CpG oligonucleotide and application thereof

Abstract

Relates to CpG oligonucleotide and application thereof. The CpG oligonucleotide has a TLR9 agonist activity.

Description

CpG oligonucleotides and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to patent application number “CN202310066663.2” filed on January 19, 2023, with invention name “CpG oligonucleotides and their applications”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention generally relates to the field of biomedical technology, and in particular to a CpG oligonucleotide and applications thereof. Background Art

[0004] Toll-like receptors (TLRs) are transmembrane proteins that primarily act as sensors for microbial components, recognizing bacterial and viral components. TLRs constitute a major defense mechanism in mammalian infections and some non-infectious diseases. To date, 10 human (TLRs 1-10) and 12 mouse TLRs (TLRs 1-9 and TLRs 11-13) have been identified. Among them, TLR9 is expressed on human plasmacytoid dendritic cells (pDCs) and B cells, as well as on various cells of the mouse myeloid lineage (including monocytes, macrophages, and conventional dendritic cells).

[0005] TLR9 specifically recognizes CpG motifs contained in bacteria, viruses, plasmids, or synthetic double-stranded or single-stranded oligonucleotides. CpG ODNs are immunomodulatory synthetic oligonucleotides specifically designed to stimulate TLR9. To date, four types of synthetic CpG ODNs have been described, including K-type ODN (also known as B-type), D-type ODN (also known as A-type), C-type ODN, and P-type ODN, each with distinct structural and biological properties.

[0006] Currently, the only approved CpG ODN is CpG 1018, and the number of vaccines containing this adjuvant is relatively small, with only vaccines for hepatitis B and COVID-19 approved. Furthermore, no CpG ODN has been approved for cancer treatment. Given the heavy global burden of infectious diseases and cancer, there is a continued need to develop more active TLR9 agonists for their prevention and treatment.

[0007] Summary of the Invention

[0008] In one aspect of the present invention, it relates to a CpG oligonucleotide, the sequence of which is shown in SEQ ID NO: 1.

[0009] In another aspect, the present invention relates to an immunostimulatory composition comprising the CpG oligonucleotide as described above.

[0010] In yet another aspect of the present invention, it relates to a delivery system comprising i) a CpG oligonucleotide as described above or an immunostimulatory composition as described above, and ii) a delivery vehicle.

[0011] In another aspect of the present invention, it relates to the use of the above-mentioned CpG oligonucleotide, the above-mentioned immunostimulatory composition, or the above-mentioned delivery system in the preparation of a drug for regulating the activity of immune cells, wherein the use is carried out in vivo or in vitro.

[0012] In another aspect of the present invention, the present invention relates to the use of the above-mentioned CpG oligonucleotide, the above-mentioned immunostimulatory composition, or the above-mentioned delivery system in the preparation of a drug for treating and / or preventing at least one of the following indications: tumor, antiviral, antibacterial, antifungal, antiparasitic, reducing chemotherapy side effects, anti-fatigue or enhancing immunity, and promoting the subject's immune response to antigens in a subject in need.

[0013] In another aspect, the present invention relates to a method for eliciting a TLR9-mediated immune response in a subject, comprising administering to the subject an effective amount of the CpG oligonucleotide as described above, or the immunostimulatory composition as described above, or the delivery system as described above.

[0014] The novel CpG oligonucleotides provided by the present invention possess TLR9 agonist activity and exhibit superior pharmaceutical activity compared to existing technologies. These CpG oligonucleotides have broad-spectrum immune response modulation capabilities, increasing the release of cytokines such as IFN-α, IL-6, and TNF-α, and possess anti-tumor and anti-infection properties, making them suitable for applications in anti-tumor drugs and vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] FIG1 shows that CpG1 provided in one embodiment of the present invention has the effect of activating mouse TLR9. HEK-Blue mTLR9 cells (180 μL, 2-3×10 5Cells / mL) were seeded in a 96-well plate and cultured for about 1-24 hours. Then, 20 μL of different sequence solutions (CpG1, CpG 2395, CpG 7909, CpG 1018, and negative control FX-700) were added to the cells at final concentrations of 0, 0.25, 0.5, 1, 2, 5, 10, 20, and 40 μM, and cultured for 24 hours. The culture supernatant was collected and added to QUANTI-Blue TM The solution was tested and incubated for 15 min. The OD value at 630 nm was measured by spectrophotometer to quantify the level of embryonic alkaline phosphatase (SEAP). TM The SEAP secretion of mTLR9 cell lines was used to evaluate the activation effect of different sequences at different concentrations on mouse TLR9.

[0017] FIG2 shows that CpG1 has the ability to induce mouse spleen cell proliferation, as provided in one embodiment of the present invention. Mouse spleen cells (2×10 6 Cells / 100 μL) were seeded in a 96-well plate and incubated for approximately 1-24 hours. Then, 10 μL of different sequence solutions (CpG1, CpG 2395, CpG 7909, CpG 1018, and negative control FX-700) were added to final concentrations of 0, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, and 2 μM, and incubated for approximately 72 hours. 10 μL of CCK-8 solution was added to each well and incubated for an additional 1-4 hours. The OD value at 450 nm was measured using a microplate reader, which indirectly reflects the number of viable cells.

[0018] Figure 3 shows that sequence 1 effectively stimulates human PBMC to secrete IFN-α. Human PBMC (1-2×10 6 Cells were seeded at 10 μL / mL in a 96-well plate and incubated for at least 1 hour. Then, 10 μL of a different sequence solution (CpG1, CpG 2395, CpG 7909, CpG 1018, and the negative control FX-700) was added to final concentrations of 0, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 5, and 10 μM. The cells were incubated for 16-24 hours. The cell supernatant was collected and IFN-α levels were measured using a kit.

[0019] Figure 4 shows that CpG1 effectively stimulates human PBMC to secrete IL-6. Human PBMC (1-2×10 6Cells were seeded at 10 μL per 1 mL of a 96-well plate and incubated for at least 1 hour. Then, 10 μL of a different sequence of solutions (CpG1, CpG 2395, CpG 7909, CpG 1018, and the negative control FX-700) were added to final concentrations of 0, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 5, and 10 μM, and incubated for 16-24 hours. The cell supernatant was collected and IL-6 levels were measured using a kit.

[0020] Figure 5 shows that CpG1 effectively stimulates human PBMC to secrete TNF-α. 6 Cells were seeded at 10 μL per 1 mL of a 96-well plate and incubated for at least 1 hour. Then, 10 μL of a different sequence of solutions (CpG1, CpG 2395, CpG 7909, CpG 1018, and the negative control FX-700) were added to final concentrations of 0, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 5, and 10 μM, and incubated for 16-24 hours. The cell supernatant was collected and assayed for TNF-α using a kit.

[0021] Figure 6 shows that CpG1 effectively enhances the immunogenicity of the novel coronavirus vaccine composition. Six- to eight-week-old female BALB / c mice were immunized intramuscularly on days 0 and 28, with a 200 μL injection of either immunization saline, BA 4 / 5 + aluminum hydroxide, or BA 4 / 5 + aluminum hydroxide + CpG1, respectively. Blood was collected from the mice at the specified time points, and serum was obtained by centrifugation. Antibody titers in the blood were measured using an indirect ELISA.

[0022] Figure 7 demonstrates that CpG1 effectively enhances the immunogenicity of herpes zoster vaccine compositions. Female C57BL / 6J mice were immunized intramuscularly on D0 and D28 with a 50 μL injection of PBS solution, 5 μg gE, 5 μg gE plus varying amounts of CpG1, and 5 μg gE plus 8 μg CpG1 plus 40 μg Alum. Blood was collected from mice at the designated time points, serum was separated, and antibody titers were measured by ELISA.

[0023] Figure 8 demonstrates that CpG1 effectively enhances the immunogenicity of hepatitis B vaccine compositions. Female BALB / c mice were immunized once intraperitoneally on days 0 and 28, with a 1 mL injection of PBS solution, hepatitis B antigen, or hepatitis B antigen plus aluminum hydroxide plus varying amounts of CpG1. Blood was collected from mice at the designated time points, serum was separated, and antibody levels in the blood were measured using a kit.

[0024] Figures 9A-9D demonstrate that CpG1 effectively enhances the immunogenicity of quadrivalent influenza split vaccine compositions for types A1, A3, By, and Bv. Female BALB / c mice were immunized once in the inner thigh on D0 with a 0.1 mL injection of PBS solution, influenza antigen, or influenza antigen plus varying amounts of CpG1. Blood was collected from mice at the designated time points, serum was isolated, and antibody levels were measured by ELISA. DETAILED DESCRIPTION

[0025] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0026] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0027] Terminology Description

[0028] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0029] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0030] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0031] Concentration values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for 2%, fluctuations within ±0.1% are permitted. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are permitted. For example, for 100 mM, fluctuations within ±1%, ±2%, ±5%, etc. are permitted. Regarding molecular weight, fluctuations within ±10% are permitted.

[0032] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0033] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0034] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleotide sequence," and "polynucleotide" refer to RNA or DNA that is linear or branched, single-stranded or double-stranded, or a hybrid thereof. The term also includes RNA / DNA hybrids. The heterocyclic bases or nucleic acid bases incorporated into CpG oligonucleotides can be the naturally occurring major purine and pyrimidine bases (i.e., uracil, thymine, cytosine, adenine, and guanine), as well as naturally occurring and synthetic modifications of the major bases. Thus, a CpG-C oligonucleotide can include one or more of inosine, 2'-deoxyuridine, and 2-amino-2'-deoxyadenosine. When CpG is produced synthetically, less common bases can also be used in the synthesis. The terms "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "nucleic acid construct," "oligonucleotide," "ODN," and "polynucleotide" are also used interchangeably herein. Nucleic acid molecules and / or nucleotide sequences provided herein are shown herein in 5' to 3' orientation from left to right and are represented using the standard code for representing nucleotide symbols as specified in the World Intellectual Property Organization (WIPO) ST.26 standard.

[0035] As used herein, the term "TLR9 agonist" generally refers to an oligonucleotide-based compound that is capable of enhancing, inducing or modulating immune stimulation mediated by TLR9.

[0036] As used herein, the term "CpG or CpG motif" refers to a nucleic acid having a cytosine followed by a guanine linked by a phosphate bond, wherein the pyrimidine ring of the cytosine is unmethylated. "Methylated CpG" refers to a cytosine whose pyrimidine ring is methylated, typically at the 5-position of the pyrimidine ring. A CpG motif is a base pattern comprising an unmethylated central CpG and at least one base on the 3' and 5' sides of the central CpG. The flanking bases of the CpG confer a significant portion of the activity of a CpG ODN.

[0037] As used herein, the term "CpG ODN" refers to a CpG oligodeoxynucleotide having a length of at least about ten nucleotides and including one unmethylated CpG. The CpG ODN is single-stranded. The entire CpG ODN may be unmethylated or partially unmethylated. CpG ODN includes D-type (also referred to as A-type), K-type (also referred to as B-type), C-type, and P-type ODNs.

[0038] As used herein, the terms "patient," "subject," or "subject" are intended to refer to any animal, particularly mammals, and the disclosed methods can be used to treat any type of avian, mammalian, or aquatic species, particularly including humans and mammalian veterinary patients such as cows, sheep, goats, horses, dogs, pigs, cats, pandas, elephants, rabbits, rats, and mice.

[0039] As used herein, "antigen" (Ag) refers to any substance capable of inducing an immune response. Specifically, it refers to substances that are specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, producing immune response products (sensitized lymphocytes or antibodies), and capable of specifically binding to these products in vivo or in vitro. This includes, but is not limited to, xenoantigens, such as pathogenic microorganisms, viruses, toxoids, and other antigens from different species; alloantigens; autoantigens; and heterophilic antigens.

[0040] As used herein, the term "antibody" includes polyclonal antibodies and monoclonal antibodies, and the term "antibody fragment" includes antigen compound binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, Fab'-SH, scFv, bispecific antibodies and antibody minimum recognition units, as well as single-chain derivatives of these antibodies and fragments, such as scFv-Fc, etc. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, humanized antibodies and human antibodies, as well as related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0041] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description in this application.

[0042] Detailed Description of the Invention

[0043] CpG oligonucleotides

[0044] The present invention unexpectedly discovered a CpG oligonucleotide with excellent performance and conducted research on it.

[0045] The first aspect of the present invention relates to a CpG oligonucleotide, the sequence of which is shown in SEQ ID NO: 1.

[0046] The CpG oligonucleotides described herein include CpG oligonucleotides having one or more chemical modifications. Modifications include, but are not limited to, modifications of 3'OH or 5'OH groups, modifications of nucleotide bases, modifications of sugar components, modifications of backbones and modifications of phosphate groups. Such modifications can make CpG oligonucleotides more stable and / or less susceptible to degradation under certain conditions. For example, in some embodiments, CpG oligonucleotides are resistant to nucleases. Such modifications can be performed during the synthesis of the oligonucleotide or after synthesis, and the modifications can occur on the phosphodiester bridge bond between nucleosides, on the ribose unit and / or on natural nucleobases (i.e., adenine, guanine, cytosine and thymine). When modified during the synthesis of the oligonucleotide, the modified base can be incorporated into the interior of the oligonucleotide or located at the end of the oligonucleotide. When modified after the synthesis of the oligonucleotide, the modification can be performed using an active group, for example, by an amino-modified component, by 3' or 5'OH, or by a phosphate group.

[0047] CpG oligonucleotide can contain naturally occurring or modified non-natural base, and can contain modified sugar, phosphate and / or end.For example, except phosphodiester bond, phosphate modification includes but is not limited to: methyl phosphonate, phosphorothioate, phosphoramidate (bridged or non-bridged), phosphotriester and phosphorodithioate, and can be used in any combination.In certain embodiments, CpG oligonucleotide has only thiophosphate bond, only phosphodiester bond or the combination of phosphodiester bond and thiophosphate bond.

[0048] CpG oligonucleotides can contain one or more ribonucleotides (containing ribose as the sole or main sugar component), deoxyribonucleotides (containing deoxyribose as the main sugar component), modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can also be a pentose, a deoxypentose, a hexose, a deoxyhexose, glucose, arabinose, xylose, lyxose and the sugar analog cyclopentyl. The sugar can be in the form of a pyranosyl or furanosyl group. In CpG oligonucleotides, the sugar moiety is preferably a furanoside of ribose, deoxyribose, arabinose or 2'-O-alkylribose, and the sugar can be linked to various heterocyclic bases in an anomeric configuration.

[0049] Sugar modifications known in the art, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA and 2'-alkoxy- or amino-RNA / DNA chimeras and others described herein, can also be made and combined with any phosphate modification. Examples of base modifications include, but are not limited to, the addition of electron-withdrawing moieties to C-5 and / or C-6 of cytosine (e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) of CpG oligonucleotides and C-5 and / or C-6 of uracil (e.g., 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil) of CpG oligonucleotides. The use of base modifications in palindromic sequences of CpG oligonucleotides should not interfere with the self-complementarity of the bases involved in Watson-Crick base pairing. However, outside of palindromic sequences, modified bases can be used without this limitation. For example, 2'-O-methyl-uridine and 2'-O-methyl-cytidine can be used outside of a palindrome, whereas 5-bromo-2'-deoxycytidine can be used inside and outside of a palindrome. Other modified nucleotides that can be employed inside and outside of a palindrome include 7-deaza-8-aza-dG, 2-amino-dA, and 2-thio-dT.

[0050] In some embodiments, the CpG oligonucleotide comprises one or more modified phosphate groups. Modification of the phosphate group can be understood as a polynucleotide derivative in which at least a portion of the phosphodiester bonds of the nucleotide are replaced with phosphorothioate bonds.

[0051] In some embodiments, in the CpG oligonucleotide shown in SEQ ID NO: 1, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the nucleotides are modified with phosphate groups.

[0052] In some embodiments, the modification of the phosphate group comprises one or more of a phosphorothioate internucleotide linkage, a methylphosphonate linkage, and a boranophosphate linkage.

[0053] In some embodiments, the CpG oligonucleotides of the present invention have a homogeneous backbone (e.g., complete phosphodiester or complete phosphorothioate) or a heterogeneous (or chimeric) backbone. The phosphorothioate backbone modification can reduce the sensitivity of the oligonucleotide to nucleases, thereby being more stable under certain conditions (compared to natural phosphodiester backbone nucleic acids). Other bonds that can provide higher stability for the nucleic acids of the present invention include but are not limited to dithiophosphate bonds, methylphosphonate bonds, methylphosphorothioate bonds, boron phosphonate bonds, peptide bonds, alkyl bonds, and dephosphorylation bonds. Therefore, in some embodiments, the CpG oligonucleotides have a non-naturally occurring backbone. In some embodiments, the CpG oligonucleotides have a backbone that is completely phosphorothioate.

[0054] Immunostimulatory compositions

[0055] A second aspect of the invention relates to an immunostimulatory composition comprising a CpG oligonucleotide as described above.

[0056] In some embodiments, the immunostimulatory composition further comprises another adjuvant in addition to the CpG oligonucleotide.

[0057] In some embodiments, the adjuvant includes one or more of alum, complete Freund's adjuvant, incomplete Freund's adjuvant, squalene, squalane, muramyl dipeptide, MF59, AS03, AS04, monophosphatidyl lipid A, flagellin, Poly (I: C), aluminum salt and calcium salt.

[0058] Among them, complete Freund's adjuvant, incomplete Freund's adjuvant, squalene, squalane and alum are generally not used in humans.

[0059] Preferably, the adjuvant includes an aluminum salt or a calcium salt. The aluminum salt may be aluminum sulfate, aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, or the like.

[0060] In some embodiments, the immunostimulatory composition is typically in the form of a vaccine adjuvant.

[0061] In some embodiments, the immunostimulatory composition comprises 1 μg-2000 μg / mL of the above-described CpG oligonucleotide; preferably 5 μg-500 μg / mL of the above-described CpG oligonucleotide. The immunostimulatory composition further comprises an aluminum salt, such as aluminum hydroxide adjuvant. The aluminum salt adjuvant is present in an amount of 100 μg-1 mg / mL, preferably 400-800 μg / mL.

[0062] In some embodiments, the immunostimulatory composition further comprises at least one antigen.

[0063] A typical product form of such immunostimulatory composition is a vaccine.

[0064] In some embodiments, the vaccine is a water-in-oil emulsion having an aqueous phase and an oily phase.

[0065] In some embodiments, the vaccine is an oil-in-water emulsion having an aqueous phase and an oily phase.

[0066] Vaccines are typically formulated for parenteral administration. Typical immunizations are by nasal route, but the invention also contemplates oral and subcutaneous (SC), intramuscular (IM), intravenous (IV), intraperitoneal (IP) or intradermal (ID) injections.

[0067] The above-mentioned vaccine is administered in a manner compatible with the dosage formulation, and in an amount such as a therapeutically effective amount and an immunogenic effective amount. The dosage depends on the subject being treated, the ability of the subject's immune system to synthesize antibodies, and the expected degree of protection. The exact amount of active ingredient to be administered depends on the physician's judgment, and the dosage varies from individual to individual. The appropriate regimen for initial administration and booster vaccination may also vary, but typically, an injection is given or administered in another manner after a certain interval (several weeks or months) after the first administration.

[0068] In some embodiments, the antigen is a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, or a parasite antigen.

[0069] Herein, exemplary tumor antigens are well known to those skilled in the art, and include tumor-specific antigens (TSA) and tumor-associated antigens (TAA), for example, any one or more selected from the group consisting of the following antigens or their functional fragments: α-fetoprotein (AFP), α-actinin-4, A3, antigens specific for A33 antibodies, ART-4, B7, Ba 733, BAGE, BrE3 antigen, BMCA, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD21, C D23, CD25, CD29, CD30, CD32b, CD37, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD126, CD 132, CD133, CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CLDN family proteins, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen p (CSAp), CEA (CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast activation protein α (FAP), fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, GD2, gp100, GRO-β, HLA-DR, HM1.24. Human chorionic gonadotropin (HCG) and its subunits, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18 , IL-23, IL-25, insulin-like growth factor 1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1α, MIP-1β, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac , MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, mesothelin (MSLN), pancreatic cancer mucin, prostate stem cell antigen (PSCA), placental growth factor, p53, PLAGL2, prostatic acid phosphatase, trophoblast cell surface antigen 2 (TROP2), PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RAN TES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Fredenreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, Kras, oncogene markers, and oncogene products. Preferred solid tumor-specific antigens are selected from the group consisting of CLDN family proteins, Ep-CAM, FAP, PSCA, TROP2, and MSLN. The CLDN family protein may be selected from CLDN1, CLDN2, CLDN3, CLDN4, CLDN5, CLDN6, CLDN7, CLDN8, CLDN9, CLDN10, CLDN11, CLDN12, CLDN15, CLDN16, CLDN18 (CLDN18.1 or CLDN18.2), CLDN20, CLDN23.

[0070] In certain embodiments, the tumor is a carcinoma, sarcoma, myeloma, leukemia, lymphoma, and mixed tumors. Non-limiting examples of tumors that can be treated by the methods and compositions described herein include cancer cells from the bladder, blood, bone, bone marrow, brain, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus. Furthermore, the cancer may be of the following histological types, in particular, but not limited to: malignant neoplasms; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; bile duct carcinoma; mixed hepatocellular and bile duct carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial polyposis coli; malignant carcinoid tumors; bronchiolar-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granulosa cell carcinoma; Granulocyte carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenal cortical carcinoma; skin adnexal carcinoma; apocrine carcinoma; sebaceous gland carcinoma; cerumenic gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma cell tumor; malignant granulosa cell tumor; and malignant fibroblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor of the testis; malignant lipid cell tumor; malignant Paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mixed Müllerian tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner's tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian thyroid carcinoma Adenoma; Choriocarcinoma; Malignant mesonephroma; Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Lymphangiosarcoma; Osteosarcoma; Parosteal osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloblastic odontosarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; Malignant pinealoma; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrillary astrocytoma; Astroblastoma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroma; Neuroblastoma; Retinoblastoma;Olfactory neurogenic tumors; malignant meningioma; neurofibrosarcoma; malignant neurilemoma; malignant granular cell tumor; paragranuloma; small lymphocytic malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myeloid sarcoma; plasmacytoma, colorectal cancer, and hairy cell leukemia.

[0071] Herein, exemplary viruses may include: adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, flaviviridae, hepatitis delta virus, hepeviridae, mononegavirales, nidovirales, picornaviridae, orthomyxoviridae, papillomaviridae, parvoviridae, polyomaviridae, One or more of the families poxviridae, reoviridae, retroviridae, coronaviridae, paramyxovirinae, herpesviridae, or togaviridae.

[0072] It also includes double-stranded DNA viruses (dsDNA) such as adenovirus, herpes virus, and poxvirus; single-stranded DNA viruses (ssDNA) such as parvovirus; double-stranded RNA viruses (dsRNA) such as rotavirus and reovirus; positive-sense single-stranded RNA viruses (+ssRNA) such as new coronavirus, norovirus, hepatitis C, hepatitis A virus, dengue fever, etc.; negative-sense single-stranded RNA viruses (-ssRNA) such as influenza virus, rabies virus, etc.; single-stranded RNA retroviruses (ssRNA-RT) such as HIV virus; double-stranded DNA retroviruses (dsDNA-RT) such as hepatitis B virus, etc.

[0073] When the immunostimulatory composition is a vaccine, the viral antigen can be a coronavirus antigen, a norovirus antigen, an Ebola virus antigen, an HIV antigen, an influenza virus antigen, a rabies virus antigen, a herpes virus antigen, a rotavirus antigen, a hepatitis virus antigen, an HIV antigen, an HPV antigen, or an RSV antigen, specifically a novel coronavirus antigen, a herpes zoster virus antigen, a hepatitis B virus antigen, an influenza A virus antigen, or an influenza B virus antigen. The influenza virus antigen can be an influenza A1 virus antigen, an influenza A3 virus antigen, an influenza Bv virus antigen, or an influenza By virus antigen.

[0074] In one embodiment, in the vaccine, the mass ratio of the viral antigen to the CpG oligonucleotide is 10:1-1:150, and the mass ratio is preferably 5:1-1:50.

[0075] In one embodiment, the vaccine comprises 1 μg-2000 μg / mL of the CpG oligonucleotide according to any one of claims 1 to 4 and 1-100 μg / mL of viral antigen; preferably comprises 5 μg-500 μg / mL of the CpG oligonucleotide according to any one of claims 1 to 4.

[0076] Herein, exemplary bacteria may include one or more of the genera Staphylococcus, Streptococcus, Listeria, Erysipelothrix, Nephrobacterium, Bacillus, Clostridium, Mycobacterium, Actinomyces, Nocardia, Corynebacterium, and Rhodococcus; and may further include one or more of Bacillus anthracis, Bacillus erysipelothrix, Bacillus tetani, Listeria, Bacillus anthracis, Mycobacterium tuberculosis, Escherichia coli, Proteus, Shigella dysenteriae, Klebsiella pneumoniae, Brucella, Bacillus perfringens, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Acinetobacter, Yersinia, Legionella pneumophila, Bordetella pertussis, Bordetella parapertussis, Shigella, Pasteurella, Vibrio cholerae, and Bacillus parahaemolyticus.

[0077] Herein, exemplary fungi may include one or more of Coccidioides immitis, Coccidioides spp., Histoplasma capsulatum, Histoplasma durnei, Blastomyces lobateus, Paracoccidioides brasiliensis, Blastomyces dermatitidis, Sporothrix schenckii, Penicillium marneffei, Candida albicans, Candida glabrata, Candida tropicalis, Candida lusutica, Aspergillus, Exophiala zhenni, Chromomyces peijensis, Chromomyces compactus, Chromomyces verrucosum, Chromomyces dermatitidis, Geotrichum candidum, Pedostreatus boydii, Cryptococcus neoformans, Trichosporon, Rhizopus oryzae, Mucor indica, Absidia corymbifera, Coprosporon racemosus, Frog feces mold, Coronary ear mold, Heterosporium, Rhinosporium sibiricum, Hyaluromyces, and Pseudomonas aeruginosa.

[0078] Herein, exemplary parasites may include one or more of digestive tract parasites (such as ascaris, hookworms, tapeworms, Entamoeba histolytica and Giardia lamblia, etc.), cavity parasites (such as Trichomonas vaginalis), liver parasites (such as liver flukes, echinococcosis), lung parasites (such as Paragonimus westermannii), brain tissue parasites (such as cysticercosis, Toxoplasma gondii), intravascular parasites (such as schistosomes), intralymphatic parasites (such as filarial worms), muscle tissue parasites (such as Trichinella spiralis larvae), intracellular parasites (such as Plasmodium, Leishmania), bone tissue parasites (such as hydatids; skin parasites, such as scabies mites, hair follicle mites), and intraocular parasites (such as sucking nematodes, pork cysticercosis).

[0079] According to another aspect of the present invention, the immunostimulatory composition is a tumor therapeutic agent.

[0080] In some embodiments, the immunostimulatory composition further comprises at least one antibody targeting a tumor antigen.

[0081] The tumor antigen may be as defined above.

[0082] The immunostimulatory compositions provided by the present invention may also include pharmaceutically acceptable excipients including, for example, solvents, fillers, buffers, tonicity modifiers, and preservatives (see, for example, Pramanick et al., Pharma Times, 45:65-77, 2013). In certain embodiments, the pharmaceutical composition may include excipients that act as one or more of the solvents, fillers, buffers, and tonicity modifiers (e.g., sodium chloride in saline can serve as an aqueous vehicle and tonicity modifier).

[0083] In some embodiments, the immunostimulatory composition further comprises one or more of immune cell therapy drugs, chemical drugs, substances that promote mucosal immune absorption or mucosal adhesion, immunomodulators, ligands of pattern recognition receptors, pharmaceutically acceptable salts or excipients.

[0084] The immune cell therapy drug can be selected from one or more of tumor infiltrating lymphocytes, dendritic cells, cytokine-induced killer cells, dendritic cell-cytokine-induced killer cells, natural killer cells, γδT cells, CD3AK, CAR-T and TCR-T.

[0085] Examples of such chemotherapeutic drugs include: alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trihydroxymethylmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin ; CC-1065 (including its synthetic analogs adolesin, kazelesin, and bitrazesin); Nostoc cyclic peptides (particularly Nostoc cyclic peptide 1 and Nostoc cyclic peptide 8); dolastatin; duocarmycin (including its synthetic analogs KW-2189 and CBI-TMI); chrysoprolol; hyoscyamine; sarcodictyin; spongestatin; nitrogen mustards such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, nitrogen oxide hydrochloride, melphalan, new nitrogen mustard, phenylephrine, prednimustine, trofosfamide, uramustine; nitroureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; anti- and phenotypes thereof. The present invention also includes antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma and calicheamicin phi, see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemycins, including dynemycin A; bisphosphonates, such as clodronate; esperamicins; and the neocarcinogen chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, carabicin, caminomycin, carmomycin, chromomycin, and more. Biotin, daunorubicin, detoxorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexicomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivomycin, peplomycin, potfiromycin, puromycin, triferon-adriamycin, rhodorubicin, streptozocin, streptozocin, tuberculin, ubenimex, zoloft, daunorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as leucovorin, methotrexate, pteropterin, and trimetrexate;Purine analogs such as fludarabine, 6-mercaptopurine, thioamidine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as captestosterone, drostanolone propionate, cyclothiocarbamate, melastane, and testosterone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; and folic acid supplements such as folinic acid. acid); aceglucuronolide; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; colcemid; diazocine; elformithine; elliptonium acetate; epothilone; etoglucagon; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansines such as maytansine and ansamitocin; mitoxantrone; mitoxantrone; mopidarol; nivalridine; pentostatin; methambucil; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxane; sizofuran; spirogermanium; tenuzolic acid; triazinon; 2,2′,2″-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, baculocin A, and serpentin); urethan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; noxoline; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0086] The substance promoting mucosal immune absorption or mucosal adhesion can be selected from one or more of special surfactants, chelating agents, and adhesives, and is further preferably selected from one or more of polylactic acid-glycolic acid copolymer, dextran, and polysaccharide.

[0087] The immunomodulator can be selected from one or more of chemokines, stem cell growth factors, lymphotoxins, and hematopoietic factors, and further preferably one or more of colony stimulating factor (CSF), interferon, erythropoietin, thrombopoietin, tumor necrosis factor (TNF), interleukin (IL), granulocyte-colony stimulating factor (G-CSF), and granulocyte macrophage-colony stimulating factor (GM-CSF).

[0088] The pattern recognition receptor ligand can be selected from the group consisting of a TLR receptor ligand, a RLR receptor ligand, a CLR receptor ligand, and a NLR receptor ligand.

[0089] It is easy to understand that the concept of CpG oligonucleotides well known to those skilled in the art includes its pharmaceutically acceptable salt form, unless otherwise indicated. Exemplary alkali salts include: ammonium salts, alkali metal salts, such as sodium, lithium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, zinc salts, and organic bases (e.g., organic amines), such as N-Me-D-reduced glucosamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline, tromethamine, dicyclohexylamine, tert-butylamine, and salts formed with amino acids such as arginine, lysine, etc. In addition, the CpG oligonucleotides can be provided in a pharmaceutical solution comprising a pharmaceutically acceptable excipient. Alternatively, the CpG oligonucleotides can be provided as a lyophilized solid, which is then reconstituted in sterile water, saline or a pharmaceutically acceptable buffer before administration.

[0090] Delivery system

[0091] According to yet another aspect of the present invention, it also relates to a delivery system comprising i) the CpG oligonucleotide as described above or the immunostimulatory composition as described above, and ii) a delivery vehicle.

[0092] In some embodiments, the delivery vehicle comprises one or more liposomes, one or more exosomes, one or more microvesicles, one or more dendrimers, one or more nanocomplexes, one or more nanogels, one or more gold nanoparticles, polylactic-co-glycolic acid, one or more cell-penetrating peptides, and a group consisting thereof.

[0093] Liposomes can be cationic liposomes or neutral liposomes, which can be prepared or modified by known methods, such as adding polyethylene glycol (PEG)-modified liposomes to effectively prevent the aggregation of liposome carriers and increase their stability. Liposomes or lipid transfection preparations can be prepared by methods known to those skilled in the art. Such methods are described in, for example, WO 2016205764 and U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, each of which is incorporated herein by reference in its entirety.

[0094] Dendrimers, a family of specialized polymers with well-defined molecular structures, precisely controlled chemical structures, and unique multivalent properties, are increasingly becoming non-viral vectors for gene delivery. Typical dendrimers include poly(amidoamine) (PAMAM) dendrimers, which can be further modified, such as by modifying the PAMAM surface with the nucleobase analog 2-amino-6-chloropurine to create AP-PAMAM derivatives or by coupling chondroitin sulfate (CS) to PAMAM to create CS-PAMAM.

[0095] A preferred example of the nanocomplex is a nanocomplex prepared by electrostatic interaction between CpG and polyethyleneimine (PEI).

[0096] A preferred example of the nanogel is a polyethylene glycol (PEG)-based nanogel.

[0097] The delivery system can usually improve the stability of CpG and enhance the cellular endocytosis function, and can generally enhance the immune activation effect of CpG.

[0098] Treatment methods and applications

[0099] The present invention also relates to the use of the above-mentioned CpG oligonucleotide, the above-mentioned immunostimulatory composition, or the above-mentioned delivery system in the preparation of a drug for regulating immune cell activity, wherein the use is performed in vivo or in vitro.

[0100] In some embodiments, the immune cell is selected from the group consisting of a macrophage, a lymphocyte, and a dendritic cell.

[0101] In some embodiments, the immune cells are present in human PBMCs.

[0102] In some embodiments, regulating the activity of immune cells is promoting the release of inflammatory factors by the immune cells.

[0103] In some embodiments, the inflammatory factor comprises at least one of IFN-α, TNF-α, and IL-6.

[0104] The present invention also relates to the use of the above-mentioned CpG oligonucleotide, the above-mentioned immunostimulatory composition, or the above-mentioned delivery system in the preparation of a medicament for treating and / or preventing at least one of the following indications: tumor, antiviral, antibacterial, antifungal, antiparasitic, reducing chemotherapy side effects, anti-fatigue or enhancing immunity, and promoting the subject's immune response to antigens in a subject in need.

[0105] In some embodiments, the indication is related to a TLR9-mediated immune response.

[0106] In some embodiments, the drug is in the form of an injectable dosage form, a respiratory tract dosage form, nasal drops, a skin dosage form, a mucosal dosage form, or a cavity dosage form.

[0107] In some embodiments, the antigen comprises a tumor, viral, bacterial, fungal, or parasitic antigen.

[0108] In some embodiments, the drug is a vaccine adjuvant or a vaccine.

[0109] In some embodiments, the medicament is a vaccine adjuvant or vaccine for coronavirus, herpes virus, hepatitis virus or influenza virus.

[0110] In some embodiments, the drug is a vaccine adjuvant or vaccine for the novel coronavirus, herpes zoster virus, hepatitis B virus, influenza A virus, or influenza B virus. Specifically, it is a vaccine adjuvant or vaccine for influenza A1 virus, influenza A3 virus, influenza Bv virus, or influenza By virus, or a quadrivalent vaccine adjuvant or vaccine for influenza A1 virus, influenza A3 virus antigen, influenza Bv virus, or influenza By virus.

[0111] In some embodiments, the subject is a mammal.

[0112] In some embodiments, the subject is a primate.

[0113] In some embodiments, the subject is a human.

[0114] The present invention also relates to the use of the above-mentioned CpG oligonucleotide as a TLR9 agonist.

[0115] The present invention further provides a method for eliciting a TLR9-mediated immune response in a subject, comprising administering to the subject an effective amount of the CpG oligonucleotide as described above, or the immunostimulatory composition as described above.

[0116] The term "effective amount" as used in the present invention refers to the dosage of the component to which the term corresponds to achieve the purpose of treating, preventing, alleviating and / or relieving the disease or condition described in the present invention in a subject.

[0117] In some embodiments, wherein the administering is performed intravenously, intramuscularly, intramammarily, intradermally, intraperitoneally, subcutaneously, by spray, by aerosol, in ovo, mucosally, transdermally, by immersion, orally, intraocularly, intratracheally, or intranasally.

[0118] In some embodiments, wherein the administering is performed via a delivery system as described above.

[0119] Delivery or administration can be via single dose or multiple doses.

[0120] In some embodiments, the disease associated with TLR9-mediated immune response is at least one of tumor, antiviral, antibacterial, antifungal, antiparasitic, reducing chemotherapy side effects, anti-fatigue or enhancing immunity, and promoting the subject's immune response to antigens.

[0121] In some embodiments, the CpG oligonucleotide described above, or the immunostimulatory composition described above, or the delivery system described above is used as a vaccine adjuvant or vaccine.

[0122] In some embodiments, the CpG oligonucleotide as described above, or the immunostimulatory composition as described above, or the delivery system as described above is used as a vaccine adjuvant or vaccine for coronavirus, herpes virus, hepatitis virus or influenza virus.

[0123] In some embodiments, the CpG oligonucleotide described above, or the immunostimulatory composition described above, or the delivery system described above is used as a vaccine adjuvant or vaccine for a novel coronavirus, herpes zoster virus, hepatitis B virus, influenza A virus, or influenza B virus. Specifically, it is used as a vaccine adjuvant or vaccine for influenza A1 virus, influenza A3 virus, influenza Bv virus, or influenza By virus, or as a quadrivalent vaccine adjuvant or vaccine for influenza A1 virus, influenza A3 virus antigen, influenza Bv virus, or influenza By virus.

[0124] In some embodiments, the subject suffers from an infectious disease, and the CpG oligonucleotide as described above, or the immunostimulatory composition as described above, is administered to elicit an immune response against the pathogen causing the infectious disease.

[0125] In some embodiments, the method can be used in combination with surgery, radiotherapy, chemotherapy, and various immunotherapies, or can be used in combination with traditional therapies for patients with viral infections, bacterial infections, or parasitic infections.

[0126] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0127] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0128] Example 1 Preparation of artificially synthesized CpG-containing single-stranded deoxyoligonucleotides

[0129] The CpG-containing single-stranded deoxyoligonucleotides (CpG-ODN) shown in Table 1 were synthesized using the solid-phase phosphoramidite triester method.

[0130] 1. Reagents and materials:

[0131] Trichloroacetic Acid (TCA), controllable solid phase carrier, DMT (dimethoxytrityl), tetrazolium activator, acetic anhydride, N-methylimidazole, four nucleotide monomers A, T, C, and G, DNA synthesizer, purifier, ultrafiltration, vacuum dryer, high performance liquid chromatography, etc.

[0132] 2. Methods:

[0133] Deprotection

[0134] Trichloroacetic Acid (TCA) was used to remove the dimethoxytrityl (DMT) protecting group of the nucleotide attached to the controlled solid phase support (Controlled Pore Glass) to obtain a free 5'-hydroxyl end for the next condensation reaction.

[0135] activation

[0136] The phosphoramidite-protected nucleotide monomer is mixed with a tetrazolium activator and introduced into a synthesis column to form a phosphoramidite tetrazolium-active intermediate (whose 3′-end has been activated, but the 5′-end is still protected by DMT). This intermediate will undergo a condensation reaction with the deprotected nucleotide on a controllable solid phase support.

[0137] connect

[0138] When the phosphoramidite tetrazole active intermediate encounters the nucleotide with the deprotected group on the controllable solid phase support, it will undergo a nucleophilic reaction with its 5'-hydroxyl group, condense and remove the tetrazole. At this time, the synthesized oligonucleotide chain is extended forward by one base.

[0139] Closed

[0140] After the condensation reaction, in order to prevent the 5′-hydroxyl group connected to the controllable solid phase support from being extended in the subsequent cyclic reaction, this terminal hydroxyl group is often blocked by acetylation. Generally, the acetylation reagent is formed by mixing acetic anhydride and N-methylimidazole.

[0141] Oxidation

[0142] During the condensation reaction, the nucleotide monomers are connected to the oligonucleotides attached to the controllable solid phase support through phosphite bonds. However, the phosphite bonds are unstable and easily hydrolyzed by acids and bases. At this time, iodine-tetrahydrofuran solution is often used to convert phosphite into phosphotriester to obtain stable oligonucleotides.

[0143] After these five steps, a deoxynucleotide is attached to the nucleotide on the controllable solid-phase support. Trichloroacetic acid is then used to remove the DMT protecting group on the 5'-hydroxyl group of the newly attached deoxynucleotide. Repeating the activation, ligation, blocking, and oxidation processes yields a crude DNA fragment. Finally, post-synthesis processing, including cleavage, deprotection (typically, A and C bases are protected with a benzoyl group; G bases are protected with an isobutyryl group; T bases do not need to be protected; and phosphorous acid is protected with a cyanoethyl group), purification (commonly used methods include HAP, PAGE, HPLC, C18, and OPC), and quantification, yields an oligonucleotide fragment that meets experimental requirements.

[0144] Unsulfurized CpG single-stranded deoxyoligonucleotides were synthesized on an ABI 3900 DNA synthesizer; fully and partially sulfurized CpG single-stranded deoxyoligonucleotides were synthesized on an ABI 394 DNA synthesizer using the displacement method.

[0145] Table 1: Artificially synthesized CpG-containing single-stranded deoxyoligonucleotides

[0146] Note: Capital letters indicate phosphorothioate backbones; lowercase letters indicate phosphodiester backbones.

[0147] Example 2 Active CpG Screening

[0148] The active sequences in CpG1-15 prepared in Example 1 were screened by testing the proliferation effect of CpG on human PBMC and mouse spleen cells T and B cells.

[0149] Type A CpG 2216, type B CpG 1018, and type C CpG 2395 were used as positive controls, and FX-700 was used as a negative control. The CpGs used in Example 2 and all subsequent examples were prepared by our company.

[0150] 1. Reagents and materials:

[0151] Human peripheral blood leukocyte concentrate, normal saline (Dupont, China), PBS (BI, Israel), Ficoll-Paque PLUS (GE, USA), RPMI-1640 medium (Corning, USA), FBS (Clark, USA), penicillin / streptomycin (Quanshijin, China), trypan blue (Sigma, USA), CellTrace TM CFSE Cell Proliferation Kit (InvitroGen, USA), Human TruStain FcX TM (Fc receptor blocking solution) (BD, USA), human CD45-PerCP-Cy5.5 (BD, USA), human CD3-APC (BD, USA), human CD19-V450 (BD, USA); cell strainer (BD Falcon, USA), red blood cell lysis buffer (BD, USA), LIVE / DEAD TM fixable aqua dead cell stain kit (InvitroGen, USA), mouse CD16 / CD32 antibody (2.5G2) (BD, USA), mouse CD45-PerCP-Cy5.5 (30-F11) (BD, USA), mouse CD3e-PE-Cy7 (145-2C11) (BD, USA), mouse CD19-APC (1D3) (BD, USA);

[0152] RPMI-1640 complete medium: RPMI-1640 medium containing 10% fetal bovine serum and 100 U / mL-100 μg / mL Pen-Strep (penicillin).

[0153] Erythrocyte lysis buffer: Dilute 10× erythrocyte lysis buffer to 1× using sterile water. That is, add 4500 μL of sterile water to 500 μL of erythrocyte lysis buffer.

[0154] Hydroxyfluorescein diacetate succinimidyl ester (CFSE) solution: Dissolve CFSE in DMSO to a 1 mg / mL stock solution. When used, dilute with serum-free PBS to a working concentration of 5 μg / mL.

[0155] 2. Methods:

[0156] Isolation of mouse spleen cells. BALB / c mice were euthanized and immediately immersed in 75% ethanol. In a cleanroom, a small incision was made on the left side of the mouse's abdomen. The spleen was removed with forceps and placed in a culture dish containing a small amount of pre-chilled RPMI-1640 medium. Gently triturate the spleen with the plunger of a 1 mL syringe until a chylomicronized fluid formed. Filter the cells through a 100 μm filter. Collect the single-cell suspension from the mouse spleen and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 2-3 mL of red blood cell lysis buffer. Incubate on ice for 2 minutes. Terminate the reaction by adding 15-20 mL of complete RPMI-1640 medium and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant and resuspend the mouse spleen cells in PBS. Count the cells using trypan blue staining. Add 10 mL of PBS and centrifuge at 1500 rpm for 5 minutes.

[0157] Isolation of hPBMC. Dilute concentrated human peripheral blood leukocytes 3-fold with normal saline. Slowly add the diluted concentrated human peripheral blood leukocytes to the upper layer of Ficoll-Paque PLUS at a 1:1 ratio, increase 8 and drop 1, and centrifuge at 2500 rpm for 25 minutes. Aspirate the buffy coat layer, add 20 mL of normal saline, and centrifuge at 1800 rpm for 10 minutes. Discard the supernatant, resuspend the cells in 20 mL of normal saline, and centrifuge at 1500 rpm for 8 minutes. Discard the supernatant, resuspend the cells in 20 mL of normal saline, and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, resuspend the mononuclear cells in PBS, and count the cells using trypan blue staining. Add PBS to make up to 45 mL and centrifuge at 1500 rpm for 5 minutes.

[0158] CFSE-labeled cells. Resuspend human PBMC or mouse spleen cells in PBS to a density of 1×10 7 10 cells / mL. Add 5 μL CFSE to each 1 mL of resuspension for staining. Incubate at room temperature in the dark for 7 minutes (human PBMC) or 5 minutes (mouse spleen cells). Add pre-chilled RPMI-1640 complete medium to terminate the reaction and centrifuge at 1500 rpm for 5 minutes. Add 10-20 mL PBS to wash twice and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant and resuspend the cells in RPMI-1640 complete medium to a density of 5×10 6 pieces / mL.

[0159] CpG ODN stimulation. Prepared cell suspension was added at 100 μL / well (about 5×10 5Cells were plated into a 96-well U-shaped plate. CpG 1-15, CpG 2216, CpG 1018, CpG 2395, and FX-700 solutions were then added to the plate at 100 μL / well to achieve a final CpG concentration of 1 μM. Separate unstimulated wells containing only cells and culture medium (stimulation concentration = 0) were set up. The plates were incubated in a 37°C, 5% CO2, saturated humidity incubator for 5 days (hPBMC) or 3 days (mouse spleen cells).

[0160] Flow cytometry was used to detect T and B cell proliferation. Cells were collected in flow cytometry tubes and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in 2 mL of PBS. 2 μL of Human TruStain FcX TM (for hPBMCs) or mouse CD16 / CD32 antibodies (for mouse spleen cells) were incubated at room temperature in the dark for 10 minutes for nonspecific blocking. 0.5 μL of Aqua was added to distinguish between live and dead cells. Anti-CD45, anti-CD3, and anti-CD19 mAbs were added for cell surface staining, mixed well, and incubated at 4°C for 25-30 minutes. The cells were washed twice with 1-2 mL of PBS and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in 300 μL of PBS. The proliferation of T and B cells was then detected using an LSRFortessa™ flow cytometer (BD, USA).

[0161] 3. Results:

[0162] The results are shown in Table 2.

[0163] Table 2 CpG-induced B cell proliferation in humans and mice

[0164] The results in Table 2 show that among CpG1-15, only CpG1 has the activity of stimulating B cell proliferation, which is effective in both human PBMC and mouse spleen cells. In addition, all CpGs have no significant stimulating effect on T cell proliferation in human PBMC and mouse spleen cells.

[0165] Example 3 Detection of CpG ODN Biological Activity in HEK Blue mTLR9 Cells

[0166] By detecting HEK-Blue TM The secretion of embryonic alkaline phosphatase (SEAP) of mTLR9 cell line was used to evaluate the activation effect of different concentrations of CpG on mouse TLR9. HEK-Blue was stimulated with certain concentrations of CpG1, CpG 2395, CpG 7909, CpG 1018, and negative control FX-700 (non-CpG). TMmTLR9 cells (expressing mouse TLR9) activate NF-κB and AP-1, thereby inducing the production of SEAP, which can be expressed by Quanti-Blue TM Reagents for detection, Quanti-Blue TM The reagent binds to SEAP and displays a blue color. The OD value at 630 nm is measured by a spectrophotometer to quantify the level of SEAP, thereby evaluating the activation of mouse TLR9 by different CpGs.

[0167] 1. Reagents and materials:

[0168] HEK-Blue TM mTLR9 cells (WCB reservoir cells);

[0169] DMEM (Gibco, 11995-065); FBS (Gibco, 10099-141C); Zeocin TM (ant-zn-05, InvivoGen, USA); Blasticidin (ant-b1-05, InvivoGen, USA); Normocin TM (ant-nr-1, InvivoGen, USA), Pen-Strep (Gibco, 15140-122); Quanti-Blue (Invivogen, rep-qbl); 96-well plate (Costar, 3599).

[0170] Growth medium: Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / L glucose, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 100 μg / mL normocin, and Pen-Strep (100 U / mL to 100 μg / mL). After two passages of revived cells, the growth medium should be supplemented with the selective antibiotics 100 μg / mL Zeocin and 30 μg / mL Blasticidin.

[0171] QUANTI-Blue TM Test solution: Add 1 mL of QB reagent and 1 mL of QB buffer to a 250 mL sterile culture flask. Make up to 100 mL with sterile water and vortex to mix. Incubate at room temperature for 10 minutes before use.

[0172] Test sample solution: Weigh an appropriate amount of lyophilized powder of the test sample (CpG1, CpG 2395, CpG 7909, CpG 1018, negative control FX-700), dissolve it with sterile PBS, and then dilute it to the required concentration with growth medium without supplementation of selective antibiotics.

[0173] 2. Methods:

[0174] HEK-Blue mTLR9 cells were cultured in vitro as adherent monolayers in growth medium supplemented with selective antibiotics in a 37°C, 5% CO2 incubator. The medium was changed twice a week, and cells were typically cultured at a rate of at least 0.5 × 10 cells / week. 6 Cells / mL were passaged (cell density should not exceed 6×10 6 cells / mL), not exceeding 15 generations.

[0175] Take HEK-Blue in logarithmic growth phase TM mTLR9 cells were washed and digested to prepare single cells, and resuspended in fresh growth medium supplemented with selective antibiotics to adjust the cell density to 2-3 × 10 5 pieces / mL.

[0176] Add 180 μL / well of the prepared cell suspension to a U-shaped 96-well plate. Incubate the plate in a 37°C, 5% CO2, saturated humidity incubator for approximately 1-24 hours, then top up each well to 180 μL. Add 20 μL / well of the test sample solution of varying concentrations to the corresponding wells of the U-shaped 96-well plate, with final concentrations of 0, 0.25, 0.5, 1, 2, 5, 10, 20, and 40 μM. Set up three replicates for each concentration and continue incubation for 24 hours.

[0177] After the incubation, centrifuge the U-shaped 96-well plate at 1000 rpm for 5 min, aspirate 40 μL of supernatant from each well and place it in a flat-bottomed 96-well plate, and quickly add 160 μL of QUANTI-Blue TM The test solution was shaken briefly and incubated in a 37°C, 5% CO2 incubator for 15 minutes. The 96-well test plate was removed and the OD value at a wavelength of 630 nm was measured using a spectrophotometer.

[0178] 3. Results:

[0179] See Figure 1. Figure 1 shows that all sequences showed significant responses, with a dose-dependent relationship. CpG1 showed a relatively strong response, followed by CpG 2395, CpG 1018, and CpG 7909. The reverse sequence, FX-700, had no effect on mTLR9 activation.

[0180] Example 4 CCK-8 assay for the effect of CpG ODN stimulation on mouse splenocyte proliferation

[0181] The CCK-8 (Cell Counting Kit-8) assay was used with a multifunctional microplate reader to examine the effects of different CpGs on mouse splenic lymphocyte proliferation. The assay principle is that WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazolium monosodium salt) is reduced by mitochondrial dehydrogenases in the presence of the electron coupling agent 1-Methoxy PMS to produce an orange-yellow, water-soluble formazan. The color of the resulting formazan is directly proportional to cell proliferation and inversely proportional to cytotoxicity. OD values ​​were measured at a wavelength of 450 nm using a microplate reader, indirectly reflecting the number of viable cells.

[0182] 1. Reagents and materials:

[0183] 96-well plate (Costar, 3599);

[0184] CCK-8 (Tongren Chemical, CK04); FBS (Gibco, 10099-141C); Pen-Strep (Gibco, 15140-122); RPMI-1640 (Sigma, R8758-500 mL);

[0185] RPMI-1640 complete medium: RPMI-1640 medium containing 10% fetal bovine serum and 100 U / mL-100 μg / mL Pen-Strep (penicillin).

[0186] 2. Methods:

[0187] Mouse spleen cell isolation. Mice were sacrificed by cervical dislocation and completely immersed in a container containing 75% alcohol for 3 minutes. The mice were secured in a biosafety cabinet. The abdominal cavity was opened, fat and fascia were trimmed, and the spleen was removed. Blood was washed with saline. An appropriate amount of saline solution was added to a culture dish, and a 100μm cell sieve was placed. The spleen was removed and placed in the sieve. Using a clean 10mL or 20mL syringe, the tip of the syringe was used to press the tissue. The cells within the membrane will gradually dissociate. After passing through the sieve, they were suspended in the culture dish solution and rinsed with saline until the tissue color faded. A 70μm pore size cell sieve was placed on a 50mL centrifuge tube and rinsed with 2mL of saline. Cells were pipetted into the 70μm sieve and filtered. After filtration, the sieve was rinsed twice with 2mL of saline. The entire cell suspension was collected into a 50mL centrifuge tube. Centrifuge at 319g for 5 minutes at room temperature, discard the supernatant, and collect the cell pellet. Add pre-chilled 1X red blood cell lysis buffer to the cell pellet. Gently disperse the cells by blowing them apart. Incubate on ice, shaking the tube several times by inverting it every 1 minute. After 5 minutes, add 20 mL of normal saline. Centrifuge at 319g for 5 minutes. Discard the supernatant and collect the cell pellet.

[0188] Cell plate. Resuspend mouse spleen cells in complete culture medium and adjust the cell density to 2×10 based on the cell count results. 7 cells / mL, added into 96-well plates at 100 μL / well, and cultured in a 37°C, 5% CO2 incubator for about 1-24 h.

[0189] CpG ODN stimulation. Dilute the test samples (CpG1, CpG 2395, CpG 7909, CpG 1018, and negative control FX-700) in complete culture medium and add 10 μL per well to a 96-well plate. Set up three replicates for each concentration. Each cell plate should also have three control wells containing cells but no CpG ODN and three blank wells containing no cells or CpG. The working concentrations of CpG ODN are 0, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, and 2 μM, respectively. Mix thoroughly and incubate in a 37°C, 5% CO2 incubator for approximately 72 hours.

[0190] CCK-8 assay: Add 10 μL of CCK-8 solution to each well, incubate the plate in an incubator for 1-4 hours, and measure the absorbance at 450 nm using a microplate reader.

[0191] 3. Results

[0192] See Figure 2. As shown in Figure 2, FX-700 failed to induce mouse spleen cell proliferation, and as the concentration of FX-700 increased, GC inhibited mouse spleen cell proliferation. With the exception of FX-700, all other tested CpG ODNs demonstrated the ability to induce mouse spleen cell proliferation. The peak induction ability of most CpG ODNs occurred at 0.25 μM. At 0.25 μM, the order of CpG ODN proliferation induction, from strongest to weakest, was: CpG1 > CpG 1018 > CpG 7909 > CpG 2395.

[0193] Example 5 Cytokine activity after stimulation of human PBMC with different CpGs

[0194] After normal human PBMCs were stimulated in vitro with different concentrations of different CpGs for 16-24 hours, the levels of cytokines such as IFN-α, TNF-α, and IL-6 in the cell supernatant were measured to evaluate the immunostimulatory activity.

[0195] 1. Reagents and materials:

[0196] RPMI-1640 (Gibco, 11875-093); FBS (Gibco, 10099-141C); Pen-Strep (Gibco, 15140-122); Ficoll-Paque TM PLUS (GE, 17144003-1); Human TNF-αPrecoated ELISA kit (Dayu, 1117202); Human IFN-αPrecoated ELISA kit (Dayu, 1110012); Human IL-6 Precoated ELISA kit (Dayu, 1110602);

[0197] Fresh healthy human whole blood;

[0198] Complete culture medium: RPMI-1640 medium containing 10% fetal bovine serum and 100 U / mL-100 μg / mL Pen-Strep.

[0199] 2. Methods:

[0200] Human PBMC isolation: Transfer 30 mL of peripheral blood to a 100 mL sterile bottle, add 30 mL of normal saline at a ratio of 1:1, and mix well. Take 10 15 mL sterile centrifuge tubes and add 5 mL of Ficoll-Paque TMPLUS, and slowly add 6mL of diluted blood on it, being careful not to damage the interface. Transfer to a centrifuge, centrifuge at 960g for 30min at room temperature. Aspirate the buffy coat layer and place it in a 50mL centrifuge tube, add 20mL of normal saline, centrifuge at 319g for 10min at room temperature. Discard the supernatant and resuspend the cells in 40mL of normal saline, centrifuge at 460g for 5min at room temperature. Resuspend the cells in 5mL of complete culture medium and adjust the suspension concentration to 1-2×10 according to the number of cells. 6 / mL for future use.

[0201] Seed the cells. Add the adjusted cell concentration to a 96-deep-well plate at 1 mL / well according to the well plate layout. Incubate in a 37°C, 5% CO2 incubator for at least 1 hour.

[0202] CpG ODN stimulation: Dilute CpG ODN in complete medium and add 10 μL per well to the 96-deep-well plate described above. Do not replicate for a single concentration. The working concentrations of CpG ODN are 0, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 5, and 10 μM. Incubate in a 37°C, 5% CO2 incubator for approximately 16-24 hours.

[0203] ELISA cytokine content detection: According to the detection method provided in the instructions of each kit, the content of IFN-α, IL-6, and TNF-α in the cell supernatant was detected.

[0204] 3. Results:

[0205] See Figures 3-5. As shown in Figure 3, CpG1 is comparable to CpG 2395 in stimulating human PBMC to secrete IFN-α, exhibiting a strong stimulating effect and significantly superior to CpG 1018 and CpG 7909. As shown in Figure 4, CpG1 is significantly more potent than CpG 1018, CpG 2395, and CpG 7909 in stimulating human PBMC to secrete IL-6 at CpG ODN concentrations of 0.25-2 μM. As shown in Figure 5, each CpG ODN exhibits a certain stimulating effect on TNF-α secretion in human PBMC compared to the negative control.

[0206] Example 6 Effect of CpG on the Immunogenicity of the Novel Coronavirus Vaccine Composition

[0207] The effect of CpG1 on the immunogenicity of the new coronavirus recombinant subunit vaccine composition was evaluated by detecting the anti-BA 4 / 5 antibody titer in the serum of immunized mice.

[0208] 1. Reagents and materials:

[0209] Female BALB / c mice aged 6–8 weeks were purchased from Weitonglihua Laboratory Animal Co., Ltd.

[0210] Normal saline was purchased from Qingdao High-Tech Industrial Park Haiao Biotechnology Co., Ltd.;

[0211] Antigen Omicron BA.4 / BA.5 RBD protein (hereinafter referred to as BA 4 / 5), catalog number SPD-C522r, purchased from ACRO; CpG 1: source is the same as in Example 2;

[0212] Aluminum hydroxide adjuvant (Sinopharm Group).

[0213] 2. Methods:

[0214] A novel coronavirus vaccine composition was prepared using physiological saline as the solvent. Each 200 μl of the vaccine composition contained 5 μg of BA 4 / 5, 90 μg of Al(OH)3 (calculated as aluminum), 1 μg of CpG 1 (if available), and the balance was physiological saline.

[0215] Mice were randomly divided into groups of 24 mice each and immunized as shown in Table 3. BALB / c mice in each group received a single intramuscular injection of 200 μL on D0 and D28 (the day of the first immunization was designated as immunization day 0, i.e., D0). Immune saline was used as a control. Blood samples were collected from 12 mice per group on D7, D29, and D56. Blood was collected from the retroorbital venous plexus on D7, and from the eyeballs on D29 and D56.

[0216] Table 3: Mouse grouping

[0217] Blood was collected from mice at the prescribed time points, and serum was obtained by centrifugation. The anti-BA 4 / 5 antibody titer was determined using an indirect ELISA (the coating antigen was Omicron BA.4 / BA.5 RBD protein).

[0218] 3. Results:

[0219] As shown in Figure 6, neither the saline group nor the aluminum hydroxide single adjuvant group showed immunogenicity. The dual-adjuvant group showed higher antibody titers. Antibody titers were positively correlated with the number of days of treatment. The dual-adjuvant group maintained high antibody titers on Day 56. At Day 56, the BA4 / 5 + aluminum hydroxide + CpG 1 group had the highest antibody titer, 2-5 times that of the same-dose group.

[0220] Example 7 Effect of CpG on the Immunogenicity of Herpes Zoster Vaccine Composition in Mice

[0221] The effect of CpG1 on the immunogenicity of the herpes zoster vaccine composition was evaluated by detecting the gE protein-specific antibody titer in the serum of immunized mice.

[0222] 1. Reagents and materials:

[0223] Female C57BL / 6J mice were purchased from Weitonglihua Laboratory Animal Co., Ltd.

[0224] PBS phosphate buffered saline dry powder, purchased from Solarbio;

[0225] gE protein, a self-produced protein, the sequence of which is shown in SEQ ID NO: 21 ();

[0226] CpG 1: Source is the same as in Example 2; aluminum hydroxide adjuvant (Alum) (Sinopharm Group).

[0227] 2. Methods:

[0228] Herpes zoster vaccine compositions were prepared using PBS solution as the solvent. Each 50 μl of the vaccine composition contained 5 μg of gE protein. The dosages of aluminum adjuvant and CpG1 (if any) in each group were as shown in Table 4, with the remainder being PBS solution.

[0229] Eight-week-old female C57BL / 6J mice were randomly divided into groups of 12 mice each and immunized as shown in Table 4. Each group of mice received a single intramuscular injection of 50 μL on D0 and D28 (the day of the first immunization was designated as immunization day 0, i.e., D0). An immune PBS solution was used as a control. Orbital blood was collected on D21, D28 (before the second immunization), D31, and D56, with 200-300 μL of blood drawn from each mouse. Serum was isolated and assayed for binding antibody titers using ELISA. Antibody titers were expressed as geometric mean titers (GMTs). The mouse groupings are shown in Table 4.

[0230] Table 4: Mouse grouping

[0231] 3. Results:

[0232] See Figure 7. As shown in Figure 7, CpG 1 can enhance the immunogenicity of the antigen. Furthermore, within the 1-72 μg dose range, the immunopotentiating effect of CpG 1 initially increases and then decreases with increasing dose. Furthermore, compared with the CpG 1 alone vaccine group, the CpG 1 + Alum dual-adjuvant vaccine group significantly increased VZV gE-specific antibody titers (by more than 100-fold).

[0233] Example 8 Effect of CpG on the Immunogenicity of Hepatitis B Vaccine Composition in Mice

[0234] The effect of CpG 1 on the immunogenicity of the hepatitis B vaccine composition was evaluated by detecting the anti-HBs antibody level in the serum of immunized mice.

[0235] 1. Reagents and materials:

[0236] Female BALB / c mice were purchased from Weitonglihua Laboratory Animal Co., Ltd.;

[0237] PBS phosphate buffered saline dry powder (Solarbio) Hepatitis B stock solution; (Yunnan Watson)

[0238] CpG 1; source is the same as in Example 2; aluminum hydroxide adjuvant (Sinopharm Group).

[0239] 2. Methods:

[0240] Hepatitis B vaccine compositions were prepared using PBS solution as the solvent. Each 1 mL of the vaccine composition contained 1 μg of antigen, 0.43 mg of aluminum hydroxide (calculated as aluminum), 5, 15, 45, and 135 μg of CpG 1 (if any), with the remainder being PBS solution.

[0241] Female BALB / c mice aged 6-8 weeks were randomly divided into groups of 10 mice per group and immunized as shown in Table 5. On D0 and D28 (the day of the first immunization was recorded as immunization day 0, i.e., D0), each group of mice was immunized with an intraperitoneal injection of 1 mL, with an immune PBS solution as a control. Blood was collected on D21, D28 (before the second immunization), D42, and D56, with 200-300 μL of blood collected from each mouse each time. Serum was separated and the anti-HBs antibody levels in the serum samples were detected using a commercial hepatitis B surface antibody detection kit. The antibody titer was expressed as the geometric mean titer (GMT). The mouse grouping is shown in Table 5.

[0242] Table 5: Mouse grouping

[0243] Note: The hepatitis B in this table refers to the hepatitis B stock solution, which contains hepatitis B antigen.

[0244] 3. Results:

[0245] See Figure 8. As shown in Figure 8, CpG 1 can enhance the immunogenicity of the antigen in a dose-dependent manner within the dose range of 5-45 μg per dose. When the CpG 1 dose is further increased to 135 μg, the immunogenicity of the vaccine composition decreases.

[0246] Example 9 Effect of CpG on the Immunogenicity of Influenza Vaccine Compositions in Mice

[0247] The effect of CpG 1 on the immunogenicity of the quadrivalent influenza vaccine composition was evaluated by detecting the levels of anti-A1 antigen antibodies, anti-A3 antigen antibodies, anti-Bv antigen antibodies, and anti-By antigen antibodies in the serum of immunized mice.

[0248] 1. Reagents and materials:

[0249] Female BALB / c mice were purchased from Weitonglihua Laboratory Animal Co., Ltd.;

[0250] PBS phosphate buffered saline dry powder (Solarbio);

[0251] Influenza A1, A3, By, and Bv antigens (SinoBiological);

[0252] CpG 1: Source is the same as in Example 2.

[0253] 2. Methods:

[0254] A quadrivalent influenza vaccine composition was prepared using PBS as the solvent. Each 0.1 mL of the vaccine composition contained 3.5 μg of each antigen (A1, A3, Bv, By) and 10, 20, 40, and 80 μg of CpG 1, with the remainder being PBS.

[0255] Female BALB / c mice aged 6-8 weeks were randomly divided into groups of 20 mice per group (10 mice in the PBS group) and immunized as shown in Table 6. Each group of mice received a single immunization on D0, injected into the inner thigh of the hind limb, with an injection volume of 0.1 mL. An immune PBS solution was used as a control. Blood was drawn from the mice at the prescribed times, with 200-300 μL of blood drawn from each mouse. Serum was separated and the geometric mean titers of four antibodies (anti-A1 antigen antibody, anti-A3 antigen antibody, anti-Bv antigen antibody, and anti-By antigen antibody) in the mouse serum were measured by ELISA. Antibody titers were expressed as geometric mean titers (GMT). The mouse groupings are shown in Table 6.

[0256] Table 6: Mouse grouping

[0257] 3. Results:

[0258] See Figures 9A-9D. As shown, CpG 1 rapidly induced the production of protective antibodies; significantly increased antibody titers in response to quadrivalent influenza vaccine (QIV), maintaining elevated antibody titers for a prolonged period; and exhibited a dose-dependent relationship within a certain range, with the immunopotentiating effect of CpG 1 initially increasing and then decreasing with increasing dose. The strongest immunostimulatory effect was achieved at a dose of 40 μg per mouse.

[0259] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A CpG oligonucleotide, the sequence of which is shown in SEQ ID NO:

1. The CpG oligonucleotide according to claim 1 , comprising a chemical modification.

3. The CpG oligonucleotide according to claim 2, comprising modification of one or more than one phosphate groups.

4. The CpG oligonucleotide according to claim 3, wherein the modification of the phosphate group comprises one or more of a phosphorothioate internucleotide bond, a methylphosphonate bond, and a boranophosphate bond.

5. An immunostimulatory composition comprising the CpG oligonucleotide according to any one of claims 1 to 4. The immunostimulatory composition according to claim 5 , further comprising another adjuvant other than the CpG oligonucleotide.

7. The immunostimulatory composition according to claim 6, wherein the adjuvant comprises one or more of alum, complete Freund's adjuvant, incomplete Freund's adjuvant, squalene, squalane, muramyl dipeptide, MF59, AS03, AS04, monophosphatidyl lipid A, flagellin, Poly (I: C), aluminum salt and calcium salt.

8. The immunostimulatory composition according to any one of claims 5 to 7, further comprising at least one antigen.

9. The immunostimulatory composition according to claim 8, wherein the antigen is a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.

10. The immunostimulatory composition according to any one of claims 5 to 7, which is a tumor therapeutic agent.

11. The immunostimulatory composition according to claim 10, further comprising at least one antibody targeting a tumor antigen.

12. The immunostimulatory composition according to claim 9 or 11, wherein the tumor antigen is selected from any one or more of the group consisting of the following antigens or their functional fragments: α-fetoprotein, α-actinin-4, A3, antigens specific for A33 antibodies, ART-4, B7, Ba 733, BAGE, BrE3 antigen, BMCA, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD1 9. CD21, CD23, CD25, CD29, CD30, CD32b, CD37, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, C D83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CLDN family proteins, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen p, CEA, CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, fibroblast activation protein α, fibroblast growth factor, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, GD2, gp100, GRO-β, HLA-DR, HM1.

24. Human chorionic gonadotropin and its subunits, HMGB-1, hypoxia-inducible factor, HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL- 18, IL-23, IL-25, insulin-like growth factor 1, KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor, MAGE, MAGE-3, MART1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1α, MIP-1β, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, mesothelin, pancreatic cancer mucin, prostate stem cell antigen, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, trophoblast cell surface antigen 2, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RAN TES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Fredenreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bc1-2, bc1-6, and Kras. .

13. The immunostimulatory composition according to any one of claims 5 to 12, further comprising one or more of immune cell therapy drugs, chemical drugs, substances that promote mucosal immune absorption or mucosal adhesion, immunomodulators, ligands of pattern recognition receptors, and pharmaceutically acceptable salts or excipients.

14. The immunostimulatory composition according to any one of claims 5 to 7, which is a vaccine adjuvant.

15. The immunostimulatory composition according to claim 14, comprising 1 μg-2000 μg / mL of the CpG oligonucleotide according to any one of claims 1 to 4; preferably comprising 5 μg-500 μg / mL of the CpG oligonucleotide according to any one of claims 1 to 4.

16. The immunostimulatory composition according to claim 15, further comprising an aluminium salt adjuvant, preferably an aluminium hydroxide adjuvant.

17. The immunostimulatory composition according to claim 16, wherein the content of the aluminum salt adjuvant is 100 μg-1 mg / mL, preferably 400-800 μg / mL.

18. The immunostimulatory composition according to claim 8, which is a vaccine.

19. The immunostimulatory composition according to claim 18, wherein the antigen is a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.

20. The immunostimulatory composition according to claim 19, wherein the viral antigen is a coronavirus antigen, a herpes virus antigen, a hepatitis virus antigen or an influenza virus antigen.

21. The immunostimulatory composition according to claim 20, wherein the viral antigen is a novel coronavirus antigen, a herpes zoster virus antigen, a hepatitis B virus antigen, an influenza A virus antigen or an influenza B virus antigen; preferably an influenza A1 virus antigen, an influenza A3 virus antigen, an influenza Bv virus antigen, or an influenza By virus antigen.

22. according to claim 20 or 21 described immunostimulatory compositions, wherein the mass ratio of viral antigen and CpG oligonucleotide is 10:1-1:150, and the preferred mass ratio is 5:1-1:

50.

23. The immunostimulatory composition according to claim 20 or 21, comprising 1 μg-2000 μg / mL of the CpG oligonucleotide according to any one of claims 1 to 4, and 1-100 μg / mL of a viral antigen; preferably comprising 5 μg-500 μg / mL of the CpG oligonucleotide according to any one of claims 1 to 4.

24. A delivery system comprising i) the CpG oligonucleotide of any one of claims 1 to 4 or the immunostimulatory composition of any one of claims 5 to 23, and ii) a delivery vehicle.

25. The delivery system of claim 24, wherein the delivery vehicle comprises one or more liposomes, one or more exosomes, one or more microvesicles, one or more dendrimers, one or more nanocomplexes, one or more nanogels, one or more nanogold particles, polylactic-co-glycolic acid, one or more cell-penetrating peptides, and a group consisting of the same.

26. Use of the CpG oligonucleotide according to any one of claims 1 to 4, the immunostimulatory composition according to any one of claims 5 to 23, or the delivery system according to any one of claims 24-25 in the preparation of a medicament for regulating immune cell activity, wherein the use is performed in vivo or in vitro.

27. The use according to claim 26, wherein the immune cells are selected from macrophages, lymphocytes and dendritic cells.

28. The use according to claim 26 or 27, wherein regulating the activity of immune cells is promoting the immune cells to release inflammatory factors.

29. The use according to claim 28, wherein the inflammatory factor comprises at least one of IFN-α, TNF-α and IL-6.

30. Use of the CpG oligonucleotide of any one of claims 1 to 4, or the immunostimulatory composition of any one of claims 5 to 23, or the delivery system of any one of claims 24-25 in the preparation of a medicament for treating and / or preventing at least one of the following indications: tumor, viral infection, bacterial infection, fungal infection, parasitic infection, chemotherapy side effects, fatigue or low immunity, and low immune response of the subject to an antigen in a subject in need thereof.

31. The use according to claim 30, wherein the drug is in the form of an injection, a respiratory tract administration, a nasal drop, a skin administration, a mucosal administration or a cavity administration.

32. The use according to claim 30, wherein the antigen comprises a tumor, virus, bacteria, fungus or parasite antigen.

33. The use according to claim 30, wherein the medicine is a vaccine adjuvant or a vaccine.

34. The use according to claim 33, wherein the medicine is a vaccine adjuvant or vaccine for coronavirus, herpes virus, hepatitis virus or influenza virus.

35. The use according to claim 34, wherein the drug is a vaccine adjuvant or vaccine for a novel coronavirus, herpes zoster virus, hepatitis B virus, influenza A virus, or influenza B virus; preferably, a vaccine adjuvant or vaccine for influenza A1 virus, influenza A3 virus, influenza Bv virus, or influenza By virus, or a quadrivalent vaccine adjuvant or vaccine for influenza A1 virus, influenza A3 virus, influenza Bv virus, or influenza By virus.

36. The use according to any one of claims 30 to 35, wherein the subject is a mammal.

37. The use according to claim 36, wherein the subject is a primate.

38. The use according to claim 37, wherein the subject is a human.

39. A method for eliciting a TLR9-mediated immune response in a subject, comprising administering to the subject an effective amount of a CpG oligonucleotide according to any one of claims 1-4, or an immunostimulatory composition according to any one of claims 5-23, or a delivery system according to any one of claims 24-25.

40. The method of claim 39, wherein the administering is intravenous, intramuscular, intramammary, intradermal, intraperitoneal, subcutaneous, by spray, by aerosol, in ovo, mucosal, transdermal, by immersion, orally, intraocularly, intratracheally, or intranasally.

41. The method of claim 39 or 40, wherein the administration is performed by single dose or multiple doses.

42. The method according to claim 39, wherein the disease associated with TLR9-mediated immune response is at least one of tumors, viral infection, bacterial infection, fungal infection, parasitic infection, chemotherapy side effects, fatigue or low immunity, and low immune response of the subject to an antigen.

43. The method according to claim 39, the CpG oligonucleotide according to any one of claims 1-4, or the immunostimulatory composition according to any one of claims 5-23, or the delivery system according to any one of claims 24-25 is used as a vaccine adjuvant or vaccine.

44. The method of claim 43, wherein the vaccine adjuvant or vaccine is a vaccine adjuvant or vaccine for a coronavirus, herpes virus, hepatitis virus or influenza virus.

45. The method according to claim 44, wherein the vaccine adjuvant or vaccine is a vaccine adjuvant or vaccine for a novel coronavirus, herpes zoster virus, hepatitis B virus, or influenza A virus, or influenza B virus; preferably a vaccine adjuvant or vaccine for influenza A1 virus, influenza A3 virus, influenza Bv virus, or influenza By virus, or a quadrivalent vaccine adjuvant or vaccine for influenza A1 virus, influenza A3 virus, influenza Bv virus, or influenza By virus.

46. ​​The method according to any one of claims 39 to 45, wherein the subject is a mammal.

47. The method of claim 46, wherein the subject is a primate.

48. The method of claim 47, wherein the subject is a human.

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