Immunologic adjuvant composition and preparation method and application thereof

Abstract

The invention provides an immunologic adjuvant composition as well as a preparation method and application thereof. The immunologic adjuvant composition comprises an immunologic active agent and a liposome for loading the immunologic active agent, wherein the immunologic active agent comprises saponin and CpG oligodeoxynucleotide. The immunologic adjuvant composition can induce immune response of mammals to human herpes virus and / or hepatitis B virus, and can induce generation of a relatively high cellular immune response level. The QS21 and the CpG oligodeoxynucleotide are simultaneously loaded by using the liposome, so that the QS21 and the CpG oligodeoxynucleotide have a better synergistic effect in the aspect of promoting cellular immunity, and compared with a Shingrix commercially available vaccine, the vaccine can induce generation of a higher cellular immune response level.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals. Specifically, the present invention relates to an immunoadjuvant composition for inducing an immune response, a preparation method thereof, and uses thereof. Background Art

[0002] Hepatitis B virus (HBV) infection is one of the serious public health problems worldwide. HBV infection is an important cause of chronic hepatitis B, liver cirrhosis, and hepatocellular carcinoma. Commonly used drugs for clinical treatment of chronic HBV infection mainly include nucleoside analogs and interferons. However, nucleosides cannot completely clear cccDNA in hepatocytes, and long-term use is prone to the emergence of drug-resistant mutant strains and rebound after drug withdrawal; while interferons are not suitable for asymptomatic HBV virus carriers, and in chronic HBV patients, the HbeAg seroconversion rate after using for half a year is only 33%, the treatment effect is limited and the side effects are large.

[0003] Herpes zoster is a viral skin disease caused by varicella-zoster virus (VZV). When VZV is initially infected, it enters the local lymph nodes through the respiratory mucosal epithelium for replication. The lymphocytes infected with the virus then enter the blood circulation through the lymphatic circulation to infect peripheral blood mononuclear leukocytes, and the virus spreads to the skin along with the blood flow, and the clinical manifestation is chickenpox. Vaccine preparations are the most effective and economical means for preventing and controlling such diseases.

[0004] Liposomes are composed of a phospholipid bilayer, and their structure is similar to that of cell membranes, with both adjuvant and carrier functions, and can be used as carriers for antigens that induce hapten-specific immune responses. Encapsulating antigens in liposomes can protect the encapsulated antigens from degradation, thereby reducing the dose and number of vaccinations of antigens, reducing the toxicity of the encapsulated antigens, and enhancing the ability of animals to tolerate high-dose pathogenic microorganisms or their toxin attacks, so it has become an ideal adjuvant carrier.

[0005] Saponins are triterpenoid glycosides extracted from the bark of soap trees. Quil-A is a partially purified aqueous extract of saponin materials, and QS21 is a non-toxic fraction of Quil-A purified by HPLC. It is a complex glycolipid structure that contains a quillaic acid triterpene ring structure substituted by a branched-chain trisaccharide and a linear tetrasaccharide, and the linear tetrasaccharide is connected to the acyl chain.

[0006] The chemical nature of CpG oligodeoxynucleotides is deoxyoligonucleotides containing cytosine-guanine dinucleotides, and they have an immune response similar to that of natural CpG pattern recognition receptors. They can bind to Toll-like receptors on the cell membrane and effectively trigger mammalian immune responses through the TLR9 signaling pathway.

[0007] 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.

[0008] In the prior art, patent application CN101330924 A of GlaxoSmithKline Biopharmaceuticals Ltd. (hereinafter referred to as Shingrix) discloses a composite preparation comprising QS21, MPL and liposomes, which is a new type of herpes zoster vaccine that can induce an immune response against herpes zoster virus in mammals. The vaccine uses liposomes as delivery carriers to coordinate QS21 and MPL to achieve adjuvant delivery, but does not record how QS21, MPL and liposomes interact with each other, and how to coordinate QS21 and MPL to achieve adjuvant delivery. Therefore, the structure of liposomes and the interaction between liposomes and adjuvants are currently the focus of liposomes.

[0009] In the prior art, Exquisite 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 records that the mode of action of liposomes and oligonucleotides is to anchor the oligonucleotides to the hydrophobic region of the phospholipid bilayer of the liposome by conjugating them through a linker molecule. Specifically, the patent application discloses a nanostructure having a liposome core with a lipid bilayer, and an oligonucleotide located outside the liposome core, wherein an immunostimulant is combined with the lipid bilayer, and the oligonucleotide forms an oligonucleotide shell, which is anchored to the surface of the liposome core by conjugating it with a linker molecule, the linker molecule is tocopherol or cholesterol, and wherein all oligonucleotides have a 5'-end exposed to the outer surface of the nanostructure. However, there are various types of oligonucleotides, and the structures formed by different types of oligonucleotides and liposomes and the direct interactions between the two are also very different.

[0010] Therefore, the direct interaction between specific adjuvants and liposomes and the resulting immune effects remain an issue that needs to be studied urgently. Summary of the invention

[0011] In view of the above problems, the purpose of the present invention is to provide an immune adjuvant composition and its preparation method and use. The immune adjuvant composition can induce an immune response of mammals against human herpes virus and / or hepatitis B virus, and can induce a stronger cellular immune response level.

[0012] definition:

[0013] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the definitions of terms in this field, those skilled in the art can specifically refer to Current Protocols in Molecular Biology (Ausubel).

[0014] Although the present invention shows numerical ranges and parameter approximations in a broad scope, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently necessarily contains certain errors, which are caused by the standard deviations existing in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "2 to 40" recited should be considered to include any and all sub-ranges between the minimum value of 2 and the maximum value of 40 (including the endpoints), that is to say, all sub-ranges starting from the minimum value of 2 or greater, such as 2 to 6.1, and sub-ranges ending with the maximum value of 40 or less, such as 5.5 to 40. In addition, any reference cited as "incorporated herein" should be understood to be incorporated in its entirety.

[0015] The term "or" used herein may be interchangeably used with the term "and / or", unless the context clearly indicates otherwise.

[0016] The term "immunoadjuvant composition" used herein may be interchangeably used with "combined immunoadjuvant" and "immunoadjuvant combination", which refers to a combination of at least one drug and optionally a pharmaceutically acceptable excipient or auxiliary combined together to achieve a specific purpose.

[0017] The term "mammal" used herein refers to humans or other animals, such as wild animals (such as herons, storks, cranes, etc.), domestic animals (such as ducks, geese, etc.) or experimental animals (such as orangutans, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0018] The term "quasi-binary mixture" used herein refers to a complex formed by the binding of an adjuvant such as saponin and a sterol such as cholesterol in a liposome by van der Waals forces or hydrophobic forces.

[0019] The above object of the present invention is achieved by providing the following technical solutions:

[0020] In a first aspect, the present invention provides an immunoadjuvant composition, which comprises an immunostimulatory agent and a liposome for loading the immunostimulatory agent, and the immunostimulatory agent comprises saponin and CpG oligodeoxynucleotide.

[0021] Preferably, the liposome comprises phospholipid and sterol.

[0022] Further preferably, the phospholipid is selected from one or more of dioleoyl phosphatidyl base, egg yolk phosphatidylcholine, phosphocholine or natural phospholipid derivatives; more preferably, the phospholipid is dioleoyl phosphatidyl base (DOPC).

[0023] Further preferably, the sterol is selected from one or more of cholesterol, stigmasterol or ergosterol; more preferably, the sterol is cholesterol.

[0024] 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 binds to the external hydrophilic surface; further preferably, the CpG oligodeoxynucleotide binds to the external hydrophilic surface through van der Waals forces and hydrophobic interactions.

[0025] Preferably, the sterol is embedded in the internal hydrophobic region of the phospholipid bilayer structure.

[0026] Preferably, the saponin and the sterol form a pseudo-binary mixture of a worm-like micelle assembly. Further preferably, the pseudo-binary mixture is formed by the hydrophobic interaction between the hydrophobic groups of the sterol in the liposome and the triterpene part of the saponin and the hydrogen bond interaction between the hydrophilic group of the sterol and the glycosyl part of the saponin.

[0027] Preferably, the CpG oligodeoxynucleotide has two or more copies of the 5'-TTCGTT-3' motif or the 5'-TCGTCGTCG-3' motif.

[0028] Preferably, the CpG oligodeoxynucleotide comprises a sequence selected from or consisting of one of the following:

[0029] (1) an amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13;

[0030] (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13;

[0031] (3) an amino acid sequence having one or more, such as 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.

[0032] 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.

[0033] More preferably, the CpG oligodeoxynucleotide comprises or consists of an amino acid sequence as shown in SEQ ID NO: 1.

[0034] Preferably, the saponin is selected from one or more of quillaia saponin, ginsenoside, platycodin, astragaloside, notoginsenoside, glycyrrhizin, albizia bark saponin, ophiopogon saponin, bupleurum saponin or panax japonicus saponin.

[0035] Preferably, the quillaia saponin is selected from one or more of QS-7, QS-17, QS-18 or QS-21; more preferably, the quillaia saponin is QS-21.

[0036] Preferably, the weight ratio of the phospholipid to the sterol is 20 - 4000:50 - 1000, preferably 20 - 2000:50 - 500.

[0037] Preferably, the weight ratio of the sum of the weights 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.

[0038] Preferably, the weight ratio of the saponin to the liposome is 0.1 - 5:10 - 150, preferably 0.2 - 5:15 - 150.

[0039] Preferably, the immunoadjuvant composition is in the form of nanoparticles; more preferably, the particle size of the nanoparticles is 20 - 120 nm.

[0040] 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 of them.

[0041] Preferably, the antigen is selected from one or more of hepatitis A, B, C or E virus antigens, human herpes antigen, human immunodeficiency virus antigen, varicella - zoster virus antigen, human cytomegalovirus antigen, respiratory syncytial virus antigen, human papillomavirus antigen, influenza virus antigen or Mycobacterium tuberculosis antigen; more preferably, the antigen is hepatitis B antigen or herpes antigen.

[0042] Preferably, the hepatitis B antigen is hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg).

[0043] Preferably, the hepatitis B surface antigen comprises or consists of an amino acid sequence selected from one of the following:

[0044] (1) The amino acid sequence shown in SEQ ID NO: 14;

[0045] (2) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 14;

[0046] (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.

[0047] Further preferably, the hepatitis B surface antigen comprises or consists of the amino acid sequence shown in SEQ ID NO: 14.

[0048] Preferably, the hepatitis B core antigen comprises or consists of an amino acid sequence selected from one of the following:

[0049] (1) The amino acid sequence shown in SEQ ID NO: 15;

[0050] (2) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 15;

[0051] (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.

[0052] Further preferably, the hepatitis B core antigen comprises or consists of the amino acid sequence shown in SEQ ID NO: 15.

[0053] Preferably, the herpes antigen is herpes gE protein.

[0054] Preferably, the herpes gE protein comprises or consists of an amino acid sequence selected from one of the following:

[0055] (1) The amino acid sequence shown in SEQ ID NO: 16;

[0056] (2) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 16;

[0057] (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.

[0058] Further preferably, the herpes gE protein comprises the amino acid sequence shown in SEQ ID NO: 16 or consists thereof.

[0059] Preferably, the weight ratio of the herpes antigen, the sum of the weights 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, and more preferably 1:1:10.

[0060] Preferably, the weight ratio of the hepatitis B antigen, the sum of the weights of the liposome and the 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.

[0061] In a second aspect, the present invention provides a method for preparing the immunoadjuvant composition according to the first aspect of the present invention, which comprises the following steps:

[0062] Dissolve the saponin in an aqueous solution, add the liposome, perform a first stirring, and then add the CPG oligodeoxynucleotide for a second stirring to obtain the immunoadjuvant composition.

[0063] Preferably, the stirring speeds of the first stirring and the second stirring are each independently 50 - 150 rpm, preferably 50 - 100 rpm.

[0064] Preferably, the aqueous solution comprises phosphate and sodium chloride.

[0065] 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.

[0066] Preferably, the preparation method further comprises the step of adding an antigen after the second stirring.

[0067] In a third aspect, the present invention provides an immune kit, which comprises the immunoadjuvant composition according to the first aspect of the present invention.

[0068] In a fourth aspect, the present invention provides the use of the immunoadjuvant 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:

[0069] (1) A drug for preventing and / or treating human herpesvirus and / or hepatitis B virus infection or related diseases; preferably, the drug is a vaccine;

[0070] (2) A kit for diagnosing human herpesvirus and / or hepatitis B virus infection; or

[0071] (3) An immunogen for developing antibodies against human herpesvirus and / or hepatitis B virus;

[0072] Preferably, the human herpesvirus is one or more selected from varicella-zoster virus, herpes simplex virus type 1 (HSV1), or herpes simplex virus type 2 (HSV2).

[0073] The present invention has at least the following beneficial effects:

[0074] In the immunoadjuvant composition provided by the present invention, saponins such as QS21 can interact with CpG oligodeoxynucleotides and liposomes. Among them, QS21 is embedded in the inner hydrophobic region of the phospholipid bilayer structure and forms a quasi-binary mixture of worm-like micelle assemblies with sterols such as cholesterol. This assembly with a quasi-binary mixture structure is formed by the hydrophobic interaction between the hydrophobic groups of cholesterol in the liposome and the triterpene part of QS21, as well as the hydrogen bond interaction between the hydrophilic group of cholesterol and the glycosyl part of QS21. Its hydrophobic interaction and hydrogen bond interaction can improve the binding rate and drug loading capacity of QS21 with liposomes, and further improve the stability of QS21 liposomes.

[0075] The CpG oligodeoxynucleotides in the immunoadjuvant composition provided by the present invention are negatively charged and have van der Waals forces and hydrophobic forces with phospholipid molecules such as DOPC, so that CpG can directly bind to the liposome surface, improving the binding rate and drug loading capacity of CpG with liposomes, and further improving the stability of CPG-QS21 liposomes.

[0076] The immunoadjuvant composition provided by the present invention can induce an immune response in mammals against human herpesvirus and / or hepatitis B virus, and can induce a strong level of cellular immune response. Specifically, the immunoadjuvant composition provided by the present invention can induce mammals to produce a relatively high level of gE antigen-specific IFN-γ and gE antigen-specific IgG / IgG1 / IgG2a titers, and after a long time, the induced gE antigen-specific IFN-γ and gE antigen-specific IgG / IgG1 / IgG2a titers still remain at a relatively high level; the immunoadjuvant composition provided by the present invention can effectively reduce the HBsAg level in HBV-infected model mice, and can induce a relatively high level of HBsAg and HBcAg-specific IFN-γ.

[0077] By using liposomes to simultaneously load QS21 and CpG oligodeoxynucleotides, the present invention enables the two to have a good synergistic effect in promoting cellular immunity. Compared with the commercially available Shingrix vaccine, the immunoadjuvant composition of the present invention can induce a stronger level of immune response. Description of the Drawings

[0078] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, wherein:

[0079] Figure 1 It is a cryo-electron microscopy image of the CPG-QS21 liposome nanocomposite preparation obtained in Example 2 of the present invention;

[0080] Figure 2 It is a schematic structural diagram of CPG-QS21 liposomes;

[0081] Figure 3 It is a particle size diagram of the CPG-QS21 liposome nanocomposite preparation obtained in Example 2 of the present invention;

[0082] Figure 4 It shows the effect of different ratios of CpG oligodeoxynucleotides on the level of T lymphocyte spot numbers of herpes gE antigen-specific IFN-γ in Example 3 of the present invention;

[0083] Figure 5 It shows the effect of different ratios of CpG oligodeoxynucleotides on the level of T lymphocyte spot numbers of herpes gE antigen-specific IFN-γ in Example 4 of the present invention;

[0084] Figure 6 It shows the effect 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;

[0085] Figure 7 It shows the effect of different ratios of herpes gE antigen on the level of gE-specific IFN-γ lymphocyte spot numbers secreted by splenocytes in Example 5 of the present invention;

[0086] Figure 8 It shows the effect of the herpes zoster vaccine on the level of gE-specific IFN-γ lymphocyte spot numbers secreted by mouse splenocytes in Example 6 of the present invention;

[0087] Figure 9 It shows the effect of the herpes zoster vaccine on the levels of gE-specific IgG and its subtypes and the IgG2a / IgG1 ratio in the serum of mice in Example 6 of the present invention;

[0088] Figure 10Show the effect of hepatitis B vaccine on the levels of HBsAg in the sera of mice at different weeks after immunization in Example 7 of the present invention;

[0089] Figure 11 Show the effect of hepatitis B vaccine on the level of the number of gE-specific IFN-γ lymphocyte spots secreted by splenocytes of mice in Example 7 of the present invention. Detailed implementation manners

[0090] The present invention will be further described in detail below in conjunction with the detailed implementation manners. The provided examples are only for clarifying the present invention and not for limiting the scope of the present invention.

[0091] Example 1 Preparation and preparation of biological materials

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

[0093] 2. HBcAg stock solution: The amino acid sequence of the HBcAg protein in the present invention, SEQ ID NO: 15, refers to SEQ ID NO: 1 of Chinese Patent CN104673760B. The HBcAg antigen protein is prepared from recombinant yeast cells of the HBcAg gene to obtain a crude HBcAg fermentation broth. The specific preparation method refers to paragraphs 0049 to 0068 of Chinese Patent CN104673760B. After cell disruption, filtration, and column chromatography, the HBsAg stock solution is obtained. The specific purification method refers to paragraphs 0069 to 0080 of Chinese Patent CN104673760B.

[0094] 3. gE stock solution: The amino acid sequence of the gE protein, SEQ ID NO: 16, refers to SEQ ID NO: 1 of Chinese Patent CN112972671A. After codon optimization, PCR amplification, ligation and transformation of the nucleic acid sequence of the gE protein into the target vector, the target plasmid is obtained through screening. After transfection into CHO cells and fermentation, the gE stock solution is obtained through filtration and purification. The specific preparation method refers to the literature Thomsson E, Persson L, etc., "Journal of Virological Methods" 202011, Vol. 175, No. 1, pp. 53-59.

[0095] 4, PBS solution: Purchased from Hyclone, product number: SH30256.01.

[0096] 5, Serum-free medium: Serum-Free Medium, purchased from Dakewei Biotechnology Co., Ltd., product number: 6015012.

[0097] 6, QS21: Purchased from BRENNTAG, CAS.NO.A010-023.

[0098] 7, Preparation method of CPG oligodeoxynucleotide raw material: Prepared according to the conventional solid-phase phosphoramidite triester chemical synthesis method. Starting from the 3' end, first deprotect the group, then perform activation, connection, oxidation, and capping processes to obtain a crude DNA fragment. Finally, perform post-synthesis treatments such as cleavage, deprotection, purification, and quantification. For the specific preparation method, refer to paragraphs 0063 to 0064 of patent CN200810004736.0. The CPG oligodeoxynucleotide used in the present invention is CpG T1, and the specific sequence is shown in Table 1.

[0099] 8, Shingrix vaccine: Purchased from GlaxoSmithKline Biologicals Co., Ltd.

[0100] Table 1 Specific sequence of CPG oligodeoxynucleotide artificial sequence

[0101]

[0102]

[0103] Example 2 Preparation and detection of liposome nanocomposite preparation

[0104] 1, Preparation of liposome nanocomposite preparation

[0105] (1) Preparation of blank liposomes

[0106] Dissolve dioleoyl phosphatidylcholine and cholesterol with a weight ratio of 1000:250 in an organic solvent. Remove the organic solvent by rotary evaporation. Set the rotary evaporation temperature to 50°C, the temperature of the low-temperature circulation device to -10°C, control the rotary evaporation vacuum to be maintained at 20 - 120 hPa, control the rotary evaporation speed to be maintained at 30 - 180 rpm, form a lipid film on the wall of the rotary evaporation flask, dissolve the lipid film with an aqueous solution, hydrate to form a lipid suspension, and then homogenize 6 times. Control the homogenization pressure to be maintained at 150 bar. Extrude the obtained lipid solution through two polycarbonate membranes 6 times. Control the pore size of the first polycarbonate membrane to be 200 nm and the pore size of the second polycarbonate membrane to be 100 nm to obtain blank liposomes.

[0107] Among them, the organic solvent is absolute ethanol, and the aqueous solution is an aqueous solution of phosphate and sodium chloride. Among them, 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.

[0108] (2) Preparation of CPG-QS21 liposomes

[0109] Dissolve QS21 in the aqueous solution, add blank liposomes, stir evenly, control the stirring speed to be maintained at 50 - 150 rpm to obtain QS21 liposomes, then add CPG oligodeoxynucleotides, and stir evenly with the QS21 liposomes, control the stirring speed to be maintained at 50 - 150 rpm to obtain CPG-QS21 liposomes. Among them, the aqueous solution is an aqueous solution of phosphate and sodium chloride. Among them, the concentration of phosphate is 20 mmol / L, and the concentration of sodium chloride is 150 mmol / L. The weight ratio of QS21 to liposomes is 50∶1250, and the ratio of the two remains unchanged. The two are combined and recorded as QS21 liposomes.

[0110] Mix the CPG-QS21 liposomes prepared in this example with the required antigen stock solution for injection in the following examples.

[0111] 2. Detection of liposome nano-composite preparation

[0112] (1) Detection of encapsulation efficiency of QS21

[0113] Use a high-performance liquid chromatograph to detect the encapsulation efficiency of CPG-QS21 liposomes. Among them, the liposome nano-composite preparation used for this detection has a vaccine formulation for human use, 0.5 mL per dose, which contains 1300 μg of QS21 liposomes (including 50 μg of QS21), 500 μg of CpG, and 100 μg of gE antigen.

[0114] Encapsulation efficiency detection method: Use a high-performance liquid chromatograph to detect the encapsulation efficiency of QS21. Octadecylsilane-bonded silica gel is used as the filler; formic acid-water (1∶1000) is used as mobile phase A, and formic acid-acetonitrile (1∶1000) is used as mobile phase B for linear gradient elution; the flow rate is 1.0 mL per minute; the column temperature is 40 °C; the injection volume is 100 μL; detector: CAD (nebulization temperature: 50 °C; acquisition frequency: 10 Hz).

[0115]

[0116] In the formula: A L is the sum of the peak areas of QS-21a and QS-21b encapsulated in liposomes;

[0117] A F is the sum of the peak areas of free QS-21a and QS-21b;

[0118] 100 is the dilution factor of the encapsulated part of the solution;

[0119] 20 is the dilution factor of the free part of the solution.

[0120] (2) Detection of the microscopic morphology and particle size of the liposome nano - composite preparation

[0121] The microscopic morphology of CPG - QS21 liposomes was observed by cryo - electron microscopy.

[0122] The average particle size of CPG - QS21 liposomes was detected by laser dynamic light scattering method. Laser dynamic light scattering detection method: First, the measurement cell was washed once with filtered double - distilled water. The sample was first ultrasonically treated in water bath (100W, ultrasonic treatment for 5 - 7 times) and then filtered through a 0.22μm needle filter (Pall Corporation). 1mL of the sample was taken and put into the measurement cell (the height of the sample in the measurement cell was 10mm - 15mm). According to the instrument instructions, open the sample cell cover, put in the measurement cell (with the side with the V symbol facing the operator), and click Start to start the measurement.

[0123] 3. Detection results

[0124] Table 2 Encapsulation efficiency of QS21 in CpG - QS21 liposomes

[0125]

[0126] The encapsulation efficiency of QS21 in CPG - QS21 liposomes detected by high - performance liquid chromatography is shown in Table 2. With the increase of the concentration of QS21, the encapsulation efficiency of QS21 in CPG - QS21 liposomes gradually increases. When it increases to 100μg / mL, the encapsulation efficiency of QS21 reaches the highest. Then, when the amount of QS21 continues to increase, the encapsulation efficiency of QS21 shows a downward trend. However, when the amount of QS21 is less than 200μg / mL, it can reach a level higher than the industry - recognized standard (85%), which may be related to the encapsulation capacity of the liposome.

[0127] The particle size of CPG - QS21 liposomes detected by laser dynamic light scattering method is as Figure 3 shown, and its average particle size is 93.08nm.

[0128] The microscopic morphological characterization of CPG - QS21 liposomes is as Figure 1 shown. The structural schematic diagram of CPG - QS21 liposomes is as Figure 2As shown. It can be seen that blank liposomes, QS21 liposomes, and CPG-QS21 liposomes all have a phospholipid bilayer structure. Each phospholipid molecule has a hydrophilic head and a lipophilic tail. Among them, two layers of phospholipid molecules face each other, forming an external hydrophilic surface and an internal hydrophobic region. Cholesterol is located in the internal hydrophobic region, and QS21 is embedded in the internal hydrophobic region, forming a pseudo-binary mixture with cholesterol in a worm-like micelle assembly. This assembly with a pseudo-binary mixture structure is formed by the combined action of the hydrophobic interaction between the hydrophobic part of cholesterol composition in the liposome and the triterpene part of QS21, and the hydrogen bond interaction between the hydrophilic part of cholesterol and the glycosyl part of QS21. Its hydrophobic interaction and hydrogen bond interaction can improve the binding rate and drug loading capacity of QS21 with liposomes, and further improve the stability of QS21 liposomes.

[0129] Since the CpG oligodeoxynucleotide is negatively charged and there are van der Waals forces and hydrophobic forces between it and the liposome, the CpG oligodeoxynucleotide binds to the phospholipid molecule DOPC on the surface of the liposome. Due to the van der Waals forces and hydrophobic forces between CpG and the phospholipid molecule DOPC, CpG and the liposome can directly bind on the surface of the liposome, improving the binding rate and drug loading capacity of CpG with the liposome, and further improving the stability of CPG-QS21 liposomes.

[0130] Patent application CN 106535876 A discloses a liposome nanostructure, which has a liposome core with a lipid bilayer and an oligonucleotide containing a class B CpG motif located outside the liposome core. All the oligonucleotides form an oligonucleotide shell, and the oligonucleotide shell is anchored to the surface of the liposome core by conjugating with a linker molecule, where the linker molecule is tocopherol or cholesterol, and the surface of the liposome core is the hydrophobic region surface of the liposome. That is to say, the oligonucleotide in the liposome of patent application CN106535876 A needs to enter the interior of the liposome phospholipid bilayer through the gap on the outer surface between phospholipid molecules and then can be anchored to the surface of the hydrophobic region of the liposome through the linker molecule. Obviously, the linker molecule is located in the liposome core region, that is, the hydrophobic region of the liposome, and the oligonucleotide needs to enter the interior of the liposome phospholipid bilayer from the gap on the outer surface of the phospholipid molecules and then contact the linker molecule to be further anchored to the surface of the hydrophobic region of the liposome. Compared with the liposome structure of patent application CN106535876 A, on the one hand, the CpG in the liposome structure of the present invention can directly bind to the liposome on the surface of the liposome, which is more direct and efficient, and can significantly improve the CpG binding rate; on the other hand, the binding of CpG to the liposome in the liposome structure of the present invention relies on its own van der Waals forces and can achieve effective binding without a linker molecule, further improving the binding rate of CpG with the liposome.

[0131] Example 3 Primary Screening Experiment of CPG-QS21 Liposome Vaccine

[0132] 1. Experimental animals: C57BL / 6 mice, female, 6 weeks old, 60 in number, from Shanghai Lingchang Biotechnology Co., Ltd., animal license number: SCXK(Shanghai)2018-0003.

[0133] 2. Animal grouping: The specific animal grouping is shown in Table 3, and the injection volume per mouse is 100 μL each time. Among them, before each injection, the components shown in the following table are dissolved and diluted to 100 μL with PBS solution. The dosages of CpG and QS21 liposomes in CpG-QS21 liposomes for each group are shown in the following table.

[0134] Table 3 Grouping of SM21053 animal experiments

[0135]

[0136]

[0137] 3. Experimental procedures: Immunization by intramuscular injection, 100 μL per mouse in each group, immunized once. Detection of the level of T lymphocyte spot number secreting gE-specific IFN-γ in splenocytes was carried out at wk01 (one week after the first immunization). Among them, before the spot level detection, groups A-L were injected with serum-free medium containing the gE-specific stimulatory peptide library; at the same time, a medium negative control group (medium group) was set for each group. In the medium group: Splenocytes of non-immunized mice were isolated and injected with the same amount of serum-free medium without the above-mentioned stimulatory peptide library.

[0138] 4. Experimental results: The level of T lymphocyte spots secreting gE-specific IFN-γ in splenocytes was detected by the ELISpot method, and the results are shown in Figure 4 , and the results show that:

[0139] a) Comparing groups A-F immunized with 2 μg gE antigen combined with CpG-QS21 liposomes containing 2 μg QS21 liposomes and different doses of CpG (2 - 100 μg), with the increase of the CpG dose, the level of gE antigen-specific IFN-γ induced also showed an increasing trend. However, in group E (containing 50 μg CpG) and group F (containing 100 μg CpG) with high doses of CpG, the splenocytes of mice produced a certain level of non-specific IFN-γ response, and the state of splenocytes also showed a tendency of being too large; in addition, the level of gE antigen-specific IFN-γ secreted by splenocytes in group C containing 10 μg CpG was relatively high (about 1200 SFC / 10 6 splenocytes), and the splenocytes were normal.

[0140] b) When comparing the G-L group immunized with 2 μg of gE antigen combined with CPG-QS21 liposomes containing 1 μg of QS21 liposomes and different doses of CpG (2 - 100 μg), the splenocytes of mice in the K group (containing 50 μg of CpG) and the L group (containing 100 μg of CpG) with high doses of CpG also produced a certain level of non-specific IFN-γ response, and the state of splenocytes also showed a tendency of being overactivated, similar to that of the E group and the F group; in addition, the level of gE antigen-specific IFN-γ secreted by the splenocytes of the J group immunized with 20 μg of CpG was the highest (about 1500 SFC / 10 6 splenocytes), and the splenocytes were normal.

[0141] c) In CPG-QS21 liposomes, different doses of QS21 liposomes (1 μg, 2 μg) combined with CpG can induce a strong level of gE antigen-specific IFN-γ.

[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 to CpG is 1:20; when the dose of QS21 liposomes is 2 μg, the preferred weight ratio of QS21 liposomes to CpG is 1:5.

[0143] Table 4 Positive conversion rate of gE-specific IFN-γ secreted by splenocytes %

[0144]

[0145] Example 4 Screening experiment of vaccines containing CPG-QS21 liposome nanocomposite preparations

[0146] 1. Experimental animals: C57BL / 6 mice, female, 4 - 5 weeks old, 72 in number, from Shanghai Lingchang Biotechnology Co., Ltd., animal license number: SCXK (Shanghai) 2018 - 0003.

[0147] 2. Animal grouping: The specific animal grouping is shown in Table 5, and the injection volume per time is 100 μL / mouse. Among them, before each injection, the components shown in the following table were dissolved in PBS solution and diluted to 100 μL. The dosages of CpG and QS21 liposomes in each group of CPG-QS21 liposomes are shown in the following table. Group A is the negative control, injecting 100 μL / mouse of PBS solution containing 2 μg of gE antigen; Group F was given the commercially available Shingrix vaccine, with the antigen being 2 μg / mouse, and the dosages of QS21 liposomes and MPL both being 2 μg / mouse.

[0148] Table 5 Grouping of SM21056 animal experiments

[0149]

[0150] 3. Experimental procedures: Immunization by intramuscular injection was performed twice at wk00 and wk02. Groups A - E were injected with 100 μL per mouse, and Group F was injected with 20 μL per mouse. Half of the mice were tested at wk01, and the remaining half were given a second immunization at wk02 and tested at wk03, including the detection of the number of T lymphocyte spots secreting IFN-γ by splenocytes and the detection of gE antigen-specific antibodies in serum. Among them, before the spot level detection, Groups A - F were injected with serum-free medium containing a gE-specific stimulatory peptide library. At the same time, a medium negative control group (medium group) was set up for each group. In the medium group: Splenocytes of non-immunized mice were isolated and injected with the same amount of serum-free medium without the above-mentioned stimulatory peptide library.

[0151] 4. Experimental results:

[0152] The ELISpot method was used to detect the T lymphocyte spot level of splenocytes secreting gE-specific IFN-γ. The results are shown in Figure 5 , and the results showed that:

[0153] a) At wk01 (one week after the first immunization), Group A immunized with 2 μg of gE antigen did not produce gE antigen-specific IFN-γ levels; Groups B and C immunized with 2 μg of gE antigen combined with CPG-QS21 liposomes containing 2 μg of QS21 liposomes and QS21 liposomes:CpG = 1:10 or 1:20 could produce obvious gE antigen-specific IFN-γ levels. However, due to the higher dose of CpG (40 μg) in Group C, its non-specific reaction was stronger, and the spleens of the mice enlarged. In comparison, the dose condition of Group B was better (about 630 SFC / 10 6 splenocytes); Groups D and E immunized with 2 μg of gE antigen combined with CPG-QS21 liposomes containing 1 μg of QS21 liposomes and QS21 liposomes:CpG = 1:10 or 1:20 could also produce gE antigen-specific IFN-γ levels comparable to those of Group B, and the IFN-γ level corresponding to Group D (1 μg of QS21 liposomes + 10 μg of CpG) was relatively higher (about 700 SFC / 10 6 splenocytes); Although the commercially available Shingrix vaccine group immunized with the same antigen dose could produce gE antigen-specific IFN-γ levels (about 200 SFC / 10 6 splenocytes), the overall level was lower than that of Groups B - E.

[0154] b) wk03 (one week after the second immunization), the levels of gE antigen-specific IFN-γ induced in each immunization group increased. The IFN-γ level in Group A immunized with 2 μg of gE antigen was still relatively low. When immunized with 2 μg of gE antigen combined with CPG-QS21 liposomes containing QS21 liposomes:CpG = 1:10 or 1:20, at the same dose of QS21 liposomes, the level of cellular immunity induced by the ratio of QS21 liposomes:CpG = 1:10 was higher than that of the 1:20 ratio (i.e., Group B was higher than Group C, and Group D was higher than Group E). The levels of gE antigen-specific IFN-γ induced in Group B and Group D reached 5000 and 2000 SFC / 10 6 splenocytes.

[0155] The levels of gE antigen-specific IgG / IgG1 / IgG2a antibodies in the sera of mice were detected by ELISA method, and the results are shown in Figure 6 , and the results showed that:

[0156] wk03 (one week after the second immunization), Group A immunized with simple antigen could induce a certain level of IgG / IgG1 titer, but the IgG2a titer was relatively low. After combining with CPG-QS21 liposomes containing QS21 liposomes:CpG = 1:10 or 1:20 (i.e., Groups B - E), it could induce higher levels of antigen-specific IgG / IgG1 / IgG2a titers (around 5.0 Lg, 5.0 Lg, and 4.0 Lg respectively), and there was no significant difference among the groups. The commercially available Shingrix vaccine group (Group F) immunized with the same antigen dose could also induce relatively high levels of IgG / IgG1 titers (around 4.7 Lg and 5.3 Lg respectively), but the IgG2a titer was relatively low (only about 1.9 Lg), and there were significant differences compared with Groups B - E (p < 0.001).

[0157] Overall comparison, when immunizing with different ratios of CpG adjuvant and QS21 adjuvant in the CPG-QS21 liposome nanocomposite preparation, the induced immune response level is more biased towards the Th1 pathway, and the commercially available Shingrix vaccine is more biased towards the Th2 pathway.

[0158] In summary, among the induced antigen-specific cellular immunity levels, the ratio of QS21 liposomes:CpG = 1:10 is the highest. It can also induce high levels of antigen-specific IgG / IgG1 / IgG2a titers, and the levels of cellular immunity and humoral immunity induced are higher than those of the commercially available Shingrix vaccine. Therefore, the preferred ratio of QS21 liposomes and CpG adjuvant in the CPG-QS21 liposome nanocomposite preparation is 1:10.

[0159] Antigen Ratio Screening Experiment of the Vaccine Containing CPG-QS21 Liposome Nanocomposite Preparation

[0160] 1. Experimental animals: C57BL / 6 mice, female, 6 weeks old, 32 in number, from Shanghai Lingchang Biotechnology Co., Ltd., animal license number: SCXK(Shanghai)2018-0003.

[0161] 2. Animal grouping: The specific animal grouping is shown in Table 6. The injection volume per time is 50 μL per mouse. Group D uses a different batch of gE antigen from groups A - C. Among them, before injection in each group, the components shown in the following table are dissolved with PBS solution and diluted to 50 μL.

[0162] Table 6 Animal Experiment Grouping of SM22016

[0163]

[0164] 3. Experimental procedure: Immunize by intramuscular injection at 50 μL per mouse, immunize once in total. Detect the level of the number of T lymphocyte spots secreting gE-specific IFN-γ in splenocytes at wk01 (one week after the first immunization). Among them, before the spot level detection, groups A - D are injected with serum-free medium containing a gE-specific stimulatory peptide library; at the same time, a medium negative control group (medium group) is set in each group. Medium group: Isolate splenocytes from non-immunized mice and inject the same amount of serum-free medium without the above stimulatory peptide library.

[0165] 4. Experimental results: Based on the comprehensive research results of 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 induced level of gE antigen-specific IFN-γ is dose-dependent with the gE antigen dose. Among them, when 1 μg of gE antigen is combined with CPG-QS21 liposome nanocomposite preparation (the doses of QS21 liposome and CpG adjuvant are 1 μg and 10 μg respectively), a relatively high level of antigen-specific IFN-γ can be induced. Considering the antigen dose and cost comprehensively, (1 μg of gE antigen + CPG-QS21 liposome containing 1 μg of QS21 liposome and 10 μg of CpG) is preferentially selected as the immunization dose for the mouse efficacy test, that is, it is preliminarily determined that the ratio of QS21 liposome and CpG in the gE antigen and CPG-QS21 liposome nanocomposite preparation is 1∶1∶10 as the optimal ratio.

[0166] Example 6 Experimental Group Setting and Immunization Process of Herpes Zoster Vaccine

[0167] 1. Experimental animals: C57BL / 6 mice, female, 6 weeks old, 30 in number, from Shanghai Lingchang Biotechnology Co., Ltd., animal license number: SCXK(Shanghai)2018-0003.

[0168] 2. Animal grouping: The specific animal grouping is shown in Table 1. The injection volume each time is 100 μL. Before injection for each group, the components shown in the following table are dissolved in PBS solution and diluted to 100 μL.

[0169] Table 7 Animal grouping and dosing for the experiment

[0170]

[0171] *In the CPG-QS21 liposome, CPG is 10 μg and QS21 liposome is 1 μg.

[0172] 3. Animal immunization

[0173] Immunization was repeated twice at wk00 and wk02 by intramuscular injection at the left / right posterior thigh as the injection site.

[0174] 4. Evaluation of the cellular immune effect of the herpes zoster vaccine

[0175] (1) Detection method: The T lymphocyte spot number level detection method. Among them, before the spot level detection, groups A - F were injected with serum-free medium containing the gE-specific stimulatory peptide library; at the same time, a medium negative control group (medium group) was set for each group. In the medium group: Spleen cells of non-immunized mice were isolated and injected with the same amount of serum-free medium without the above-mentioned stimulatory peptide library.

[0176] (2) Evaluation index: When the number of spots in the control well ≤ 5 SFC, the number of spots in the sample well should ≥ 10 SFC; when the number of spots in the control well is between 5 SFC and 10 SFC, the ratio of the number of spots in the sample well to the number of spots in the control well should ≥ 2; when the number of spots in the control well > 10 SFC, the ratio of the number of spots in the sample well to the number of spots in the control well should ≥ 3.

[0177] 5. Experimental results

[0178] Table 8 Positive conversion rate % of gE-specific IFN-γ secreted by spleen cells

[0179]

[0180] Result analysis: The T lymphocyte spot number level of spleen cells secreting gE-specific IFN-γ in mice of each immunized group is as Figure 8 shown, and the positive conversion rate results of gE-specific IFN-γ are shown in Table 8. At wk02 (two weeks after the first immunization), group E can induce a relatively high level of gE-specific IFN-γ cellular immune response, and the spot level is about 900 SFC / 10 6Spleen cells had a higher level of gE-specific IFN-γ cellular immunity after secondary immunization, and the spot level could reach 2200 SFC / 10 6 Spleen cells, significantly higher than the simple antigen group, any other single adjuvant group, and the commercially available Shingrix vaccine group. As time extended, the induced level of gE-specific IFN-γ decreased, but at wk24, a relatively high level of gE-specific IFN-γ (~900 SFC / 10 6 Spleen cells) could still be maintained, and the positive conversion rate remained 100%.

[0181] The results showed that the QS21 liposome adjuvant and CpG adjuvant contained in the injectable CPG-QS21 liposome nano-composite preparation had good synergistic effects in promoting cellular immunity, and compared with the commercially available Shingrix vaccine, the TVAX-006 vaccine could induce a stronger cellular immune response level.

[0182] 6. Detection of gE-specific antibodies in serum for herpes zoster

[0183] (1) Detection method: ELISA

[0184] (2) Experimental results: The titers of gE-specific IgG / IgG1 / IgG2a antibodies in the sera of each immunized group after immunization were as Figure 9 shown: Each group could induce the titers of gE antigen-specific IgG / IgG1 / IgG2a antibodies at wk02 (two weeks after the first immunization). Among them, the titers of gE antigen-specific IgG / IgG1 / IgG2a induced by group E and group F were higher than those of any single adjuvant group and the simple antigen group; after secondary immunization, the titers of gE-specific IgG / IgG1 / IgG2a antibodies induced by each group further increased. The titers of gE-specific IgG / IgG1 / IgG2a produced by group E and group F maintained relatively high levels from wk04 to wkl2. Among them, the level of group E decreased at wk24 but still remained relatively high (the titers of IgG / IgG1 / IgG2a antibodies were 3.798 Lg, 3.110 Lg, and 3.395 Lg respectively), and group F could still maintain a relatively high level at wk24 (the titers of IgG / IgG1 / IgG2a antibodies were 4.043 Lg, 4.537 Lg, and 3.277 Lg respectively); The IgG2a / IgG1 ratio indicated that the immune responses induced by group E and the gE antigen combined with CpG adjuvant group were more biased towards the Th1 pathway, while the commercially available Shingrix vaccine, the single antigen group, and the gE antigen combined with QS21 liposome adjuvant group were more biased towards the Th2 pathway.

[0185] Example 7 Experimental group settings and immunization procedures for hepatitis B vaccine

[0186] 1. Experimental animals: C57BL / 6 mice, female, 6 weeks old, 48 in number, from Shanghai Lingchang Biotechnology Co., Ltd., animal license number: SCXK(Shanghai)2018-0003.

[0187] 2. Animal grouping: The specific animal grouping is shown in Table 9. The injection volume per mouse is 100 μL each time. Before injection for each group, the components shown in the following table were dissolved in PBS solution and diluted to 100 μL. Group A was the negative control, injecting 100 μL of PBS solution per mouse.

[0188] Table 9 Grouping and dosing

[0189]

[0190] * In the CPG-QS21 liposome, CPG is 10 μg and QS21 liposome is 1 μg.

[0191] 3. Animal immunization

[0192] rAAV8-1.3HBV ayw was injected into the tail vein of C57BL / 6 mice to establish a mouse model of persistent rAAV8-HBV infection in C57BL / 6. This HBV infection model was used to evaluate different types of antiviral drugs, TVAX-008 injection (HBsAg + HBcAg + CpG, i.e., Group B), TVAX-028 injection (HBsAg + HBcAg + CPG-QS21 liposome, i.e., Group C), antigen group (HBsAg + HBcAg, i.e., Group D), antigen liposome group (HBsAg + HBcAg + blank liposome, i.e., Group E), antigen-free adjuvant group (HBsAg + HBcAg + QS21 + CpG, i.e., Group F), and at the same time, a PBS group was set as the negative control.

[0193] Both TVAX-008 injection (HBsAg + HBcAg + CpG) and TVAX-028 injection (HBsAg + HBcAg + CPG-QS21 liposome) were administered intramuscularly once every two weeks.

[0194] Dosing frequency and duration: Immunization was performed by intramuscular injection for each group at wk06, wk08, wk10, wk12, wk14, and wk16 respectively, for a total of 6 immunizations. The intramuscular injection site was the posterior thigh.

[0195] 4. Detection of HBsAg level in serum by hepatitis B vaccine

[0196] (1) Detection steps: Refer to the BD TM ELISPOT Mouse IFN-γ ELISPOT Set instruction manual.

[0197] (2) Detection results: The levels of HBsAg in the sera of mice in each group at different detection times are as Figure 10 shown. The results indicate 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) can all effectively reduce the HBsAg level in HBV-infected model mice, and the decreasing trend of Group C (HBsAg + HBcAg + CPG-QS21 liposome) is higher than that of Group B (HBsAg + HBcAg + CpG) and Group F (HBsAg + HBcAg + QS21 + CpG).

[0198] 5. Evaluation of the cellular immune effect of the hepatitis B vaccine

[0199] (1) Detection method: The method for detecting the level of T lymphocyte spot numbers. Before the spot level detection, in Groups A-C, peptide library stimulation was performed during the activation of splenocytes, and the PS4 and PCP peptide libraries diluted and dissolved with serum-free medium were used for stimulation detection respectively; at the same time, a medium negative control group (medium group) was set in each group. In the medium group: The splenocytes of non-immunized mice were isolated and injected with the same amount of serum-free medium.

[0200] (2) Detection results: The levels of T lymphocyte spot numbers secreting HBsAg- and HBcAg-specific IFN-γ in the splenocytes of mice in each group at the end of the experiment are as Figure 11 shown. The results indicate that compared with the PBS control group, the antigen group, and the antigen liposome group, higher levels of HBsAg- and HBcAg-specific IFN-γ can be detected in Group B (HBsAg + HBcAg + CpG), Group F (HBsAg + HBcAg + QS21 + CpG), and Group C (HBsAg + HBcAg + CPG-QS21 liposome) (stimulated with the PS4 and PCP peptide libraries respectively), and the level of IFN-γ secreted by the administration group of Group C (HBsAg + HBcAg + CPG-QS21 liposome) is significantly higher than that of the administration groups of Group B (HBsAg + HBcAg + CpG) and Group F (HBsAg + HBcAg + QS21 + CpG).

[0201] The above are only several exemplary embodiments of the present invention and do not impose any form of limitation on the present invention. Although the present invention is disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any equivalent or equivalent embodiments obtained by making some changes or modifications to the disclosed technical content by those skilled in the relevant art without departing from the technical solution of the present invention all fall within the scope of the present invention.

Claims

1. An immunoadjuvant composition comprising an immunostimulatory agent and liposomes for loading the immunostimulatory agent, wherein the immunostimulatory agent comprises saponin and CpG oligodeoxynucleotide.

2. The immunoadjuvant composition according to claim 1, wherein, The liposome contains phospholipids and sterols; Preferably, the phospholipids are selected from one or more of dioleoyl phosphatidyl base, egg yolk phosphatidylcholine, phosphocholine or natural phospholipid derivatives; More preferably, the phospholipids are dioleoyl phosphatidyl base; Preferably, the sterols are 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 the 5'-TTCGTT-3' motif or the 5'-TCGTCGTCG-3' motif; Preferably, the CpG oligodeoxynucleotide contains a sequence selected from one of the following or consists of the same: (1) The amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13; (2) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 13; (3) An amino acid sequence having one or more, such as 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 contains or consists of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; More preferably, the CpG oligodeoxynucleotide contains 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 saponins are selected from one or more of quillaia saponins, ginsenosides, platycodin saponins, astragalus saponins, notoginseng saponins, glycyrrhizin saponins, albizia bark saponins, ophiopogon japonicus saponins, bupleurum saponins or japonicum saponins; Preferably, the quillaia saponins are selected from one or more of QS-7, QS-17, QS-18 or QS-21; More preferably, the quillaia saponin is QS-21.

5. The immunoadjuvant composition according to any one of claims 1 to 4, wherein, The weight ratio of the phospholipids to the sterols is 20-4000:50-1000, preferably 20-2000:50-500; Preferably, the weight ratio of the sum of the weights 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 the saponin to the liposome is 0.1-5:10-150, preferably 0.2-5:15-150.

6. The immunoadjuvant composition according to any one of claims 1 to 5, the immunoadjuvant composition being in the form of nanoparticles; preferably, the nanoparticles have a particle size of 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 of them; Preferably, the antigen is selected from one or more of hepatitis A, B, C or E virus antigens, human herpes antigen, human immunodeficiency virus antigen, varicella-zoster virus antigen, human cytomegalovirus antigen, respiratory syncytial virus antigen, human papillomavirus antigen, influenza virus antigen or Mycobacterium tuberculosis antigen; More preferably, the antigen is hepatitis B antigen or herpes antigen; Preferably, the hepatitis B antigen is 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 weights 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 weights of the liposome and the saponin, and the CpG oligodeoxynucleotide is 5-100:0.1-5:0.5-120, preferably 15-50:1:5-20, more preferably 30:1:

10.

9. A method for preparing the immunoadjuvant composition according to any one of claims 1 to 8, comprising the following steps: Dissolve saponin in an aqueous solution, add liposomes, perform a first stirring, and then add CPG oligodeoxynucleotide for a second stirring to obtain the immunoadjuvant composition.

10. The method for preparing the immunoadjuvant composition according to claim 9, wherein,The stirring speeds of the first stirring and the second stirring are each independently 50-150 rpm, preferably 50-100 rpm; Preferably, the aqueous solution contains 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 immunoadjuvant composition according to claim 9 or 10, wherein, The preparation method further includes a step of adding an antigen after the second stirring.

12. An immune kit, which comprises the immunoadjuvant composition according to any one of claims 1 to 8.

13. Use of the immunoadjuvant 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 herpesvirus and / or hepatitis B virus infection or related diseases; preferably, the drug is a vaccine; (2) A kit for diagnosing human herpesvirus and / or hepatitis B virus infection; or (3) An immunogen for developing human herpesvirus and / or hepatitis B virus antibodies; Preferably, the human herpesvirus 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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