CpG ODN with immunomodulatory function and its use

Abstract

Provided are immunomodulatory CpG ODNs chemically modified with the structure represented by general formula I, and uses thereof. The CpG ODNs have immunostimulatory activity, can stimulate B cell proliferation, and can produce specific cytokines. The CpG ODNs can be used alone as vaccine adjuvants or in combination with other adjuvants to exert synergistic effects. They can also be used in the preparation of drugs for preventing or treating tumors, infections, and allergies.

Description

[Technical Field]

[0001] The present invention relates to a CpG ODN with immunomodulatory function and its use. The CpG ODN has good immunostimulatory activity, can stimulate B cell proliferation, and can produce cytokines. It can be used alone as a vaccine adjuvant or in combination with other adjuvants to achieve synergistic effects, and can also be used in the preparation of drugs for preventing or treating tumors, infections, and allergies. [Background technology]

[0002] In 1984, the nucleic acid component of Bacillus Calmette-Guerin (BCG) was discovered to have antitumor activity in a mouse tumor-bearing model, and it was successfully used to treat human tumors (Tokunaga T, Yamamoto H, Shimada S, et al. Antitumor activity of deoxyribonucleic acid fraction from mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity. J Natl Cancer Inst, 1984, 72(4):955-962). After degradation analysis using ribonucleases and deoxyribonucleases, it was proven that the actual active component was bacterial deoxyribonucleic acid (Tokunaga T, Yamamoto S, Namba KA. Synthetic single-stranded DNA, Poly(dG, dC), induces interferon-alpha / beta and gamma, augments natural killer activity, and suppresses tumor growth. Jpn J Cancer Res. 1988;79(6):682-686; Tokunaga T, Yano O, Kuramoto E, et al. Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of mycobacterium bovis BCG induce interferon and activate natural killer cells. Microbiol Immunol. 1992;36(1):55-66). Sequence analysis showed that immunoreactive oligodeoxynucleotides (ODNs) contain at least one or more CG dinucleotides in their sequences, with the CGs linked by phosphorus (P). Therefore, oligodeoxynucleotides (ODNs) were collectively called CpG oligodeoxynucleotides (CpG ODNs), also known as immune stimulatory sequences (ISSs).

[0003] The immunostimulatory activity of CpG ODNs is influenced by their structure. CG dinucleotides are abundant in the genomes of pathogenic microorganisms such as bacteria. However, CG dinucleotides are rarely found in the genomes of humans and vertebrates. Even if CG dinucleotides are present in the genomes of humans and vertebrates, the cytosine and guanine nucleotides within them are usually methylated. Deletion, inversion, and methylation of CpG dinucleotides can result in loss of activity, indicating that the presence of unmethylated CpG dinucleotides in CpG ODNs is the basis for their immunostimulatory activity (Ling Shih-hwan. Research Progress on Immunoactive Oligodeoxynucleotides CpG [J]. Chinese Journal of Microbiology and Immunology, 2008, 28(6):571-576). In CpG ODNs, multiple deoxynucleotides are regularly arranged in diverse combinations. However, the immunoactivity of CpG ODNs with different sequence structure features varies significantly, and alterations of one or several nucleotides in the sequence can significantly affect their immunoactivity. Therefore, understanding and grasping the relationship between structure and activity will help us design more sequences with immune activity.

[0004] For CpG ODNs that have the activity of stimulating B lymphocytes in mice, their base sequences generally follow the following rule: the two bases closest to the 5' end of the CpG dinucleotide are generally purines, preferably GpA, and the two bases closest to the 3' end are generally pyrimidines, preferably TpC or TpT (Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation [J]. Nature, 1995, 374(6522):546-549). C or G near the 5' end of the CpG dinucleotide can significantly inhibit the activity of CpG ODN in stimulating NK lymphocytes in mice, while C near the 3' end has little effect on activity. The 5'-terminal GA GACGTT / C CpG motif has a strong effect on mice, whereas the 5'-terminal GA GACGTT / C or GGCGTT / C motifs, obtained by substituting GC or GG, show reduced immunoreactivity. Human peripheral blood mononuclear cells can be activated by motifs containing "GTCGTT," "TTCGTT," or "AACGTT," with the most potent motif being GTCGTT (Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA [J]. J Immunol, 1996, 157(5):1840-1845). Multiple TCG repeats help enhance the stimulatory activity of CpG ODNs on human B cells and NK cells (Hartmann G, Krieg AM. Mechanism and function of a newly identified CpG DNA motif in human primary B cells [J]. J Immunol, 2000, 164(2):944-953). T7 and T8, which contain multiple TCG repeats, also have good immunostimulatory activity on human PBMCs.Multiple TCG repeats form a GTCGTC motif, which has been shown to have strong immunostimulatory activity in humans when the 3' end of a CpG dinucleotide is TpC (Xu Honglin, Wang Siqing, Wang Shifeng. Two CpG motifs highly activate human immune cells [J]. Chinese Journal of Microbiology and Immunology, 2001, 21(5):471-475). CpG ODN sequences contain two or more copies of the 5'-NTCGTT-3' motif, which is 15-35 nucleotides in length, where N does not represent A or G. Such CpG ODNs have good immunostimulatory activity in human and mouse immune cells in vitro (Xu Honglin. Thioligodeoxynucleotides with immunostimulatory activity and their uses, CN 101492672 A). The core sequence of CpG ODN is a six-nucleotide motif with the general formula: 5'-X1-X2-CG-Y1-Y2-3', where X1 is a purine base nucleotide, X2 is either purine or thymine (T), and Y1 and Y2 are both pyrimidines. In addition to this six-nucleotide motif, the surrounding sequences and sequences between multiple CpGs also affect the activity of CpG ODN (Krieg AM, Hartmann G, Yi AK. Mechanism of action of CpG ODN A. Curt Top Microbiol, 2000. 247(1):1-21). Other studies have found that the sequences of bases flanking both sides of CpG mostly follow the 5'PurPurCGPyrPyr3' rule, i.e., two purines at the 5' end and two pyrimidines at the 3' end (G. Mutwiri, R. Pontarollo, S. BaBIUK. Biological activity of immunostimulatory CpG ODN motif in domestic animals. Veterinary Immunopathology, 2003, 91:89-103).

[0005] TLR9 is a member of the Toll-like receptor (TLR) family, which primarily recognizes CpG motifs in bacterial DNA. CpG ODN can stimulate an individual's innate immune response in a TLR9-dependent manner. CpG ODN in viral and bacterial genomes is a natural agonist of TLR9. Therefore, when cells are infected with bacteria or bacteria are ingested, TLR9 initiates a Th1-dominant immune response (AHLERS JD, BELYAKOV I M. Memories that last forever: Strategies for optimizing vaccine T-cell memory [J] Blood, 2010, 115(9);1678-1689).

[0006] CpG ODN not only stimulates TLR9-expressing cells, triggering an immunoregulatory cascade that ultimately leads to the production of pro-inflammatory cytokines and chemokines, but also improves the antigen-presenting function of dendritic cells, monocytes, and macrophages, induces B cell proliferation, stimulates the immunoprotective activity of NK cells, and induces in vivo immune responses. Therefore, CpG ODN is a highly efficient and low-toxicity immunoadjuvant and is highly valuable for the treatment of diseases (Sun SQ, Zhang XH, Tough D F. Type I interferon-mediated stimulation of T cells by CpG DNA [J] J. Exp Med. 1998, 188(12):2335-2342).

[0007] The vaccine adjuvant activity of CpG ODN has been demonstrated in numerous animal studies of prophylactic and therapeutic vaccines. In mouse models, CpG ODN has been used in combination with a variety of vaccines, including those against papillomavirus, hepatitis B virus, brucellosis, chlamydia, HIV, aspergillus, mycobacteria, trypanosomes, myxovirus, hepatitis C virus, cytomegalovirus, dengue virus, rabies virus, and influenza virus. Since 2000, more than 20 vaccines using CpG ODN as adjuvants have been clinically investigated (KRIEG AM. Therapeutic potential of Toll-like receptor 9 activation [J]. Nat Rev Drug Discov. 2006;5(6):471-484; Dennis M Klinman. CpG DNA as a vaccine adjuvant [J]. Expert Rev Vaccines. 2003;2(2):305-315; KRIEG AM. CpG still rocks! Update on an accidental drug [J]. Nucleic Acid Ther. 2012;22(2):77-89). Type B CpG ODN has been the primary subject of experimental research, and it is being used as a vaccine adjuvant for the prevention and treatment of infectious diseases. The HEPLISAV-B hepatitis B vaccine by Dynavax was approved by the FDA in November 2017.

[0008] The inhibitory effect of CpG ODN on viruses has been demonstrated in experiments with respiratory syncytial virus, hepatitis virus, HIV, etc. (Liu S, Sun W, Cao Y. Study on anti-HBV effects by antisense oligodeoxynucleotides in vitro. China Journal of Preventive Medicine, 2001, 35(5), 338-340; Lund OS, Hansen JE. Inhibition of HIV-1 replication by chimeric phosphorothioate oligodeoxynucleotides applied in free solution. Intervirology, 1998, 41(2-3), 63-68).

[0009] CpG ODN has antitumor activity in many mouse models, and when tumors are relatively small, CpG ODN alone can effectively induce T cell-mediated tumor rejection. However, for large tumors, CpG ODN must be combined with other treatments, such as monoclonal antibodies, radiation therapy, surgery, and chemotherapy, to achieve a strong synergistic effect. CpG ODN-mediated tumor regression can be T cell-dependent and NK cell-independent, or NK cell-dependent and T cell-independent. As an adjuvant for melanoma polypeptide antigen vaccines, CpG 7909 can significantly improve survival in tumor-bearing patients and induce strong melanoma protein antigen-specific CD8+ T cell responses (van Ojik, H. et al. Phase I / II study with CPG 7909 as an adjuvant to vaccination with MAGA-3 protein in patients with MAGA-3 positive tumors. Ann Oncol 2002, 13, 157; Speiser, DE et al. Rapid and strong human CD8(+) T cell responses to vaccination with peptide, IFA, and CPG oligodeoxynucleotide 7909. J Clin Invest 2005, 115, 739-746).

[0010] CpG ODN, either alone or in combination with antitumor antibodies, can induce Th1 cytokine secretion and enhance ADCC activity (Hartmann, E. et al. Identification and functional analysis of tumor-infiltrating plasmacytoid dendritic cells in head and neck cancer. Cancer Res 2003:63, 6478-6487). The combination of type B 1018 ISS and rituximab has been highly effective in the treatment of non-Hodgkin's lymphoma and is currently in clinical trials (Friedberg, J. et al. Combination immunotherapy with a CPG oligonucleotide (1018 ISS) and rituximab in patients with non-Hodgkin's lymphoma: increased interferon-α / β-inducible gene expression, without significant toxicity. Blood 2005:105, 489-495).

[0011] In clinical trials, cancer cell growth and metastasis were inhibited to a certain extent in some patients with recurrent glioblastoma who received peritumoral injections of CpG ODN followed by radiation therapy (Senti G, Johansen P, Haug S, et al. Use of A-type CpG oligodeoxynucleotides as an adjuvant in allergen-specific immunotherapy in humans: a phase I / IIa clinical trial[J]. Clinic Experim Allergy, 2009, 39(4):562-570; Carpentier A, Laigle-Donadey F, Zohar S, et al. Phase 1 trial of a CpG oligodeoxynucleotide for patients with recurrent glioblastoma[J]. Neuro-Oncol, 2006, 8(1):60-66). Other clinical trials have shown that intratumoral injection of CpG 7909 achieved complete regression of melanoma tumors but failed to inhibit distant tumor growth in five patients with metastatic melanoma (Pashenkov M, Goess G, Wagner C, et al. Phase II trial of a toll-like receptor 9-activating oligonucleotide in patients with metastatic melanoma[J]. Clinical Oncol Official J American Society Clinic Oncol, 2006. 24(36):5716-5724). Data from two phase 3 clinical trials of CpG 7909 showed a failure to improve clinical outcomes compared with chemotherapy alone. The field needs to continue discovering novel immunomodulatory polynucleotides.

[0012] CpG ODN is a highly efficient and low-toxicity immune adjuvant with great potential value in the treatment of infectious diseases, immunodeficiency diseases, tumors, and allergic diseases. However, its current practical use is limited, and more in-depth research is needed to design more effective sequence structures so that they can more widely exert their potential and be used in clinical practice more safely and efficiently. Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention provides a series of CpG ODNs with immunomodulatory functions. The structures of these CpG ODNs are novel, and they have immunostimulatory effects in both mice and humans, making them of great clinical value. Specifically, the present invention solves the problems in the field through the following technical solutions: [Means for solving the problem]

[0014] 1. An immunomodulatory CpG ODN comprising or consisting of a nucleotide sequence selected from SEQ ID NOs: 1 to 6, wherein at least one nucleotide in the nucleotide sequence is a chemically modified nucleotide having a structure shown in general formula I: [ka] (wherein Y is S or O, R is H or a positively charged counterion, B is independently an unmodified or modified nucleobase, and R1 is H, F, Cl, OH, OMe, Me, O-ethyloxymethyl).

[0015] 2. The immunomodulatory CpG ODN according to item 1, wherein Y is S.

[0016] 3. The immunomodulatory CpG ODN according to item 1 or 2, wherein all nucleotides in the nucleotide sequence of the CpG ODN are chemically modified nucleotides having the structure shown in general formula I.

[0017] 4. The immunomodulatory CpG ODN according to Item 3, wherein the sequence of the immunomodulatory CpG ODN is selected from SEQ ID NOs: 1 to 6, preferably SEQ ID NOs: 1 to 6 which are all phosphorothioated, and more preferably SEQ ID NO: 3 or 6 which are all phosphorothioated.

[0018] 5. A pharmaceutical composition comprising the immunomodulatory CpG ODN according to any one of items 1 to 4 and a pharmaceutically acceptable carrier.

[0019] 6. Use of an immunomodulatory CpG ODN according to any one of items 1 to 4 in the preparation of a vaccine adjuvant.

[0020] 7. The use according to item 6, wherein the vaccine is a rabies vaccine, and the amount of CpG ODN is preferably 0.01 μg to 1000 μg / ml, more preferably 1 to 10 μg / ml, for example, 1, 3, or 10 μg / ml.

[0021] 8. The use according to item 6, wherein the vaccine is a SARS-COV-2 vaccine, preferably an inactivated SARS-COV-2 vaccine, and the amount of CpG ODN is 0.01 μg to 1000 μg / ml.

[0022] 9. The use according to any one of items 6 to 8, wherein the vaccine adjuvant further comprises one or more other adjuvants that act in synergy with the immunomodulatory CpG ODN, such as insoluble aluminum salt colloids, oil-water emulsions, microorganisms and metabolites, nucleic acids and their analogs, cytokines, immune stimulating complexes, propolis, and liposomes.

[0023] 10. Use of an immunomodulatory CpG ODN according to any one of items 1 to 4 or a pharmaceutical composition according to item 5 in the preparation of a medicament for preventing or treating a tumor, microbial infection or allergy in a subject.

[0024] 11. The subject is a human or an animal, for example, a livestock such as a dog, pig, cow, or horse, a poultry such as a chicken, duck, or goose, a mouse, or a rat. 10 Use as described in.

[0025] 12. A vaccine comprising the immunomodulatory CpG ODN according to any one of items 1 to 4 and an antigen, wherein the antigen is a rabies antigen or a SARS-COV-2 antigen.

[0026] 13. The vaccine according to Item 12, wherein the vaccine is for human or animal use, the immunomodulatory CpG ODN is a phosphorothioated immunomodulatory CpG ODN shown in SEQ ID NO: 3, and the amount of the CpG ODN is 0.01 μg to 1000 μg / ml, more preferably 1 to 10 μg / ml, for example, 1, 3, or 10 μg / ml.

[0027] 14. The vaccine according to item 12, wherein the vaccine is a SARS-COV-2 vaccine, preferably an inactivated SARS-COV-2 vaccine, and the vaccine further comprises an aluminum adjuvant, such as an aluminum hydroxide adjuvant.

[0028] 15. The vaccine of item 14, wherein the immunomodulatory CpG ODN is a fully phosphorothioated immunomodulatory CpG ODN as set forth in SEQ ID NO: 6.

[0029] 16. The vaccine according to item 15, wherein the content of the antigen is 1 to 10 μg / mL, for example, 2, 4, or 8 μg / mL; the content of the aluminum hydroxide is 1 to 1000 μg / mL, preferably 300 to 500 μg / mL; and the content of the immunomodulatory CpG ODN is 1 to 1000 μg / mL, preferably 2 to 500 μg / mL, for example, 5 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, or 400 μg / mL. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 shows the effect of CpG ODN on the proliferation of mouse splenic T and B cells. [Figure 2] FIG. 2 shows the effect of CpG ODN on stimulating mouse splenocytes to produce the cytokine IFN-α. [Figure 3] FIG. 3 shows the effect of CpG ODN on stimulating mouse splenocytes to produce the cytokine IL-6. [Figure 4] FIG. 4 shows the effect of CpG ODN on stimulating mouse splenocytes to produce the cytokine TNF-α. [Figure 5] FIG. 5 shows the effect of CpG ODN on human PBMC T and B cell proliferation. [Figure 6] FIG. 6 shows the effect of CpG ODN to stimulate the secretion of IFN-α in human PBMCs. [Figure 7] FIG. 7 shows the effect of CpG ODN to stimulate IL-6 secretion in human PBMCs. [Figure 8] FIG. 8 shows the effect of CpG ODN to stimulate the secretion of TNF-α in human PBMCs. [Figure 9] FIG. 9 shows the results of detecting CpG stimulation of HEK-Blue hTLR9 cells. [Figure 10] FIG. 10 shows the detection results of CpG stimulating HEK-Blue mTLR9 cells. [Figure 11]FIG. 11 shows the results of detecting CpG stimulation of Ramos-Blue cells. [Figure 12] FIG. 12 shows the effect of ODN3 on rabies virus neutralizing antibody titers. [Figure 13] FIG. 13 shows the immune enhancing effect of different dosages of ODN3 in combination with rabies vaccine. [Figure 14] FIG. 14 shows the immune enhancing effect of different dosages of rabies vaccine in combination with ODN3. [Figure 15] FIG. 15 shows the effect of different CpG ODN and rabies vaccine combinations on rabies virus neutralizing antibody titers. [Figure 16] Figure 16 shows the effect of ODN6 in combination with an inactivated SARS-CoV-2 vaccine on mouse anti-SARS-CoV-2 S protein-specific IgG antibody titers at D6-28. [Figure 17] Figure 17 shows the effect of ODN6 in combination with an inactivated SARS-COV-2 vaccine on mouse anti-SARS-CoV-2 S protein-specific IgG antibody titers at D28. [Figure 18] Figure 18 shows the effect of ODN6 in combination with an inactivated SARS-COV-2 vaccine on mouse anti-SARS-CoV-2 neutralizing antibody titers. DETAILED DESCRIPTION OF THE INVENTION

[0031] definition CpG ODN

[0032] The CpG ODN of the present invention is an unmethylated dinucleotide linked by a phosphodiester bond and has immunostimulatory activity. CpG ODN can promote the proliferation and differentiation of B cells and the secretion of IL-6, thereby inducing the secretion of antibodies and activating presenting cells such as monocytes, macrophages, and dendritic cells to secrete various cytokines (e.g., IL-12, IL-6, TNF-α, IFN-α, and IFN-β). Cytokines indirectly promote the activity of killer T cells (CTLs) and natural killer cells (NK cells), induce cellular immunity against intracellular pathogens, and induce the secretion of IFN-γ from NK cells and T cells. In addition to inducing natural immune responses, CpG ODN can also enhance antigen-specific responses for the following reasons: (1) there is a strong synergistic effect between the signaling pathway initiated by B cell antigen receptors and the B cell signaling pathway initiated by CpG; (2) CpG ODN can increase antigen-specific T helper Th1-like cytokines, thereby enhancing the antigen-specific responses of B cells and T cells; and (3) cellular responses require positive regulation by costimulatory molecules.

[0033] As early as the 1890s, it was discovered that injecting bacterial extracts into cancer patients significantly reduced their disease. Subsequent studies showed that bacterial DNA had direct immunostimulatory and antitumor effects. Experimental studies with synthetic oligodeoxynucleotides revealed that the immunostimulatory effects of bacterial DNA were related to the unmethylated CpG dinucleotides contained within it.

[0034] CpG ODNs with immunostimulatory activity have the following basic structural features:

[0035] a. The CpG motif is the basic structure by which CpG ODN produces immunostimulatory activity, and is composed of a CpG dinucleotide and two bases at its 5' and 3' ends.

[0036] b. The purines and pyrimidines on either side of the CpG and the spacing of the CpG can affect the immunostimulatory activity and characteristics of action of CpG ODN.

[0037] c. Regarding the number of CpG motifs contained in an ODN, 2 to 4 CpG motifs is usually optimal, and the interval between CpG motifs is usually at least 2 bases (preferably thymine).

[0038] d. CpG ODN containing a poly-G sequence (consisting of three or more guanines) has a strong effect on stimulating plasmacytoid dendritic cells (pDCs) to produce interferon-α. All-thio-modified CpG ODN is the most stable and has the best stimulatory effect on B cells, but the effect of all-thio-modified CpG ODN on stimulating pDCs to produce IFN-α is weaker than that of partially-thio-modified CpG ODN.

[0039] Based on their functional characteristics, CpG ODNs can be divided into three types (Tomoki Ito, et al., Blood, 2006, Vol. 107, Num. 6:2423-2431):

[0040] (1) Type A CpG ODNs, which are synthesized using a chimeric backbone with phosphorothioate at the 5' and 3' ends and phosphodiester in the middle CpG region, can effectively activate natural killer cells (NK cells) and plasmacytoid dendritic cells (pDC cells) to produce large amounts of IFN-α, but can only activate B cells to a limited extent.

[0041] (2) Type B CpG ODN, synthesized via a nuclease-resistant phosphorothioate backbone, effectively activates B cells and pDC cells, induces IL-12 production, and induces antibody secretion, but only activates NK cells to a limited extent. Type B CpG ODN is generally effective as a vaccine adjuvant.

[0042] (3) C-type CpG ODN, which is synthesized via a phosphorothioate backbone and has a stimulatory activity between that of A-type CpG ODN and that of B-type CpG ODN. For example, it can effectively activate B cells, but also NK cells and pDC cells.

[0043] The immunomodulatory CpG ODN used in the present invention comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 1 to 6, wherein at least one nucleotide in the nucleotide sequence is a chemically modified nucleotide having the structure shown in general formula I: [ka] wherein Y is S or O, particularly S; R is H or a positively charged counterion; B is independently an unmodified or modified nucleobase; and R is H, F, Cl, OH, OMe, Me, O-ethyloxymethyl, where Me represents methyl.

[0044] The bases in the CpG ODNs of the present invention may be unmodified, partially modified, or fully modified nucleobases (wherein natural nucleobases include adenine, guanine, cytosine, and thymine). Modification of the CpG ODN backbone may include partial or complete phosphorothioate modification of the bases in the CpG ODNs of the present invention. The modification may be performed during or after the synthesis of the oligonucleotide, and may occur on the phosphodiester bridge between nucleosides, on the ribose unit, and / or on the natural nucleobases (i.e., adenine, guanine, cytosine, and thymine). When modified during the synthesis of the oligonucleotide, the modified base may be incorporated into the oligonucleotide or at the end of the oligonucleotide. When modified after the synthesis of the oligonucleotide, the modification may be performed using an active group, for example, an amino group-modifying moiety, a 3' or 5' hydroxyl group, or a phosphate group.

[0045] The chemical modification in the present invention may include a backbone modification in the CpG ODN of the present invention, which is a stable sugar phosphate backbone of a nucleic acid molecule, in which a sulfur replaces the oxygen of a non-bridging phosphate on at least one internucleotide bond, or a sulfur replaces the oxygen of a non-bridging phosphate on each or every other internucleotide bond, including, but not limited to, modifying the backbone with phosphorothioate to obtain a phosphorothioated backbone.Other modifications to the oligonucleotide backbone can also be made, for example, by using nonionic DNA analogs, such as alkyl phosphates and aryl phosphates, to modify the oligonucleotide backbone, in which the oxygen in the charged phosphate is replaced with an alkyl or aryl group, or by using phosphodiesters and alkylphosphotriesters to modify the backbone, in which the charged oxygen is alkylated.

[0046] The immunomodulatory CpG ODN of the present invention has a novel sequence structure and has immunostimulatory effects in both mice and humans, making it of great clinical value.

[0047] In certain embodiments, the sequence of the immunomodulatory CpG ODN of the present invention is ODN3 or ODN6, which comprises at least one chemically modified nucleotide having the structure shown in general formula I, wherein the substituents in general formula I are as defined above.

[0048] In one embodiment, the present invention also provides a pharmaceutical composition comprising an immunomodulatory CpG ODN described herein and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical formulation that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0049] subject

[0050] As used herein, the term "subject" refers to animals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, pigs, cats, rabbits, rats, mice, fish, birds, poultry, such as chickens, ducks, geese, etc. Preferably, the animal is a mammal. In a preferred embodiment, the subject is a human.

[0051] immune cells

[0052] In the present invention, immune cells refer to all cells and their precursor cells that are involved in and related to immune responses, including T cells (e.g., CD4+ cells, CD8+ cells, and various other T cell subtypes), B cells (e.g., CD19), natural killer cells (NK cells), macrophages, monocytes, dendritic cells, and neutrophils.

[0053] Specific T lymphocytes and specific B lymphocytes expressing specific antigen receptors mediate adaptive immune responses. After antigen-specific stimulation, B lymphocytes are activated, proliferate, differentiate into plasma cells, produce specific antibodies, and mediate humoral immune responses. After antigen-specific stimulation, T lymphocytes are activated, proliferate, and differentiate into effector T cells, mediating cellular immune responses and supporting humoral immune responses. In addition, during the initiation phase of adaptive immune responses, dedicated APCs such as dendritic cells and monocyte macrophages are involved, presenting antigens and activating T cells. During the effector phase of adaptive immune responses, monocyte macrophages, NK cells, and other cells are involved, working in cooperation with T cells and antibodies to eliminate antigens.

[0054] Cells involved in the innate immune response mainly include monocyte macrophages, granulocytes, dendritic cells, NK cells, endothelial cells, mast cells, erythrocytes, platelets, etc., as well as a small number of T and B lymphocyte subgroups. NK cells are the third type of lymphocyte, which have nonspecific cytotoxic activity and play an important role in the innate immune response against viral infections and tumors. Monocyte macrophages, granulocytes, etc. have strong phagocytic and killing functions and are involved in inflammatory responses by releasing a number of active products.

[0055] The synergistic effect of an antigen and the CpG ODN of the present invention induces both humoral and cellular immune responses, enhances the immune function of Th1-type T cells, and significantly enhances the immune response of T cells.

[0056] vaccine

[0057] The term "vaccine" in the present invention refers to a vaccine well known to those skilled in the art. It generally refers to any biological preparation that, after administration by injection or mucosal route, can induce the production of specific antibodies and / or cellular immunity against a particular pathogen in an individual, thereby conferring on the individual the ability to protect against or eliminate the pathogen. This includes proteins, polysaccharides, nucleic acids, live vectors, and infectious agents. Vaccines are autoimmune preparations for the prevention of infectious diseases, prepared from pathogenic microorganisms (e.g., bacteria, rickettsia, viruses, etc.) and their metabolic products that have been artificially attenuated, inactivated, or by genetic engineering and other methods. Vaccines retain the properties of pathogenic bacteria that stimulate the immune system of an animal. When an animal is exposed to such a harmless pathogen, its immune system produces some protective substances, such as immune hormones, active physiological substances, and special antibodies. When the animal is again exposed to such a pathogen, the animal's immune system, following its original memory, produces more protective substances to prevent the damage caused by the pathogenic bacteria.

[0058] As used herein, a "vaccine" refers to a preparation designed to induce an immune response against an antigen. A vaccine may be therapeutic, administered during treatment to enhance the immune response or drive a specific response, or prophylactic, administered before or shortly after the onset of disease. A vaccine may be both therapeutic and prophylactic at the same time, treating an existing disease and preventing future recurrence of the disease. A vaccine can be administered to a subject by conventional administration methods in the art. As used herein, the terms "administration" or "administering" include all appropriate means of providing a substance to a patient. Conventional routes include oral, sublingual, transmucosal, transdermal, rectal, vaginal, subcutaneous, intramuscular, intravenous, intraarterial, intrathecal, administration via a catheter, administration via an implant, etc.

[0059] The antigens of the present invention can be used to prepare a medicament for inducing an immune response against the antigen in a subject. In one embodiment, the antigens of the present invention can be used to prepare a rabies vaccine. In a preferred embodiment of the present invention, the antigens of the present invention can be used to prepare rabies vaccines for animals and humans. Rabies vaccines for animals include inactivated vaccines, attenuated vaccines, and genetically engineered vaccines. Rabies vaccines for humans include neural tissue-derived vaccines, avian embryo culture vaccines, cell culture vaccines, subunit and purified vaccines, and genetically engineered vaccines. In one embodiment, the antigens of the present invention can be used to prepare a SARS-COV-2 vaccine.

[0060] Vaccine adjuvants

[0061] The term "vaccine adjuvant" or "adjuvant" in the present invention refers to vaccine adjuvants well known to those skilled in the art. The word "adjuvant" comes from the Latin word "Aduvare," which means to assist or enhance. A vaccine adjuvant is an additive to a vaccine. When it is injected into the body prior to antigen injection or mixed with the antigen, it can enhance the immune response to the antigen or change the type of immune response; it is a non-specific immune enhancer and is not antigenic itself.

[0062] Currently, there is no unified international standard for classifying adjuvants. Commonly used adjuvants mainly include insoluble aluminum salt colloids, oil-water emulsions, microorganisms and their metabolites, nucleic acids and their analogs, cytokines, immune stimulating complexes, propolis, liposomes, etc. The immunomodulatory CpG ODNs of the present invention can also be used as vaccine adjuvants and exhibit excellent adjuvant function. The immunomodulatory CpG ODNs of the present invention can be used alone as adjuvants for vaccines (e.g., rabies vaccines or SARS-COV-2 vaccines) or in combination with other commonly used adjuvants as adjuvants for vaccines (e.g., rabies vaccines or SARS-COV-2 vaccines). The immunomodulatory CpG ODNs and these commonly used adjuvants can exert additive or synergistic effects to improve the immunogenicity of antigens, thereby reducing vaccine doses or improving vaccine efficacy (e.g., reducing vaccine doses or the number of vaccine administrations). Therefore, in one embodiment, the present invention also provides the use of an immunomodulatory CpG ODN in the preparation of a vaccine adjuvant, preferably the vaccine is a rabies vaccine or a SARS-COV-2 vaccine. In one embodiment, the vaccine adjuvant herein further comprises one or more other substances that function synergistically with the immunomodulatory CpG ODN.When used as an adjuvant in a rabies vaccine or a SARS-COV-2 vaccine, the immunomodulatory CpGs described herein An effective amount of ODN can be determined by one skilled in the art through routine experimentation. For example, an effective amount may be 0.01 μg to 1000 μg / ml vaccine, including any value within the range of 0.01 μg to 1000 μg / ml, such as 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1.0 μg / ml, 1.1 μg / ml, 1.2 μg / ml, 1.3 μg / ml, 1.4 μg / ml, 1.5 μg / ml, 1.6 μg / ml, 1.7 μg / ml, 1.8 μg / ml, 1.9 μg / ml, 2.0 μg / ml, 3.0 μg / ml, 4.0 μg / ml, 5.0 μg / ml, 6.0 μg / ml, 7.0 μg / ml, 8.0 μg / ml, 9.0 μg / ml, 10.0 μg / ml, 11.0 μg / ml, 12.0 μg / ml, 13.0 μg / ml, 14.0 μg / ml, 15.0 μg / ml, 16.0 μg / ml, 17.0 μg / ml, 18.0 μg / ml, 19.0 μg / ml, 20.0 μg / ml, 21.0 μg / ml, 22.0 μg / ml, 23.0 μg / ml, 24.0 μg / ml, 25.0 μg / ml, 26.0 μg / ml, 27.0 μg / ml, 28.0 μg / ml, 29. / ml, 3.0μg / ml, 4.0μg / ml, 5.0μg / ml, 6.0μg / ml, 7.0μg / ml, 8.0μg / ml, 9.0μg / ml, 10.0μg / ml, 20.0μg / ml, 30.0μg / ml, 40.0μg / ml, 50.0μg / ml, 60.0μg / ml, 70.0μg / ml, 80.0μg / ml, 90.0μg / ml, 100.0μg / ml, 200.0μg / ml, 300.0μg / ml, 400.0μg / ml, 500.0μg / ml, 600.0μg / ml, 700.0μg / ml, 800.0μg / ml, 900.0μg / ml, and 1000.0μg / ml vaccines.

[0063] Pharmaceuticals

[0064] The term "medicament" or "pharmaceutical formulation" refers to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. In one embodiment, the present invention relates to the use of an immunomodulatory CpG ODN or pharmaceutical composition in the preparation of a medicament for preventing or treating tumors, microbial infections, or allergies in a subject. The subject may be a human or an animal, such as a mouse, rat, domestic animal, such as a dog, pig, cow, horse, or poultry, such as a chicken, duck, or goose. Those skilled in the art can determine the effective amount of an immunomodulatory CpG ODN in a medicament or pharmaceutical formulation according to conventional methods, and can determine the method of administering the medicament according to conventional methods.

[0065] [Detailed Description of the Invention] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific examples and drawings. The following examples are for illustrative purposes only, and the protection scope of the present invention is based on the appended claims. [Example]

[0066] Materials and Methods

[0067] All CpG ODNs were synthesized by Suzhou Ruibo Bioscience and Technology Co., Ltd., China, and they include ODN1 (5'-tcgcgacgttcgcgggacgttcccta-3', SEQ ID NO: 1), ODN2 (5'-tcgcgacgttcgcgcgacgttcgcta-3', SEQ ID NO: 2), ODN3 (5'-tcgcgacgttcgccgacgttcgta-3', SEQ ID NO: 3), ODN4 (5'-tg The CpG ODNs included ODN1 (5'-tgactgtgaacgttcgagatga-3', SEQ ID NO: 4), ODN5 (5'-tcgtcgttcgtcgttcgacgttc-3', SEQ ID NO: 5), and ODN6 (5'-tcgaggttcgtcgttcctcgttc-3', SEQ ID NO: 6), where ODN1, ODN2, ODN3, ODN4, ODN5, and ODN6 were all phosphorothioated. HP3004 was a positive control CpG ODN (5'-tgactgtgaacgttcgagatga-3', SEQ ID NO: 7, all phosphorothioated), and HP0000 was a negative control CpG ODN (5'-tggccaagcttgggccccttgcaagggcc-3', SEQ ID NO: 8, all phosphorothioated). All CpG ODNs were dissolved in sterile / endotoxin-free water (InvivoGen, USA) and stored at -40°C for use. Rabies vaccines were obtained from Changchun Biological Products, China (Vero cell rabies vaccine) / Chengdu Kanghua Biological Products Co., Ltd., China (human diploid cell rabies vaccine). Inactivated SARS-CoV-2 vaccine was provided by Zhejiang Tianyuan Biological Pharmaceutical Co., Ltd., China.

[0068] Human peripheral blood leukocyte concentrates and experimental animals:

[0069] Human peripheral blood enriched leukocytes were purchased from Changchun Central Blood Station, China. Six- to eight-week-old female BALB / c mice were purchased from Changchun Biological Products Research Institute Co., Ltd., China.

[0070] Isolation and culture of human peripheral blood mononuclear cells (PBMCs):

[0071] Human peripheral blood-derived leukocytes were diluted with two volumes of saline and added 1:1 to the surface of Ficoll separation solution (Corning, USA) in a plastic centrifuge tube. The tube was centrifuged at 2800 rpm for 20 minutes (8 increase, 0 decrease) to collect the mononuclear cell layer suspension. The suspension was washed three times with 1x PBS and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cells were suspended in RPMI-1640 (Corning, USA) complete medium supplemented with 10% fetal bovine serum (Clark, USA), 1% penicillin / streptomycin (Hyclone, USA), and 1% HEPES (Invivogen, USA). 2x10 cells were collected. 5 CpG was added at different concentrations (0.03, 0.1, 0.3, 1, and 3 μM), and the cells were cultured in an incubator at 37° C. and 5% CO 2 .

[0072] Cytokine secretion assay:

[0073] Human PBMCs (2 × 10 5 / well) and mouse spleen cells (1 × 10 6 Cells (1 / well) were seeded into a 96-well U-shaped plate, and different concentrations of CpG (0.03, 0.1, 0.3, 1, and 3 μM) were added. After incubation in an incubator at 37°C and 5% CO for 16 hours, the supernatants were collected and the levels of human IFN-α (Mabtech, Sweden), mouse IFN-α (eBioscience, Australia), mouse IL-6 (Mabtech, Sweden), and mouse TNF-α (Mabtech, Sweden) in the supernatants were detected according to the ELISA kit instructions.

[0074] Example 1 Preparation of CpG ODN

[0075] CpG ODNs were synthesized using an automated DNA synthesizer via the solid-phase phosphoramidite triester method, with steps of deprotection, activation, thiolation, and capping. The synthesized oligonucleotides were deprotected with concentrated ammonia, then purified and desalted. The purified oligonucleotides were lyophilized in the form of sodium salts and characterized by MS before use. CpG ODN sequences with purity >90% were then obtained. CpG ODNs designated ODN1, ODN2, ODN3, ODN4, ODN5, and ODN6 were used in subsequent experiments.

[0076] 5'-DMT dA, dG, dC, dT, and other phosphoramidite monomers were purchased from Shanghai Jiaowei Science and Technology Development Co., Ltd. The corresponding vectors were purchased from Chemgenes (Wilmington, MA). 2'-substituted ribonucleoside phosphoramidites were purchased from Shanghai Jiaowei Science and Technology Development Co., Ltd. and Promega (Obispo, CA).

[0077] Example 2 Effect of CpG ODN on the proliferation of mouse splenic T and B cells

[0078] Isolation and culture of mouse splenocytes:

[0079] Mouse spleens were isolated under sterile conditions, and BALB / c mouse spleen cell suspensions were prepared after polishing and filtration. Cells were suspended in RPMI-1640 complete medium at a concentration of 5 × 10 5 or 1 x 10 6 Cells were seeded onto a 96-well U-shaped plate at 100 cells / well, and different concentrations of CpG (0.03, 0.1, 0.3, 1, and 3 μM) were added, followed by incubation in an incubator at 37° C. and 5% CO 2 .

[0080] After 16 hours of culture, the cells were harvested, washed twice with 1x PBS, and centrifuged at 1500 rpm for 5 minutes. The cells were resuspended in 1x PBS, and anti-CD4, anti-CD8, and anti-CD19 antibodies (BD, USA) were added to the cell suspension, followed by incubation at 4°C for 30 minutes in the dark. The cells were washed twice with 1x PBS, centrifuged at 1500 rpm for 5 minutes, resuspended in 1x PBS, and analyzed by flow cytometry using a BD LSRFortessa™ flow cytometer (BD, USA).

[0081] The supernatant was collected for ELISA analysis. The levels of CD4, CD8, and CD19 in the supernatant were measured by sandwich ELISA to obtain the effect of CpG ODN on the proliferation of mouse splenic T and B cells. The results are shown in Figure 1.

[0082] Conclusion: CpG ODN can significantly stimulate the activation of mouse splenic B cells (indicated by CD19 levels), induce the proliferation of mouse splenic B cells, and then upregulate the expression of costimulatory molecules and the secretion of cytokines (e.g., IL-6, TNF-α).

[0083] Example 3 Cytokine induction in mouse splenocyte cultures

[0084] Splenocytes from 4- to 8-week-old C57BL / 6 mice were prepared and cultured in RPMI complete medium. 5 × 10 6Cells were seeded into 24-well Petri dishes at 1000 cells / ml. CpG ODN in PBS buffer was added to the cell cultures to final concentrations of 0.03, 0.1, 0.3, 1, and 3 μM, respectively. The cells were then incubated at 37°C for 24 hours, and the supernatants were collected for ELISA analysis. IFN-α, IL-6, and TNF-α levels in the supernatants were determined by sandwich ELISA. Reagents used in the examples, including cytokines, antibodies, and standards, were purchased from BD PharMingen. The results are shown in Figures 2, 3, and 4. Figure 2 shows that different CpG ODNs effectively stimulated IFN-α secretion by pDCs, with HP3004 as a positive control. Figure 3 shows that different CpG ODNs stimulated IL-6 secretion by B cells, with HP3004 as a positive control. FIG. 4 shows that different CpG ODNs stimulated the production of TNF-α by B cells, where HP3004 was a positive control.

[0085] Conclusion: Different CpG ODN sequences upregulated and stimulated the secretion of cytokines IL-6 and TNF-α.

[0086] Example 4 Effect of CpG ODN on T and B cell proliferation induced by human PBMC

[0087] The medium used for the assay was RPMI 1640 medium supplemented with 1.5 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 50 μM 2-mercaptoethanol, 100 IU / ml penicillin-streptomycin mixture, and 10% heat-inactivated fetal bovine serum. A total of 0.5 × 10 per ml was used. 6 B cells (i.e., 0.1 × 10 6 The cells (200 μl / well) were stimulated with different concentrations of the CpG ODN to be tested (0.03, 0.1, 0.3, 1, and 3 μM) in triplicate in a 96-well flat-bottom plate for 72 hours. After 66 hours, 0.75 μCi of [ 3Cells were pulsed with [H]-thymidine (1 Ci = 37 GBq, Perkin Elmer Life Science) and then harvested 8 hours later. Plates were harvested using a cell harvester, and radioactivity incorporation was determined using standard liquid scintillation techniques. Results are presented as mean cpm + / - SD or proliferation index (cpm treated group / cpm medium control). Results are shown in Figure 5, where HP3004 was the positive control.

[0088] Conclusion: CpG ODN can activate human PBMC cells, induce B cell proliferation, and then upregulate the expression of costimulatory molecules and the secretion of cytokines (e.g., IL-6, TNF-α).

[0089] Example 5 Cytokine induction by CpG ODN in PBMC cultures

[0090] 5 x 10 human PBMCs 6 Cells were seeded into 96-well plates at 1000 cells / ml. CpG ODN in phosphate-buffered saline (PBS, pH 7.4, Mediatech) was added to the cell culture to a final concentration of 10.0 μg / ml. The cells were then incubated at 37°C for 24 hours, and the supernatant was collected for ELISA analysis. Each experiment was performed in triplicate. IFN-α, IL-6, and TNF-α levels were determined by sandwich ELISA. Reagents used in the examples, including cytokines, antibodies, and standards, were purchased from PharMingen. The results are shown in Figures 6 to 8.

[0091] Conclusion: CpG ODN sequences can significantly increase the levels of IL-6 and TNF-α.

[0092] Example 6: HEK-BLUE detection

[0093] Cell passaging

[0094] Cells were maintained and subcultured in growth medium supplemented with 10 μg / ml Blasticidin and 100 μg / ml Zeocin™.

[0095] Growth medium: DMEM, 4.5 g / L glucose, 10% (v / v) fetal bovine serum, 50 U / ml penicillin, 50 U / ml streptomycin, 100 μg / ml Normocin™, 2 mM glutamine.

[0096] Subculture medium [Table 1]

[0097] After reaching 70-80% confluency, the cells should be passaged and the original medium replaced with PBS. Then, the cells should be detached by tapping the ampoule or using a cell scraper. The detached cells were collected and centrifuged for 5 minutes. The cells were counted and plated in a 96-well plate at 2-4 x 10 cells. 4 Cells were seeded and then treated 2-3 days later.

[0098] The final concentrations of the positive control (HP3004), negative control (HP0000), and CpG ODN were 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 μg / ml, respectively, and the medium concentrations were >90%. 10 μl of the negative control, positive control, and test substance were added to the corresponding wells. The cells were incubated for 24 hours in a 5% CO2 incubator. The medium was poured into a 250 ml narrow-neck flask, 100 ml of water was added, the mixture was mixed evenly, and the mixture was heated at 37°C for 30 minutes. 50 μl of the cell supernatant was taken and centrifuged for 5 minutes. 20 μl of sample was added to the supernatant, and 180 μl of QUANTI-Blue was added to each well of a 96-well plate. The mixture was then incubated at 37°C for 6 hours. The absorbance at 655 nm was measured using a microplate reader. See Figures 9-11.

[0099] Results: As can be seen from Figures 9 to 11, CpG ODN had a favorable effect on the activity of HEK-Blue hTLR9 cells, HEK-Blue mTLR9 cells, and Ramos-Blue cells.

[0100] Example 7 Effect of the combination of ODN3 and rabies vaccine on anti-rabies virus neutralizing antibody titers in mice

[0101] The mice were divided into eight groups, each with eight mice. Two days before immunization, background serum was collected from each group of mice. The vaccine was injected (intramuscularly) on days 0, 3, and 7. On days 4, 6, 8, 10, 14, 28, and 56, the eyes were removed, blood was collected, and serum was separated. The anti-rabies virus neutralizing antibody titers in the mouse serum were determined for each mouse using the RFFIT method. The results are shown in Figure 12.

[0102] The specific groupings were as follows: [Table 2]

[0103] Results: As can be seen from Figure 12, the antibody levels produced in the groups receiving rabies vaccine and different doses of ODN3 varied over time, but showed an overall increasing trend. The antibody levels in the rabies vaccine + 1 μg ODN3 group (RV+1), the rabies vaccine + 3 μg ODN3 group (RV+3), and the rabies vaccine + 10 μg ODN3 group (RV+10) were higher than those in the vaccine group without adjuvant (RV). 14 days after immunization, the antibody levels in the four groups (rabies vaccine, rabies vaccine + 1 μg ODN3, rabies vaccine + 3 μg ODN3, and rabies vaccine + 10 μg ODN3) peaked and then declined. Among these, the rabies vaccine + 10 μg ODN3 group had the highest antibody levels within all detection times. Based on the time to neutralizing antibody production, peak, and duration of neutralizing antibody, the experimental results of the 10 μg ODN3 group were superior to the other dose groups.

[0104] Example 8 Comparison of different doses of ODN3 to enhance the effect of rabies vaccine in inducing antibody production

[0105] In this study, 112 mice (56 females and 56 males, weighing 18-22 grams per mouse), rabies vaccine (1 ml / dose) (containing 2.5 IU), and ODN3 were used. The mice were divided into groups of 8 mice (4 males and 4 females). Vaccine groups included rabies vaccine, rabies vaccine + 0.3 μg ODN3, rabies vaccine + 1 μg ODN3, rabies vaccine + 3 μg ODN3, and rabies vaccine + 10 μg ODN3. Rabies vaccine and CpG ODN were both dissolved in PBS. Mice were immunized according to the different groups on days 0, 3, 7, 14, and 28. Immunization was performed intraperitoneally. Blood was collected from the tail vein of the mice 4, 6, and 8 days after immunization, and serum was separated. The rabies vaccine antibody titer in mouse serum was detected using the Rapid Fluorescent Focus Inhibition Test (RFFIT). Blood was collected from the tail vein of mice 2 days before immunization, and the resulting serum was used as a negative control.

[0106] Results: The immune effect of the rabies vaccine in each group increased over time. The immune effect of the rabies vaccine increased with increasing ODN3. The results are shown in Figure 13.

[0107] Conclusion: ODN3 can significantly enhance the immune effect of rabies vaccine.

[0108] Example 9 Use of ODN3 as a rabies vaccine adjuvant to reduce rabies vaccine dosage

[0109] A total of 128 mice (64 female and 64 male, weighing 18-22 g / mouse) were used, each containing 2.5 IU of rabies vaccine (1 ml / dose). The mice were divided into eight groups (four male and four female). The vaccine groups were: rabies vaccine, rabies vaccine + 1 μg ODN3, 1 / 2 rabies vaccine + 1 μg ODN3, 1 / 4 rabies vaccine + 1 μg ODN3, and 1 / 8 rabies vaccine + 1 μg ODN3.

[0110] Both the rabies vaccine and ODN3 were dissolved in PBS. Mouse immunization: Mice were immunized according to different groups on days 0, 3, 7, 14, and 21. Immunization was performed by intraperitoneal injection. On day 28, blood was collected from the tail vein of the mice, and serum was separated. Rabies vaccine antibody titers in the mouse serum were detected using a rapid fluorescent focus inhibition test (RFFIT) for rabies vaccine. Blood was collected from the tail vein of the mice two days before immunization, and the resulting serum was used as a negative control.

[0111] Results: The combination of reduced doses of rabies vaccine and ODN3 was still able to stimulate mice to produce high levels of rabies virus-specific antibodies. The antibody titers (GMTs) in the rabies vaccine + 1 μg ODN3, 1 / 2 rabies vaccine + 1 μg ODN3, 1 / 4 rabies vaccine + 1 μg ODN3, and 1 / 8 rabies vaccine + 1 μg ODN3 groups all achieved higher levels than those achieved when rabies vaccine was used alone, indicating that CpG ODN can reduce the dosage of rabies vaccine. The results are shown in Figure 14.

[0112] Conclusion: ODN3 can reduce the dosage of rabies vaccine.

[0113] Example 10 Different CpG ODN sequences used as rabies vaccine adjuvants

[0114] BALB / c mice were randomly divided into eight groups: human rabies vaccine group, human rabies vaccine + ODN1 (10 μg / mouse) group, human rabies vaccine + ODN2 (10 μg / mouse) group, human rabies vaccine + ODN3 (10 μg / mouse) group, human rabies vaccine + ODN4 (10 μg / mouse) group, human rabies vaccine + ODN5 (10 μg / mouse) group, human rabies vaccine + ODN6 (10 μg / mouse) group, and human rabies vaccine + HP0000 (10 μg / mouse) group. Each group was immunized three times via hind leg muscle on days 0, 7, and 21, respectively. Each immunization dose was 0.2 ml per mouse. On days 14, 28, and 56 after immunization, the eyeballs were removed, blood was collected, and serum was separated to detect the content of anti-rabies virus antibodies in the serum. The neutralizing antibody titers are shown in FIG.

[0115] Conclusion: CpG ODN can improve the level of antibody titers.

[0116] Example 11: Effect of ODN6 in combination with an inactivated SARS-CoV-2 vaccine on anti-SARS-CoV-2 S protein-specific IgG and virus-neutralizing antibody titers in mice

[0117] BALB / c mice weighing 18–20 g were selected and divided into groups of 9 or 10 mice (half male and half female). Each group was immunized intraperitoneally with 0.5 ml of each injection on days 0 and 14 according to the designed immunization program. Blood was collected on days 0, 6, 13, 21, and 28 according to the designed time points, and serum was separated. All serum was tested for S protein-specific IgG and virus-neutralizing antibody. The geometric mean titers of serum IgG and virus-neutralizing antibody for each group were statistically calculated. The results are shown in Figures 16–18.

[0118] The specific groupings were as follows: [Table 3]

[0119] Results: As can be seen from Figures 16-18, antigen-specific S1 antibodies were barely detectable in the double adjuvant control group of D28. Except for the 2 μg / mL antigen + 450 μg / mL aluminum hydroxide adjuvant group and the 2 μg / mL antigen + 450 μg / mL aluminum hydroxide adjuvant + 400 μg / mL CpG group, which had relatively low S1 antibody levels, the other groups induced higher S1 antibody titers. As can be seen from the results of the neutralizing antibody titers in the serum of mice in each group, the neutralizing antibody levels were relatively low in the 2 μg / mL antigen + 450 μg / mL aluminum hydroxide adjuvant group, the 2 μg / mL antigen + 450 μg / mL aluminum hydroxide adjuvant + 400 μg / mL CpG group, and the 4 μg / mL antigen + 450 μg / mL aluminum hydroxide adjuvant + 80 μg / mL CpG group, but higher neutralizing antibody titers were induced in the other groups.

[0120] Conclusions: The combination of an inactivated SARS-CoV-2 vaccine with ODN6 and aluminum adjuvant can induce higher titers of SARS-CoV-2 S protein-specific IgG and virus-neutralizing antibodies. On D28, the 4 μg / mL antigen + 450 μg / mL aluminum hydroxide adjuvant + 40 μg / mL CpG group induced the highest S1 antibody titer. The 8 μg / mL antigen + 450 μg / mL aluminum hydroxide adjuvant + 400 μg / mL CpG group induced the highest neutralizing antibody titer.

[0121] Equivalent technical solutions

[0122] The specific examples described above further describe the objectives, technical solutions and beneficial effects of the present invention in detail, but they should not be construed as limiting the scope of the invention. It should be noted that any modifications, equivalent substitutions and improvements made by those skilled in the art within the spirit and scope of the invention shall be included in the protection scope of the present invention.

Claims

1. An immunomodulatory CpG ODN comprising a nucleotide sequence selected from SEQ ID NOs: 1 to 6, all of which are phosphorothioated, or consisting of a nucleotide sequence selected from SEQ ID NOs: 1 to 6, all of which are phosphorothioated.

2. The immunomodulatory CpG ODN described in claim 1, wherein the sequence of the immunomodulatory CpG ODN is SEQ ID NO: 3 or 6, all of which are phosphorothioated.

3. A pharmaceutical composition comprising the immunomodulatory CpG ODN of claim 1 or 2 and a pharmaceutically acceptable carrier.

4. 3. The immunomodulatory CpG ODN of claim 1 or 2 for use as a vaccine adjuvant.

5. The immunomodulatory CpG ODN of claim 4 , wherein the vaccine is a rabies vaccine.

6. The immunomodulatory CpG ODN described in claim 5, wherein the amount of the CpG ODN used is 0.01 μg to 1000 μg / ml.

7. The immunomodulatory CpG ODN described in claim 6, wherein the amount of the CpG ODN used is 1 to 10 μg / ml, for example, 1, 3, or 10 μg / ml.

8. The immunomodulatory CpG ODN of claim 4, wherein the vaccine is a SARS-COV-2 vaccine.

9. The immunomodulatory CpG ODN of claim 8, wherein the vaccine is an inactivated SARS-COV-2 vaccine.

10. The immunomodulatory CpG ODN described in claim 8, wherein the amount of the CpG ODN used is 0.01 μg to 1000 μg / ml.

11. The immunomodulatory CpG ODN according to any one of claims 4 to 10, wherein the vaccine adjuvant further comprises one or more other adjuvants that act in cooperation with the immunomodulatory CpG ODN, such as insoluble aluminum salt colloids, oil-water emulsions, microorganisms and their metabolic products, nucleic acids and their analogs, cytokines, immune stimulating complexes, propolis, and liposomes.

12. An immunomodulatory CpG ODN as described in claim 1 or 2 or a pharmaceutical composition as described in claim 3 for preventing or treating a tumor, microbial infection or allergy in a subject.

13. The immunomodulatory CpG ODN or pharmaceutical composition of claim 12, wherein the subject is a human or an animal, for example, livestock such as dogs, pigs, cows, and horses, poultry such as chickens, ducks, and geese, mice, and rats.

14. A vaccine comprising the immunomodulatory CpG ODN of claim 1 or 2 and an antigen, wherein the antigen is a rabies antigen or a SARS-COV-2 antigen.

15. The vaccine according to claim 14, wherein the vaccine is a rabies vaccine for humans or animals, the immunomodulatory CpG ODNs are all phosphorothioated immunomodulatory CpG ODNs represented by SEQ ID NO: 3, and the amount of the CpG ODN used is 0.01 μg to 1000 μg / ml.

16. The vaccine of claim 15, wherein the amount of CpG ODN used is 1 to 10 μg / ml, for example, 1, 3, or 10 μg / ml.

17. 15. The vaccine of claim 14, wherein the vaccine is a SARS-COV-2 vaccine, and the vaccine further comprises an aluminum adjuvant, such as an aluminum hydroxide adjuvant.

18. The vaccine of claim 17, wherein the vaccine is an inactivated SARS-COV-2 vaccine.

19. The vaccine of claim 17 or 18, wherein the immunomodulatory CpG ODN is an immunomodulatory CpG ODN represented by SEQ ID NO: 6 that is all phosphorothioated.

20. 20. The vaccine of claim 19, wherein the antigen content is 1 to 10 μg / mL, for example 2, 4 or 8 μg / mL, the aluminum hydroxide content is 1 to 1000 μg / mL, and the immunomodulatory CpG ODN content is 1 to 1000 μg / mL.

21. The vaccine described in claim 20, wherein the aluminum hydroxide content is 300 to 500 μg / mL.

22. The vaccine of claim 20, wherein the content of the immunomodulatory CpG ODN is 2 to 500 μg / mL, for example, 5 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, or 400 μg / mL.

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