Cpg oligodeoxynucleotides and uses thereof

Abstract

The application relates to the technical field of biological medicine, and particularly discloses CpG oligodeoxynucleotides and application thereof. The CpG oligodeoxynucleotide has a sequence as shown in any one of SEQ ID NO. 3-6, 9, and preferably has a sequence as shown in SEQ ID NO. 3-6. The CpG oligodeoxynucleotide can effectively activate the immune system of a human body or an animal, has the functions of preventing and treating pathogenic microorganism infection in a broad spectrum, has the function of improving the immunogenicity of a common vaccine, and can be applied as a human or animal vaccine adjuvant.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to CpG oligodeoxynucleotides and their applications. Background Technology

[0002] Vaccines can be divided into whole-pathogen vaccines and subunit vaccines. Whole-pathogen vaccines are traditional vaccines, composed of viral particles, bacteria, or other pathogens grown in a culture medium, either attenuated or inactivated to destroy their pathogenicity. Subunit vaccines mainly include recombinant protein vaccines, viral vector vaccines, and nucleic acid vaccines, and are usually classified as innovative vaccines. Innovative vaccines have become more attractive due to their safety and ease of scalability. However, these vaccines, because they use only a specific part of the organism, can only produce a weak immune response. Therefore, adjuvants and delivery systems are needed to deliver antigens in an appropriate manner and elicit a strong immune response.

[0003] Adjuvants are substances added to vaccine antigens to enhance or modulate their immunogenicity. Adjuvants offer numerous advantages, such as reducing the number of immunization injections, decreasing antigen dosage, and enhancing immune responses. Traditional aluminum salt adjuvants, including aluminum hydroxide and aluminum phosphate, and their combinations, are the most widely used. Discovered in 1926, they have been used for nearly 100 years and can activate Th2-type immune responses, i.e., humoral immune responses. However, in practical applications, the immune-enhancing effect of low-dose aluminum salt adjuvants alone is limited, while higher doses often cause side effects such as injection site swelling, granulomas, fever, pain, and allergic reactions. Therefore, the development of a more broad-spectrum, safe, effective, and mass-producible ideal adjuvant is urgently needed.

[0004] CpG-ODN is a synthetically produced oligodeoxynucleotide (ODN) containing unmethylated cytosine-guanine dinucleotide (CpG). It can mimic bacterial DNA to stimulate immune cells in various mammals, including humans. Based on different chemical structures and biological characteristics, different types of CpG-ODN exhibit varying structural features and immune effects, generally classified into three categories: A, B, and C. Category A CpG-ODN has a palindromic sequence containing CpG dinucleotides as its core, with polyG tails at both ends. The phosphodiester backbone is partially thiolated. The palindromic sequence and polyG form a higher-order structure, which can activate plasmacytoid dendritic cells and induce the production of large amounts of type I interferon, but has weak activity against B cells. Category B CpG-ODN is a fully thiolated linear CpG ODN, exhibiting strong immunostimulatory activity against B cells but unable to activate plasmacytoid dendritic cells. C-type CpG-ODN is a fully thiolated CpGODN that can form dimers through palindromic sequences. It possesses the activities of both type A and type B CpG-ODN, and can activate both plasmacytoid dendritic cells and B cells.

[0005] The immunostimulatory activity of CpG ODNs is structure-specific, and many factors influence its activity, with many aspects still requiring further investigation. Current research focuses on the backbone structure, flanking sequences, terminal PolyG modifications, and the number and position of motifs. Backbone structure includes the length of the oligonucleotide chain and backbone modifications, but the optimal chain length for strong immunostimulatory activity remains inconclusive. For example, Yamamot et al. suggested a minimum stimulatory sequence length of 30 bases, while Zuhai K et al. proposed a minimum of 15 bases. Other reports indicate stronger immunostimulatory activity at chain lengths of 20 bp, 21 bp, and 24 bp, and weaker at 16 bp, 27 bp, and 30 bp, but actual studies have revealed various deviations from this pattern. Furthermore, the immunostimulatory effect of CpG ODN is closely related to its free 5' end. Most researchers believe that the recognition and binding of CpG ODN by the TLR9 receptor is 5' end dependent. However, some researchers have found that the activity remains even after linking the 5' and 3' ends of the ODN to form a ring structure, indicating that the relationship between the immunostimulatory effect of CpG ODN and its structure is not conclusive. Moreover, although it is generally accepted in the field that the number, distance, spatial connection, and 5' base composition of CpG motifs in the sequence all affect the immunostimulatory activity of CpG ODN, a systematic analysis has yet to determine which design principles necessarily enhance the immunostimulatory effect.

[0006] Existing CpG oligodeoxynucleotides, such as CpG 1018, CpG7909, and CpG55.2, have been used clinically as immune enhancers or vaccine adjuvants, but their immune-enhancing effects still need further improvement. Summary of the Invention

[0007] The main objective of this invention is to provide a CpG oligodeoxynucleotide sequence that can effectively activate the human or animal immune system and has broad-spectrum prevention and treatment functions against pathogenic microorganisms.

[0008] The specific technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides CpG oligodeoxynucleotides having a sequence as shown in any one of SEQ ID NO. 3-6 or 9. Preferably, it has a sequence as shown in SEQ ID NO. 3-6.

[0010] The CpG oligodeoxynucleotide of this invention is an immunostimulatory oligonucleotide containing unmethylated cytosine guanine, with a size between 10 and 100 bases. This oligonucleotide is composed of unmodified or modified nucleotide monomers. It has broad-spectrum functions for the prevention and treatment of diseases caused by pathogenic microorganisms, including but not limited to viruses, bacteria, fungi, and parasites; it functions as an adjuvant in human or animal anti-infective vaccines; and it can achieve the purpose of preventing and treating diseases through systemic or local administration.

[0011] The CpG oligodeoxynucleotide of this invention is a synthetically produced, white, off-white to pale yellow, loosely purified CpG ODN product. This invention verifies its efficacy on B cells and T cells by detecting the activation effect of CpG ODN on IgG based on antibody levels in mouse serum, the effect of CpG ODN on cell proliferation based on mouse spleen cells, and the stimulatory effect of CpG ODN on the release of cytokines such as IFNγ and IL-2+ based on mouse spleen lymphocytes.

[0012] The CpG oligodeoxynucleotides (CpG ODN sequences) of the present invention may contain one or more chemical modifications, the types of which include, but are not limited to, one or more of thiomodification, methoxymodification, fluorination, and nanoparticle modification. Nanoparticle modification includes, but is not limited to, one or more of PLGA, chitosan, and liposomes.

[0013] Preferably, the CpG oligodeoxynucleotide is fully thiolated.

[0014] Unmethylated oligonucleotides are easily degraded in the body. This invention enhances the resistance of CpG ODN to nuclease degradation by modifying it with thiophosphate, thus prolonging its action time.

[0015] The CpG ODN sequence of the present invention may also include purification and desalting steps after artificial synthesis.

[0016] Secondly, the present invention provides the application of the above-mentioned CpG oligodeoxynucleotides in any of the following aspects:

[0017] (1) To prepare drugs that enhance the body's immune function;

[0018] (2) To prepare drugs that enhance the proliferation function of immune cells;

[0019] (3) Prepare drugs that enhance the release of cytokines from immune cells;

[0020] (4) Prepare drugs for the prevention and treatment of viral infections of the body;

[0021] (5) To prepare drugs for the prevention and treatment of respiratory bacterial infections;

[0022] (6) To prepare drugs for the prevention and treatment of respiratory fungal infections;

[0023] (7) To prepare drugs for the prevention and treatment of respiratory parasitic infections;

[0024] (8) To prepare drugs for treating tumors;

[0025] (9) Preparation of human vaccines;

[0026] (10) Prepare animal vaccines.

[0027] In the application of this invention, the virus is COVID-19 virus, influenza virus, human papillomavirus, or herpes zoster virus.

[0028] In the application of the present invention, the CpG oligodeoxynucleotide in the medicament for treating tumors is either the sole active ingredient, one of the active ingredients, or an adjuvant to the active ingredient.

[0029] In the application of this invention, the drug is administered either systemically or locally.

[0030] In the application of this invention, the administration methods of the drug include, but are not limited to, one or more of the following: nasal spray, pulmonary inhalation, oral administration, rectal administration, genital tract administration, subcutaneous injection, intradermal injection, intramuscular injection, intratumoral injection, intravenous injection, and mucosal application.

[0031] The CpG ODN sequence (immunomodulatory nucleic acid) of this invention can be either double-stranded or single-stranded. Generally, double-stranded molecules are more stable in vivo, while single-stranded molecules can enhance immune activity. Therefore, in this invention, single-stranded nucleic acid molecules are generally the preferred choice, but double-stranded molecules can also be selected according to other requirements.

[0032] When used for treatment, the immunomodulatory nucleic acid of the present invention can be used alone or in combination with other therapeutic agents. For example, the immunomodulatory nucleic acid can be used simultaneously with antigens to induce antigen-specific humoral or mucosal immune responses that can reduce or eliminate infectious pathogens.

[0033] The immunomodulatory nucleic acid of the present invention can also be used in combination with other therapeutic agents, including antimicrobial agents, adjuvants, cytokines, anticancer agents, therapeutic drugs, therapeutic drugs and other analogues.

[0034] When used for therapeutic purposes, an effective dose of immunomodulatory nucleic acid is administered by any method of delivering nucleic acid locally or systemically.

[0035] When administered orally, the immunomodulatory nucleic acid of this invention can be combined with pharmaceutically acceptable active compound excipients (e.g., fillers, dispersants, binders, etc.) to form formulations. These excipients enable the components of this invention to form tablets, pills, sugar-coated pills, capsules, liquid formulations, gels, syrups, ointments, suspensions, and similar formulations suitable for oral ingestion by the therapeutic subjects.

[0036] When administered by inhalation, the immunomodulatory nucleic acid of the present invention can be administered using a sealing device or a nebulizer containing a suitable propellant gas (such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).

[0037] When administered by injection, the injection solution may be a suspension, solution, or emulsion in an oil or water medium. It may contain preservatives, suspending agents, stabilizers, and / or dispersants.

[0038] The beneficial effects of this invention are at least as follows:

[0039] The novel CpG oligodeoxynucleotide sequence designed and synthesized in this invention can effectively activate the human or animal immune system, and has the function of broad-spectrum prevention and treatment of pathogenic microbial infections; it can enhance the immunogenicity of shared vaccines and can be used as an adjuvant for human or animal vaccines; compared with the already marketed CpG 1018, it can significantly improve the immune enhancement effect. Attached Figure Description

[0040] Figure 1 The results of the activation of IgG by different CpG ODNs in Example 2 of the present invention;

[0041] Figure 2 The results of the effect of different CpG ODNs on cell proliferation in Example 2 of this invention;

[0042] Figure 3 The results of the stimulatory effects of different CpG ODNs on the release of IFNγ and IL-2 cytokines in Example 2 of the present invention;

[0043] Figure 4 This illustrates the effects of different vaccine and adjuvant combinations on IgG activation in Example 3 of the present invention.

[0044] Figure 5 The results of the stimulatory effects of different vaccine and adjuvant combinations on IFNγ release in Example 3 of the present invention;

[0045] Figure 6 The results of the stimulation of IL-2 release by different combinations of vaccines and adjuvants in Example 3 of the present invention. Detailed Implementation

[0046] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Where specific techniques or conditions are not specified in the following examples, they are performed according to the techniques or conditions described in the literature in the art, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0047] Example 1: Design and Synthesis of CpG ODN Molecules

[0048] This invention designed a total of 9 CpG ODN sequences (of which sequences 1, 2, 7, and 8 are comparison sequences) and commissioned a gene synthesis company to artificially synthesize them.

[0049] Sequences 1-8 are B-type CpG ODNs without palindromic structures and fully thiolated, sequence 9 is a C-type CpG ODN with palindromic structures and fully thiolated, and CpG1018 is a control CpG.

[0050] Sequence 1: 5'-TCGTCGTTTTTGTTTTGTCGT-T-3' (SEQ ID NO.1);

[0051] Sequence 2: 5'-TCGTGCGTTTTTGTTTTGTCG-TTT-3' (SEQ IDNO.2);

[0052] Sequence 3: 5'-TCGTGCCGTGCCGTTTTGTCG-CGT-3' (SEQ IDNO.3);

[0053] Sequence 4: 5'-TCGTGCGTTTTTGTTTTGTCG-CGT-3' (SEQ IDNO.4);

[0054] Sequence 5: 5'-TCGTGCCGTTTTTGTCGCGTCG-CGT-3' (SEQ IDNO.5);

[0055] Sequence 6: 5'-TCGTCGTTTTTGTCGTTTGTCG-CGT-3' (SEQ IDNO.6);

[0056] Sequence 7: 5'-TCGTCGTTTTGTCGTT-3' (SEQ ID NO.7);

[0057] Sequence 8: 5'-TCGACGTTTTTGTTTTGACGT-TCT-3' (SEQ IDNO.8);

[0058] Sequence 9: 5'-TCGTCGTTTTTAAAAACGACG-A-3' (SEQ ID NO. 9);

[0059] CpG1018: 5'-TGACTGTGAACGTTCGAGATG-A-3' (SEQ ID NO. 10).

[0060] Example 2 Animal Experiment 1

[0061] This embodiment uses an animal model to evaluate the immune-boosting effect of the 10 CpG ODN sequences in Example 1 on the vaccine.

[0062] I. Mouse Immunization

[0063] C57BL / 6J mice (6-8 weeks old, 14-20 g, purchased from the Laboratory Animal Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences) were randomly divided into an experimental group and a control group, with 10 mice in each group. The experimental group received a subcutaneous injection of VZV (herpes zoster virus) vaccine containing different CpG ODNs sequences (the active ingredient was partially derived from GlaxoSmithKline) in the abdomen. The injection dose was 50 μl / mouse, the antigen immunization dose was 5 μg / mouse, and the CpG immunization dose was 10 μg / mouse. The control group received 50 μl of VZV vaccine without CpG ODNs sequences per mouse, and the antigen immunization dose was 5 μg / mouse. A booster immunization was given 14 days later.

[0064] II. ELISA kit for detecting specific antibody IgG

[0065] 1. Fourteen days after booster immunization, blood was collected from the eyes of C57BL / 6J mice. Blood from each mouse was collected in a 1.5ml EP tube and the blood sample was quickly placed in a 4°C freezer. After 4 hours, the serum was separated and centrifuged at 3500rpm for 15 minutes at 4°C using a refrigerated centrifuge. The supernatant was transferred to a new tube with a serial number and stored at -20°C for antibody detection.

[0066] 2. Remove the reaction plate and set up the experimental wells and control wells.

[0067] 3. Coat the 96-well plate (Corning) with antigen at a concentration of 10 μg / well and incubate overnight at 4°C.

[0068] 4. Discard the solution in the wells, seal the wells with PBS containing 2% bovine serum albumin, 200 μl / well, and incubate at 37°C for 2 hours.

[0069] 5. Spin dry and wash the board 3 times.

[0070] 6. Add 100 μl of PBS to each control well; add 100 μl of serum sample diluted 1:200 to each experimental well, and incubate in an incubator for 1.5 hours.

[0071] 7. Wash the plate, add 100 μl of horseradish peroxidase-labeled IgG detection antibody (Thermo Fisher Scientific) to each well, and incubate in an incubator for 1 hour.

[0072] 8. Discard the liquid, wash the plate 5 times, add 100 μl of colorimetric reagent (purchased from BD) to each well, mix gently for 10 seconds, and incubate at room temperature for 20 minutes.

[0073] 9. Add 100 μl of 0.2 M H2SO4 to each well to stop the reaction. Read the absorbance at 450 nm using a microplate reader within 30 minutes.

[0074] See results Figure 1 .from Figure 1 It can be seen that CpG ODNs have varying degrees of ability to promote IgG antibody secretion, with sequences 3-6 showing significant effects and sequence 6 exhibiting the best effect. The VZV antigen + sequence 6 group showed a 378% increase compared to the VZV antigen alone group and a 150% increase compared to the VZV antigen + CpG1018 group.

[0075] III. Preparation of Mouse Spleen Cells

[0076] Fourteen days after booster immunization, C57BL / 6J mice were euthanized by enucleation, blood was collected, and the spleens were aseptically harvested. In a petri dish containing a small amount of cell culture medium, the spleen was squeezed through a 200-mesh wire mesh using a syringe plunger to obtain a single-cell suspension. The cells were washed twice with washing buffer. The mixture was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and 1 ml of erythrocyte lysis buffer was added. The precipitate was bounced back, and the mixture was incubated at room temperature for 3-5 minutes. 4 ml of 1640 culture medium was added, mixed, and centrifuged at 1500 rpm for 5 minutes. The white precipitate at the bottom of the tube was resuspended in culture medium and washed once. The cells were bounced back, and 10% FBS 1640 culture medium was added. The mixture was gently agitated with a pipette. After trypan blue staining, the number of mononuclear cells was counted, and the percentage of viable cells was calculated.

[0077] IV. Determination of the proliferative activity of CpG ODNs on mouse spleen cells using the CCK-8 kit (GLPBIO)

[0078] 1. Seed 100 μl of cell suspension per well in a 96-well plate, resulting in a cell concentration of 1 × 10⁻⁶ cells. 5 The culture plate was placed in an incubator and pre-cultured for 24 hours with cells / ml.

[0079] 2. Add 10 μl of VZV vaccine containing different CpG sequences to the culture plate. The CpG concentration is 0.2 mg / ml and the antigen concentration is 0.1 mg / ml. A separate control group containing a VZV vaccine without CpG ODNs sequences has an antigen concentration of 0.1 mg / ml.

[0080] 3. Incubate the culture plate in an incubator for 96 hours.

[0081] 4. Add 10 μl of CCK-8 solution to each well.

[0082] 5. Place the culture plate in an incubator and incubate for 4 hours.

[0083] 6. Measure the absorbance at 450 nm using an ELISA reader.

[0084] See results Figure 2 .Depend on Figure 2 It can be seen that sequences 2, 7, and 8 have no significant promoting effect on cell viability. Sequences 3-6 have a more significant promoting effect on cell viability, with sequence 3 being the most significant. The absorbance of the VZV antigen + sequence 3 group is 5.5 times that of the VZV antigen alone group and 1.4 times that of the VZV antigen + CpG1018 group.

[0085] V. Detection of cytokine IFNγ and IL-2 levels in the supernatant of mouse spleen cell culture stimulated by CpG ODNs

[0086] 1. Dilute the capture antibody according to the instructions of the kit (IFNγ kit: U-CyTech; IL-2 kit: mabtech), 100 μl / well, and coat overnight at 4°C.

[0087] 2. The next day, pour out the coating solution, wash three times with sterile PBS, and then block with 1640 medium containing 10% FBS, 200 μl / well, at room temperature for 2 hours.

[0088] 3. Dilute mouse spleen cells to the required concentration using 10% FBS RPMI-1640 medium (IFNγ detection: 2 × 10⁻⁶). 5 Cells / ml; IL-2 detection: 5×10 5 (pcs / ml).

[0089] 4. Discard the blocking solution and add spleen lymphocytes (cell concentration: IFNγ detection: 2×10⁻⁶). 5 Cells / ml; IL-2 detection: 5×10 5 VZV vaccine containing CpG (0.2 mg / ml) and antigen (0.1 mg / ml) were added to each well. No irritant was added to the negative control well. ConA was used as a positive control. A VZV vaccine group without CpG ODNs sequence was set up as a control with an antigen concentration of 0.1 mg / ml. The mixture was incubated in an incubator for 48 hours.

[0090] 5. Pour off the culture medium and lyse the cells with 200 μl / well of ice-cold deionized water, incubate at 4°C for 20 minutes.

[0091] 6. Wash the plate 4 times with PBST, add 100 μl of biotin-labeled antibody per well, and incubate at room temperature in the dark for 1 hour.

[0092] 7. Wash the plate 4 times with PBST, add 100 μl of enzyme-labeled avidin to each well, and incubate at room temperature in the dark for 1 hour.

[0093] 8. Wash the plate 3 times with PBST, then wash it 2 times with PBS. Add 100 μl of substrate development solution per well and develop the color at room temperature in the dark for 15-20 minutes.

[0094] 9. The color reaction is stopped with deionized water, and the spots are counted using a spot analyzer.

[0095] See results Figure 3 From the cytokine expression level map ( Figure 3As can be seen from the results, the expression levels of IFNγ and IL-2 in the splenic lymphocytes of mice immunized with sequences 3-6 were significantly higher. IFNγ was 7.9–16.6 times higher than the unadjuvanted group and 3.5–7.2 times higher than the CpG1018 group; IL-2 was 6.0–7.6 times higher than the unadjuvanted group and 4.0–5.1 times higher than the CpG1018 group. In addition to sequences 3-6, sequence 9 also showed a significant promoting effect on the cytokines IFNγ and IL-2.

[0096] Example 3 Animal Experiment 2

[0097] As shown in Example 2, the immune-promoting effect of CpG ODN sequence 3-6 is significant. Therefore, this example further evaluates the immune-promoting effect of CpG ODN sequence 3-6 in Example 1 combined with different vaccines using an animal model.

[0098] I. Mouse Immunization

[0099] C57BL / 6J mice (6-8 weeks old, 14-20 g, purchased from the Laboratory Animal Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences) were randomly divided into 9 experimental groups and 3 control groups, with 10 mice in each group. The experimental groups received subcutaneous injections of different vaccine and adjuvant combinations in the abdomen: COVID-19 vaccine (Zhifei Biological), 100 μl / mouse, antigen immunization dose 5 μg / mouse; influenza vaccine (Hualan Biological's quadrivalent (H1N1, H3N2, Bv, and By) influenza virus split vaccine), 160 μl / mouse, antigen immunization dose 5 μg / mouse / subtype; HPV vaccine (Merck, bivalent (HPV16, HPV18)), 100 μl / mouse, antigen immunization dose HPV16: 8 μg / mouse, HPV18: 4 μg / mouse; CpG The immunization doses of ODN sequence 3-6 were 10 μg / animal; the immunization dose of CpG1018 was 10 μg / animal; and the immunization dose of aluminum hydroxide adjuvant was 50 μg / animal. The control group received the same amount of the corresponding vaccine without adjuvant as the experimental group, and a booster immunization was given 14 days later.

[0100] II. ELISA kit for detecting specific antibody IgG

[0101] 1. Fourteen days after booster immunization, blood was collected from the eyes of C57BL / 6J mice. Blood from each mouse was collected in a 1.5ml EP tube and the blood sample was quickly placed in a 4℃ freezer. After 4 hours, the serum was separated and centrifuged at 3500rpm for 15min at 4℃ using a refrigerated centrifuge. The supernatant was transferred to a new tube with a serial number and stored at -20℃ for antibody detection.

[0102] 2. Remove the reaction plate and set up the experimental wells and control wells.

[0103] 3. Coat the 96-well plate (Corning) with antigen at a concentration of 10 μg / well and incubate overnight at 4°C.

[0104] 4. Discard the solution in the wells, seal the wells with PBS containing 2% bovine serum albumin, 200 μl / well, and incubate at 37°C for 2 hours.

[0105] 5. Spin dry and wash the board 3 times.

[0106] 6. Add 100 μl of PBS to each control well; add 100 μl of serum sample diluted 1:200 to each experimental well, and incubate in an incubator for 1.5 hours.

[0107] 7. Wash the plates, add 100 μl of horseradish peroxidase-labeled IgG detection antibody (Thermo Fisher Scientific) to each well, and incubate in an incubator for 1 hour.

[0108] 8. Discard the liquid, wash the plate 5 times, add 100 μl of colorimetric reagent (purchased from BD) to each well, mix gently for 10 seconds, and incubate at room temperature for 20 minutes.

[0109] 9. Add 100 μl of 0.2 M H2SO4 to each well to stop the reaction. Read the absorbance at 450 nm using a microplate reader within 30 minutes.

[0110] See results Figure 4 .from Figure 4 The results show that CpG ODN sequence 3-6, when combined with COVID-19, influenza, and HPV vaccines, significantly boosted IgG immunity. In the COVID-19 vaccine group, the combination of CpG ODN sequence 3-6 with the vaccine increased IgG efficacy by 4–6 times, 3.5–5.4 times, and 0.7–1.4 times compared to the combination of the vaccine alone, CpG1018, and aluminum adjuvant with the vaccine, respectively. In the influenza vaccine group, the combination of CpG ODN sequence 3-6 with the influenza vaccine increased IgG efficacy by 7.8–11.1 times, 0.3–0.8 times, and 0.2–0.7 times compared to the combination of the vaccine alone, CpG1018, and aluminum adjuvant with the vaccine, respectively. In the HPV vaccine group, the combination of CpG ODN sequence 3-6 with the HPV vaccine increased IgG efficacy by 7.6–9.8 times, 1–1.6 times, and 2.4–3.3 times compared to the combination of the vaccine alone, CpG1018, and aluminum adjuvant with the vaccine, respectively. The effect of promoting IgG secretion was significantly improved compared to that of the clinically approved CpG1018 and aluminum adjuvant.

[0111] III. Preparation of Mouse Spleen Cells

[0112] Fourteen days after booster immunization, C57BL / 6J mice were euthanized by enucleation, blood was collected, and the spleens were aseptically harvested. In a petri dish containing a small amount of cell culture medium, the spleen was squeezed through a 200-mesh wire mesh using a syringe plunger to obtain a single-cell suspension. The cells were washed twice with washing buffer. The mixture was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and 1 ml of erythrocyte lysis buffer was added. The precipitate was bounced back, and the mixture was incubated at room temperature for 3-5 minutes. 4 ml of 1640 culture medium was added, mixed, and centrifuged at 1500 rpm for 5 minutes. The white precipitate at the bottom of the tube was resuspended in culture medium and washed once. The cells were bounced back, and 10% FBS 1640 culture medium was added. The mixture was gently agitated with a pipette. After trypan blue staining, the number of mononuclear cells was counted, and the percentage of viable cells was calculated.

[0113] IV. Detection of cytokine IFNγ and IL-2 levels in mouse spleen cell culture supernatant stimulated by different vaccine and adjuvant combinations

[0114] 1. Dilute the capture antibody according to the instructions of the kit (IFNγ kit: U-CyTech; IL-2 kit: mabtech), 100 μl / well, and coat overnight at 4°C.

[0115] 2. The next day, pour out the coating solution, wash three times with sterile PBS, and then block with 1640 medium containing 10% FBS, 200 μl / well, at room temperature for 2 hours.

[0116] 3. Dilute mouse spleen cells to the required concentration using 10% FBS RPMI-1640 medium (IFNγ detection: 2 × 10⁻⁶). 5 Cells / ml; IL-2 detection: 5×10 5 (pcs / ml).

[0117] 4. Discard the blocking solution and add spleen lymphocytes (cell concentration: IFNγ detection: 2×10⁻⁶). 5 Cells / ml; IL-2 detection: 5×10 5 (samples / ml), 100 μl / well. Vaccines with different adjuvants were added to the experimental wells. The concentrations of CpG ODN sequences 3-6 were 0.2 mg / ml, CpG1018 was 0.2 mg / ml, aluminum hydroxide was 1.0 mg / ml, and the antigen concentrations were 0.05 mg / ml for COVID-19, 0.03 mg / ml for all four influenza subtypes, 0.08 mg / ml for HPV16, and 0.04 mg / ml for HPV18. No irritant was added to the negative control wells. ConA was used as a positive control. A separate control group without adjuvants was set up, with the same antigen concentration as the experimental groups. The mixtures were incubated for 48 hours.

[0118] 5. Pour out the culture medium and lyse the cells with 200 μl / well of ice-cold deionized water, incubate at 4°C for 20 minutes.

[0119] 6. Wash the plate 4 times with PBST, add 100 μl of biotin-labeled antibody per well, and incubate at room temperature in the dark for 1 hour.

[0120] 7. Wash the plate 4 times with PBST, add 100 μl of enzyme-labeled avidin to each well, and incubate at room temperature in the dark for 1 hour.

[0121] 8. Wash the plate 3 times with PBST, then wash it 2 times with PBS. Add 100 μl of substrate development solution per well and develop the color at room temperature in the dark for 15-20 minutes.

[0122] 9. The color reaction is stopped with deionized water, and the spots are counted using a spot analyzer.

[0123] See results Figures 5-6 From the expression levels of IFNγ and IL-2 Figures 5-6 The results show that sequences 3-6, when combined with COVID-19 vaccines, influenza vaccines, and HPV vaccines, significantly increased the stimulatory effect on IFNγ and IL-2 secretion. Figure 4 Analysis showed that sequences 3-6 are more suitable for use in combination with COVID-19 vaccines, influenza vaccines, and HPV vaccines compared to CpG1018 and aluminum adjuvants.

[0124] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. CpG oligodeoxynucleotides, characterized in that, The nucleotide sequence is shown in SEQ ID NO.3; the CpG oligodeoxynucleotide has undergone full thiolation modification.

2. The use of the CpG oligodeoxynucleotide of claim 1 in any of the following aspects: (1) To prepare drugs that enhance the body's immune function; (2) To prepare drugs that enhance the proliferation function of immune cells; (3) Prepare drugs that enhance the release of cytokines from immune cells; (4) Prepare drugs for the prevention and treatment of viral infections of the body; the virus is COVID-19 virus, influenza virus, human papillomavirus or herpes zoster virus; (5) Preparation of human vaccines; (6) Prepare animal vaccines.

3. The application according to claim 2, characterized in that, The drug can be administered systemically or locally.

4. The application according to claim 3, characterized in that, The administration methods of the drug include, but are not limited to, one or more of the following: nasal spray, pulmonary inhalation, oral administration, rectal administration, genital administration, subcutaneous injection, intradermal injection, intramuscular injection, intratumoral injection, intravenous injection, and mucosal application.

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