Application of CpG ODN in preparation of medicine for regulating animal immune cell activity

Abstract

The invention discloses application of CpG ODN in preparation of a medicine for regulating animal immune cell activity, and relates to the technical field of animal vaccines. In the invention, the regulation of the animal immune cell activity is to promote the immune response reaction of immune cells; the step of promoting the immune response reaction of the immune cells comprises the step of promoting the immune cells to generate interferon. The CpG ODN provided by the invention can well and specifically stimulate immune cells of animals, especially pigs to release cytokines and promote the animals to generate rapid and strong immune response to immunogens; when being applied to an animal vaccine, the vaccine can stimulate animals to generate a higher antibody level in a shorter time, and can maintain the high-level antibody for a long time, and the immunogenicity of the vaccine is remarkably improved.

Description

[0001] This application claims priority to Chinese Patent Application No. 202411254664.0, filed on September 9, 2024, entitled "Application of CpG ODN in the Preparation of a Drug for Regulating the Activity of Animal Immune Cells", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of animal vaccine technology, and more specifically, to the application of CpG ODN in the preparation of drugs that regulate the activity of animal immune cells. Background Technology

[0003] With the rapid expansion of the livestock farming industry, research on animal vaccines has received increasing attention. Animal vaccines are an important tool for preventing and controlling infectious diseases in animals. One of the most important steps in animal vaccine development is the selection of a suitable adjuvant. Developing safe, highly purified subunit and inactivated vaccines requires safe and effective adjuvants because the antigens in these types of vaccines often have lower immunogenicity compared to modified live microorganisms.

[0004] Adjuvants can be broadly classified into two categories: immunostimulants (saponins, Toll-like receptor (TLR) agonists, cytokines) and delivery agents (emulsions, microparticles, mineral salts).

[0005] Synthetically produced oligodeoxynucleotides containing a cytosine-guanine dinucleotide motif (CpG ODN) are TLR9 stimulants. Examples of TLR agonists used in livestock vaccines include the use of Pseudomonas aeruginosa membrane lipoprotein OprI in a classical swine fever (CSF) subunit vaccine. OprI promotes T cell and humoral responses through TLR2-mediated APC activation and maturation. Bacterial flagellin, a TLR5 agonist, also exhibits adjuvant properties in foot-and-mouth disease vaccine formulations. Intranasal inoculation of chickens with CpG ODN, along with inactivated avian influenza virus, induces a stronger immune response than when the antigen is used alone. Other examples of TLR agonists include bacterial ghosts (BG). Pigs vaccinated with the chimeric vector vaccine rAdV-SFV-E2, adjuvanted with BG, are completely unaffected by CSF virus challenge, while pigs vaccinated without BG receive only limited protection. However, pigs that received the vaccine with BG as an adjuvant also experienced temporary allergic reactions.

[0006] While TLR agonists have garnered significant attention in veterinary vaccine technology, their application faces challenges. Overactivation of the innate immune system via pathogen recognition receptors (PRRs) can lead to septic shock. Furthermore, CpGs exhibit strong species specificity; the same ODN sequence can have varying effects on different animal species, or even fail to elicit an immune response. Therefore, a suitable balance between safety and efficacy must be struck, and practical adjuvants must be developed to specifically promote the immune response in these animals, thereby enhancing vaccine efficacy. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide an application of CpG ODN in the preparation of a drug for regulating the activity of animal immune cells. The CpG ODN provided by the present invention can specifically stimulate the release of cytokines such as interferon by animal immune cells, especially pigs, thereby enhancing the rapid and strong immune response of animals to immunogens.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] Application of CpG ODN in the preparation of drugs that regulate the activity of animal immune cells.

[0010] Furthermore, the regulation of animal immune cell activity is to promote the immune response of immune cells.

[0011] Furthermore, the promotion of the immune response of immune cells includes promoting the production of interferon by immune cells.

[0012] Furthermore, the interferon is INF-α.

[0013] Furthermore, the promotion of immune cell immune response also includes promoting the production of IL-6 and TNF-α by immune cells.

[0014] The immune cells are selected from at least one of macrophages, lymphocytes, and dendritic cells.

[0015] Furthermore, the nucleotide sequence of the CpG ODN contains the sequences "TCG" and / or "ACG"; preferably, the nucleotide sequence of the CpG ODN contains at least two repeats of "TCG" and / or "ACG"; more preferably, the nucleotide sequence of the CpG ODN contains both "TCG" and "ACG"; particularly preferably, the nucleotide sequence of the CpG ODN contains at least two repeats of "TCG" and at least one repeat of "ACG".

[0016] Furthermore, at least one set of "ACG" sequences is followed by a "TT" sequence.

[0017] Furthermore, the nucleotide sequence of the CpG ODN is the sequence described in SEQ ID NO: 1-6.

[0018] Furthermore, the animal is a domestic animal or poultry, preferably a pig.

[0019] Furthermore, the drug is a vaccine, the CpG ODN is a vaccine adjuvant, and the regulation of animal immune cell activity is to promote the production of immunity against the antigen in animals.

[0020] Vaccines are typically formulated for parenteral administration. Typical immunization methods include intramuscular injection (IM), oral immunization (PO), and subcutaneous injection (SC).

[0021] The vaccines described above are administered in a dosage-compatible manner, and at amounts such as therapeutically effective doses and immunogenically effective doses. The dosage depends on the recipient, the recipient's immune system's ability to synthesize antibodies, and the expected level of protection. The exact amount of active ingredient to be administered depends on the veterinarian's judgment and varies from person to person. The appropriate regimen for initial administration and booster vaccinations may also vary, but typically one or more injections or other methods are administered at certain intervals (weeks or months) after the initial administration.

[0022] Furthermore, the antibody titer of the vaccine using CpG ODN adjuvant is at least 20% higher than that of the vaccine without adjuvant within 2 weeks of immunization, preferably at least 50%, and more preferably at least 100%.

[0023] Furthermore, the antibody titer of the vaccine using CpG ODN adjuvant is at least 20% higher than that of the vaccine without adjuvant after 4 weeks of immunization, and more preferably, the antibody titer is at least 50% higher.

[0024] The CpG ODN adjuvant provided by this invention can not only generate high levels of antibodies earlier, but also maintain high levels of antibodies for a long time, thus significantly improving the immunogenicity of the vaccine.

[0025] Furthermore, the nucleotide sequence of the CpG ODN is a sequence having SEQ ID NO: 1-6.

[0026] Furthermore, the antigen is a virus-based antigen, which includes viruses, viral nucleic acids, or viral proteins.

[0027] Furthermore, the animal is a mammal, and in particular, the animal is a pig.

[0028] Furthermore, the antigens include: PRRSV (Nidovirales) arteritis virus, Coronavirus, or antigens based on such viruses.

[0029] Furthermore, the antigen includes: porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), or antigens based on such viruses.

[0030] Furthermore, the nucleic acid sequence of the CpG ODN is the sequence described in any one of SEQ ID NO: 1-3.

[0031] In this invention, the nucleic acid sequence of CpG ODN is any one of SEQ ID NO: 1-3, especially SEQ ID NO: 1, which can significantly stimulate the secretion of IFN-α by porcine PBMCs, thereby enhancing the activity of the immune response. When used, the optimal concentration of CpG ODN is between 0.15 μM and 1.5 μM, with 0.5 μM being the most preferred.

[0032] Furthermore, the animals mentioned are birds, especially poultry.

[0033] Furthermore, the antigens include: influenza viruses, paramyxoviridae viruses, Avibirnavirus genus viruses, adenoviruses, or antigens based on such viruses.

[0034] Furthermore, the antigens include: influenza A virus, avian influenza virus, Newcastle disease virus, infectious bursal disease virus, egg drop syndrome virus, or antigens based on the above viruses.

[0035] Furthermore, the nucleic acid sequence of the CpG ODN is the sequence described in any one of SEQ ID NO: 3-6.

[0036] Furthermore, the vaccine also includes the combination of the CpG ODN with at least one of an emulsion adjuvant, a mineral salt adjuvant, a TLR agonist, a cytokine, a saponin, and a polymer.

[0037] Furthermore, the emulsion adjuvant is selected from Montanide ISA206, Montanide ISA201, white oil adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, MF59, etc. At least one of the following: the mineral salt adjuvant is selected from aluminum adjuvant or calcium phosphate adjuvant; the aluminum adjuvant is at least one of aluminum hydroxide, aluminum phosphate, aluminum sulfate, and potassium aluminum sulfate; the TLR agonist is selected from at least one of Poly(I:C), monophosphatidyllipoprotein A, flagellin, Pseudomonas aeruginosa membrane lipoprotein OprI, Escherichia coli heat-labile enterotoxin, and sclerotium slough; the cytokine is selected from at least one of IFN-γ, IL-6, IL-7, IL-18, and fish type I interferon; the saponin is selected from at least one of Quil-A, QS-21, and immunostimulatory complex; the polymer is selected from carbomer, etc. Polyphosphazene, At least one of them.

[0038] In this invention, exemplary pathogenic microorganisms causing poultry diseases may include: Newcastle disease virus (NDV), avian influenza virus (H5, H7, H9 subtypes), avian adenovirus, infectious bursal disease virus (IBDV), avian infectious bronchitis virus, Haemophilus paragallinarum, infectious laryngotracheitis virus, egg drop syndrome virus, and fowlpox virus (APV).

[0039] In this article, exemplary pathogens that cause livestock diseases may include: porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus (PCV), Mycoplasma hyopneumoniae, foot and mouth disease virus (FMDV), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), and pseudorabies virus (PRV).

[0040] In this article, exemplary pathogens that cause diseases in pets may include: feline panleukopenia virus, feline calicivirus (FCV), feline herpesvirus, rabies virus (RV), and feline parvovirus (FPV).

[0041] Compared with the prior art, the present invention has the following advantages:

[0042] The CpG ODN provided by this invention can specifically stimulate the release of cytokines such as interferon from immune cells in animals, especially pigs, promoting a rapid and strong immune response to immunogens. When applied to animal vaccines, it can stimulate animals to produce higher antibody levels in a shorter time and maintain high antibody levels for a long period, significantly improving vaccine immunogenicity.

[0043] When the antigen is PRRSV GP5 protein antigen, the combination of CpG ODN and ISA 206 adjuvant can achieve an antibody titer of over 1.4 within 2 weeks after immunization, while the antibody titer in the group without CpG ODN is only about 0.4 within 2 weeks after immunization. In addition, it can maintain a high level of antibodies for a long time, and the immunogenicity of the vaccine is significantly improved. Attached Figure Description

[0044] Figure 1The levels of porcine reproductive and respiratory syndrome virus (PRRSV) specific antibodies in serum samples after immunization with saline, antigen subunit + ISA206, and antigen subunit + ISA206 + ODN1, respectively, in Example 2.

[0045] Figure 2 The levels of neutralizing antibodies against the CV777 strain in serum samples after immunization with CV777 antigen, CV777 antigen + ODN3, and CV777 antigen + ODN3 + aluminum adjuvant in Example 3 are shown.

[0046] Figure 3 The antibody levels in chicken serum samples from the New Zealand influenza bivalent vaccine group, the New Zealand influenza bivalent vaccine + ODN1 group, and the saline control group in Example 4 are shown.

[0047] Figure 4 The antibody levels in chicken serum samples from the infectious bursal disease vaccine group, the infectious bursal disease vaccine + 2μg CpG ODN6 group, and the non-immunized control group in Example 7 are shown. Detailed Implementation

[0048] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0050] General technical and definition descriptions

[0051] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0052] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0053] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0054] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM may fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0055] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0056] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0057] As used herein, the term "CpG or CpG motif" refers to a nucleic acid having a cytosine and a guanine linked by a phosphate ester bond, wherein the pyrimidine ring of the cytosine is unmethylated. "Methylated CpG" refers to a methylated pyrimidine ring of the cytosine, typically occurring at the 5-position of the pyrimidine ring. A CpG motif is a base pattern comprising an unmethylated central CpG with at least one base flanking it at both the 3' and 5' positions. The flanking bases of the CpG confer a significant portion of the activity of the CpG ODN.

[0058] As used herein, the term "CpG ODN" refers to a CpG oligodeoxynucleotide that is at least about ten nucleotides in length and includes one unmethylated CpG. CpG ODNs are single-stranded. The entire CpG ODN can be unmethylated or partially unmethylated. CpG ODNs include D-type (also known as A-type), K-type (also known as B-type), C-type, and P-type ODNs.

[0059] As used in this article, "antigen" (Ag) refers to any substance that can induce an immune response in the body. Specifically, it refers to substances that can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, producing immune response products (sensitized lymphocytes or antibodies), and that can specifically bind to these products in vivo and in vitro. This includes, but is not limited to, heteroantigens, such as pathogenic microorganisms, viruses, toxoids, and other antigens between different species; allogeneic antigens, autoantigens, and heterophilic antigens.

[0060] As used herein, the terms "GP5 protein" and "major envelope glycoprotein" in the term "Porcine reproductive and respiratory syndrome" (PRRS) refer to the polypeptide encoded by ORF5 of the PRRSV genome as understood in the art. Without being bound by theory but for the purpose of facilitating understanding of the invention, the GP5 protein encoded by ORF5 of the PRRSV genome is believed to be a receptor recognition protein (RRP) in PRRSV.

[0061] As used in this article, "animal vaccine" refers to an immunizing agent used to prevent infectious diseases, made from pathogenic microorganisms (such as viruses, bacteria, rickettsiae, etc.) and their metabolites through artificial attenuation, inactivation, or genetic engineering. Vaccines retain the characteristic of pathogens stimulating the animal's immune system. When an animal comes into contact with this harmless pathogen, the immune system produces certain protective substances, such as immune hormones, active physiological substances, and specific antibodies. When the animal is exposed to the same pathogen again, its immune system, following its previous memory, produces more protective substances to prevent harm from the pathogen. "Animal" here includes any type of poultry, livestock, pets, or aquatic species, especially including animals such as chickens, ducks, geese, cattle, sheep, goats, pigs, dogs, cats, and fish.

[0062] Example 1: Cytokine production activity of porcine PBMCs stimulated by different CpG ODNs

[0063] After stimulating porcine PBMCs with different concentrations of different CpG ODNs in vitro for 16-24 hours, the levels of cytokines such as IFN-α, TNF-α, and IL-6 in the cell supernatant were measured to screen for porcine-sensitive CpG ODN sequences.

[0064] 1. Reagents and materials:

[0065] CpG ODN was provided by Huapu Biotechnology (Hebei) Co., Ltd. ODN1-20 are shown below:

[0066] ODN1 (SEQ ID NO: 1): 5'-TCGCGAACGTTCGCCGCGTACGTACGCGG-3';

[0067] ODN2 (SEQ ID NO: 2): 5'-TCGTCGAACGTTCGAGATGAT-3';

[0068] ODN3 (SEQ ID NO: 3): 5'-TCGACGTTCGTCGTTCGTCGTTC-3';

[0069] ODN4 (SEQ ID NO: 4): 5'-TGACTGTGAA CGTTCGAGATGA-3';

[0070] ODN5 (SEQ ID NO: 5): 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3';

[0071] ODN6 (SEQ ID NO: 6): 5'-TCGCAACGTTGCCTTCGAAGG-3';

[0072] ODN7(SEQ ID NO:7):5'-TCGTCGTTACCGATGACGTCGCCGT-3';

[0073] ODN8(SEQ ID NO:8):5'-TCGCCCACCGGTGGGGGGGG-3';

[0074] ODN9(SEQ ID NO:9):5'-TCGAGGACAAGATTCTCGTGC-3';

[0075] ODN10(SEQ ID NO:10):5'-TCGGCACGCGACGTGCTGGCCGTCGTTTC C-3';

[0076] ODN11(SEQ ID NO:11):5'-TCGTTGCCGTCGGCCCCCCC-3';

[0077] ODN12(SEQ ID NO:12):5'-TCGAGGACAAGATTCTCGT-3';

[0078] ODN13(SEQ ID NO:13):5'-TCGGCACGCGACGTGCTGGCCGTCGTT-3';

[0079] ODN14(SEQ ID NO:14):5'-TCGTCGCGCCGTCACGGGGGG-3';

[0080] ODN15(SEQ ID NO:15):5'-TCGTGACGTCCTCGAGTT-3';

[0081] ODN16(SEQ ID NO:16):5'-TCGTCTTTCGACTCGTTCTC-3';

[0082] ODN17(SEQ ID NO:17):5'-TCGACTTTCGTCGTTCTGTT-3';

[0083] ODN18(SEQ ID NO:18):5'-TCGTCGTTTCGTCGTTCTC-3';

[0084] ODN19(SEQ ID NO:19):5'-TCGTCGTTTTCGTCGTTTTCGTCGTT-3';

[0085] ODN20 (SEQ ID NO: 20): 5'-TGGCCAAGCTTGGGCCCCTTGCAAGGGCC-3', GC-ODN, used as a negative control.

[0086] RPMI-1640 (Gibco, 11875-093); FBS (Gibco, 10099-141C); Pen-Strep (Gibco, 15140-122);

[0087] In the sequence, lowercase letters represent bases linked by thiophosphate bonds, and uppercase letters represent bases linked by phosphate diester bonds.

[0088] Complete culture medium: RPMI-1640 medium containing 10% fetal bovine serum and 100 U / mL-100 μg / mL Pen-Strep.

[0089] INF-α Detection Kit (Huamei Biotechnology, CSB-E07328p-48T);

[0090] TNF-α detection kit (Huamei Biotechnology, CSB-E16980p);

[0091] IL-6 Detection Kit (Huamei Biotechnology, CSB-E06786p);

[0092] Pig PBMCs (Miaoshun Biotechnology, pPB100C-BP);

[0093] 2. Method:

[0094] Cell seeding plate. The concentration of porcine PBMC cells was adjusted to 5 × 10⁻⁶. 6 Add 0.5 mL / well to a 96-well plate according to the well plate layout design, and incubate statically at 37°C and 5% CO2 for at least 1 hour.

[0095] CpG ODN stimulation. The CpG ODN was diluted with complete culture medium, and 100 μL was added to each well of a 96-well plate, with three replicates per concentration. The working concentrations of CpG ODN were 0.019, 0.056, 0.167, 0.5, 1.5, and 4.5 μM. The group with added culture medium (without ODN) served as the blank control, and the group with added PMA served as the positive control. The plates were incubated statically at 37°C and 5% CO2 for approximately 16-24 hours.

[0096] ELISA method for cytokine content detection. The IFN-α content in the cell supernatant was detected according to the detection method provided in the instructions of each kit.

[0097] 3. Results:

[0098] See Table 1. As shown in Table 1, not all ODNs can stimulate IFN-α secretion by porcine PBMCs. ODN1 has the best ability to stimulate IFN-α secretion by porcine PBMCs; followed by ODN2; then ODN6; in addition, ODN3, ODN4, and ODN5 also have a certain degree of stimulating effect, but weaker than ODN6; in addition, ODN8 and ODN14 have a weak stimulating effect; while other sequences have no stimulating effect, consistent with the negative control (ODN20) and the group without added ODN (culture medium control group).

[0099] Table 1: Results of different sequences stimulating IFN-α secretion from porcine PBMCs

[0100]

[0101]

[0102]

[0103]

[0104] Example 2: Effect of CpG ODN on the immunogenicity of porcine reproductive and respiratory syndrome (PRRS) vaccine composition

[0105] The effect of CpG ODN on the immunogenicity of the porcine reproductive and respiratory syndrome (PRRS) vaccine composition was evaluated by detecting the level of PRRS virus-specific antibodies in immunized porcine serum. ODN1 was randomly selected in this embodiment.

[0106] 1. Reagents and materials:

[0107] One-month-old piglets, purchased from Huayu Pig Farm in Dezhou, Huanshan District, Shandong Province;

[0108] ODN1 was provided by Huapu Biotechnology (Hebei) Co., Ltd.

[0109] ISA 206 adjuvant, from SEPPIC;

[0110] PRRSV GP5 protein antigen, from Nanjing Anyan Biotechnology;

[0111] The saline solution was sourced from Zhejiang Dubang Pharmaceutical Co., Ltd.

[0112] 2. Method:

[0113] A porcine reproductive and respiratory syndrome (PRRS) vaccine composition was prepared using physiological saline as the solvent. Each 2 mL of the vaccine composition contained 500 μg of antigen, 25 μg of ODN1 (if available), 1 mL of ISA 206 adjuvant, and the remainder being physiological saline.

[0114] One-month-old piglets were randomly divided into three groups of five piglets each, and immunized according to Table 2. On day 0 (the day of immunization was designated as day 0), each group received a single intramuscular injection of 2 mL, with saline solution serving as a control. Blood samples were collected before immunization and at 1, 2, 3, 4, 5, and 6 weeks post-immunization. Serum was separated, and the levels of porcine reproductive and respiratory syndrome virus (PRRSV)-specific antibodies in the serum samples were detected using ELISA. Animal grouping is shown in Table 2.

[0115] Table 2: Animal Grouping

[0116]

[0117] 3. Results:

[0118] See Figure 1 .Depend on Figure 1 It can be seen that ODN1 combined with ISA 206 can significantly enhance the immunogenicity of the porcine reproductive and respiratory syndrome (PRRS) subunit vaccine composition. Compared with the ISA206 adjuvant vaccine group alone, the antibody in the combined adjuvant vaccine group reached its peak 2 weeks earlier and maintained a high level for 2-6 weeks after immunization.

[0119] Example 3: Effect of CpG on the Immunogenicity of Porcine Epidemic Diarrhea Vaccine Composition

[0120] The effect of CpG ODN on the immunogenicity of the porcine epidemic diarrhea virus (PEDV) vaccine composition was assessed by detecting the level of neutralizing antibodies against PEDV in the serum of immunized pigs using ODN3.

[0121] 1. Reagents and materials:

[0122] One-month-old piglets, purchased from Huayu Pig Farm in Dezhou, Huanshan District, Shandong Province;

[0123] ODN3 was provided by Huapu Biotechnology (Hebei) Co., Ltd.

[0124] Aluminum hydroxide gel adjuvant, from Invivogen;

[0125] The porcine epidemic diarrhea vaccine virus (PEDV, strain CV777) was provided by the Harbin Veterinary Research Institute. The inactivated virus antigen was prepared using conventional methods. First, the porcine epidemic diarrhea virus PEDV CV777 was proliferated in Vero cells, and the viral fluid was harvested. Then, the viral fluid was added to an inactivating agent for inactivation.

[0126] The saline solution was sourced from Zhejiang Dubang Pharmaceutical Co., Ltd.

[0127] 2. Method:

[0128] A porcine epidemic diarrhea vaccine composition was prepared using physiological saline as the solvent. Each 2 mL of the vaccine composition contained 375 μg of PEDV CV777 antigen, 50 μg of ODN3, 20% v / v aluminum hydroxide gel adjuvant (if available), and the balance being physiological saline.

[0129] One-month-old piglets were randomly divided into three groups of five piglets each, and immunized according to Table 2. On day 0 (the day of immunization was designated as day 0), each group received a single intramuscular injection of 2 mL. Blood samples were collected at 1, 2, 3, 4, and 5 weeks post-immunization, and serum was separated to detect the level of CV777 strain neutralizing antibodies in the serum samples. Animal grouping is shown in Table 3.

[0130] Table 3: Animal Grouping

[0131]

[0132] 3. Results:

[0133] See Figure 2 ,Depend on Figure 2 It can be seen that ODN3 alone can induce high levels of neutralizing antibodies in the porcine epidemic diarrhea vaccine composition earlier; while the ODN3 + aluminum adjuvant dual adjuvant vaccine group can not only induce high levels of neutralizing antibodies earlier, but also maintain high levels of neutralizing antibodies for a long time, and the immunogenicity of the vaccine is significantly improved.

[0134] Example 4: Effect of CpG on the Immunogenicity of Avian Newcastle Disease Bivalent Vaccine

[0135] The impact of CpG ODN on the immunogenicity of a bivalent avian influenza vaccine for poultry was assessed by detecting antibody levels in post-immunization chicken serum. Antibody hemagglutination inhibition titers (HI) were expressed as the logarithm (log2) of the reciprocal of the serum dilution factor.

[0136] 1. Reagents and materials:

[0137] Specific Pathogen Free (SPF) chickens were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0138] ODN1 was provided by Huapu Biotechnology (Hebei) Co., Ltd.

[0139] The commercially available Newcastle disease and avian influenza (H9 subtype) bivalent inactivated vaccine (La Sota strain + F strain) contains white oil and aqueous and oil phase emulsifiers, and is produced by Sinopharm Yangzhou Weike Biotechnology Co., Ltd.

[0140] The saline solution was sourced from Zhejiang Dubang Pharmaceutical Co., Ltd.

[0141] 2. Method:

[0142] In this experiment, 15-day-old chickens were vaccinated subcutaneously in the neck at a dose of 0.5 mL per chicken. Three groups were established: a NSF-MS bivalent vaccine group, a NSF-MS bivalent vaccine + 2.5 μg ODN1 group, and a saline control group. Each group contained 10 chickens.

[0143] Blood samples were collected at 14, 21, and 28 days post-immunization to separate serum and detect antibody levels in the chicken serum samples.

[0144] 3. Results:

[0145] See Figure 3 A, Figure 3 B, by Figure 3 A and Figure 3 As shown in B, ODN1 adjuvant can enhance the immunogenicity of the new influenza bivalent vaccine.

[0146] Example 5: Effect of CpG ODN on the Immunogenicity of Newcastle Disease Vaccine in Poultry

[0147] The impact of CpG ODN on the immunogenicity of Newcastle disease vaccines in poultry was assessed by detecting antibody levels in chicken serum after immunization.

[0148] 1. Reagents and materials:

[0149] White-feathered broiler chickens were purchased from a hatchery in Baihai'an.

[0150] ODN2 was provided by Huapu Biotechnology (Hebei) Co., Ltd.

[0151] Newcastle disease inactivated vaccine for chickens, trade name Xinbeian, purchased from Zhaoqing Dahua Agricultural Biopharmaceutical Co., Ltd.

[0152] Physiological saline was purchased from Zhejiang Dubang Pharmaceutical Co., Ltd.

[0153] 2. Method:

[0154] In this experiment, 7-day-old chickens were inoculated subcutaneously via the neck at a dose of 0.2 mL per chicken.

[0155] The effect of CpG on the immunogenicity of Newcastle disease vaccine was investigated in broiler chickens. Broiler chickens were divided into three groups: Newcastle disease vaccine group, Newcastle disease vaccine + 2 μg CpG ODN2 group, and saline control group, with 10 chickens in each group.

[0156] Blood samples were collected at 1, 7, 14, 21, 28, and 35 days after immunization, serum was separated, and antibody levels in the chicken serum samples were detected.

[0157] 3. Results:

[0158] See Table 4. As shown in Table 4, ODN2 adjuvant can enhance the immunogenicity of Newcastle disease vaccine.

[0159] Table 4:

[0160]

[0161]

[0162] Example 6: Effect of CpG ODN on the Immunogenicity of Avian Influenza Vaccine in Poultry

[0163] The impact of CpG ODN on the immunogenicity of avian influenza vaccines in poultry was assessed by detecting antibody levels in chicken serum after immunization.

[0164] 1. Reagents and materials:

[0165] White-feathered broiler chickens were purchased from a hatchery in Baihai'an.

[0166] ODN5 was provided by Huapu Biotechnology (Hebei) Co., Ltd.

[0167] Avian influenza inactivated vaccine (H9 subtype), trade name Gan'an, purchased from Sinopharm Yangzhou Weike Biotechnology Co., Ltd.;

[0168] The saline solution was sourced from Zhejiang Dubang Pharmaceutical Co., Ltd.

[0169] 2. Method:

[0170] In this experiment, 7-day-old chickens were inoculated subcutaneously via the neck at a dose of 0.2 mL per chicken.

[0171] The effect of CpG ODN on the immunogenicity of avian influenza vaccine was investigated in broiler chickens. Broiler chickens were divided into three groups: avian influenza vaccine group, avian influenza vaccine + 2 μg ODN5 group, and non-immunized control group, with 10 chickens in each group.

[0172] Blood samples were collected at 1, 7, 14, 21, 28, and 35 days after immunization, serum was separated, and antibody levels in the chicken serum samples were detected.

[0173] 3. Results:

[0174] See Table 5. As shown in Table 5, ODN5 adjuvant can enhance the immunogenicity of avian influenza vaccines.

[0175] Table 5:

[0176]

[0177]

[0178] Example 7: Effect of CpG ODN on the Immunogenicity of Infectious Bursal Disease Vaccine in Poultry

[0179] The impact of CpG ODN on the immunogenicity of infectious bursal disease vaccine in poultry was assessed by detecting antibody levels in chicken serum after immunization.

[0180] 1. Reagents and materials:

[0181] White-feathered broiler chickens were purchased from a hatchery in Baihai'an.

[0182] ODN6 was provided by Huapu Biotechnology (Hebei) Co., Ltd.

[0183] The genetically engineered subunit vaccine for infectious bursal disease in chickens was purchased from Qingdao Yibang Biotechnology Co., Ltd.

[0184] The saline solution was sourced from Zhejiang Dubang Pharmaceutical Co., Ltd.

[0185] 2. Method:

[0186] In this experiment, 7-day-old chickens were inoculated subcutaneously via the neck at a dose of 0.25 mL per chicken.

[0187] According to the vaccine instructions from the Yibang official website: [Function and Use] Used for the prevention of infectious bursal disease in chickens.

[0188] [Usage and Dosage] Subcutaneous or intramuscular injection in the neck. For chicks, administer 0.25 ml per chick at 1-3 weeks of age, with an immunity period of 3 months; for breeding chickens, administer 0.5 ml per chick 2 weeks before laying, with an immunity period of 6 months.

[0189] The effect of CpG ODN on the immunogenicity of infectious bursal disease (IBD) vaccine was investigated in broiler chickens. Broiler chickens were divided into three groups: an IBD vaccine group, an IBD vaccine + 2 μg CpG ODN group, and a non-immunized control group, with 10 chickens in each group.

[0190] Blood samples were collected at 1, 7, 14, 21, 28, and 35 days after immunization, serum was separated, and antibody levels in the chicken serum samples were detected.

[0191] 3. Results:

[0192] See Figure 4 .Depend on Figure 4 It is known that ODN6 adjuvant can significantly improve the immunogenicity of infectious bursal disease vaccine.

[0193] Example 8: Effect of CpG ODN on the immunogenicity of poultry egg drop syndrome vaccine

[0194] The impact of CpG ODN on the immunogenicity of poultry egg drop syndrome vaccine was assessed by detecting antibody levels in chicken serum after immunization.

[0195] 1. Reagents and materials:

[0196] The laying hens were purchased from a chicken farm in Puyang City, Henan Province.

[0197] ODN4 was provided by Huapu Biotechnology (Hebei) Co., Ltd.

[0198] Inactivated vaccine against egg drop syndrome (Jing 911 strain) was purchased from Qingdao Yibang Biotechnology Co., Ltd.

[0199] The saline solution was sourced from Zhejiang Dubang Pharmaceutical Co., Ltd.

[0200] 2. Method:

[0201] In this trial, commercial laying hens were vaccinated 14 to 28 days before the start of egg production, at a dose of 0.5 mL per hen via intramuscular or subcutaneous injection in the neck.

[0202] According to the vaccine instructions from the Yibang official website, [Function and Use] it is used to prevent egg drop syndrome in chickens.

[0203] [Usage and Dosage] Intramuscular or subcutaneous injection in the neck. Administer 0.5 ml per animal 14–28 days before the onset of labor.

[0204] The effect of CpG ODN on the immunogenicity of egg drop syndrome vaccine was investigated in commercial laying hens. Commercial laying hens were divided into three groups: an egg drop syndrome vaccine group, an egg drop syndrome vaccine + 2 μg ODN group, and a saline control group, with 10 hens in each group.

[0205] Blood samples were collected on the day of immunization and at 14, 21, 28, 35, and 48 days after immunization. Serum was separated, and antibody levels in the chicken serum samples were detected.

[0206] 3. Results:

[0207] See Table 6. As shown in Table 6, ODN4 adjuvant can enhance the immunogenicity of egg drop syndrome vaccine.

[0208] Table 6:

[0209]

[0210] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0211] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of CpG ODN in the preparation of a medicament for modulating immune cell activity in an animal.

2. Use according to claim 1, characterized in that, The modulating immune cell activity is promoting immune response of immune cells.

3. Use according to claim 2, characterized in that, The promoting immune response of immune cells includes promoting interferon production by immune cells.

4. Use according to claim 3, characterized in that, The interferon is INF-α.

5. Use according to any one of claims 1 to 4, characterized in that, The nucleotide sequence of the CpG ODN is having a sequence as set forth in any one of SEQ ID NO: 1-6.

6. Use according to any one of claims 1 to 4, characterized in that, The animal is livestock or poultry, preferably, the livestock is pig.

7. Use according to claim 1, characterized in that, The medicament is vaccine, the CpG ODN is vaccine adjuvant, and the modulating immune cell activity in an animal is promoting immune response of the animal against an antigen.

8. Use according to claim 7, characterized in that, The antibody titer of the vaccine using CpG ODN adjuvant is at least 20% higher than that of the vaccine without adjuvant at 2 weeks after immunization, preferably, the antibody titer is at least 50% higher, more preferably, the antibody titer is at least 100% higher.

9. Use according to claim 7, characterized in that, The antibody titer of the vaccine using CpG ODN adjuvant is at least 20% higher than that of the vaccine without adjuvant at 4 weeks after immunization, preferably, the antibody titer is at least 50% higher, more preferably, the antibody titer is at least 100% higher.

10. Use according to any one of claims 7 to 9, characterized in that, The nucleotide sequence of the CpG ODN is having a sequence as set forth in any one of SEQ ID NO: 1-6.

11. Use according to any one of claims 7 to 9, characterized in that, The antigen is a virus-based antigen, which includes virus, viral nucleic acid or viral protein.

12. Use according to any one of claims 7 to 11, characterized in that, The animal is mammal, in particular, the animal is pig.

13. Use according to claim 12, characterized in that, The antigen includes PRRSV (Nidovirales), arterivirus, Coronavirus, or antigen based on the virus.

14. Use according to claim 13, characterized in that, The antigen includes porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, or antigen based on the virus.

15. Use according to claims 13-14, characterized in that, The nucleotide sequence of the CpG ODN is having a sequence as set forth in any one of SEQ ID NO: 1-3.

16. The use according to any one of claims 7 to 11, characterized in that, The animal is bird, in particular, poultry.

17. Use according to claim 16, characterized in that, The antigen includes influenza virus, paramyxovirus, Avibirnavirus, adenovirus, or antigen based on the virus.

18. The use according to claim 17, characterized in that, The antigen includes influenza A virus, avian influenza virus, Newcastle disease virus, fowl inclusion body disease virus, poultry egg drop syndrome virus, or antigen based on the virus.

19. Use according to any one of claims 17-18, characterized in that, The nucleotide sequence of the CpG ODN is having a sequence as set forth in any one of SEQ ID NO: 3-6.

20. The use according to claim 7, characterized in that, The vaccine further comprises combining the CpG ODN with at least one of emulsion adjuvant, mineral salt adjuvant, TLR agonist, cytokine, saponin, and polymer.

21. The use according to claim 20, characterized in that, the emulsion adjuvant is selected from at least one of Montanide ISA 206, Montanide ISA 201, white oil adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, MF59, the mineral salt adjuvant is selected from at least one of an aluminium adjuvant or a calcium phosphate adjuvant; the aluminium adjuvant is at least one of aluminium hydroxide, aluminium phosphate, aluminium sulphate, potassium aluminium sulphate; the TLR agonist is selected from at least one of Poly (I:C), monophosphoryl lipid A, flagellin, Pseudomonas aeruginosa outer membrane lipoprotein OprI, E. coli heat-labile enterotoxin, blebs; the cytokine is selected from at least one of IFN-γ, IL-6, IL-7, IL-18, fish type I interferon; the saponin is selected from at least one of Quil-A, QS-21, immunostimulating complex; the polymer is selected from at least one of carbomer such as polyphosphazene, polyphosphazine.

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