Immunologic adjuvant composition, preparation method therefor and use thereof

Abstract

Provided are an immunologic adjuvant composition, a preparation method therefor and a use thereof. The immunologic adjuvant composition comprises an immune activator and a liposome used for loading the immune activator, wherein the immune activator comprises a saponin and a CpG oligodeoxynucleotide. The immunologic adjuvant composition can induce an immune response of a mammal against a human herpesvirus and / or hepatitis B virus, and can induce a strong level of cellular immune response. By using the liposome to simultaneously load a QS21 and the CpG oligodeoxynucleotide, the two have a better synergistic effect in the aspect of promoting cellular immunity, and can induce a stronger level of cellular immune response compared to a commercially available Shingrix vaccine.

Description

Immune adjuvant composition and its preparation method and use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311732221.3 filed on December 15, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biopharmaceuticals and specifically relates to an immune adjuvant composition for inducing an immune response, and a preparation method and use thereof. Background Art

[0004] Hepatitis B virus (HBV) infection is a serious public health problem worldwide. HBV infection is a major cause of chronic hepatitis B, cirrhosis, and hepatocellular carcinoma. Commonly used drugs for the clinical treatment of chronic HBV infection include nucleoside analogs and interferon. However, nucleosides cannot completely eliminate cccDNA in liver cells, and long-term use can easily lead to the emergence of drug-resistant mutant strains and rebound after discontinuation of the drug. Interferon is not suitable for asymptomatic HBV carriers, and in chronic HBV patients, the HBeAg seroconversion rate after six months of use is only 33%, resulting in limited therapeutic efficacy and significant side effects.

[0005] Herpes zoster (shingles) is a viral skin disease caused by the varicella-zoster virus (VZV). During primary infection, VZV enters the local lymph nodes through the respiratory mucosal epithelium and replicates. Infected lymphocytes then enter the bloodstream via the lymphatic system and infect peripheral blood mononuclear leukocytes. The virus then spreads through the bloodstream to the skin, resulting in the clinical manifestation of varicella. Vaccines are the most effective and cost-effective means of preventing and controlling this disease.

[0006] Liposomes are composed of a phospholipid bilayer with a structure similar to that of a cell membrane. They function as both adjuvants and carriers, serving as carriers for antigens that induce hapten-specific immune responses. Encapsulating antigens in liposomes protects them from degradation, thereby reducing the dose and frequency of administration, lowering the toxicity of the encapsulated antigen, and enhancing an animal's ability to tolerate high doses of pathogens or their toxins. This makes them an ideal adjuvant carrier.

[0007] Saponins are triterpene glycosides extracted from the bark of the Quillaja tree. Quil-A is a partially purified aqueous extract of the saponin material. QS21 is the HPLC-purified non-toxic fraction of Quil-A. It is a complex sugar ester structure consisting of a sapote triterpene ring structure substituted with branched trisaccharides and linear tetrasaccharides, in which the linear tetrasaccharides are linked to acyl chains.

[0008] The chemical nature of CpG oligodeoxynucleotides is a deoxyoligonucleotide containing cytosine guanine dinucleotides. It has an immune response similar to that of natural CpG pattern recognition receptors, can bind to Toll-like receptors on the cell membrane, and effectively trigger mammalian immune responses through the TLR9 signaling pathway.

[0009] Monophosphoryl lipid (MPL) is a lipopolysaccharide derivative with low toxicity and adjuvant activity. It is a TLR4 agonist that can stimulate macrophages to produce TNF and, together with the produced TNF, promote NK cells to produce IFN-γ, selectively activating Th1 cells.

[0010] Patent application CN 101330924 A from GlaxoSmithKline Biopharmaceuticals Ltd. (hereinafter referred to as Shingrix) discloses a novel shingles vaccine comprising QS21, MPL, and liposomes. Shingrix's novel shingles vaccine is designed to induce an immune response against the shingles virus in mammals. This vaccine utilizes liposomes as a delivery vehicle, synergizing QS21 and MPL to deliver the adjuvant. However, the invention does not describe how QS21, MPL, and liposomes interact, or how they synergize to deliver the adjuvant. Therefore, the structure of the liposomes and the interaction between them and the adjuvant remain key issues in the liposome field.

[0011] Exxiquer Co., Ltd. has conducted in-depth research on the structure of liposomes and the interaction between liposomes and adjuvants. The company's patent application CN 106535876 A describes the interaction between liposomes and oligonucleotides by conjugating the oligonucleotides to the hydrophobic region of the liposome's phospholipid bilayer via a linker molecule. Specifically, the patent application discloses a nanostructure comprising a liposome core with a lipid bilayer and an oligonucleotide positioned outside the liposome core. The immunostimulant is bound to the lipid bilayer, and the oligonucleotides form an oligonucleotide shell that is anchored to the surface of the liposome core via conjugation with a linker molecule, such as tocopherol or cholesterol. All oligonucleotides have 5'-ends exposed on the outer surface of the nanostructure. However, there are many different types of oligonucleotides, and the structures formed by different types of oligonucleotides with liposomes, as well as the direct interactions between the two, vary greatly.

[0012] Therefore, the direct interaction between specific adjuvants and liposomes and the resulting immune effects remain an issue that needs to be studied urgently.

[0013] Summary of the Invention

[0014] In response to the above problems, the present invention aims to provide an immune adjuvant composition, a preparation method thereof, and its use. The immune adjuvant composition can induce an immune response against human herpes virus and / or hepatitis B virus in mammals and can induce a strong cellular immune response.

[0015] definition:

[0016] Unless otherwise defined, all technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For the definition of terms in this field, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel).

[0017] Although the present invention displays numerical ranges and parameter approximations in a broad sense, the numerical values ​​shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to encompass any and all sub-ranges contained therein. For example, the range of "2 to 40" recorded should be considered to include any and all sub-ranges between the minimum value 2 and the maximum value 40 (including endpoints), that is, all sub-ranges starting with a minimum value of 2 or greater, such as 2 to 6.1, and sub-ranges terminating with a maximum value of 40 or less, such as 5.5 to 40. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0018] As used herein, the term "or" is used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0019] The term "immune adjuvant composition" used herein is used interchangeably with "combination immune adjuvant" and "immune adjuvant combination", which refers to a combination of at least one drug and optional pharmaceutically acceptable excipients or adjuvants combined together to achieve a specific purpose.

[0020] The term "mammal" as used herein refers to humans or other animals, such as wild animals (e.g., herons, storks, cranes, etc.), domestic animals (e.g., ducks, geese, etc.) or experimental animals (e.g., gorillas, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0021] The term "pseudo-binary mixture" as used herein refers to a complex formed by an adjuvant such as saponin and a sterol such as cholesterol in a liposome bound by van der Waals forces or hydrophobic forces.

[0022] The above object of the present invention is achieved by providing the following technical solutions:

[0023] In a first aspect, the present invention provides an immune adjuvant composition comprising an immunologically active agent and a liposome for loading the immunologically active agent, wherein the immunologically active agent comprises saponin and CpG oligodeoxynucleotide.

[0024] Preferably, the liposomes comprise phospholipids and sterols.

[0025] Further preferably, the phospholipid is selected from one or more of dioleoylphosphatidylcholine, egg yolk phosphatidylcholine, phosphorylcholine or natural phospholipid derivatives; more preferably, the phospholipid is dioleoylphosphatidylcholine (DOPC).

[0026] Further preferably, the sterol is selected from one or more of cholesterol, stigmasterol or ergosterol; more preferably, the sterol is cholesterol.

[0027] Preferably, the liposome has a phospholipid bilayer structure, the phospholipid bilayer structure includes an external hydrophilic surface and an internal hydrophobic region, the saponin is embedded in the internal hydrophobic region, and the CpG oligodeoxynucleotide is bound to the external hydrophilic surface; further preferably, the CpG oligodeoxynucleotide is bound to the external hydrophilic surface through van der Waals forces and hydrophobic forces.

[0028] Preferably, the sterol is embedded in the inner hydrophobic region of the phospholipid bilayer structure.

[0029] Preferably, the saponin and the sterol form a pseudo-binary mixture of worm-like micellar assemblies. Further preferably, the pseudo-binary mixture is formed based on the hydrophobic interaction between the hydrophobic groups of the sterol and the triterpene portion of the saponin within the liposome, and the hydrogen bonding between the hydrophilic groups of the sterol and the glycosyl portion of the saponin.

[0030] Preferably, the CpG oligodeoxynucleotide has two or more copies of a 5'-TTCGTT-3' motif or a 5'-TCGTCGTCG-3' motif.

[0031] Preferably, the CpG oligodeoxynucleotide comprises or consists of a sequence selected from one of the following:

[0032] (1) the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13;

[0033] (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in one of SEQ ID NO: 1 to SEQ ID NO: 13;

[0034] (3) An amino acid sequence having one or more, for example, 2, 3, 4 or 5, amino acid substitutions, deletions or insertions in the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13.

[0035] Further preferably, the CpG oligodeoxynucleotide comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0036] Further preferably, the CpG oligodeoxynucleotide comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0037] Preferably, the saponin is selected from one or more of Quillaja saponins, ginsenosides, platycodon saponins, astragaloside saponins, notoginseng saponins, glycyrrhizin saponins, julibrissin, ophiopogon saponins, bupleurum saponins or panax japonicus saponins.

[0038] Preferably, the Quillaja saponin is selected from one or more of QS-7, QS-17, QS-18 or QS-21; further preferably, the Quillaja saponin is QS-21.

[0039] Preferably, the weight ratio of the phospholipid to the sterol is 20-4000:50-1000, preferably 20-2000:50-500.

[0040] Preferably, the weight ratio of the sum of the weight of the liposome and saponin to the weight of the CpG oligodeoxynucleotide is 0.1-5:0.5-120, preferably 1:5-20, more preferably 1:10.

[0041] Preferably, the weight ratio of the saponin to the liposome is 0.1-5:10-150, preferably 0.2-5:15-150.

[0042] Preferably, the immune adjuvant composition is in the form of nanoparticles; further preferably, the particle size of the nanoparticles is 20-120 nm.

[0043] Preferably, the immunoadjuvant composition further comprises an antigen, a fragment of the antigen, a variant of the antigen, or a mixture of at least two thereof.

[0044] Preferably, the antigen is selected from one or more of hepatitis A, B, C or E virus antigens, human herpes antigens, human immunodeficiency virus antigens, varicella-zoster virus antigens, human cytomegalovirus antigens, respiratory syncytial virus antigens, human papillomavirus antigens, influenza virus antigens or Mycobacterium tuberculosis antigens. Further preferably, the antigen is a hepatitis B antigen or a herpes antigen.

[0045] Preferably, the hepatitis B antigens are hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg).

[0046] Preferably, the hepatitis B surface antigen comprises or consists of an amino acid sequence selected from one of the following:

[0047] (1) the amino acid sequence shown in SEQ ID NO: 14;

[0048] (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 14;

[0049] (3) An amino acid sequence having one or more amino acid substitutions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 14.

[0050] Further preferably, the hepatitis B surface antigen comprises or consists of the amino acid sequence shown in SEQ ID NO: 14.

[0051] Preferably, the hepatitis B core antigen comprises or consists of an amino acid sequence selected from one of the following:

[0052] (1) the amino acid sequence shown in SEQ ID NO: 15;

[0053] (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 15;

[0054] (3) An amino acid sequence having one or more amino acid substitutions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 15.

[0055] Further preferably, the hepatitis B core antigen comprises or consists of the amino acid sequence shown in SEQ ID NO: 15.

[0056] Preferably, the herpes antigen is herpes gE protein.

[0057] Preferably, the herpes gE protein comprises or consists of an amino acid sequence selected from one of the following:

[0058] (1) the amino acid sequence shown in SEQ ID NO: 16;

[0059] (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 16;

[0060] (3) An amino acid sequence having one or more amino acid substitutions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 16.

[0061] Further preferably, the herpes gE protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 16.

[0062] Preferably, the weight ratio of the herpes antigen, the sum of the weight of the liposome and saponin, and the CpG oligodeoxynucleotide is 0.1-5:0.1-5:0.5-120, preferably 1-2:1:5-20, and more preferably 1:1:10.

[0063] Preferably, the weight ratio of the hepatitis B antigen, the sum of the weight of liposomes and saponin, and CpG oligodeoxynucleotide is 5-100:0.1-5:0.5-120, preferably 15-50:1:5-20, and more preferably 30:1:10.

[0064] In a second aspect, the present invention provides a method for preparing the immune adjuvant composition according to the first aspect of the present invention, comprising the following steps:

[0065] The saponin is dissolved in an aqueous solution, liposomes are added, and a first stirring is performed. Then, CPG oligodeoxynucleotide is added and a second stirring is performed to obtain the immune adjuvant composition.

[0066] Preferably, the stirring speeds of the first stirring and the second stirring are independently 50-150 rpm, preferably 50-100 rpm.

[0067] Preferably, the aqueous solution comprises phosphate and sodium chloride.

[0068] Further preferably, the concentration of phosphate in the aqueous solution is 10-50 mmol / L, preferably 20 mmol / L; the concentration of sodium chloride in the aqueous solution is 100-350 mmol / L, preferably 150 mmol / L.

[0069] Preferably, the preparation method further comprises the step of adding an antigen after the second stirring.

[0070] In a third aspect, the present invention provides an immune kit comprising the immune adjuvant composition according to the first aspect of the present invention.

[0071] In a fourth aspect, the present invention provides use of the immune adjuvant composition according to the first aspect of the present invention or the immune kit according to the third aspect of the present invention in the preparation of the following products:

[0072] (1) A drug for preventing and / or treating human herpes virus and / or hepatitis B virus infection or diseases related thereto; preferably, the drug is a vaccine;

[0073] (2) a kit for diagnosing human herpes virus and / or hepatitis B virus infection; or

[0074] (3) Immunogens for developing antibodies against human herpes virus and / or hepatitis B virus;

[0075] Preferably, the human herpes virus is one or more selected from varicella-zoster virus, herpes simplex virus type 1 (HSV1) or herpes simplex virus type 2 (HSV2).

[0076] The present invention has at least the following beneficial effects:

[0077] The immune adjuvant composition provided by the present invention interacts with saponins such as QS21, CpG oligodeoxynucleotides, and liposomes. QS21 is embedded within the hydrophobic region of the phospholipid bilayer structure and forms a pseudo-binary mixture with sterols such as cholesterol, forming a worm-like micelle assembly. This assembly, with a pseudo-binary mixture structure, is formed through hydrophobic interactions between the hydrophobic groups of cholesterol and the triterpene moiety of QS21 within the liposome, as well as hydrogen bonding between the hydrophilic groups of cholesterol and the glycosyl moiety of QS21. These hydrophobic interactions and hydrogen bonding can increase the binding rate of QS21 to the liposomes and drug loading, thereby improving the stability of the QS21 liposomes.

[0078] The CpG oligodeoxynucleotides in the immune adjuvant composition provided by the present invention are negatively charged, and there are van der Waals forces and hydrophobic forces between them and phospholipid molecules such as DOPC, so that CpG can directly bind to the liposome surface, thereby increasing the binding rate of CpG to the liposome and the drug loading capacity, thereby improving the stability of the CpG-QS21 liposome.

[0079] The immunoadjuvant composition provided by the present invention can induce an immune response against human herpes virus and / or hepatitis B virus in mammals, and can induce a strong cellular immune response. Specifically, the immunoadjuvant composition provided by the present invention can induce mammals to produce high levels of gE antigen-specific IFN-γ and gE antigen-specific IgG / IgG1 / IgG2a titers, and after a long period of time, the induced gE antigen-specific IFN-γ and gE antigen-specific IgG / IgG1 / IgG2a titers remain at a high level. The immunoadjuvant composition provided by the present invention can effectively reduce HBsAg levels in HBV-infected model mice and can induce high levels of HBsAg- and HBcAg-specific IFN-γ.

[0080] The present invention uses liposomes to simultaneously load QS21 and CpG oligodeoxynucleotides, so that the two have a good synergistic effect in promoting cellular immunity. Compared with the commercially available Shingrix vaccine, the immune adjuvant composition of the present invention can induce a stronger immune response level.

[0081] BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0083] FIG1 is a cryo-electron micrograph of the CPG-QS21 liposome nanocomposite preparation prepared in Example 2 of the present invention;

[0084] Figure 2 is a schematic structural diagram of CPG-QS21 liposomes;

[0085] FIG3 is a particle size diagram of the CPG-QS21 liposome nanocomposite preparation prepared in Example 2 of the present invention;

[0086] FIG4 shows the effect of different ratios of CPG oligodeoxynucleotides on the level of T lymphocyte spot counts of herpes gE antigen-specific IFN-γ in Example 3 of the present invention;

[0087] FIG5 shows the effect of different ratios of CPG oligodeoxynucleotides on the level of T lymphocyte spot counts of herpes gE antigen-specific IFN-γ in Example 4 of the present invention;

[0088] FIG6 shows the effects of different CPG oligodeoxynucleotides on the levels of herpes gE antigen-specific IgG and its subtypes and the IgG2a / IgG1 ratio in Example 4 of the present invention;

[0089] FIG7 shows the effect of different ratios of herpes gE antigen on the number of gE-specific IFN-γ lymphocyte spots secreted by splenocytes in Example 5 of the present invention;

[0090] FIG8 shows the effect of herpes zoster vaccine on the level of gE-specific IFN-γ lymphocyte spot counts secreted by mouse spleen cells in Example 6 of the present invention;

[0091] FIG9 shows the effect of the herpes zoster vaccine on the levels of gE-specific IgG and its subtypes and the IgG2a / IgG1 ratio in mouse serum in Example 6 of the present invention;

[0092] FIG10 shows the effect of the hepatitis B vaccine on the serum HBsAg levels of mice at different weeks after immunization in Example 7 of the present invention;

[0093] FIG11 shows the effect of hepatitis B vaccine on the level of lymphocyte spot counts of gE-specific IFN-γ secreted by mouse spleen cells in Example 7 of the present invention.

[0094] Best Mode for Carrying Out the Invention

[0095] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0096] Example 1 Preparation and Preparation of Biomaterials

[0097] 1. HBsAg Stock Solution: The amino acid sequence of the HBsAg protein in the present invention, SEQ ID NO: 14, is found in SEQ ID NO: 1 of Chinese Patent CN108330145B. The HBsAg antigen protein is prepared from yeast cells recombinant with the HBsAg gene to obtain a crude HBsAg fermentation broth. The specific preparation method is described in paragraphs 0044 to 0091 of Chinese Patent CN108330145B. After bacterial disruption, filtration, and column chromatography, the HBsAg stock solution is obtained. The specific purification method is described in paragraphs 0092 to 0136 of Chinese Patent CN108330145B.

[0098] 2. HBcAg Stock Solution: The amino acid sequence of the HBcAg protein in the present invention, SEQ ID NO: 15, is referenced to SEQ ID NO: 1 in Chinese Patent CN104673760B. The HBcAg antigen protein is prepared from recombinant yeast cells bearing the HBcAg gene to obtain a crude HBcAg fermentation broth. The specific preparation method is described in paragraphs 0049 to 0068 of Chinese Patent CN104673760B. After bacterial disruption, filtration, and column chromatography, a HBsAg stock solution is obtained. The specific purification method is described in paragraphs 0069 to 0080 of Chinese Patent CN104673760B.

[0099] 3. gE stock solution: The amino acid sequence of gE protein, SEQ ID NO: 16, refers to SEQ ID NO: 1 in Chinese Patent CN112972671A. The nucleic acid sequence of the gE protein was codon-optimized, amplified by PCR, and ligated into a target vector. The target plasmid was screened and transfected into CHO cells. After fermentation, the gE stock solution was purified by filtration. The specific preparation method is described in Thompson E, Persson L, et al., Journal of Virological Methods, 2020, Vol. 175, No. 1, pp. 53-59.

[0100] 4. PBS solution: purchased from Hyclone, product number: SH30256.01.

[0101] 5. Serum-free medium: Serum-free medium, purchased from Dakoway Biotechnology Co., Ltd., catalog number: 6015012.

[0102] 6. QS21: purchased from BRENNTAG, CAS.NO.A010-023.

[0103] 7. Preparation of CPG oligodeoxynucleotides: Prepared using conventional solid-phase phosphoramidite triester chemical synthesis, starting from the 3' end, by first deprotecting the DNA fragments, followed by activation, ligation, oxidation, and blocking to obtain a crude DNA fragment. Finally, post-synthesis processing such as cleavage, deprotection, purification, and quantification is performed. For detailed preparation methods, see paragraphs 0063-0064 of patent CN200810004736.0. The CPG oligodeoxynucleotide used in this invention is CpG T1, the specific sequence of which is shown in Table 1.

[0104] 8. Shingrix vaccine: purchased from GlaxoSmithKline Biopharmaceuticals Ltd.

[0105] Table 1 Specific sequences of CPG oligodeoxynucleotides

[0106] Example 2 Preparation and Detection of Liposome Nanocomposite Preparations

[0107] 1. Preparation of liposome nanocomposite preparations

[0108] (1) Preparation of blank liposomes

[0109] Dioleoylphosphatidylserine and cholesterol in a weight ratio of 1000:250 were dissolved in an organic solvent, and the organic solvent was removed by rotary evaporation. The rotary evaporation temperature was set to 50°C, the temperature of the low-temperature circulation device was set to -10°C, the rotary evaporation vacuum was controlled to maintain at 20-120hPa, and the rotary evaporation speed was controlled to maintain at 30-180rpm. A lipid film was formed on the wall of the rotary evaporation bottle, and the lipid film was dissolved with an aqueous solution and hydrated to form a lipid suspension. The film was then homogenized 6 times, and the homogenization pressure was controlled to maintain at 150bar. The resulting lipid solution was extruded through two polycarbonate filter membranes 6 times, and the pore size of the first polycarbonate filter membrane was controlled to be 200nm and the pore size of the second polycarbonate filter membrane was controlled to be 100nm to obtain blank liposomes.

[0110] The organic solvent is anhydrous ethanol, and the aqueous solution is an aqueous solution of phosphate and sodium chloride, wherein the concentration of phosphate (i.e., sodium dihydrogen phosphate and disodium hydrogen phosphate) is 20 mmol / L, and the concentration of sodium chloride is 150 mmol / L.

[0111] (2) Preparation of CPG-QS21 liposomes

[0112] QS21 was dissolved in an aqueous solution, blank liposomes were added, and the mixture was stirred uniformly at a stirring speed of 50-150 rpm to obtain QS21 liposomes. CPG oligodeoxynucleotides were then added and the mixture was stirred uniformly with the QS21 liposomes at a stirring speed of 50-150 rpm to obtain CPG-QS21 liposomes. The aqueous solution was a phosphate and sodium chloride solution, with a phosphate concentration of 20 mmol / L and a sodium chloride concentration of 150 mmol / L. The weight ratio of QS21 to liposomes was 50:1250, and the ratio of the two remained constant. The two were combined and recorded as QS21 liposomes.

[0113] The CPG-QS21 liposomes prepared in this example were mixed with the desired antigen stock solution and used for injection in the following examples.

[0114] 2. Detection of liposome nanocomposite preparations

[0115] (1) QS21 encapsulation efficiency test

[0116] The encapsulation efficiency of CPG-QS21 liposomes was measured using high-performance liquid chromatography. The liposome nanocomposite formulation used for this test had a vaccine formulation for human use, with each 0.5 mL dose containing 1300 μg of QS21 liposomes (including 50 μg of QS21), 500 μg of CpG, and 100 μg of gE antigen.

[0117] Encapsulation efficiency detection method: The encapsulation efficiency of QS21 was detected by high performance liquid chromatography, with octadecylsilane bonded silica gel as the filler; formic acid-water (1:1000) as mobile phase A, and formic acid-acetonitrile (1:1000) as mobile phase B, with linear gradient elution; the flow rate was 1.0 mL per minute; the column temperature was 40°C; the injection volume was 100 μL; the detector was CAD (nebulization temperature: 50°C; acquisition frequency: 10 Hz).

[0118] The encapsulation efficiency was calculated as

[0119] Where: A L is the sum of the peak areas of QS-21a and QS-21b encapsulated in liposomes;

[0120] A F is the sum of the peak areas of free QS-21a and QS-21b;

[0121] 100 is the dilution factor of the encapsulated part solution;

[0122] 20 is the dilution factor of the free portion solution.

[0123] (2) Micromorphology and particle size detection of liposome nanocomposite preparations

[0124] The micromorphology of CPG-QS21 liposomes was observed using cryo-electron microscopy.

[0125] The average particle size of CPG-QS21 liposomes was determined using laser dynamic light scattering. Laser dynamic light scattering detection method: First rinse the measuring cell with filtered double-distilled water. Ultrasonicate the sample through a water barrier (100W, 5-7 cycles) and then filter it through a 0.22 µm syringe filter (Pall Corporation). Place 1 µm of sample into the measuring cell (the sample should be 10-15 mm high). Following the instrument's instructions, open the sample cell cap, place the sample into the measuring cell (with the V symbol facing the measuring instrument), and click Start to begin the measurement.

[0126] 3. Test results

[0127] Table 2 Encapsulation efficiency of QS21 in CpG-QS21 liposomes

[0128] The encapsulation efficiency of QS21 in CPG-QS21 liposomes, as measured by high-performance liquid chromatography, is shown in Table 2. When the QS21 concentration increased to 100 μg / mL, the encapsulation efficiency of QS21 decreased with further increases in the amount of QS21. However, when the amount of QS21 was 10-200 μg / mL, it reached a level higher than the industry-recognized standard (85%), which may be related to the encapsulation capacity of the liposomes.

[0129] The particle size of CPG-QS21 liposomes detected by laser dynamic light scattering is shown in FIG3 , and the average particle size is 93.08 nm.

[0130] The microscopic morphology of CPG-QS21 liposomes is shown in Figure 1. A schematic diagram of the CPG-QS21 liposome structure is shown in Figure 2. As can be seen, blank liposomes, QS21 liposomes, and CPG-QS21 liposomes all possess a phospholipid bilayer structure, with each phospholipid molecule possessing a hydrophilic head and a lipophilic tail. The two layers of phospholipid molecules face each other, forming an outer hydrophilic surface and an inner hydrophobic region. Cholesterol resides in this inner hydrophobic region, while QS21 is embedded within it, forming a pseudo-binary mixture with cholesterol, a worm-like micelle assembly. This pseudo-binary mixture structure is based on the hydrophobic interactions between the hydrophobic portion of cholesterol and the triterpene portion of QS21 within the liposomes, as well as hydrogen bonding between the hydrophilic portion of cholesterol and the glycosyl portion of QS21. These hydrophobic interactions and hydrogen bonding enhance the binding efficiency of QS21 to the liposomes and drug loading, thereby improving the stability of the QS21 liposomes.

[0131] Because the CpG oligodeoxynucleotides are negatively charged, they interact with the liposomes through van der Waals and hydrophobic forces. Consequently, the CpG oligodeoxynucleotides bind to the phospholipid DOPC on the liposome surface. These van der Waals and hydrophobic forces allow direct binding of CpG to the liposome surface, increasing the binding efficiency and drug loading, and thus improving the stability of the CpG-QS21 liposomes.

[0132] Patent application CN 106535876 A discloses a liposome nanostructure comprising a liposome core with a lipid bilayer and oligonucleotides containing a class B CpG motif located outside the liposome core, wherein all oligonucleotides form an oligonucleotide shell that is anchored to the surface of the liposome core by conjugation with a linker molecule, wherein the linker molecule is tocopherol or cholesterol, and the surface of the liposome core is the surface of the hydrophobic region of the liposome. In other words, the oligonucleotides in the liposomes of patent application CN 106535876 A need to enter the interior of the liposome phospholipid bilayer from the gaps on the outer surface between phospholipid molecules and then pass through the linker molecule to be anchored to the surface of the hydrophobic region of the liposome. Obviously, the linker molecule is located in the liposome core region, i.e., the hydrophobic region of the liposome. The oligonucleotides need to enter the interior of the liposome phospholipid bilayer from the gaps on the outer surface of the phospholipid molecules to contact the linker molecule and further anchor to the surface of the hydrophobic region of the liposome. Compared with the liposome structure of patent application CN 106535876 A, on the one hand, the CpG in the liposome structure of the present invention can be directly bound to the liposome on the liposome surface, which is more direct and efficient and can significantly improve the CpG binding rate; on the other hand, the binding of CpG in the liposome structure of the present invention to the liposome relies on its own van der Waals force, and effective binding can be achieved without the need for linker molecules, further improving the binding rate of CpG to the liposome.

[0133] Example 3 Preliminary screening experiment of CPG-QS21 liposome vaccine

[0134] 1. Experimental animals: 60 female C57BL / 6 mice, 6 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd., animal license number: SCXK(Shanghai)2018-0003.

[0135] 2. Animal Grouping: Specific animal groupings are shown in Table 3. Each injection volume was 100 μL per animal. Prior to injection, the components listed in the table below were dissolved in PBS and diluted to 100 μL. The amounts of CPG and QS21 liposomes used in each CPG-QS21 liposome group are shown in the table below.

[0136] Table 3 SM21053 animal experiment groups

[0137] 3. Experimental procedures: Immunization was performed by intramuscular injection, with 100 μL / mouse injected per group for a total of one immunization. The number of T lymphocyte spots secreting gE-specific IFN-γ by splenocytes was detected on wk01 (one week after the first immunization). Before the spot level detection, the AL group was injected with serum-free culture medium containing a gE-specific stimulating peptide library. At the same time, a culture medium negative control group (medium group) was set up in each group. In the medium group, splenocytes from non-immunized mice were isolated and injected with the same amount of serum-free culture medium without the above-mentioned stimulating peptide library.

[0138] 4. Experimental results: The ELISpot method was used to detect the T lymphocyte spot level of gE-specific IFN-γ secreted by splenocytes. The results are shown in Figure 4. The results show that:

[0139] a) Comparison was made between groups AF, which were immunized with 2 μg gE antigen combined with 2 μg QS21 liposomes and CPG-QS21 liposomes containing different doses of CpG (2-100 μg). The levels of gE antigen-specific IFN-γ induced by the AF group increased with the increase in CpG dose. However, the spleen cells of mice in groups E (containing 50 μg CpG) and F (containing 100 μg CpG) containing high doses of CpG produced a certain level of nonspecific IFN-γ response, and the spleen cell state also showed an over-large trend. In addition, the spleen cells of group C, which was immunized with 10 μg CpG, secreted a higher level of gE antigen-specific IFN-γ (approximately 1200 SFC / 10 6 splenocytes), and the splenocytes were normal.

[0140] b) Compared with the GL group, which was immunized with 2 μg gE antigen combined with 1 μg QS21 liposomes and CPG-QS21 liposomes of different doses of CpG (2-100 μg), the splenocytes of mice in the K group (containing 50 μg CpG) and the L group (containing 100 μg CpG), which contained high doses of CpG, also produced a certain level of nonspecific IFN-γ response, and the splenocyte status also showed an over-expression trend, similar to that of the E and F groups. In addition, the splenocytes of the J group, which was immunized with 20 μg CpG, secreted the highest level of gE antigen-specific IFN-γ (approximately 1500 SFC / 10 6 splenocytes), and the splenocytes were normal.

[0141] c) In CpG-QS21 liposomes, different QS21 liposome doses (1 μg, 2 μg) combined with CpG could induce strong gE antigen-specific IFN-γ levels.

[0142] Based on the above results, in CPG-QS21 liposomes, when the dose of QS21 liposomes is 1 μg, the preferred weight ratio of QS21 liposomes:CpG is 1:20; when the dose of QS21 liposomes is 2 μg, the preferred weight ratio of QS21 liposomes:CpG is 1:5.

[0143] Table 4 gE-specific IFN-γ positive conversion rate (%) secreted by splenocytes

[0144] Example 4 Screening experiment of vaccines containing CPG-QS21 liposome nanocomposite formulations

[0145] 1. Experimental animals: 72 female C57BL / 6 mice, 4–5 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd. (animal license number: SCXK(Shanghai)2018-0003).

[0146] 2. Animal Grouping: Specific animal groupings are shown in Table 5. Each injection volume was 100 μL per animal. Prior to injection, the components listed in the table below were dissolved in PBS and diluted to 100 μL. The dosages of CPG and QS21 liposomes in each CPG-QS21 liposome group are shown in the table below. Group A served as the negative control and received an injection of 100 μL of PBS solution containing 2 μg of gE antigen per animal. Group F received the commercially available Shingrix vaccine, with 2 μg of antigen per animal, and both the QS21 liposome and MPL doses were 2 μg per animal.

[0147] Table 5 SM21056 animal experiment grouping

[0148] 3. Experimental procedures: Immunization was performed by intramuscular injection twice, at wk00 and wk02, with 100 μL / mouse injected in group AE and 20 μL / mouse injected in group F. Half of the mice were tested at wk01, and the remaining half were immunized twice at wk02 and tested at wk03, including detection of the number of T lymphocyte spots secreting IFN-γ by splenocytes and detection of gE antigen-specific antibodies in serum. Before the spot level detection, group AF was injected with serum-free culture medium containing a gE-specific stimulating peptide library. At the same time, a culture medium negative control group (medium group) was set up in each group. In the medium group, splenocytes from non-immunized mice were isolated and injected with an equal amount of serum-free culture medium without the above-mentioned stimulating peptide library.

[0149] 4. Experimental results:

[0150] The ELISpot method was used to detect the T lymphocyte spot level of gE-specific IFN-γ secreted by splenocytes. The results are shown in Figure 5. The results show that:

[0151] a) At wk01 (one week after the first immunization), group A, which was immunized with 2 μg of gE antigen, did not produce gE antigen-specific IFN-γ levels. Groups B and C, which were immunized with 2 μg of gE antigen combined with 2 μg of QS21 liposomes and CPG-QS21 liposomes with a QS21 liposome:CpG ratio of 1:10 or 1:20, both produced significant gE antigen-specific IFN-γ levels. However, group C had a higher dose of CpG (40 μg), resulting in a stronger nonspecific reaction and enlarged spleens. In comparison, group B had a better dose condition (approximately 630 SFC / 10 6 Groups D and E, which were immunized with 2 μg gE antigen combined with 1 μg QS21 liposomes and CPG-QS21 liposomes with a QS21 liposome:CpG ratio of 1:10 or 1:20, also produced gE antigen-specific IFN-γ levels comparable to those in group B, and the IFN-γ level in group D (1 μg QS21 liposomes + 10 μg CpG) was relatively higher (approximately 700 SFC / 10 6 splenocytes); while the Shingrix commercial vaccine group immunized with the same antigen dose could produce gE antigen-specific IFN-γ levels (about 200 SFC / 10 6 splenocytes), but the overall level was lower than that in the BE group.

[0152] b) At wk03 (one week after the second immunization), the levels of gE antigen-specific IFN-γ induced by each immunization group increased. However, the IFN-γ level in group A, which was immunized with 2 μg of gE antigen, was still low. In the case of immunization with 2 μg of gE antigen combined with CPG-QS21 liposomes containing QS21 liposomes:CpG at a ratio of 1:10 or 1:20, the cellular immunity level induced by the ratio of QS21 liposomes:CpG was higher than that of the ratio of 1:20 (i.e., group B was higher than group C, and group D was higher than group E). The gE antigen-specific IFN-γ levels induced by groups B and D reached 5000 and 2000 SFC / 10, respectively. 6 Spleen cells.

[0153] The ELISA method was used to detect the levels of gE antigen-specific IgG / IgG1 / IgG2a antibodies in mouse serum. The results are shown in Figure 6. The results show that:

[0154] At wk03 (one week after the second immunization), group A immunized with a single antigen could induce a certain level of IgG / IgG1 titer, but the IgG2a titer was low. After combined with CPG-QS21 liposomes containing QS21 liposomes: CpG = 1:10 or 1:20 (i.e., group BE), higher levels of antigen-specific IgG / IgG1 / IgG2a titer were induced (around 5.0 Lg, 5.0 Lg, and 4.0 Lg, respectively), and there was no significant difference between the groups; while the group immunized with the Shingrix commercial vaccine (group F) with the same antigen dose also induced higher levels of IgG / IgG1 titer (around 4.7 Lg and 5.3 Lg, respectively), but the IgG2a titer was relatively low (only around 1.9 Lg), and there was a significant difference between the two groups (p < 0.001).

[0155] Comprehensive comparison shows that the immune response levels induced by different ratios of CpG adjuvant and QS21 adjuvant in the immune CPG-QS21 liposome nanocomposite preparation are more inclined to the Th1 pathway, while the Shingrix commercially available vaccine is more inclined to the Th2 pathway.

[0156] In summary, the induced antigen-specific cellular immunity level was highest when the ratio of QS21 liposomes:CpG = 1:10, and it could also induce high levels of antigen-specific IgG / IgG1 / IgG2a titers, and the levels of cellular immunity and humoral immunity induced by the Shingrix commercial vaccine were higher. Therefore, the preferred ratio of QS21 liposomes to CpG adjuvant in the CPG-QS21 liposome nanocomposite preparation was 1:10.

[0157] Example 5 Antigen ratio screening experiment of vaccine containing CPG-QS21 liposome nanocomposite formulation

[0158] 1. Experimental animals: 32 female C57BL / 6 mice, 6 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd., animal license number: SCXK(Shanghai)2018-0003.

[0159] 2. Animal Grouping: Specific animal groupings are shown in Table 6. Each injection volume was 50 μL per animal. Group D used gE antigen from a different batch than Groups AC. Prior to injection, each group was treated with the following components dissolved in PBS and diluted to 50 μL.

[0160] Table 6 SM22016 animal experiment grouping

[0161] 3. Experimental procedures: Immunization was performed once with an intramuscular injection of 50 μL per mouse. The T lymphocyte spot count level of gE-specific IFN-γ secreted by splenocytes was detected on wk01 (one week after the first immunization). Before the spot level detection, the AD group was injected with serum-free culture medium containing a gE-specific stimulating peptide library. At the same time, a culture medium negative control group (medium group) was set up in each group. In the medium group, splenocytes from non-immunized mice were isolated and injected with the same amount of serum-free culture medium without the above-mentioned stimulating peptide library.

[0162] 4. Experimental results: Based on the results of the study on the ratio of gE antigen and CPG-QS21 liposome nanocomposite preparation (see Figure 7), after a single immunization with different doses of gE antigen combined with CPG-QS21 liposome nanocomposite preparation (QS21 liposome: CpG = 1:10), the level of gE antigen-specific IFN-γ induced was dependent on the gE antigen dose. Among them, 1 μg gE antigen combined with CPG-QS21 liposome nanocomposite preparation (QS21 liposome and CpG adjuvant doses were 1 μg and 10 μg, respectively) could induce higher antigen-specific IFN-γ levels. Considering the antigen dose and cost, the preferred choice was (1 μg gE antigen + 1 μg QS21 liposome and 10 μg CpG adjuvant). The CpG-QS21 liposomes were used as the immune dose for the efficacy test in mice, and the optimal ratio of gE antigen to QS21 liposomes and CpG in the CPG-QS21 liposome nanocomposite preparation was preliminarily determined to be 1:1:10.

[0163] Example 6 Experimental Group Setup and Immunization Process of Herpes Zoster Vaccine

[0164] 1. Experimental animals: 30 female C57BL / 6 mice, 6 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd., animal license number: SCXK(Shanghai)2018-0003.

[0165] 2. Animal grouping: Specific animal groupings are shown in Table 1. The injection volume for each time was 100 μL. Before injection, each group was given the following components dissolved in PBS solution and diluted to 100 μL.

[0166] Table 7 Experimental animal groups and dosages

[0167] *CPG in CPG-QS21 liposomes is 10 μg, and QS21 liposomes is 1 μg.

[0168] 3. Animal immunization

[0169] Immunization was repeated twice at wk00 and wk02, with intramuscular injection in the left / right hind thigh.

[0170] 4. Evaluation of the cellular immune effect of herpes zoster vaccine

[0171] (1) Detection method: T lymphocyte spot number level detection method, wherein, before the spot level detection, the AF group was injected with serum-free culture medium containing gE-specific stimulating peptide library; at the same time, each group set up a culture medium negative control group (medium group), medium group: non-immunized mouse spleen cells were separated and injected with the same amount of serum-free culture medium without the above-mentioned stimulating peptide library.

[0172] (2) Evaluation index: When the number of spots in the control well is ≤5SFC, the number of spots in the sample well should be ≥10SFC; when the number of spots in the control well is between 5SFC and 10SFC, the number of spots in the sample well / the number of spots in the control well should be ≥2; when the number of spots in the control well is >10SFC, the number of spots in the sample well / the number of spots in the control well should be ≥3.

[0173] 5. Experimental results

[0174] Table 8 The positive conversion rate of gE-specific IFN-γ secreted by splenocytes (%)

[0175] Results Analysis: The number of T lymphocyte spots secreting gE-specific IFN-γ from the spleen cells of mice in each immunization group is shown in Figure 8, and the gE-specific IFN-γ positive conversion rate is shown in Table 8. At Wk02 (two weeks after the first immunization), the E group induced a high level of gE-specific IFN-γ cellular immune response, with a spot level of approximately 900 SFC / 10 6 Splenocytes, after secondary immunization, produce higher levels of gE-specific IFN-γ cell immunity, with spot levels reaching 2200 SFC / 10 6 The level of gE-specific IFN-γ induced by the immune system decreased with time, but it could still maintain a high level of gE-specific IFN-γ (~900 SFC / 10 6 splenocytes), the positive conversion rate was still 100%.

[0176] The results showed that the QS21 liposome adjuvant and CpG adjuvant contained in the CPG-QS21 liposome nanocomposite preparation for injection have a good synergistic effect in promoting cellular immunity, and compared with the Shingrix commercially available vaccine, the TVAX-006 vaccine can induce a stronger level of cellular immune response.

[0177] 6. Detection of gE-specific antibodies in serum for herpes zoster

[0178] (1) Detection method: ELISA detection method.

[0179] (2) Experimental results: The gE-specific IgG / IgG1 / IgG2a antibody titers in the serum of each immunization group after immunization are shown in Figure 9: Each group could induce the production of gE antigen-specific IgG / IgG1 / IgG2a antibody titers at wk02 (two weeks after the first immunization), among which the gE antigen-specific IgG / IgG1 / IgG2a antibody titers induced by Group E and Group F were higher than those of any single adjuvant group and the simple antigen group; the gE-specific IgG / IgG1 / IgG2a antibody titers induced by each group after the second immunization further increased, and the gE-specific IgG / IgG1 / IgG2a antibody titers produced by Group E and Group F were higher than those of any single adjuvant group and the simple antigen group at wk04-wk12. The titers of IgG / IgG1 / IgG2a antibodies in group E declined at wk24 but still maintained a high level (3.798 Lg, 3.110 Lg, and 3.395 Lg, respectively). The titers of IgG / IgG1 / IgG2a antibodies in group F still maintained a high level at wk24 (4.043 Lg, 4.537 Lg, and 3.277 Lg, respectively). The IgG2a / IgG1 ratio showed that the immune response induced by group E and the gE antigen combined with CpG adjuvant group was more inclined to the Th1 pathway, while that induced by the Shingrix commercial vaccine, the single antigen group, and the gE antigen combined with QS21 liposome adjuvant group was more inclined to the Th2 pathway.

[0180] Example 7 Experimental Group Setup and Immunization Process of Hepatitis B Vaccine

[0181] 1. Experimental animals: 48 female C57BL / 6 mice, 6 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd., animal license number: SCXK(Shanghai)2018-0003.

[0182] 2. Animal grouping: Specific animal groupings are shown in Table 9. The injection volume was 100 μL per animal. Before injection, the components shown in the table below were dissolved and diluted to 100 μL with PBS solution. Group A was the negative control and was injected with 100 μL of PBS solution per animal.

[0183] Table 9 Grouping and Dosage *CPG in CPG-QS21 liposomes is 10 μg, and QS21 liposomes is 1 μg.

[0184] 3. Animal immunization

[0185] RAAV8-1.3HBV ayw was injected into the tail vein of C57BL / 6 mice to establish a C57BL / 6 mouse model with persistent rAAV8-HBV infection. This HBV infection model was used to evaluate different types of antiviral drugs, TVAX-008 injection (HBsAg+HBcAg+CpG, group B), TVAX-028 injection (HBsAg+HBcAg+CPG-QS21 liposomes, group C), antigen group (HBsAg+HBcAg, group D), antigen liposome group (HBsAg+HBcAg+blank liposomes, group E), and antigen free adjuvant group (HBsAg+HBcAg+QS21+CpG, group F). A PBS group was set as a negative control.

[0186] TVAX-008 injection (HBsAg+HBcAg+CpG) and TVAX-028 injection (HBsAg+HBcAg+CPG-QS21 liposome) are injected intramuscularly every two weeks.

[0187] Dosing frequency and duration: Each group was immunized by intramuscular injection at wk06, wk08, wk10, wk12, wk14, and wk16, for a total of 6 immunizations. The intramuscular injection site was the hind thigh.

[0188] 4. Detection of HBsAg levels in serum by hepatitis B vaccine

[0189] (1) Detection steps: refer to BD TM ELISPOT Mouse IFN-γ ELISPOT Set instructions.

[0190] (2) Test results: The HBsAg levels in the mouse serum of each group at different test times are shown in Figure 10. The results showed that compared with group A (PBS control group), group D (antigen group) and group E (antigen liposome group), group B (HBsAg+HBcAg+CpG), group F (HBsAg+HBcAg+QS21+CpG) and group C (HBsAg+HBcAg+CPG-QS21 liposome) could effectively reduce the HBsAg level in HBV-infected model mice, and the reduction trend of group C (HBsAg+HBcAg+CPG-QS21 liposome) was higher than that of group B (HBsAg+HBcAg+CpG) and group F (HBsAg+HBcAg+QS21+CpG).

[0191] 5. Evaluation of the cellular immune effect of hepatitis B vaccine

[0192] (1) Detection method: T lymphocyte spot number level detection method. Before the spot level detection, the AC group performed peptide library stimulation when the spleen cells were activated, and the PS4 and PCP peptide libraries diluted and dissolved in serum-free culture medium were used for stimulation detection respectively; at the same time, each group set up a culture medium negative control group (medium group). Medium group: non-immunized mouse spleen cells were separated and injected with the same amount of serum-free culture medium.

[0193] (2) Detection results: The number of T lymphocyte spots of HBsAg and HBcAg-specific IFN-γ secreted by spleen cells of each group of mice at the end of the experiment is shown in Figure 11. The results showed that compared with the PBS control group, antigen group and antigen liposome group, higher levels of HBsAg and HBcAg-specific IFN-γ were detected in group B (HBsAg + HBcAg + CpG), group F (HBsAg + HBcAg + QS21 + CpG) and group C (HBsAg + HBcAg + CPG-QS21 liposome) (stimulated by PS4 and PCP peptide pools, respectively), and the IFN-γ secretion level of group C (HBsAg + HBcAg + CPG-QS21 liposome) was significantly higher than that of group B (HBsAg + HBcAg + CpG) and group F (HBsAg + HBcAg + QS21 + CpG).

[0194] The above descriptions are merely exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention is disclosed above with reference to preferred embodiments, they are not intended to limit the present invention. Any equivalent or equivalent embodiments obtained by any person skilled in the art, without departing from the scope of the present invention, by making slight changes or modifications to the above-disclosed technical contents, fall within the scope of the present invention.

Claims

1. An immunoadjuvant composition comprising an immunologically active agent and a liposome for loading the immunologically active agent, wherein the immunologically active agent comprises saponin and CpG oligodeoxynucleotide.

2. The immunoadjuvant composition according to claim 1, wherein The liposomes comprise phospholipids and sterols; Preferably, the phospholipid is selected from one or more of dioleoylphosphatidyl alkali, egg yolk phosphatidyl choline, phosphorylcholine or natural phospholipid derivatives; more preferably, the phospholipid is dioleoylphosphatidyl alkali; Preferably, the sterol is selected from one or more of cholesterol, stigmasterol or ergosterol; more preferably, the sterol is cholesterol.

3. The immunoadjuvant composition according to claim 1 or 2, wherein The CpG oligodeoxynucleotide has two or more copies of a 5'-TTCGTT-3' motif or a 5'-TCGTCGTCG-3' motif; Preferably, the CpG oligodeoxynucleotide comprises or consists of a sequence selected from one of the following: (1) the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in any one of SEQ ID NO:1 to SEQ ID NO:13; (3) an amino acid sequence having one or more, for example, 2, 3, 4 or 5 amino acid substitutions, deletions or insertions in the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13; More preferably, the CpG oligodeoxynucleotide comprises or consists of an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; More preferably, the CpG oligodeoxynucleotide comprises or consists of the amino acid sequence shown in SEQ ID NO:

1.

4. The immunoadjuvant composition according to any one of claims 1 to 3, wherein The saponin is selected from one or more of quillaja saponin, ginsenoside, platycodon saponin, astragaloside, notoginseng saponin, glycyrrhiza saponin, julibrissin, ophiopogon saponin, bupleurum saponin or panax japonicus saponin; Preferably, the Quillaja saponin is selected from one or more of QS-7, QS-17, QS-18 or QS-21; more preferably, the Quillaja saponin is QS-21.

5. The immunoadjuvant composition according to any one of claims 1 to 4, wherein The weight ratio of the phospholipid to the sterol is 20-4000:50-1000, preferably 20-2000:50-500; Preferably, the weight ratio of the sum of the weight of the liposome and the saponin to the weight of the CpG oligodeoxynucleotide is 0.1-5:0.5-120, preferably 1:5-20, more preferably 1:10; Preferably, the weight ratio of saponin to liposome is 0.1-5:10-150, preferably 0.2-5:15-150.

6. The immune adjuvant composition according to any one of claims 1 to 5, which is in the form of nanoparticles; preferably, the particle size of the nanoparticles is 20-120 nm.

7. The immunoadjuvant composition according to any one of claims 1 to 6, wherein The immunoadjuvant composition further comprises an antigen, a fragment of the antigen, a variant of the antigen, or a mixture of at least two thereof; Preferably, the antigen is selected from one or more of hepatitis A, B, C or E virus antigens, human herpes antigens, human immunodeficiency virus antigens, varicella-zoster virus antigens, human cytomegalovirus antigens, respiratory syncytial virus antigens, human papillomavirus antigens, influenza virus antigens or Mycobacterium tuberculosis antigens, more preferably, the antigen is a hepatitis B antigen or a herpes antigen; Preferably, the hepatitis B antigens are hepatitis B surface antigen and hepatitis B core antigen.

8. The immunoadjuvant composition according to any one of claims 1 to 7, wherein The weight ratio of the herpes antigen, the sum of the weight of the liposome and the saponin, and the CpG oligodeoxynucleotide is 0.1-5:0.1-5:0.5-120, preferably 1-2:1:5-20, more preferably 1:1:10; The weight ratio of the hepatitis B antigen, the sum of the weight of the liposome and saponin, and the CpG oligodeoxynucleotide is 5-100:0.1-5:0.5-120, preferably 15-50:1:5-20, and more preferably 30:1:

10.

9. A method for preparing an immunoadjuvant composition according to any one of claims 1 to 8, comprising the following steps: The saponin is dissolved in an aqueous solution, liposomes are added, and a first stirring is performed, and then CPG oligodeoxynucleotide is added and a second stirring is performed to obtain the immune adjuvant composition.

10. The method for preparing the immune adjuvant composition according to claim 9, wherein: The stirring speeds of the first stirring and the second stirring are independently 50-150 rpm, preferably 50-100 rpm; Preferably, the aqueous solution comprises phosphate and sodium chloride; Preferably, the concentration of phosphate in the aqueous solution is 10-50 mmol / L, preferably 20 mmol / L; the concentration of sodium chloride in the aqueous solution is 100-350 mmol / L, preferably 150 mmol / L.

11. The method for preparing the immune adjuvant composition according to claim 9 or 10, wherein: The preparation method further comprises the step of adding an antigen after the second stirring. 12 . An immunization kit comprising the immune adjuvant composition according to claim 1 .

13. Use of the immune adjuvant composition according to any one of claims 1 to 8 or the immune kit according to claim 12 in the preparation of the following products: (1) A drug for preventing and / or treating human herpes virus and / or hepatitis B virus infection or diseases related thereto; preferably, the drug is a vaccine; (2) a kit for diagnosing human herpes virus and / or hepatitis B virus infection; or (3) Immunogens for the development of antibodies against human herpes simplex virus and / or hepatitis B virus; Preferably, the human herpes virus is selected from one or more of varicella-zoster virus, herpes simplex virus type 1, and herpes simplex virus type 2.

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