Immunostimulatory compositions and uses thereof

Abstract

The present invention provides an immunostimulatory composition comprising a saponin and a CpG oligodeoxynucleotide, or consisting of a saponin-containing adjuvant and a CpG oligodeoxynucleotide, wherein the CpG oligodeoxynucleotide sequence has two or more copies of the 5'-TTCGTT-3' motif or the 5'-TCGTCGTCG-3' motif, and further provides the use of the immunostimulatory composition in the manufacture of a drug for treating a disease.

Description

[Technical Field]

[0001] The present invention belongs to the field of biopharmaceuticals. Specifically, the present invention relates to an immunostimulatory composition comprising a saponin and a CpG oligodeoxynucleotide, or consisting of a saponin-containing adjuvant and a CpG oligodeoxynucleotide. The CpG oligodeoxynucleotide sequence has two or more copies of the 5'-TTCGTT-3' motif or the 5'-TCGTCGTCG-3' motif. The present invention further relates to pharmaceutical uses of the immunostimulatory composition. [Background technology]

[0002] CpG oligodeoxynucleotides (CpG oligodeoxynucleotides) are a recently discovered novel immunostimulant. They are deoxynucleotides containing cytosine guanine dinucleotides, which have an immune response similar to that of natural CpG pattern recognition receptors (PGRs). They bind to Toll-like receptors (TLRs) on the cell membrane and can effectively induce immune responses in mammals via the TLR9 signaling pathway. CpG-induced immune responses are primarily Th1-type, and can induce the conversion of Th2-type immune responses to Th1, resulting in cellular immunity. By activating immune cells such as T cells, B cells, and NK cells, they can produce large amounts of various cytokines, strengthening the body's specific and nonspecific immune effects and playing an important role in linking innate and adaptive immunity.

[0003] Saponins are glycosides with triterpene or spirosterane aglycones and are a type of plant-derived adjuvant. Quillaja saponin (QS) is extracted from the Quillaja japonica plant. QS-21 is the most commonly reported adjuvant in the QS series, but it can induce hemolysis and has systemic and local toxic side effects. A study by Alving et al. (ALVING CR, MATYAS G, BECK Z, et al. Revue Roumaine de Chimie, 2016, 61(8):631-635.) demonstrated that ALF liposomes, combined with MPLA and QS-21 as adjuvants, effectively increased serum antibody titers against the HIV gp140 protein. Ng et al. (NG H, FERNANDO GJP, DEPELSENAIRE ACI, et al. Scientific Reports, 2016, 6(1):228-230.) used a nanopatch as a subcutaneous delivery technique to form a QS-21 and adjuvant complex. The results showed that compared with conventional intramuscular injection, the nanopatch significantly reduced the dose of antigen and QS-21, and induced higher IgG titers (Han Ziyi, Zeng Zhongliang, Modern Agricultural Science and Technology, 2019(14):220-221.).

[0004] A prior art document (WO2001051083A3) reported an immunostimulatory composition containing saponin and CpG oligodeoxynucleotides, among which the CpG oligodeoxynucleotides were CpG1826 and CpG7909. However, due to the structural diversity of CpG oligodeoxynucleotides, the effects of CpG adjuvants with different sequences vary greatly.

[0005] Therefore, there is currently a demand for adjuvants and drugs with stronger immune effects. Summary of the Invention [Problem to be solved by the invention]

[0006] The inventors have conducted extensive research into the shortcomings of the prior art and have unexpectedly discovered an immunostimulatory composition with stronger immune effects, in which saponin and CpG oligodeoxynucleotides exhibit an effective synergistic effect, mediating a stronger immune response. The immunostimulatory composition has obvious advantages when used with different antigens or antigen compositions.

[0007] Therefore, one object of the present invention is to provide an immunostimulatory composition, which can be used in the manufacture of various drugs to provide efficient immunostimulation.

[0008] Another object of the present invention is to provide a vaccine adjuvant, which is capable of inducing a potent immune response in a mammal. [Means for solving the problem]

[0009] The object of the present invention is achieved by the following technical solutions: In one aspect, the present invention provides an immunostimulatory composition comprising a saponin and a CpG oligodeoxynucleotide, or consisting of a saponin-containing adjuvant and a CpG oligodeoxynucleotide, wherein the CpG oligodeoxynucleotide sequence has two or more copies of a 5'-TTCGTT-3' motif or a 5'-TCGTCGTCG-3' motif.

[0010] In the immunostimulatory composition of the present invention, the sequence of the CpG oligodeoxynucleotide is any one selected from CpG T1: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6), CpG T2: TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 7), and CpG T3: TCG TCG TCG TCG TCG TCG TCG (SEQ ID NO: 8). Preferably, the sequence of said CpG oligodeoxynucleotide is CpG T1: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6).

[0011] In the immunostimulatory composition of the present invention, the saponin is one or more selected from Quillaja saponin, ginsenoside, platycodin, astragaloside, notoginsenoside, glycyrrhizin, julibroside, ophiopogonin, saikoside, and chikusetsusaponin. Preferably, the saponin is Quillaja saponin, ginsenoside, platycodin, or astragaloside A. More preferably, the Quillaja saponin is QS-7, QS-17, QS-18, or QS-21. Even more preferably, the Quillaja saponin is QS-21. The ginsenoside may be ginsenoside Rg1, ginsenoside Rg3, ginsenoside Rb1, or ginsenoside Re. The platycodin The saponin monomer may be platycodin D, platycodin D2, or a mixture of both; the astragaloside may be astragaloside A (astragaloside IV), astragaloside I, astragaloside II, or a mixture of two or more of these saponin monomers; the notoginsenoside may be notoginsenoside R1; the ophiopogonin may be ophiopogonin D; the saikoside may be saikoside a, saikoside d, or a mixture of both; the julibroside may be the total saponin of Anthurium sieboldii; the glycyrrhizin may be the total saponin of Licorice Root; and the chikusetsusaponin may be the total saponin of Chikusetsujin.

[0012] In the immunostimulatory composition of the present invention, the adjuvant comprising a saponin is an immunostimulatory complex adjuvant (Iscom adjuvant).

[0013] In the immunostimulatory composition of the invention, the CpG oligodeoxynucleotide comprises a phosphorothioate bond, in particular a perthio oligodeoxynucleotide, preferably a perthio oligodeoxynucleotide.

[0014] In the immunostimulatory composition of the present invention, the weight ratio of the CpG oligodeoxynucleotide to the saponin is 1-40:0.1-2, preferably 2-40:0.1-2, and more preferably 2:1.

[0015] In another aspect, the present invention further provides a pharmaceutical composition comprising the immunostimulatory composition and an antigen or antigen composition.

[0016] In the pharmaceutical composition of the present invention, the antigen or antigen composition is selected from the group consisting of human immunodeficiency virus, human herpes virus, varicella-zoster virus, human cytomegalovirus, hepatitis A, B, C, and E virus, respiratory syncytial virus, human papillomavirus, influenza virus, Mycobacterium tuberculosis, Salmonella enterica, Neisseria such as Neisseria meningitidis, and Neisseria gonorrhoeae, Borrelia such as Borrelia relapsing fever or Borrelia duttonii, Chlamydia such as Chlamydia trachomatis, Bordetella such as Bordetella pertussis, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium The parasite is any one selected from the genus Toxoplasma, such as Toxoplasma gondii, or a malaria parasite such as Toxoplasma gondii.

[0017] In the pharmaceutical composition of the present invention, the human herpesvirus is HSV1 or HSV2.

[0018] In the pharmaceutical composition according to the present invention, the antigen is a tumor antigen.

[0019] In a further aspect, the present invention provides a vaccine comprising the immunostimulatory composition.

[0020] In the vaccine according to the present invention, the vaccine is a vaccine for preventing viral, bacterial and / or parasitic infections, or the vaccine is a vaccine for treating viral, bacterial and / or parasitic infections by immunotherapy.

[0021] In a further aspect, the invention provides the use of the immunostimulatory composition in the manufacture of a medicament for eliciting a cytolytic T cell response.

[0022] In certain embodiments, the invention provides for the use of the immunostimulatory composition in the manufacture of a medicament for inducing an interferon-gamma response in a mammal.

[0023] In certain embodiments, the invention provides for the use of said immunostimulatory composition in the manufacture of a vaccine for the prevention of viral, bacterial and / or parasitic infections.

[0024] In certain embodiments, the invention provides for the use of said immunostimulatory compositions in the manufacture of a vaccine for immunotherapeutic treatment of viral, bacterial and / or parasitic infections.

[0025] In certain embodiments, the invention provides for the use of said immunostimulatory composition in the manufacture of a vaccine for immunotherapeutic treatment of tumors.

[0026] The present invention further provides a method for eliciting a cytolytic T cell response, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an immunostimulatory composition of the present invention.

[0027] The present invention further provides a method for inducing an interferon-γ response in a mammal, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an immunostimulatory composition of the present invention.

[0028] The present invention further provides a method for preventing viral, bacterial and / or parasitic infections comprising administering to a subject in need thereof a prophylactically effective amount of a vaccine comprising an immunostimulatory composition of the present invention.

[0029] The present invention further provides a method for treating viral, bacterial and / or parasitic infections with immunotherapy, comprising administering to a subject in need thereof an effective amount of a vaccine comprising an immunostimulatory composition of the present invention.

[0030] The present invention further provides a method of treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an immunostimulatory composition according to the present invention. [Effects of the Invention]

[0031] The immunostimulatory composition of the present invention achieves unexpected technical effects and can mediate stronger immune responses. Although the immunostimulatory effect of CpG T1-T3 alone is weaker than that of CpG1018, CpG7909, CpG1826, etc., when used in combination with QS-21, the immunostimulatory composition exhibits an unexpected synergistic effect, significantly enhancing the immune effect.

[0032] In the present study, we found that a hepatitis B therapeutic vaccine containing the immunostimulatory composition can break through immune tolerance in genetically modified mice and produce high titers of anti-HBsAg, anti-HBcAg, and neutralizing antibodies. All of the results showed that the vaccine can significantly eliminate hepatitis B virus in genetically modified mice after multiple immunizations. After the immunization process, HBsAb levels reached near saturation, maintaining a stable, long-term immune effect, with an average HBsAg reduction rate of approximately 92%. The hepatitis B vaccine containing the immunostimulatory composition can induce relatively strong HBsAg and HBcAg-specific IFN-γ levels, demonstrating a significantly better immune effect than either adjuvant alone or the combination of conventional CPG adjuvant and QS-21.

[0033] A varicella zoster vaccine containing this immunostimulatory composition also demonstrated its excellent immunostimulatory effect. Cellular immunity experiments demonstrated that the vaccine was able to induce strong levels of herpes gE protein-specific IFN-γ, demonstrating that the protein-mediated immune effect was significantly superior to that of adjuvants alone. Humoral immunity experiments also demonstrated that the vaccine was able to induce high levels of herpes gE protein-specific IgG / IgG1 / IgG2a antibodies, demonstrating that the effect was superior to that of adjuvants alone and significantly superior to that of the combination of conventional CPG adjuvants and QS-21.

[0034] In summary, the immunostimulatory composition of the present invention has excellent immunostimulatory effects, and compared with a single adjuvant or a combination of a conventional CPG adjuvant and QS-21, the immunostimulatory composition of the present invention exhibits an efficient synergistic effect between CpG T1-T3 and QS-21, mediating a stronger immune response. It also offers significant advantages when used with different antigens or antigen compositions. Therefore, the immunostimulatory composition of the present invention, as a novel adjuvant, is expected to have high clinical value and a broad market. [Brief explanation of the drawings]

[0035] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0036] [Figure 1] 1 shows the effect of different CPG oligodeoxynucleotides on the secretion level of HBsAg antigen-specific IFN-γ. [Figure 2] 1 shows the effect of different CPG oligodeoxynucleotides on the secretion level of HBcAg antigen-specific IFN-γ. [Figure 3] 1 shows the effect of different immunostimulatory compositions according to the present invention on the secretion level of HBsAg antigen-specific IFN-γ. [Figure 4] 1 shows the effect of different immunostimulatory compositions according to the present invention on the secretion level of HBcAg antigen-specific IFN-γ. [Figure 5]1 shows the effect of different doses of an immunostimulatory composition according to the present invention on the secretion level of HBsAg antigen-specific IFN-γ. [Figure 6] 1 shows the effect of different doses of an immunostimulatory composition according to the present invention on the secretion level of HBcAg antigen-specific IFN-γ. [Figure 7] 1 shows the effect of a hepatitis B vaccine containing an immunostimulatory composition of the present invention on serum HBsAg levels. [Figure 8] 1 shows the effect of a hepatitis B vaccine containing an immunostimulatory composition of the present invention on serum HBsAb levels. [Figure 9] 1 shows the effect of a hepatitis B vaccine containing an immunostimulatory composition according to the present invention on the secretion level of HBsAg antigen-specific IFN-γ. [Figure 10] 1 shows the effect of a hepatitis B vaccine containing an immunostimulatory composition according to the present invention on the secretion level of HBcAg antigen-specific IFN-γ. [Figure 11] 1 shows the effect of a hepatitis B vaccine containing an immunostimulatory composition of the present invention on HBsAg antigen-specific IgG antibody and its subtype levels in mouse serum. In the figure, Panel A shows the serum HBsAb IgG levels in each group of mice. Panel B shows the serum HBsAb IgG1 levels in each group of mice. Panel C shows the serum HBsAb IgG2a levels in each group of mice. Panel D shows the ratio of serum HBsAb IgG2a to IgG1 in each group of mice. [Figure 12] 1 shows the effect of a hepatitis B vaccine containing an immunostimulatory composition of the present invention on the levels of HBcAg antigen-specific IgG antibodies and their subtypes in mouse serum. In the figure, Panel A shows the serum HBcAb IgG levels in each group of mice. Panel B shows the serum HBcAb IgG1 levels in each group of mice. Panel C shows the serum HBcAb IgG2a levels in each group of mice. Panel D shows the ratio of serum HBcAb IgG2a to IgG1 in each group of mice. [Figure 13] 1 shows the effect of a varicella zoster vaccine containing an immunostimulatory composition of the present invention on the secretion level of herpes gE antigen-specific IFN-γ. [Figure 14] 1 shows the effect of a varicella zoster vaccine containing an immunostimulatory composition of the present invention on antigen-specific IgG antibody and its subtype levels in mouse serum. In the figure, Panel A shows the serum IgG levels of mice in each group, Panel B shows the serum IgG1 levels of mice in each group, Panel C shows the serum IgG2a levels of mice in each group, and Panel D shows the ratio of serum IgG2a to IgG1 of mice in each group. [Figure 15] Figure 1 shows the effect of immunostimulatory compositions containing different saponins according to the present invention on the secretion level of herpes gE antigen-specific IFN-γ.

[0037] "Definition" Unless otherwise defined, all technical terms used herein have the same meaning as understood by those skilled in the art. For definitions and terms in the art, those skilled in the art can refer to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are standard three-letter and / or one-letter codes that refer to one of the 20 L-amino acids commonly used in the art.

[0038] Although broad ranges and approximations of parameters are given in the present invention, specific examples are provided with as much precision as possible. However, all numerical values ​​necessarily contain errors, to varying degrees, due to the standard deviation of their measurement. Furthermore, any range disclosed herein is understood to encompass any and all subranges contained therein. For example, a range described as "2 to 40" is considered to include any and all subranges between a minimum value of 2 and a maximum value of 40 (including the endpoints), i.e., all subranges beginning with a minimum value of 2 or greater (e.g., 2 to 6.1) and all subranges ending with a maximum value of 40 or less (e.g., 5.5 to 40). Furthermore, when a reference is "incorporated herein," it is understood to be incorporated in its entirety.

[0039] As used herein, the singular forms "a," "an," and "the" include the plural of the referent unless expressly limited to one referent. The term "or" is used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0040] As used herein, the terms "pharmaceutical composition," "drug combination," and "drug combination" are used interchangeably to refer to a combination of at least one drug and optional pharmaceutically acceptable excipients or additives that combine to achieve a particular purpose. In some embodiments, the pharmaceutical composition includes a combination of drugs that are separated in time and / or space, so long as they function together to achieve the objectives of the invention. For example, the components included in the pharmaceutical composition (e.g., gE protein, QS-21, CpG oligodeoxynucleotide) may be administered to a subject as a whole or separately. When the components included in the pharmaceutical composition are administered to a subject separately, the components may be administered to a subject simultaneously or sequentially.

[0041] As used herein, the term "CpG oligodeoxynucleotide" or "CpG-ODN" refers to a short, single-stranded synthetic DNA molecule containing one or more "CpG" units, where C represents cytosine, G represents guanine, and p represents a phosphodiester bond. In particular, the CpG oligodeoxynucleotide is unmethylated. In some embodiments, the CpG-ODN contains phosphorothioate bonds or a phosphorothioate backbone. That is, in some embodiments, the CpG-ODN is a phosphorothioate oligodeoxynucleotide (i.e., a perthio oligodeoxynucleotide). Preferably, all bonds between nucleotides in the CpG-ODN are phosphorothioate bonds, that is, the CpG-ODN is a perthio oligodeoxynucleotide. In other embodiments, the CpG-ODN contains two or more copies of the 5'-TTCGTT-3' motif or the 5'-TCGTCGTCG-3' motif. In particular, the CpG-ODN has a sequence selected from TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6), TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 7), TCG TCG TCG TCG TCG TCG TCG (SEQ ID NO: 8), preferably TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6).

[0042] As used herein, "ginsenoside, platycodin, astragaloside, notoginsenoside, glycyrrhizin, julibroside, ophiopogonin, saikoside, or chikusetsusaponin" refers to the active ingredients present in the corresponding plants. For example, ginsenoside is a sterol compound mainly present in Ginseng herbal medicines and is the active ingredient in Ginseng. In some embodiments, the ginsenoside is preferably a monomer such as ginsenoside Rg1, ginsenoside Rg3, ginsenoside Rb1, ginsenoside Re, or a mixture of two or more saponin monomers thereof; the platycodin is preferably platycodin D, platycodin D2, or a mixture of both; and the astragaloside is preferably a monomer such as astragaloside A (astragaloside IV), astragaloside I, astragaloside II, or a mixture of two or more saponin monomers thereof. It is preferable that the saponin monomers are a mixture of the above, notoginsenoside is preferably notoginsenoside R1, etc., ophiopogonin is preferably ophiopogonin D, etc., saikoside is preferably saikoside a, saikoside d or a mixture of both, julibroside is preferably the total saponin of Gokanhi, etc., glycyrrhizin is preferably the total saponin of Glycyrrhiza glabra, etc., and chikusetsusaponin is preferably the total saponin of Chikusetsuginseng, etc.

[0043] As used herein, "Iscom adjuvant" refers to an immunostimulating complex adjuvant, specifically, an antigen-free Iscom matrix, which is a cage-like adjuvant composed of phospholipids, saponin, and cholesterol.

[0044] As used herein, a "therapeutically and / or prophylactically effective amount" or "effective amount" refers to a dose sufficient to provide benefit to the subject to which it is administered. The actual dose, rate, and duration of administration will be determined by the individual condition and extent of the disease to be treated. The treatment prescription (e.g., determination of dosage) is ultimately the sole responsibility of a general practitioner or other physician, and will generally take into account the disease being treated, the individual condition of the patient, the site of delivery, the method of administration, and other factors known to physicians.

[0045] As used herein, the term "mammal" refers to humans, and may also refer to other animals, such as wild animals (e.g., herons, storks, cranes, etc.), domestic animals (e.g., ducks, geese, etc.), or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0046] In some other embodiments, the compositions of the present invention may include other additives, such as pharmaceutically acceptable carriers or excipients, particularly when present as a drug formulation.

[0047] Particularly preferred pharmaceutical carriers are water and aqueous buffer solutions, including isotonic salt solutions such as PBS (phosphate buffer), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol, or polyalkylene glycols (e.g., polypropylene glycol), triglycerides, etc. The type of pharmaceutical carrier used will depend, inter alia, on whether the composition of the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The composition of the present invention may also contain wetting agents, emulsifiers, or liquid buffer substances as additives.

[0048] The pharmaceutical composition, vaccine or drug formulation according to the invention may be administered by any suitable route of administration, for example orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

[0049] The present invention will be further described below in specific embodiments with reference to the drawings, but these are not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the spirit of the present invention, and as long as they do not deviate from the spirit of the present invention, they will fall within the scope of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention will now be described with reference to specific examples, it being understood that these examples are merely illustrative of the invention and are not intended to limit the scope of the invention in any way.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the raw materials, reagents, etc. used in the following examples are all commercially available products.

[0052] Example 1: Preparation of immunostimulatory compositions and hepatitis B vaccines according to the present invention 1. HBsAg stock solution: The amino acid sequence of HBsAg protein is as shown in SEQ ID NO:1.

[0053] HBsAg protein was prepared from HBsAg gene-recombinant yeast cells, including H. polymorpha, S. cerevisiae, and Pichia pastoris, with H. polymorpha being preferred. For details of the preparation process, see Chinese Patent Application No. CN108330145A. HBsAg gene-recombinant H. polymorpha cells were fermented and cultured to obtain bacterial cells. Then, bacterial cell disruption and purification steps such as silica gel adsorption, column chromatography, and TFF were performed.

[0054] 2. HBcAg stock solution: The amino acid sequence of the HBcAg protein is as shown in SEQ ID NO:2.

[0055] HBcAg protein is prepared from HBcAg gene-recombinant yeast cells, including H. polymorpha, S. cerevisiae, and Pichia pastoris, preferably H. polymorpha. For details of the preparation process, see Chinese Patent Application No. CN108047316A. HBcAg gene-recombinant H. polymorpha cells are fermented and cultured to obtain bacterial cells. After cell disruption and purification steps using ammonium sulfate, column chromatography, and TFF, an HBcAg stock solution is obtained.

[0056] 3. QS-21: Purchased from BRENNTAG, CAS number A010-023.

[0057] 4. Preparation method of CPG oligodeoxynucleotide raw material: The oligodeoxynucleotides are synthetically prepared oligodeoxynucleotide sequence fragments containing one or more CpG motifs, and the oligodeoxynucleotide sequences used in this example are shown in Table 1.

[0058] [Table 1]

[0059] The details of the preparation method are as follows. They were prepared using a conventional chemical synthesis method known as the solid-phase phosphoramidite triester method. Starting from the 3' end, 1) deprotection involves first removing the protecting group DMT (dimethoxytrityl group) of the nucleotide linked to CpG with trichloroacetic acid to yield a free 5' hydroxyl group for use in the condensation reaction in the next step. 2) activation involves mixing the phosphoramidite-protected nucleotide monomer with a tetrazolium activator and placing it in a synthesis column to generate a phosphoramidite tetrazole activated intermediate, which then condenses with the deprotected nucleotide in the CpG. 3) connection involves connecting the phosphoramidite tetrazole activated intermediate to the deprotected nucleotide in the CpG, where it undergoes a nucleophilic reaction with the 5' hydroxyl group, condensing to remove the tetrazole, and extending the oligonucleotide chain forward by one base. 4) Oxidation: During the condensation reaction, the nucleotide monomer is connected to the CpG-linked oligonucleotide via a phosphite ester bond. However, because the phosphite ester bond is unstable and easily hydrolyzed by acid or base, a thio reagent is used to oxidize the phosphoramidite to form a sulfur-phosphorus double bond phosphate triester, resulting in a stable oligonucleotide. 5) Blocking: After the condensation reaction, the unreacted 5' hydroxyl group attached to the CpG is typically blocked by acetylation to prevent elongation in the next cycling reaction. Through these five steps, one deoxynucleotide is connected to the CpG nucleotide. Repeating the above deprotection, activation, connection, oxidation, and blocking processes yields a crude DNA fragment. Finally, post-synthesis processing, such as cleavage, deprotection, purification, and quantification, is performed to complete the process.

[0060] 5. HBsAg stock solution and HBcAg stock solution were diluted to 200 μg / ml and 100 μg / ml, respectively, using PBS solution (purchased from Hyclone). Each CPG raw material was dissolved and diluted to 100 μg / ml using PBS solution in preparation for use in the next step.

[0061] Example 2: Screening experiments for CPG oligodeoxynucleotides 1. Experimental animals: C57BL / 6(N) mice, male, 4 weeks old, 135 mice, provided by Shanghai Lingcheng Experimental Animal Technology Co., Ltd.

[0062] Experimental group design: See Table 2. The injection volume was 100 μL per mouse. Group A was the negative control, and received 100 μL of PBS solution per mouse.

[0063] [Table 2]

[0064] 3. Experimental steps are as follows. Seven days after immunization, spleens were removed from mice, and splenic lymphocytes were prepared using conventional methods. The spleens were removed using sterile forceps and scissors under aseptic conditions. They were placed on a plate containing 2 ml of pre-chilled 2% FBS (purchased from GIBCO)-PBS. The spleens were then ground with a pestle to allow the spleen cells to pass through the mesh and onto the plate, yielding a cell suspension. The suspension was then filtered through a 40 μm cell strainer (purchased from BD) using a Pasteur pipette and placed in a 50 ml sterile centrifuge tube. The tube was centrifuged at 500 × g for 5 minutes at 4°C. The supernatant was discarded, and the cells were resuspended in 2 ml of 1x erythrocyte disruption reagent (purchased from BD). The tube was then left to stand in the dark at 4°C for 5 minutes to disrupt the red blood cells. The red blood cell disruption reaction was stopped by adding 10 ml of 2% FBS-PBS. The cells were centrifuged at 500 × g for 5 minutes at 4°C. The supernatant was discarded, and 5 ml of 2% FBS-PBS was added to resuspend the cells. Splenocytes were stimulated using the HBsAg-specific peptide pool PS4 and the HBcAg-specific peptide pool PCP as stimulators. HBsAg and HBcAg antigen-specific IFN-γ secretion levels were detected using an ELISPOT kit (BD) according to the kit's instructions. The number of spots measured by the ELISPOT kit was read using an ImmunoSPOT Series 3 ELISPOT analyzer (see Example 7 of Chinese Patent CN104043120B for detailed operating procedures).

[0065] The sequence of the HBsAg-specific peptide pool is shown in Example 7 of Chinese Patent CN104043120B, and the sequence of the HBcAg-specific peptide pool is shown in SEQ ID NOs: 16 to 30.

[0066] 4. Experimental Results: The ELISPOT spotting results are shown in Figures 1 and 2. As shown in the results, type B CpG adjuvants with different sequences had different immune effects. CpG T1-T3, CpG1018, CpG7909, CpG 1826, and CpG 684 were generally superior to type A and type C CpG adjuvants, while CpG1618 and CPG D2 had poorer immune effects, and the levels of HBsAg and HBcAg-specific IFN-γ induced by these adjuvants were both lower than those of type A and type C CpG adjuvants.

[0067] Example 3: Screening experiments for immunostimulatory compositions 1. Experimental animals: 81 C57BL / 6(N) mice, male, 4 weeks old, provided by Shanghai Lingcheng Experimental Animal Technology Co., Ltd. 2. Experimental group design: See Table 3. The injection volume was 100 μL per mouse. Group A was the negative control, and each mouse received 100 μL of PBS solution.

[0068] [Table 3]

[0069] 3. Experimental steps: same as in Example 2.

[0070] 4. Experimental results: The ELISPOT spot results are shown in Figures 3 and 4. As shown in the results, when CpG T1-T3 were combined with QS21, an efficient synergistic effect was achieved, and the induced levels of HBsAg and HBcAg-specific IFN-γ were significantly higher than those of some other CpG adjuvants such as CpG1018 and CpG7909, demonstrating an unexpected immune effect.

[0071] Example 4: Effect of different adjuvant contents on the immune effect of pharmaceutical compositions 1. Experimental animals: C57BL / 6(N) mice, male, 4 weeks old, 60 mice, provided by Shanghai Lingcheng Experimental Animal Technology Co., Ltd.

[0072] 2. Reagent materials: 1) HBsAg protein, HBcAg protein, and CpG T1 were all prepared as described in Example 1. 2) QS-21 (CAS number A010-023, purchased from BRENNTAG). 3) To prepare for use in the next step, HBsAg stock solution and HBcAg stock solution were diluted to 200 μg / ml and 100 μg / ml, respectively, using PBS solution (purchased from Hyclone). QS21 was diluted to 5 μg / ml, 50 μg / ml, and 100 μg / ml, respectively, using PBS solution. CpG T1 was dissolved and diluted to 50 μg / ml, 100 μg / ml, and 2 mg / ml, respectively, using PBS solution. CPG7909 was dissolved and diluted to 100 μg / ml using PBS solution.

[0073] 3. Experimental group design: See Table 4. The injection volume was 100 μL per mouse. Group A was the negative control, and received 100 μL of PBS solution per mouse.

[0074] 4. Experimental steps: same as in Example 2.

[0075] 5. Experimental results: See Figures 5 and 6 for the ELISPOT spot results. As shown in the results, changes in the dose of CpG T1 and QS21 both had significant effects on the vaccine composition, which were higher than those of the immunostimulatory composition at dose 5, and the induced HBsAg and HBcAg-specific IFN-γ levels were significantly higher than those of the CPG7909 group. However, due to differences between species, no significant increase in the induction effect was observed when the adjuvant dose was further increased, which is thought to be because the immune strength of the adjuvant is not accurately reflected in mice.

[0076] Although doses 1, 2, and 4 have the same immune stimulating effect as the CPG7909 group, the amount of adjuvant used is lower than that of the comparable CPG7909 group, which is advantageous.

[0077] [Table 4]

[0078] Example 5: Experimental group design and immunization process of Hepatitis B vaccine 1. Experimental animals and model establishment: C57BL / 6(N) mice: 81 males, 4 weeks old, provided by Shanghai Lingcheng Laboratory Animal Technology Co., Ltd. rAAV8-HBV adenovirus: purchased from Beijing Wujiahe Molecular Medicine Research Institute Co., Ltd. A mouse model in which rAAV8-HBV persistently infects C57BL / 6(N) mice was established by tail vein injection of rAAV8-HBV adenovirus.

[0079] 2. Reagent materials: 1) HBsAg protein: prepared according to Example 1.

[0080] 2) HBcAg protein: prepared according to Example 1.

[0081] 3) To prepare for the next step, HBsAg stock solution, HBcAg stock solution, and QS-21 were diluted to 200 μg / ml, 100 μg / ml, and 50 μg / ml, respectively, using PBS solution (purchased from Hyclone). CpG was dissolved in PBS solution and diluted to 100 μg / ml.

[0082] 3. Experimental group design: Referring to Table 5, the injection volume for each mouse was 100 μL per mouse. Group A was the negative control, and 100 μL of PBS solution was injected per mouse.

[0083] [Table 5]

[0084] 4. Animal immunization: All groups received intramuscular injections once every two weeks into the right hind thigh, for a total of six doses. These were administered at weeks 4, 6, 8, 10, 12, and 14 after tail vein injection of rAAV8-HBV virus. Blood samples were collected every two weeks after the start of administration, at weeks 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22. All mice were sacrificed at week 22.

[0085] Example 6: Effect of Hepatitis B Vaccine on Serum HBsAg Levels 1. Serum HBsAg detection steps: The detection was carried out at Nanjing Drum Tower Hospital.

[0086] A two-step immunoassay was used. First, the detection sample was bound to paramagnetic microparticles coated with hepatitis B surface antibody, washed, and then acridinium ester-labeled hepatitis B surface antibody conjugate was added. After washing, pre-excitation solution and excitation solution were added to the reaction mixture. The relative light units (RLU) of the detection sample were measured, and the HBsAg content in the sample was positively correlated with the RLU. The ARCHTITECT HBsAg standard curve was generated to determine the HBsAg concentration in the mouse serum sample. The final HBsAg concentration in the mouse serum sample was 50-200 times higher than the measured value.

[0087] 2. Results Analysis (Figure 7): The Group H vaccine containing the immunostimulant of the present invention showed a clear tendency for the corresponding HBsAg levels to decrease, and a stable and long-term immune effect was maintained after the immunization process (from week 14), demonstrating a clear advantage over the CpG-only group (Group F) and the QS-21 group (Group G). In Group H, the HBsAg level initially exceeded 6350 IU / ml and decreased to approximately 50 IU / ml. After the second immunization (week 6), the HBsAg level in this group decreased by more than 30% and by the third immunization (week 8), the HBsAg level decreased by more than 70%, and the average decrease rate remained at approximately 92% after the end of immunization at week 14. This demonstrated excellent immune efficacy. Compared with the double-adjuvant control (Group I), Group H still maintained a stable immune effect after the end of immunization at week 14, demonstrating a significantly superior immune level compared to Group I.

[0088] Example 7: Evaluation of humoral immune effect of hepatitis B vaccine 1. Serum HBsAb detection steps: The detection was carried out at Nanjing Drum Tower Hospital.

[0089] A two-step immunoassay was used. First, the detection sample was mixed with paramagnetic microparticles coated with recombinant HBsAg (rHBsAg), washed, and then an acridinium ester-labeled rHBsAg conjugate was added. After washing, pre-excitation solution and excitation solution were added to the reaction mixture. The relative light units (RLU) of the detection sample were measured, and the HBsAb content in the sample was positively correlated with the RLU. The ARCHTITECT HBsAb standard curve was then generated to determine the HBsAb concentration in the mouse serum samples. The final HBsAb concentration in the mouse serum samples was 50-200 times higher than the measured value.

[0090] 2. Results analysis (Figure 8): The Group H vaccine containing the immunostimulant began to produce HBsAb (over 10mIU / ml) after the second immunization (week 6), and the HBsAb level continued to increase with increasing immunizations, demonstrating a clear superiority over the CpG group (Group F) and the QS-21 group (Group G). Two weeks after the end of immunization (week 16), the HBsAb level nearly saturated, reaching a logarithm of 4.0, or approximately 10,000mIU / ml, demonstrating a clear advantage in antibody production compared to the double-adjuvant control (Group I).

[0091] Example 8: Evaluation of cellular immune effect of hepatitis B vaccine 1. Detection step: same as in Example 2.

[0092] 2. Evaluation index: A positive result is when the number of spots in the control well is 5 SFC or less and the number of spots in the sample well is 10 SFC or more. A positive result is when the number of spots in the control well is more than 5 SFC but less than 10 SFC, and the number of spots in the sample well / the number of spots in the control well is 2 or more. A positive result is when the number of spots in the control well is more than 10 SFC, and the number of spots in the sample well / the number of spots in the control well is 3 or more.

[0093] 3. Experimental results:

[0094] [Table 6]

[0095] Cellular immunity level detection results: See Figures 9 and 10 for ELISPOT spot results. As shown in the analysis results, the HBsAg-specific IFN-γ seroconversion rate was 100% in groups F to I, and the HBcAg-specific IFN-γ seroconversion rate was 100% in groups F to I. The H group vaccine containing the immunostimulant was able to induce relatively high levels of HBsAg and HBcAg-specific IFN-γ, with 2350 SFC / 10 6Larger than splenocytes, 1250SFC / 10 6 The level of HBsAg and HBcAg-specific IFN-γ induced by the double adjuvant control (Group I) was approximately 1630 SFC / 10 6 Splenocytes, approximately 750SFC / 10 6 In splenocytes, it was clearly lower than in group H.

[0096] Example 9: Detection of HBsAg and HBcAg specific antibodies in serum using pharmaceutical compositions 1. Detection step: A 96-well microplate was coated with purified HBsAg and HBcAg to form solid-phase antigens. After blocking, the test serum was diluted several times from a certain starting dilution to set multiple dilutions. The diluted serum samples were added to the 96-well microplate and then bound to HRP-labeled anti-IgG / IgG1 / IgG2a antibodies to form antigen-antibody (serum)-enzyme-labeled antibody complexes. Finally, the substrate TMB was added to develop color, and the absorbance (OD value) at a wavelength of 450 nm was measured using a microplate reader. The intensity of the color development was positively correlated with the levels of HBsAg and HBcAg-specific antibody IgG / IgG1 / IgG2a in the test sample. The antibody titer was determined by fitting the relationship curve of "absorbance OD value - serum sample dilution factor (Log)".

[0097] 2.Result analysis: 1) Serum HBsAb IgG antibody and subtype detection results: The HBsAg IgG antibody and subtype levels detected in mouse serum by ELISA at different times in each group are shown in Figure 11. The group H vaccine containing the immunostimulant produced relatively high titers of anti-HBsAg-specific IgG / IgG1 / IgG2a antibodies. Furthermore, antibody levels continued to increase with increasing immunization cycles, reaching near saturation by the sixth immunization (week 14), with specific antibody titers reaching more than 5.4 logarithms. No specific antibodies were detected in groups A to D. Although groups E to G produced HBsAg-specific IgG / IgG1 / IgG2a antibody levels, these antibody levels were significantly lower than those in group H. The double adjuvant control (group I) produced significantly lower anti-HBsAg-specific IgG and IgG2a antibody levels than group H.

[0098] 2) Serum HBcAb IgG antibody and subtype detection results: The HBcAb IgG antibody and subtype levels detected in mouse serum by ELISA at different times in each group are shown in Figure 12. The group H vaccine containing the immunostimulant produced relatively high titers of anti-HBcAg-specific IgG / IgG1 / IgG2a antibodies. Furthermore, antibody levels continued to increase with increasing immunizations, reaching near saturation by the sixth immunization (week 14), with specific antibody titers reaching 4.8 logarithms or more. No specific antibodies were detected in groups A through D. Although groups E through G produced HBcAg-specific IgG / IgG1 / IgG2a antibody levels, the antibody levels were significantly lower than those in group H. Furthermore, group H showed a high tendency toward the Th1 pathway, and panel D showed a clear trend toward an increase in specific IgG2a antibody, indicating that the group H vaccine can promote subtype conversion of anti-HBcAg antibodies, with a significantly higher conversion efficiency than the dual-adjuvant control (group I).

[0099] Example 10: Shingles vaccine experimental group design 1. Experimental animals and model establishment: C57BL / 6(N) mice, female, 5 weeks old, 48 mice, purchased from Shanghai Slice Laboratory Animal Co., Ltd.

[0100] 2. Reagent materials: 1) Herpes gE protein: the amino acid sequence is as shown in SEQ ID NO:31.

[0101] The preparation steps were based on the report by Thomsson E, Persson L, et al. in Journal of Virological Methods, 2011, Vol. 175, No. 1, pp. 53-59, and the details of the steps are as follows. Based on the target protein sequence, the nucleic acid sequence was optimized for mammalian expression systems, and the target gene was synthesized. The synthesized target gene was ligated into the pcDNA3.1(+) plasmid by restriction enzyme digestion and transformation to Top10 competent. Positive single clones were selected and verified by sequencing. Single clones were then amplified in large quantities, and a large amount of plasmid suitable for cell transfection was extracted using an endotoxin-free plasmid extraction kit. CHO suspension cells were transfected with the plasmid by transient transfection. When CHO cell viability fell below 70% or the fermentation time exceeded 7 days, the fermentation broth supernatant was collected by centrifugation at 5000 rpm for 30 minutes at 4°C. The fermentation broth was dialyzed against a solution containing 50 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole in a chromatography cabinet at 4°C, with a dialysis ratio of 1:100, every 4 hours, for a total of three dialysis runs. The collected samples were purified using a nickel column, and the collected peak samples of the target protein were subjected to SDS-PAGE detection. The relatively pure purified solutions were combined and dialyzed against a solution containing 20 mM phosphate and 150 mM NaCl at 4°C in a chromatography cabinet for 24 hours at a dialysis ratio of 1:100, with the solution changed every 8 hours. The sample was then filtered through a 0.22 μm sterile filter membrane and stored in a refrigerator at 4°C.

[0102] The prepared herpes gE protein stock solution should be more than 95% pure, have a protein content of 200 μg / ml or more, and have an endotoxin level of 0.1 Eu / μg or less.

[0103] 2) Herpes gE stock solution was diluted to 50 μg / ml and 10 μg / ml using PBS solution (purchased from Hyclone), QS-21 was diluted to 50 μg / ml and 10 μg / ml using PBS solution, CpG was diluted to 100 μg / ml and 20 μg / ml using PBS solution, and CpG7909 was diluted to 100 μg / ml and 20 μg / ml using PBS solution.

[0104] 3. Experimental group design: Referring to Table 7, the injection volume for each mouse was 100 μL per mouse. Group A was the negative control, and 100 μL of PBS solution was injected per mouse.

[0105] [Table 7]

[0106] 4. Animal immunization: All groups were given intramuscular injections once every two weeks at the right hind thigh, with two consecutive injections at week 0 and week 2, respectively, and all mice were killed at week 4.

[0107] Example 11: Verification of the effectiveness of cellular immunity induced by varicella zoster vaccine 1. The detection steps and evaluation indexes were the same as in Example 2. The sequences of the gE-specific peptide pool refer to SEQ ID NOs: 32 to 46.

[0108] 2. Experimental Results: The spot count levels of gE-specific IFN-γ-secreting T lymphocytes in splenocytes from mice in each group are shown in Figure 13, and the gE-specific IFN-γ seroconversion rates are shown in Table 8. As can be seen, the spot count levels of gE-specific IFN-γ-secreting T lymphocytes in splenocytes from high-dose immunization groups E and F (over 4000 SFC / 106 splenocytes) were significantly higher than those from low-dose immunization groups G and H. The spot count levels of gE-specific IFN-γ-secreting T lymphocytes in splenocytes from groups E and G (CpG T1 + QS-21) were higher than those from groups F and H (CpG7909 + QS-21) at the same dose, and the IFN-γ seroconversion rates for groups E to H were all 100%.

[0109] [Table 8]

[0110] Example 12: Verification of the effectiveness of humoral immunity by varicella zoster vaccine 1. Detection step: Blood was collected on day 28 after immunization, and serum was separated (whole blood was placed in a 37°C incubator for 40 minutes, then centrifuged at 12,000 rpm at 4°C for 10 minutes. The supernatant was aspirated and stored frozen at -20°C). The seroconversion rate of herpes gE protein-specific antibodies was detected using an ELISA kit (Shanghai Kehua) according to the kit's instructions. A blank control, a negative control, and a test sample were prepared in two parallel wells, each containing a negative mouse serum. Except for the blank control, the negative control or test sample was added to each well, followed by the addition of an enzyme conjugate. The wells were mixed evenly and sealed, and then incubated at 37°C for 30 minutes. Each well was washed with washing solution, and color developer solution A and color developer solution B were added to each well. The mixture was mixed evenly and sealed, and then incubated at 37°C for 15 minutes. Stop solution was added to each well and mixed evenly. The OD value of each well was read at a wavelength of 450 nm using a microplate reader.

[0111] 2. Experimental results: The detection of antigen-specific IgG antibody and subtype levels in mouse serum by ELISA is shown in Figure 14. The results show that group E containing the immunostimulant of the present invention had significantly better immune effects than the CpG alone group (group C), QS-21 group (group D), and double adjuvant control (group F), and the corresponding IgG and IgG2a antibody levels were significantly different from the other two groups. This means that adding CpG to QS-21 can enhance the corresponding humoral immunity levels.

[0112] Example 13: Effect of different saponins on the efficacy of recombinant varicella zoster vaccine compositions 1. Experimental animals and model establishment: C57BL / 6(N) mice, female, 5 weeks old, 48 mice, purchased from Shanghai Slice Laboratory Animal Co., Ltd.

[0113] 2. Reagent materials: 1) Herpes gE protein was prepared according to Example 10, and CpG T1 and CpG 7909 were both prepared according to Example 1. 2) QS-21 (CAS number A010-023, purchased from BRENNTAG), ginsenoside Rg1 (CAS: 22427-39-0, purchased from Nanjing Spring and Autumn Bio-Engineering Co., Ltd.), astragaloside A (CAS: 84687-43-4, purchased from Nanjing Spring and Autumn Bio-Engineering Co., Ltd.), platycodin D (CAS: 58479-68-8, purchased from Hubei Yunbi Technology Co., Ltd.), Iscom adjuvant (purchased from Shanghai Xiyuan Bio-Technology Co., Ltd.). 3) To prepare for the next step, the herpes gE stock solution was diluted to 50 μg / mL using PBS solution (purchased from Hyclone), each saponin was diluted to 50 μg / mL using PBS solution, and CpG T1 and CpG 7909 were dissolved and diluted to 100 μg / mL using PBS solution.

[0114] 3. Experimental group design: The injection volume was 100 μL per animal, as shown in Table 9. The control group was injected with 100 μL of PBS solution per animal.

[0115] 4. Experimental steps: same as in Example 2.

[0116] 5. Experimental results: See Figure 15 for the ELISPOT spot results. As shown in the results, when CpG T1 was combined with each saponin, an effective synergistic effect was achieved, and the induced gE-specific IFN-γ level was significantly higher than that of other CpG and saponin compositions, with QS21 showing the best effect.

[0117] [Table 9]

[0118] As can be seen from the above, the immune composition of the present invention has excellent immunostimulatory effects, and compared with single adjuvants or combinations of other CPG adjuvants and QS21, CpG T1-T3 and QS-21 exhibit efficient synergistic effects, mediating stronger immune responses. When used with different antigens or antigen compositions, each has clear advantages. Therefore, as a novel adjuvant, the immune composition is expected to have high clinical value and a wide market.

[0119] Although the present invention has been described in detail above, those skilled in the art should understand that various modifications and variations can be made to the present invention without departing from the spirit and scope of the present invention. The scope of protection of the present invention is not limited to the above detailed description, and such modifications and variations also fall within the scope of the claims. Although specific embodiments of the present invention have been described above as examples, it is clear to those skilled in the art that these are given by way of example and that the scope of protection of the present invention is limited by the claims. Those skilled in the art can make various changes and variations to these embodiments without departing from the spirit of the present invention, and all such changes and variations fall within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition comprising an immunostimulatory composition and an antigen or antigen composition, wherein the pharmaceutical composition is either (a) or (b) below. (a) the immunostimulatory composition comprises a saponin and a CpG oligodeoxynucleotide; the CpG oligodeoxynucleotide sequence is any one selected from CpG T1: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6), CpG T2: TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 7), and CpG T3: TCG TCG TCG TCG TCG TCG TCG TCG (SEQ ID NO: 8); The saponin is Quillaja saponin, The antigen or antigen composition is hepatitis B virus. (b) the immunostimulatory composition comprises a saponin and a CpG oligodeoxynucleotide, or consists of a saponin-containing adjuvant and a CpG oligodeoxynucleotide; the CpG oligodeoxynucleotide sequence is CpG T1: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6), or CpG T2: TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 7); The saponin is one or more selected from Quillaja saponin, ginsenoside, platycodin, and astragaloside, or the adjuvant containing a saponin is an Iscom adjuvant; The antigen or antigenic composition is varicella-zoster virus.

2. 2. The pharmaceutical composition of claim 1, wherein the Quillaja saponin is QS-7, QS-17, QS-18 or QS-21, the ginsenoside is ginsenoside Rg1, ginsenoside Rg3, ginsenoside Rb1 or ginsenoside Re, the platycodin is platycodin D, platycodin D2 or a mixture of both, and the astragaloside is astragaloside A, astragaloside I, astragaloside II or a mixture of two or more saponin monomers thereof.

3. 3. The pharmaceutical composition of claim 2, wherein the Quillaja saponin is QS-21.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the CpG oligodeoxynucleotide comprises a phosphorothioate bond.

5. The pharmaceutical composition of claim 4, wherein the CpG oligodeoxynucleotide is a perthio oligodeoxynucleotide.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the weight ratio of the CpG oligodeoxynucleotide to the saponin is 1-40:0.1-2.

7. The pharmaceutical composition according to claim 6, wherein the weight ratio of the CpG oligodeoxynucleotide to the saponin is 2-40:0.1-2.

8. The pharmaceutical composition of claim 7, wherein the weight ratio of the CpG oligodeoxynucleotide to the saponin is 2:1.

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