in cloven-hoofed animals ("hoofed animals"), particularly cattle and pigs, causing significant losses in animal productivity and economy. Most of the clinical studies on FMD have focused on improving the efficacy of vaccines for cattle rather than for pigs. The World Organization for Animal Health (OIE) guidelines for FMD vaccine production only specify the efficacy test procedure for cattle, not for pigs. However, the severity of FMD is highly dependent on both the viral strain and the host species of infection. Acute clinical FMD in pigs is more severe than in other ruminants, and infected pigs can excrete large amounts of aerosol virus, which increases the risk of transmission of severe disease.

[0003] According to recent vaccination trends in Korea, FMD vaccines effectively induce immune responses in cattle, but in pigs, although the viral neutralizing antibody titers (virus neutralization (VN) titers) are high, it is not an immune response sufficient to completely protect against FMD virus infection. This difference can be explained by the susceptibility of each animal to the virus or the host's 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. In order to improve the immunogenicity and efficacy of the vaccine, adjuvants such as oil emulsions (adjuvants) and immune enhancers such as saponins or gels are added as vaccine components. Recently, a pattern recognition receptor (PRR) ligand including cytokines (e.g., IL-15, IL-18, and IFNα), 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 has been proposed as a novel FMD vaccine adjuvant. However, these studies were conducted at a basic level, and there is very little research on further understanding the mechanisms of FMDV antigen or FMD vaccine-mediated cellular immune responses and humoral immune responses.

[0005] The present invention aims to develop an adjuvant for enhancing immunity and an FMD vaccine composition comprising the same, which uses agonists of unconventional T cells including, for example, gamma delta (gd) T cells, invariant natural killer (iNK) T cells, and mucosal-associated invariant T (MAIT) cells, which act as a linker between innate immune responses and humoral immune responses, and are considered "new protectors" in host defense, and uses conventional T cells such as "T cells" as a vaccine adjuvant to directly stimulate T cells without stimulating antigen-presenting cells (APCs) such as dendritic cells (DCs), macrophages (MΦs), monocytes, etc., thereby inducing a faster and stronger cellular immune response, so that they play an important role in the initial defense of the host during vaccination, and at the same time stimulate both cellular and humoral immune responses, thereby inducing a strong memory response and high-titer antibodies in animals, particularly pigs.

[0006] [Related Art Documents]

[0007] [Non-Patent Literature]

[0008] Lee, M. J. 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

[0009] [Problems to be Solved by the Invention]

[0010] To solve the above problems, an object of the present invention is to provide a novel immune enhancer ("adjuvant") composition that induces a strong cellular immune response by directly activating unconventional T cells and conventional T cells in bovine and porcine immune cells.

[0011] In addition, another object of the present invention is to provide a vaccine composition comprising the immune adjuvant composition.

[0012] [Means for Solving the Problems]

[0013] To achieve the above object, the present invention provides a novel immune adjuvant composition that induces a strong cellular immune response in bovine and porcine immune cells.

[0014] The novel adjuvant comprises, as active ingredients, an unconventional T cell agonist and a conventional T cell agonist.

[0015] As used herein, an immunostimulant or adjuvant refers to a substance that enhances an immune response induced by an antigen. An immunoadjuvant can exhibit the same efficacy even with a small amount of antigen in a vaccine, and thus a vaccine can be manufactured with about half to one-third of the conventional amount of antigen.

[0016] The conventional T cell agonist in the present application includes RAR-related orphan receptor gamma t (ROR gamma t).

[0017] The unconventional T cell agonist in the present application means a T cell agonist other than the known conventional T cell agonist described above. The unconventional T cell agonist can include a gamma delta T cell agonist, an iNKT cell agonist, or a MAIT cell agonist, but is not limited thereto.

[0018] The unconventional T cell agonist or the conventional T cell agonist can be included in an amount of 0.01% to 1% by weight ("wt. %"), preferably 0.1% to 0.5% by weight, more preferably 0.2% to 0.3% by weight, based on the total weight of the immunoadjuvant composition. If the content thereof is less than the above range, an immune-enhancing effect is not expressed. On the other hand, if the content thereof exceeds the above range, toxicity can be induced.

[0019] In addition to the above ingredients, the immunoadjuvant or the immunoadjuvant composition of the present application can further include an oil (or an oil emulsion), an emulsifier, a gel, etc. known in the art.

[0020] The oil (or the oil emulsion) can include ISA 201, ISA 61, ISA 50, ISA 206, or ISA 207, but is not limited thereto.

[0021] The emulsifier includes a substance generally recognized as an emulsifier, such as or other solubilizers such as PEG-40 castor oil or other polyethoxylated hydrogenated oils, but they are not limited thereto.

[0022] Further, although the immunopotentiator (or adjuvant) composition generally has an excellent immune-enhancing effect in an oil formulation, the immunoadjuvant composition according to the present application exhibits an excellent immune-enhancing effect even in a non-oil formulation.

[0023] The vaccine composition can further include additives, excipients, carriers, etc. commonly used in the art for preparing an immunoadjuvant, and can be prepared by a conventional manufacturing method commonly used in the art for preparing an immunopotentiating formulation.

[0024] The immune-enhancing effects that occur after administration of the immunoadjuvant composition of the present application can be caused by immune mediators or cells, and in particular, these immune-enhancing effects can include cellular immune-enhancing effects, mucosal immune-enhancing effects, and humoral immune-enhancing effects, but they are not limited thereto.

[0025] The immunity can include immunity against infection by any one of viruses, fungi, bacteria, and parasites, or cancer, but is not limited thereto.

[0026] The virus can 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), parasites causing malaria, human papillomavirus (HPV), influenza virus, measles virus, mumps virus, Ebola virus, respiratory syncytial virus (RSV), West Nile virus (WNV), etc., but is not limited thereto.

[0027] In addition, the present application provides a vaccine composition comprising the adjuvant composition.

[0028] The immunoadjuvant composition can be included in an amount of 30 wt.% to 70 wt.%, preferably 40 wt.% to 50 wt.%, based on the total weight of the vaccine composition, but is not limited thereto. If the amount thereof is less than the above range, the effect of the vaccine can not be expressed. On the other hand, if the amount thereof exceeds the above range, toxicity can be induced during administration.

[0029] The vaccine composition can further include additives, excipients, carriers, etc. that are commonly used in the art for preparing vaccine compositions.

[0030] Further, the vaccine composition can be prepared by a conventional manufacturing method used in the art for preparing vaccine compositions.

[0031] In the present application, the type of vaccine is not limited, but a viral vaccine is preferred, and more preferably a foot-and-mouth disease vaccine.

[0032] When the foot-and-mouth disease vaccine composition contains the immunoadjuvant according to the present application, the foot-and-mouth disease vaccine composition can express an immune effect against foot-and-mouth disease virus O serotype, A serotype, Asia serotype, C serotype, SAT1 serotype, SAT2 serotype, SAT3 serotype, etc. Preferably, the immune effect against foot-and-mouth disease virus O serotype and A serotype can be stronger.

[0033] The vaccine composition can be administered by any one of sublingual administration, transdermal administration, rectal administration, transmucosal administration, topical administration, oral administration, pleural cavity administration, intravenous administration, arterial administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intranasal administration, intrathecal administration, and intra-articular administration, etc.

[0034] [Advantages]

[0035] The present application can provide a novel immunoadjuvant capable of more effectively enhancing a humoral immune response by enhancing the innate cellular immune response of cattle and pigs, and a vaccine composition comprising the same. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Results of proliferation of PBMC, lymphocytes, monocytes, and T cells of cattle and pigs induced with inactivated FMDV O / TWN / 97 antigen are shown.

[0037] Figure 2 a to Figure 2 d and Figure 3 Results of pro-inflammatory cytokine expression in porcine immune cells and bovine immune cells mediated by FMDV (O / TWN / 97-R) antigen are shown.

[0038] Figure 4 FMDV antigen directly stimulates cytokine expression in Mo-DC and Mo-MΦ of pigs is shown.

[0039] Figure 5 a and Figure 5 b shows that FMDV antigen is endocytosed into porcine DC and MΦ by phagocytosis to initiate cellular immunity.

[0040] Figure 6 a shows the experimental process for elucidating the difference in immune response in cattle and pigs.

[0041] Figure 6 b and Figure 6 c shows the results of inducing T cell exhaustion pathways by the abnormally overexpressed innate immune response of pigs and FMD vaccination.

[0042] Figure 7 a shows the experimental process of Experimental Example 1.

[0043] Figure 7 b to Figure 7 d shows the results of confirming that the unconventional T cell agonist and the conventional T cell agonist induce early immunity, mid-term immunity, and long-term immunity in mice.

[0044] Figure 8 a shows the experimental process of Experimental Example 2.

[0045] Figure 8 b shows the results of T cell agonist-induced cell proliferation in porcine PBMC.

[0046] Figure 9 a to Figure 9 e shows the results of Experimental Example 3, in which the unconventional and conventional T cell agonists of Experimental Example 3 improved the abnormal innate immune response of pigs and activated immune cells including T cells. DETAILED DESCRIPTION

[0047] Hereinafter, the present application will be described in detail by way of examples and experimental examples.

[0048] However, the following examples and experimental examples are merely illustrative of the present application, and the scope of the present application is not limited to the following examples and experimental examples.

[0049] <Experimental materials and experimental methods>

[0050] 1. Antigen purification and inactivation

[0051] Purified inactivated virus antigen was prepared in BHK-21 cells infected with FMDV O / TWN / 97-R (GenBank AY593823; P1) constructed for phenotypic replacement of P1 by reverse genetics (reference sequence).

[0052] For virus infection, the culture medium was replaced with serum-free Dulbecco's Modified Eagle Medium (DMEM; HyClone, Logan, UT, USA) and the cells were inoculated with the virus by incubation at 37℃ for 1 hour in 5% carbon dioxide. Then, the extracellular virus was removed. At 24 hours post-infection, the virus was inactivated by treatment with 0.003N diethyl ether for 24 hours in a shaking incubator, followed by concentration with 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.

[0053] After ultracentrifugation, the bottom of the centrifuge tube was punctured and 1 mL fractions were collected. The presence of FMDV particles in each fraction sample was confirmed by optical density using a side flow device UA-6 (BioSign FMDV Ag; Princeton BioMeditech, Princeton, NJ, USA). Before use in field experiments, the PEG-pretreated supernatant was passed through ZZ-R127 and BHK-21 cells at least twice to confirm that no cytopathic effect (CPE) occurred, thereby proving the absence of live virus in the supernatant.

[0054] 2. Isolation of PBMCs

[0055] To investigate antigen-mediated immune responses, the Gyeonggi Provincial Animal Health Laboratory donated whole blood from pigs and cattle. The whole blood (15 mL) was collected in BD Vacutainer heparin tubes (BD Biosciences, Becton, Dickinson and Company, Franklin Lake, New Jersey, USA) and then processed using Ficoll-Paque. TM PLUS gradient (GE Healthcare Bio-Sciences Corp., Piscatave, NJ, USA) and centrifugation. Residual red blood cells were then lysed by treatment with ammonium chloride-potassium (ACK) lysis buffer (Gibco, Carlsbad, CA, USA). PBMCs were resuspended in Ca2+-free buffer. 2+ and Mg 2+ Cells were isolated from Dulbecco PBS (Gibco) supplemented with 2% fetal bovine serum (FBS) (Gibco) and then counted using a volumetric flow cytometer (MACSQuant Analyzer, Miltenyi Biotec, Bergisch-Gladbach, Germany). All cells were freshly isolated immediately before use, and refrigerated 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 seeded in 25 cm² incubators. 2 The cells were placed in tissue culture flasks (Eppendorf, Hamburg, Germany) and incubated at 37°C and 5% CO2 to allow newly isolated monocytes to adhere. After 3 hours of incubation, non-adherent cells were collected for lymphocyte isolation. Remaining adherent cells were thoroughly washed with Dulbecco phosphate-buffered saline (DPBS) (Gibco) before adding 4 mL of RPMI 1640 growth medium to each flask, followed by incubation at 37°C and 5% CO2.

[0056] 3. Cell separation via magnetic activated cell sorting (MACS)

[0057] Mononuclear cells, lymphocytes and T cells were isolated from PBMC. To isolate primary immune cells, mononuclear cells from adherent PBMC and T cells from non-adherent cells were purified by MACS. Adherent and non-adherent cells from PBMC were briefly resuspended in MACS buffer (1x PBS supplemented with 0.5% BSA and 2mM EDTA). Mononuclear cells and T cells were sorted using magnetic microbeads together with the Monocyte Isolation Kit and Pan T Cell Isolation Kit (Miltenyi Biotec), respectively, according to the manufacturer’s instructions, and further classified using a fluorescence-activated cell sorter (FACS, Astrios, Beckman Coulter, Brea, CA, USA). The purity of isolated cells was confirmed by flow cytometry (MACSQuant Analyzer, Miltenyi Biotec) and analyzed with FlowJo software version vX.0.7 (TreeStar Inc., Ashland, OR, USA). The purity of sorted cells was greater than 95%. Astrios TM Beckman Coulter, Brea, CA, USA). The purity of isolated cells was confirmed by flow cytometry (MACSQuant Analyzer, Miltenyi Biotec) and analyzed with FlowJo software version vX.0.7 (TreeStar Inc., Ashland, OR, USA). The purity of sorted cells was greater than 95%.

[0058] 4. Generation of Mo-DC and Mo-MΦ

[0059] To differentiate Mo-DC, isolated mononuclear cells were cultured in RPMI 1640 complete medium (Gibco) supplemented with 10% FBS (HyClone), 3mM L-glutamine (Sigma-Aldrich), 100U / mL penicillin-streptomycin (Sigma-Aldrich), 50ng / mL GM-CSF and IL-4 (Miltenyi Biotec) for 7 days to generate immature DCs. At day 3, an equal amount of the above medium was added, and at day 7, non-adherent contaminant cells were removed by vigorous washing before cell lysis. To isolate pure DCs, anti-CD11c magnetic beads were used according to the manufacturer’s instructions (Miltenyi Biotec). CD11c + / MHC II + The purity of cells was greater than 95% and cells were maintained in a 37°C, 5% CO2 incubator.

[0060] To differentiate Mo-MΦ, isolated mononuclear cells were seeded in 10 6Cells were plated at 1 x 105cells / mL in 12-well plates and cultured for 6 days in RPMI 1640 medium (Gibco) supplemented with 10% FBS (HyClone), 1 x MEM nonessential 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 the entire medium was replaced fresh on day 4. + / F4 / 80 + Cells were kept in a 37 °C, 5% CO2incubator.

[0061] 5. Preparation of mature DCs

[0062] Mature DCs were generated by adding one of the following six treatments: no treatment (no stimulation), LPS (E. coli 055:B5, Sigma-Aldrich [100 ng / mL]) + rpIFN-g (Novus Biologicals, LLC., Littleton, CO, USA [20 ng / mL]), rpTNFa (R&D Systems, Minneapolis, MN, USA [20 ng / mL]), antigen only (antigen [1 pg / mL]), LPS + rpIFN-g + antigen (LPS [100 ng / mL], rpIFN-g [20 ng / mL], and antigen [1 pg / mL]), or rpTNFa + antigen (rpTNFa [20 ng / mL] and antigen [1 pg / mL]). At specific time points after treatment (0 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h), cell culture supernatants were harvested for ELISA.

[0063] 6. M1 / M2 MΦ polarization

[0064] After 7 days of growth, Mo-MΦ were treated with one of the following six treatments: no treatment (no stimulation), M1 MΦ (LPS [100 ng / mL] and rpIFN-γ [20 ng / mL]), M2 MΦ (rpIL-4, R&D Systems [20 ng / mL]), antigen alone (1 μg / mL), M1 MΦ + antigen (IFN-γ [20 ng / mL] and LPS [100 ng / mL] and antigen [1 μg / mL]), or M2 MΦ + antigen (IL-4 [20 ng / mL] + antigen [1 μg / mL]). At specific time points after treatment (0 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h), cell culture supernatants were harvested for ELISA.

[0065] 7. Cell culture, antigen treatment, and BrdU incorporation detection in bovine and porcine PBMCs

[0066] Isolated or differentiated cells (1 x 10 6 cells / well) were cultured in complete medium consisting of 10% fetal bovine serum (HyClone), 3 mM L-glutamine (Sigma-Aldrich), 10 mM HEPES, 100 U / mL penicillin-streptomycin (Sigma-Aldrich), and RPMI 1640 medium (Gibco) supplemented with 0.05 mM 2-mercaptoethanol (Sigma-Aldrich) in a 37°C, 5% CO2incubator (Sigma-Aldrich). To stimulate, cells were treated with 1 μg of each antigen. At specific time points after treatment (0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h, and 240 h), cell proliferation was tested during DNA synthesis using the BrdU Cell Proliferation ELISA Kit (cells Signaling Technology, MA, USA) according to the manufacturer's instructions. Briefly, 10 μM BrdU was added to the cell culture, followed by incubation at 37°C for 4 h. Cells were then fixed and incubated with an anti-BrdU mouse monoclonal antibody, followed by application of horseradish peroxidase-conjugated goat anti-mouse antibody. Tetramethylbenzidine was used as a chromogenic substrate for color development. Absorbance was measured at dual wavelengths of 450 / 550 nm. Cell Titer-Blue TM reagent kit (Promega, Madison, WI, USA) was used to monitor cell viability. This experimental treatment did not affect cell viability compared to media-only control treatment.

[0067] 8. ELISA

[0068] ELISA for bovine and porcine IL-1 b, IL-6, IL-10, IL-12 / 23p40, IL-23 and TNF a (DuoSet, R&D Systems, Minneapolis, MN, USA; Cloud-Clone Corporation, Houston, USA) were performed using cell culture supernatants according to the manufacturer’s instructions.

[0069] 9. Inhibition of phagocytosis

[0070] To inhibit phagocytosis, Mo-DC and Mo-MF were incubated with 5 pg / mL cytochalasin D (CytD) (Sigma-Aldrich) for 45 min prior to antigen treatment. Then, CytD-treated Mo-DC and Mo-MF were cultured with 1 pg / mL antigen. After 6 h, culture supernatants were collected for ELISA.

[0071] 10. Bovine and porcine

[0072] To understand the fundamental differences between the immune responses between bovine and porcine under natural conditions and FMDV O antigen-mediated immune responses and associated mechanisms, field experiments were performed using bovine and porcine according to the method described by Lee et al. FMD antibody-negative animals (bovine: 5 months old, porcine: 10-12 weeks old) were used. Bovine and porcine were divided into two groups (n = 5 per group). Animals were kept isolated during the study period. The study was approved by the Animal Experiment Ethics Committee of the General Administration of Agriculture, Forestry and Animal Husbandry (Approval No. IACUC-2018-800 and IACUC-2019-185) and conducted according to the institutional guidelines.

[0073] 11. Vaccination and sampling

[0074] O / TWN / 97-R Ag was used as FMD antigen and the vaccine composition for the positive control group was as follows: 1 mL of a single dose prepared vaccine containing 15 pg of O / TNW / 97-R antigen (one dose for each of bovine and porcine), ISA 206 (50%, w / w), 10% Al(OH)3, and 150 pg Quil-A. To isolate the initial PBMC, whole blood was collected from bovine and porcine of the initial control group (foot-and-mouth disease antibody-negative). Two vaccinations were performed every 28 days and 1 mL of vaccine (one dose) was injected into the neck of the animals of the positive control group by the deep intramuscular route. Blood samples were collected from bovine and porcine for serological analysis at 0, 14, 28, 42, 56, 70, and 84 dpv and PBMC were further isolated from bovine and porcine at 28 dpv (28 days after the first vaccination dose, before the second vaccination dose). The animals were monitored daily for body temperature, symptoms at the vaccination site, and appetite. Serum samples were stored at -80 °C until testing.

[0075] 12. RNA sequence analysis (RNA-Seq)

[0076] For RNA-Seq analysis, PBMCs from whole blood of cattle and swine from initial control (n=3 heads / group) and positive control (n=3 heads / group) (28 dpv) were subjected to density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) according to the method described in Lee, M.J. 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) (hereinafter Lee et al.). TM

[0077] 13. Construction and sequencing of libraries

[0078] Illumina-based next-generation sequencing (NGS) was performed to obtain high-throughput bovine and swine transcriptome data. Total RNA was individually extracted from bovine and swine PBMCs using TRIzol reagent (Invitrogen) and RNeasy Mini kit (QIAGEN) according to the manufacturer's protocol. The total RNA was then quantified using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and its quality was assessed by an RNA 6000 Nano test kit (Agilent Technologies, Santa Clara, CA, USA) and a Bioanalyzer 2100 (Agilent). NGS sequencing libraries were generated from 1 pg of total RNA using a TruSeq RNA Sample Prep kit (Illumina, San Diego, CA, USA) according to the manufacturer's protocol. That is, poly(A)-containing RNA molecules were purified using magnetic beads conjugated with poly-T oligonucleotides. After purification, total poly(A)+RNA was cleaved into small fragments using divalent cations at high temperature. The cleaved mRNA fragments were reverse-transcribed into first-strand cDNA using random primers. The short fragments were purified with a QiaQuick PCR extraction kit and digested with elution buffer for final recovery and addition of poly(A). The short fragments were then ligated with sequencing adapters. Each library was separated by adjacent different MID tags. The libraries were then sequenced by NovaSeq 6000 (Illumina) according to the manufacturer's protocol. TM ​6000 system (Illumina) for paired-end sequencing (2 x 101 bp).

[0079] 14. Gene expression analysis

[0080] Low quality bases (PHERD score (Q) < 20) and adapter contamination were removed by Trimmomatic v.0.36 using ILLUMINACLIP:TruSeq3-SE:2:30:10 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36’ as default parameters. After quality score verification and read length verification, RNA-Seq reads were mapped into the reference bovine genome (published in April 2018; ARS-UCD1.2; GCA_002263795.2) using STAR as base default parameters and using Expectation-Maximization (RSEM, RNA-Seq). To obtain expression values for each gene / transcriptome in the genome, expression was estimated by the Expectation-Maximization method. RSEM estimated read counts were applied to edgeR v3.22.5 to obtain differentially expressed scores with statistical significance. Further, filters (i.e. Transcripts per Million (TPM) > 0.3, read counts > 5, log2 fold change > 1) were applied to select differentially expressed transcriptomes. Finally, expressed transcripts (i.e. TPM > 0.3 and read counts > 5) were analyzed to reveal expression patterns in each disease and gene family (immune genes, T cell markers and TLRs, CDS and CLR signaling pathway genes).

[0081] 15. Ingenuity Pathway Analysis (IPA)

[0082] Then, the expression profiles were grouped into clusters with similar expression patterns. IPA (QIAGEN, https: / / www.qiagenbioinformatics.com / products / ingenuity-pathway-analysis) was then used to analyze enriched pathways, networks and functions. Finally, a binary heat map showing all genes involved in key pathways was created using a local R script.

[0083] <Assessment of the effect of unconventional T cell agonists as potent vaccine adjuvants>

[0084] 1. Mice

[0085] Age- and sex-matched wild-type C57BL / 6 mice (6-7 weeks old female) were purchased from KOSA BIO Inc. (Gyeonggi-do). All mice were placed in micro-isolation cages in an animal biosafety level 3 (ABSL3) specific pathogen-free (SPF) animal facility at the Headquarters of the Korea National Institute of Agricultural Biotechnology. This study was performed in accordance with institutional guidelines approved by the Animal Experiment Ethics Committee at the Headquarters of the Korea National Institute of Agricultural Biotechnology (Approval Nos. IACUC-2018-800 and IACUC-2019-185).

[0086] 2. Unconventional and conventional T cell agonists-mediated adjuvants and host defense

[0087] To investigate the response and potential of unconventional and conventional T cell agonists as FMD vaccine adjuvants and their protective effects against FMDV infection, experiments were performed using the proposed strategy Figure 7 a) (n = 10 / group). The O / TWN / 97-R antigen was used as an inactivated FMDV antigen. The vaccine composition of the PC group was as follows: the O / TWN / 97-R antigen (15 pg / dose / mL, 1 / 10 dose for each of cattle and swine) was used, 10% Al(OH)3 and 15 pg / Quil-A / mouse, thus the total volume was 100 pL. Unconventional or conventional T cell agonists were added to all mice in the experimental groups as adjuvants (immunological adjuvants) to achieve about 0.2 wt.% based on the total weight of the adjuvant composition, and thus to achieve about 0.1 wt.% based on the total weight of the vaccine, so that the vaccine had the same composition as the PC group.

[0088] The unconventional and conventional T cell agonists used in these experiments were purchased as follows: Sigma-Aldrich (γδ T cell agonist; isopentenyl pyrophosphate lithium trihydrate, IPP(I); (E)-1-hydroxy-2-methyl-2-butenyl 4-lithium pyrophosphate, HMP(H), Abcam (iNKT cell agonist; a-galactosylceramide; a-galcer (G), T cell agonist (RORyT (R)), and Cayman (MAIT cell agonist; 6-formylpterin (F)) and Cayman Chemical, Ann Arbor, MI, USA).

[0089] Mice in the negative control received the same volume of phosphate buffered saline (PBS, pH 7.0) by the same route. Briefly, two vaccinations were performed every 35 days and mice were intramuscularly inoculated in the thigh muscle. Thereafter, at 84 dpv or 168 dpv, mice were challenged with FMDV (100 LD50of O / VET / 2013, ME-SA topotype) by intraperitoneal injection. The mice were monitored for viability and body weight until 7 dpc (7 days post challenge). Further, sera from mice sampled at 0, 7, 14, 28, 56, 84 and 168 dpv were analyzed for induced cellular and humoral immune responses by A-type structural protein enzyme-linked immunosorbent assay (structural protein (SP) A ELISA) and virus neutralization (VN) titration.

[0090] 3. Serological analysis

[0091] To detect SP antibodies in sera, the PrioCHECK FMDV Type O ELISA kit (Prionics AG, Switzerland) was used as described by Lee et al. The absorbance in the ELISA plate was converted to a percentage of inhibition (PI) value. When the PI value was 50% or higher, the animal was considered antibody positive.

[0092] The VN test was performed according to the manual of the World Organization for Animal Health (OIE) as described by Lee et al. Sera were inactivated by heat treatment in a water bath at 56°C for 30 minutes. The cell density was adjusted to form a 70% monolayer and a 2-fold serial dilution (1 :8 to 1 :1024) of the serum sample was prepared. The diluted serum sample was then incubated with 100 tissue culture infective dose (TCID) 50 of homologous virus for 1 hour at 37°C. After 1 hour, a LF-BK (bovine kidney) cell suspension was added to all the wells. After 2-3 days, CPE was checked to determine the titer, which was expressed as the reciprocal antibody dilution Log 50 required to neutralize 100 TCID 10 calculated.

[0093] 4. PBMC isolation

[0094] FMD antibody-negative animals were used as donors for porcine PBMC isolation (n = 3 animals / group). Whole blood (15 mL / donor) was collected independently in BD Vacutainer heparin tubes. Detailed protocols for PBMC isolation are described above. All cells were freshly isolated immediately before use, and refrigerated cells were not used in any experiments. The purified PBMCs were then resuspended in RPMI 1640 medium (Gibco, Carlsbad, California, USA) supplemented with 10% FBS (HyClone), 3 mM L-glutamine (Sigma-Aldrich), and 100 U / mL penicillin-streptomycin (Sigma-Aldrich). The product was divided into 1 × 10⁻⁶ cells per well. 5 Cells were seeded in 96-well plates and incubated at 37°C with 5% CO2. After 3 hours of incubation, the medium was replaced with serum-free medium, and then the cells were stimulated with FMDV O(O / TWN / 97-R) antigen alone or in combination with various unconventional T cell agonists and T cell agonists or PRR ligands.

[0095] 5. Detection of BrdU integration in porcine PBMCs

[0096] The detailed protocol for cell proliferation assays was described above. Cell proliferation was tested according to the manufacturer's instructions at 12 and 36 hours after treatment with unconventional and conventional T-cell agonists.

[0097] 6. RNA-Seq

[0098] For RNA-Seq analysis, porcine PBMCs were isolated from whole blood of FMD serum antibody negative pigs (n=3 per group). Isolated PBMCs were treated with the following reagents: Unconventional T cell agonists including gammadelta T cell agonists (isopentenyl pyrophosphate lithium tri salt, IPP (I)), (E)-1-hydroxy-2-methyl-2-butenyl 4-lithium pyrophosphate, HMP (H)), iNKT cell agonists (a-galactosylceramide, a-Galcer (G), Abeam) and MAIT cell agonists (6-formylprine, 6-Formylpterin (F), Cayman Chemical); T cell agonists (RORyT (R), Abeam), PRR ligands (resiquimod, R848, TLR-7 / 8 agonist) and trehalose-6,6-divehenate (TDB, Mincle agonist); TDB and bis-(3'-5')cyclo-dimeric guanosine monophosphate (c-di-GMP, STING agonist, InvivoGen, San Diego, CA, USA) used in combination with FMDV O (O / TWN / 97-R). Then, after 12 hours of incubation, PBMCs were collected (prepared) and RNA was extracted for qRT-PCR.

[0099] Library construction and sequencing, gene expression analysis and IPA were performed as described above.

[0100] 7. Statistical tests

[0101] All quantitative data are expressed as mean ± SEM, unless otherwise stated. For comparison of two data points, comparison of statistical significance between groups was performed by using one-way ANOVA with the Tukey’s multiple comparison test or Student’s t-test. For statistical tests, GraphPad Prism 8.3.1 software (GraphPad Software, San Diego, CA, USA) was used.

[0102] <Example of preliminary experiments> Evaluation of the immune differences between cattle and pigs

[0103] 1. FMDV antigens induced stronger proliferation in bovine than in porcine derived PBMCs, lymphocytes, monocytes and T cells.

[0104] O / TWN / 97-R antigen-mediated proliferation of bovine and porcine PBMCs, lymphocytes, monocytes and T cells was observed using the BrdU cell proliferation assay. Proliferation of all bovine cell types was significantly higher than that of porcine cells (p<0.001) (a-d in Fig. 1). Figure 1

[0105] ​2. FMDV antigen significantly induced pro-inflammatory cytokine expression in bovine immune cells compared to porcine immune cells.

[0106] O / TWN / 97-R antigen-mediated cytokine expression assays showed that cytokine expression in porcine PBMC peaked between 12h and 48h, then sharply declined, whereas cytokine expression in bovine PBMC showed a marked increase within 24h and this level was maintained up to 240h Figure 2 a-d in a, Table 1).

[0107] In lymphocytes, O / TWN / 97-R antigen-mediated cytokine expression was significantly higher in bovine cells than in porcine cells Figure 2 e-h in b, Table 1). For O / TWN / 97-R antigen-mediated cytokine expression in porcine monocytes Figure 2 i-l in c, Table 1), IL-2, IL-6, TNFα and IFNγ were further significantly elevated in bovine monocytes. The time course of O / TWN / 97-R antigen-mediated cytokine expression in bovine T cells and porcine T cells was assessed Figure 2 m-p in d, Table 1). Cytokine expression rapidly increased in both types of T cells up to 24h, then gradually declined in porcine T cells, but remained almost constant in bovine cells up to 240h.

[0108] The kinetics of IL-1 β, IL-12 / 23 p40 and IL-10 expression were identified in porcine PBMC, lymphocytes, monocytes and T cells. IL-1 β and IL-12 / 23 p40 were highly expressed as pro-inflammatory cytokines, and IL-10 was significantly low as an anti-inflammatory cytokine Figure 3 .

[0109] [Table 1]

[0110]

[0111] 3. PMDV antigen directly stimulates cytokine expression in porcine Mo-DC and Mo-MΦ

[0112] To investigate whether the APCs that respond less in bovine than in porcine, the direct secretion of antigen-mediated cytokines by porcine Mo-DC and Mo-MΦ in monocytes was identified by polarization and stimulation with O / TWN / 97-R antigen. Mo-DC Figure 4 a-e in a, Table 2) and / or Mo-MΦ Figure 4 f and g in a, Table 2) were identified. Figure 4G to J in b, Table 3), IL-1 β, IL-6, IL-12 / 23 p40 (48h) and TNFα (24h) expression decreased after reaching peak levels. O / TWN / 97-R antigen induced significantly higher levels of expression of all these pro-inflammatory cytokines, while IL-10 (anti-inflammatory cytokine) was expressed at low levels in Mo-DC and Mo-MΦ. In particular, the response of LPS and IFNγ-stimulated M1 MΦ was more pronounced than that of IL-4-stimulated M2 MΦ.

[0113] [Table 2]

[0114]

[0115]

[0116] [Table 3]

[0117]

[0118] 4. FMDV antigens are endocytosed into porcine DC and MΦ by phagocytosis to initiate cellular immunity. To identify the pathways for initiating and amplifying the innate immune response by endocytosis of O / TWN / 97-R antigens into porcine Mo-DC and Mo-MΦ, cells were treated with antigens and co-cultured before and after treatment of cells with cytochalasin D (CytD) as an immunosuppressant, followed by observation of cytokine expression in cell cultures Figure 5 a and Figure 5 b). In Mo-DC and Mo-MΦ, cytokine expression was increased at 24h and 48h after antigen co-culturing before CytD treatment, but was significantly inhibited at antigen co-culturing after CytD treatment. Further, IL-10 expression in Mo-MΦ was slightly inhibited after CytD treatment, but was not significantly different from the level before treatment.

[0119] 5. Abnormally over-expressed innate immune response in pigs and induction of T cell exhaustion pathway by FMD vaccination

[0120] To elucidate the reason for the lower immune response in pigs compared to cattle, despite antigen-mediated stimulation of porcine APC and endocytosis on porcine DC and MΦ by phagocytosis, the inventors performed RNA-Seq after isolating bovine naive PBMC and porcine naive PBMC.

[0121] According to the above procedure, the immune responses in cattle and pigs were compared, and then the fundamental differences between the immune responses induced by in vivo FMD vaccination in cattle and pigs were confirmed Figure 6 a to Figure 6 c). In a natural state, the innate immune response in cattle is controlled and maintained well, while in pigs it is abnormally over-activatedFigure 6 b and c) in a. Thus, FMD vaccination induced normal immune responses in cattle, whereas the expression of genes involved in T cell exhaustion pathways (TBX21, NEAT1, NEAT3, NEAT5, EOMES, PRDM1, BCL6, and PDCD1) was significantly increased in pigs. Figure 6 b). In particular, according to the analysis of the expression of genes involved in TLR / CDS and CLR signaling pathways, it was demonstrated that the vaccination of FMD vaccine in cattle effectively induced the expression of IL23A and IL23R. On the other hand, IL23A showed overexpression patterns in pigs, whereas no expression of IL23R was observed Figure 6 c).

[0122] <Experimental Example 1> Unconventional T cell agonists as FMD vaccine adjuvants induce early, intermediate, and long-term immunity in mice.

[0123] To induce strong cellular immune responses by directly activating T cells without stimulating APCs, before performing experiments in pigs as a subject (target) animal, mouse usability evaluations of unconventional T cell agonists (including γδ T cells, iNKT cells, MAIT cells) and conventional T cell agonists as novel FMD vaccine adjuvants were performed. Further, it was evaluated whether there is a host protection effect during FMDV infection by effectively inducing early, intermediate, and long-term immune responses even in vaccines without oil emulsion Figure 7 a).

[0124] Compared to the control, antibody titers measured by SP-O ELISA at 7 days post-vaccination (dpv) were significantly increased after administration of γδ T cell agonists (isopentenyl pyrophosphate lithium trihydrate, IPP (I)), (E)-1-hydroxy-2-methyl-2-butenyl 4-lithium pyrophosphate, HMP (H)), and iNKT cell agonists (a-galactosylceramide, a-Gal (G)). The titers of the group administered with MAIT cell agonists (6-formylprine (6-Formylpterin (F)) were also increased at 14 dpv. For the conventional T cell agonist (RORγT (R)), the antibody titers were similar to those of the unconventional T cell agonists at 28 dpv. Further, the antibody titers of all experimental groups were significantly higher than those of the control until 168 dpv Figure 7 b).

[0125] Virus neutralization (VN) titers showed similar trends to antibody titers by SP-O ELISA. The neutralizing antibody in the γδ T cell agonist (I) administration group, the γδ T cell agonist (H) administration group, and the iNKT cell agonist (G) administration group increased about 100-fold at 7 dpv, while the MAIT cell agonist (F) administration group was also found to have high VN titers at 14 dpv. The VN titers of the γδ T cell agonist (I) administration group and the iNKT cell agonist (G) administration group were the highest at 28 dpv. The VN titers peaked at 56 dpv after the booster immunization, and all of the unconventional T cell agonist administration groups maintained significantly higher neutralizing antibody titers than the control at 168 dpv Figure 7 b). The FMDV (100 LD 50 of O / VET / 2013) challenge test showed that the survival rate was 100% in all adjuvant-treated groups Figure 7 c), and the body weight change was very small Figure 7 d).

[0126] Therefore, it was confirmed that the FMD vaccine containing the unconventional T cell agonist had an effective effect on inducing early, mid-term, and long-term immunity in mice.

[0127] <Experiment Example 2> Unconventional T cell agonists induce strong cell proliferation in porcine PBMC

[0128] Unconventional T cell agonist-mediated cell proliferation was observed in porcine naive PBMC isolated from FMD antibody-negative production animals 12 hours and 36 hours after incubation Figure 8 a). To provide similar conditions to the actual test vaccine, the FMD serotype O (O / TWN / 97-R) antigen was co-administered with the unconventional T cell agonist. The cell proliferation rate was high, and the order was as follows: 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 8 b).

[0129] <Experiment Example 3> Unconventional T cell agonists enhance abnormal innate immune responses in pigs and directly activate T cells, which induce strong immune responses without stimulating APCs.

[0130] To overcome the low immunogenicity compared to bovine by inducing strong cellular immune responses in pigs, and to provide solutions against the various problems of the proposed pigs, the system of inducing immune responses by indirectly activating T cells through stimulating APCs such as DCs and MΦs with PRR ligands was compared with the system of inducing immune responses by directly stimulating T cells with unconventional T cell agonists and T cell agonists. The primary PBMCs were isolated from FMD antibody-negative pigs, and then, antigen + PRR ligands (resiquimod (R848, TLR-7 / 8 agonist) and trehalose-6,6-divehenate (TDB, Mincle agonist); TDB and bis-(3'-5') cyclic di-GMP (c-di-GMP, STING agonist) or antigen + unconventional 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 only antigen for treatment, and the PBMCs were collected after 12 hours, total RNA was extracted using TRIzol reagent (Invitrogen) and RNeasy Mini kit (QIAGEN) according to the manufacturer's recommended method, and then RNA-Seq was performed, thereby confirming the induction of adjuvant-mediated cellular immune responses and the related gene expression profiles Figure 9 a to 9e).

[0131] The gene expression profile related to TLR / CDS signals showed that the PRR ligand induced the expression of TLR-7 / 8, cGAS, and RUNX3. Further, it was confirmed that the conventional T cell agonists and the unconventional T cell agonists significantly affected the expression of TBK1, RUNX1, IL23A, and IL23R Figure 9 a) in a). Compared to the PRR ligand treatment, the treatment with the conventional T cell agonists significantly induced the CLR signaling pathway. Further, among the unconventional T cell agonists, the specific expression of IL23A and IL23R was high in the iNKT cell agonist (G) treatment group and the conventional T cell agonist (R) treatment group Figure 9 b) in a). Further, the gene expression related to the T cell exhaustion pathway was improved by the treatment with the PRR ligand and the unconventional T cell agonist Figure 9c) in a. Gene expression in Th1, Th2, Th9, Th17, Th22, Tfh, pTreg, and tTreg cells in groups treated with unconventional and conventional T cell agonists was significantly increased compared to PRR ligand treatment groups. Further, expression of IL23A and IL23R was also enhanced, as well as expression of LTA, STAT4, CCL17, CCL22, IL10, RORA, CCL20, IL17A, IL17F, IL1a, and IL1b was increased by T cell agonist treatment Figure 9 d in a, Figure 9 e to h in b and Figure 9 i to k in c). Significantly, gene expression in M1, M2a, M2b, M2c, M2d, and DCs was significantly enhanced by treatment with unconventional and conventional T cell agonists compared to APC-stimulated PRR ligand treatment. Further, expression of CD80, CD86, CCL1, CCL2, CCL3, CCL17, CCL22, IL1b, IL23A, TNFa, IL1R2, TGM2, CXCL10, CXCL16, and CD14 was significantly increased Figure 9 l in c, Figure 9 m and n in d and Figure 9 o to q in e). Additionally, it was observed that ITGB2 and IFNAR2 gene expression in NK cells was increased by treatment with unconventional and T cell agonists Figure 9 r in e.

[0132] [Confirmation information]

[0133] Allocation identification number: 1545019609

[0134] Allocation number: B-153386-2019-21-03

[0135] Project management organization name: Headquarters of the Agricultural, Forestry and Animal Husbandry Inspection

[0136] Research business project name: Development of agricultural, forestry and animal husbandry quarantine technology

[0137] Research project name: Establishment of next-generation swine foot-and-mouth disease vaccine platform capable of inducing non-oil type long-term immunity

Claims

1. The use of an immune adjuvant composition in the preparation of a foot-and-mouth disease vaccine, wherein the immune adjuvant composition comprises any one or more selected from the group consisting of γδ T cell agonists, MAIT cell agonists, and T cell agonists as an active ingredient. The γδ T cell agonist mentioned above is isopentenyl trilithium pyrophosphate. The MAIT cell agonist mentioned above is 6-formylpterin, and The T-cell agonist mentioned therein is RORγt.

2. The application according to claim 1, wherein the amount of said active ingredient is from 0.01% to 1% by weight of the total weight of the immune adjuvant composition.

3. The application according to claim 1, wherein, in addition to the active ingredient, the immune adjuvant composition further comprises a carrier.

4. The application according to claim 1, wherein the immune adjuvant composition is in the form of an oil-based or non-oil-based formulation.

5. The application according to claim 1, wherein the amount of the immune adjuvant composition is 30% to 70% by weight of the total weight of the foot-and-mouth disease vaccine.

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