CN115120713A - Aluminum hydroxide-CpG oligonucleotide-polypeptide composite adjuvant, vaccine, preparation method and application - Google Patents

Abstract

The invention belongs to the field of vaccine preparation, and in particular relates to an aluminum hydroxide-CpG oligonucleotide-polypeptide composite adjuvant, a vaccine, and a preparation method and application thereof. The technical problem to be solved by the present invention is to provide a novel compound immune adjuvant with good performance. The technical solution to solve the technical problem is to provide a composite immune adjuvant mainly containing aluminum adjuvant, CpG oligonucleotide and polypeptide. The composite immune adjuvant of the invention has the advantages of strong immune activity and high clinical safety, and is an excellent composite immune adjuvant for multiple antigens. Experiments show that various vaccines prepared by the aluminum hydroxide-CpG-polypeptide compound adjuvant of the present invention have higher immune and therapeutic effects, can effectively stimulate the body to produce antigen-specific immune responses, and prolong immune memory. More importantly, the anti-tumor vaccine prepared by the immune adjuvant of the present invention has very good effect, which provides a new choice for the development and application of vaccines in the field.

Description

Aluminum hydroxide-CpG oligonucleotide-polypeptide composite adjuvant, vaccine, preparation method and application

Technical Field

The invention belongs to the field of vaccine preparation, and particularly relates to a composite immunologic adjuvant taking aluminum hydroxide, CpG oligonucleotide and polypeptide as main components, a prepared vaccine, a preparation method and application.

Background

Adjuvants play an important role in the efficacy of vaccines, and are one of the important factors that determine vaccine development. The purpose of adding the adjuvant into the vaccine is to enhance the immunogenicity and immune protection effect of the antigen, which is to enhance the specific humoral immunity and/or cellular immune response of the specific antigen, thereby improving the generation of specific antibodies or/and the specific cellular immune function, reducing the amount of antigen required by effective immunization, reducing the frequency of the need of enhancing immunization, improving the response success rate of early immune response and immune insufficiency, and the like. Currently, vaccine adjuvants commonly used in the market are mainly aluminum adjuvants (the main species is aluminum hydroxide gel main component al (oh)3 or ALPO3, aluminum salt for short), and aluminum hydroxide is the only vaccine adjuvant approved by FDA for clinical use. The main mechanisms by which aluminium hydroxide acts as an adjuvant to elicit an immune response are: after the antigen is wrapped by the gelatinous emulsion, the antigen is slowly released at the injection site of the body, and the half-life period of the antigen in the body is improved, so that the immune response of the vaccine is improved; it mainly induces the Th2 cell of body to act on B cell to induce the humoral immunity of body. An additional advantage of aluminum adjuvants is good safety; however, aluminium adjuvants can induce IgE antibodies produced by the body, such antibodies can induce anaphylactic reactions, and such adjuvants cannot induce Th1 immune reactions, which are involved in eliminating intracellular microbial infections, i.e., killer T lymphocyte reactions (CTL). These all limit the use of aluminium salts as adjuvants.

Therefore, it would be a significant advance to develop a novel adjuvant or adjuvant complex that is effective in enhancing or modulating an antigen-specific immune response for those vaccines that have been successfully developed for use in humans or veterinary medicine. The natural immune system of vertebrates has developed different mechanisms for recognizing antigens based on various specific antigen signaling molecules, including toll-like receptors (TLRs), NODs, and the like. In the acute immune response of the body to eliminate foreign microbial infection, a series of signal transduction based on ligand recognition of TLR receptors plays an important role; moreover, TLR receptors can also induce maturation of DC cells-this is a necessary and most important step in the initiation of adaptive immunity. Among TLR receptor recognition molecules, the most mature is the unmethylated CpG oligonucleotide (CpG oligodeoxynuclotide, CpG ODN, abbreviated as CpG) derived from escherichia coli that recognizes TLR-9. CpG ODN can be combined with TLR9 in a Toll-like receptor family to activate DC cells and induce Th1 type cytokines such as IL-12, IFN-gamma, TNF-alpha and the like; can also activate NK cells, enhance the cytotoxic effect of macrophages and NK cells and promote antigen-specific Th1 type immune response.

However, CpG ODN lacks cell specificity and has a negative charge and is not easily taken up by cells, and the CpG ODN receptor TLR9 is located in cells and has low availability as an adjuvant alone. However, studies have reported that high doses of CpG ODN also cause tissue edema and may cause autoimmune diseases. How to improve the use efficiency and the safety is a great difficulty in the application of the CpG ODN.

Some polypeptides have immunomodulatory functions and direct antibacterial capabilities. These immunoregulatory functions are mainly manifested in that they can induce enhanced expression of cytokines and chemokines, direct or indirect recruitment of granulocytes to the site of infection, stimulation of histamine release from mast cells, Dendritic Cell (DC) maturation and wound healing, and it is difficult to obtain good immunoregulatory polypeptides.

In recent years, the interest of tumor immunotherapy as a new possible way to treat tumors has been increasing, and tumor vaccines for preventing and treating tumors have also become a hot research point for tumor immunotherapy. Among them, the search for specific tumor antigens and effective antigen uptake presentation auxiliary means is one of the key problems in tumor vaccine development. However, because the immunogenicity of tumor antigens is often weak, when used alone, the immune response is not strong enough, and the body can be induced to generate effective anti-tumor immune response with the help of immune adjuvants. However, the conventional adjuvants are not ideal in their effects when used in combination with tumor antigens, and it is necessary to develop a novel antigen adjuvant suitable for tumor immunotherapy.

At present, the development of an immunologic adjuvant which can stimulate more signal paths to achieve the effect of synergistically increasing the immune response and further stimulate stronger specific immune response is urgently needed in the field, so that the immunologic adjuvant is more suitable for preparing tumor vaccines and provides a new effective choice for the research and development of the tumor vaccines in the field.

Disclosure of Invention

The invention aims to solve the technical problem of providing a novel composite immunologic adjuvant with good performance.

The technical scheme for solving the technical problem is to provide a compound immunologic adjuvant. The composite immunologic adjuvant mainly comprises aluminum adjuvant, CpG oligonucleotide and at least one polypeptide selected from SEQ ID No.7, SEQ ID No.8 and SEQ ID No. 10.

Wherein the weight ratio of the main components in the composite immunologic adjuvant is aluminum adjuvant: CpG oligonucleotide: polypeptide: 1-25: 1-4.

Wherein the preferred weight ratio of the main components in the immunological adjuvant is aluminum adjuvant: CpG oligonucleotide: polypeptide: 1-25: 1: 2.

Further, the aluminum adjuvant is aluminum hydroxide gel.

Preferably, the aluminum hydroxide gel is aluminum hydroxide gel particles with the particle size of 2-4 μm. More preferably, the particle size of the aluminum hydroxide gel is 3 μm.

Wherein, the nucleotide sequence of the CpG oligonucleotide in the composite immunologic adjuvant is as follows:

5’-TCGTCGTTTTGTCGTTTTTGTCGTT-3’(SEQ ID No.1)。

wherein, the amino acid sequence of the polypeptide SEQ ID No.7 in the immunologic adjuvant is as follows: VQWRIRVAVIRK (named DP 7); the amino acid sequence of SEQ ID No.8 is: VQLRIRVCVIRK (named DP 8); the amino acid sequence of SEQ ID No.2 is: VQLRCRVCVIRK (named DP 10).

According to another aspect of the invention, the invention also provides the use of the composite immunologic adjuvant in the preparation of vaccines.

Wherein the vaccine is a therapeutic vaccine or a prophylactic vaccine.

Meanwhile, the invention also provides an immune adjuvant-antigen compound formed by adding the antigen into the composite immune adjuvant.

Wherein the weight ratio of the antigen to the composite immunologic adjuvant in the immunologic adjuvant-antigen composite is 1: 1-400 of the weight of the antigen to the weight of the immunoregulation active peptide.

Wherein the weight of the tumor antigen is 1: 1-10 that of the immunoregulatory active peptide, and the weight of the virus antigen is 1: 200-400 that of the immunoregulatory active peptide.

Further, the immunoadjuvant-antigen complex can be prepared into a therapeutic vaccine or a prophylactic vaccine.

Wherein the antigen in the immunoadjuvant-antigen complex is at least one of a tumor antigen, a viral antigen or a bacterial antigen.

Further, the tumor antigen includes a specific protein or polypeptide expressed by a tumor cell. Such as WT1, MUC1, EGFRvIII, HER-2, MAGE-A3, NY-ESO-1, PSMA, GD2 or MART1, or individualized mutant neo-antigen combinations based on patient tumor sequence determination. The viral antigen may be a protein or polypeptide that forms part of the virus, or a specific protein or polypeptide that is expressed in a cell infected with the virus under the control of the viral expression mechanism. Such as EBV, LMP2, HPV E6E 7, adenovirus 5Hexon, HCMV pp65, HBsAg protein and other virus-associated antigens.

The bacterial antigen includes a protein or polypeptide expressed by a bacterium. Such as Pseudomonas aeruginosa antigen, tetanus bacillus antigen, Streptococcus pneumoniae, Salmonella, etc.

On the basis of the technical scheme, the invention also provides a method for preparing the composite immunologic adjuvant. The method comprises the following steps:

a. taking the polypeptide and the CpG oligonucleotide according to the proportion, mixing uniformly, and incubating for 10-20min at room temperature;

b. and adding aluminum hydroxide gel according to the proportion, and uniformly mixing to obtain the composite immunologic adjuvant.

The invention also provides a method for preparing the immune adjuvant-antigen compound.

The method is characterized by comprising the following steps:

a. taking the polypeptide and the CpG oligonucleotide according to the proportion, mixing the polypeptide and the CpG oligonucleotide evenly, and incubating the mixture for 10-20min at room temperature;

b. adding aluminum hydroxide gel according to the proportion, and uniformly mixing to obtain the composite immunologic adjuvant;

c. then adding the antigen according to the proportion, and uniformly mixing to obtain the immune adjuvant-antigen compound.

In the step a of the method, the incubation is generally carried out for about 15min at room temperature. The immunoadjuvant-antigen complex obtained in step c can be used subsequently by filling the required volume with sterile PBS.

The invention has the beneficial effects that: the invention creatively obtains a novel compound immunologic adjuvant formula consisting of aluminum hydroxide/CpG oligonucleotide/polypeptide. The composite immunologic adjuvant has the advantages of strong immunocompetence, high clinical safety and the like, and is an excellent composite immunologic adjuvant aiming at various antigens. Experiments prove that various vaccines prepared by the aluminum hydroxide/CpG oligonucleotide/polypeptide adjuvant have higher immune effect and treatment effect, can effectively stimulate organisms to generate antigen specific immune response, and prolong immunological memory. More importantly, the anti-tumor vaccine prepared by the immune adjuvant has very good effect, and provides a new choice for the development and application of the vaccine in the field.

Drawings

FIG. 1 determination of CpG/immunoregulatory active peptide complex ratio and toxicity detection. Gel retardation experiment lane 1: CpG (5 μ g) + DP7(20 μ g); lane 2: CpG (5 μ g) + DP7(10 μ g); lane 3, CpG (5 μ g) + DP7(5 μ g); lane 4: CpG (5 μ g) + DP7(2.5 μ g); lane 5 CpG (5 μ g) + DP7(1.25 μ g); lane 6, CpG (5 μ g); b-the release of heme from erythrocytes stimulated by the CpG/DP (2-11) complex and the release of LDH from PBMCs stimulated by the CpG/DP (2-11) complex.

FIG. 2 CpG/immunomodulatory active peptide complexes screening. a, after the CpG ODN/defense oligopeptide DP 2-DP 11 compound stimulates the human PBMC, MCP-1 is released for detection. b, detecting the immune response triggered by HbsAg/alum/CpG/DP 2-DP 11. DP7, DP8 and DP10 chemotaxis in vivo, in each group from left to right, physiological saline, DP7, DP8 and DP10, respectively. P < 0.05; p < 0.01; p < 0.001.

FIG. 3 is a graph showing the detection of the CpG/DP7 complex adjuvant in stimulating the production of cytokines IL-1 beta, IFN-gamma, TNF-alpha and IL-10 by human peripheral blood mononuclear cells. P < 0.05; p < 0.01; p < 0.001.

FIG. 4CpG/DP7 activates BMDC activity. CpG/DP7 promotes antigen uptake by BMDCs; cpg/DP7 promotes BMDCs maturation; CpG/DP7 upregulated pERK1/2 and p-p65 expression.

FIG. 5 shows the antitumor effect of tumor vaccine prepared with NY-ESO-1 protein or OVA protein and aluminum hydroxide gel/CpG oligonucleotide// immunoregulatory active peptide mixture. Tumor growth in groups of mice in a melanoma preventative model. Tumor growth in groups of mice in the melanoma therapeutic model. Tumor growth in groups of mice in a prophylactic model of T lymphoma. Tumor growth in groups of mice in therapeutic models of T lymphoma. e, tumor growth of mice in each group in the breast cancer preventive model. And f, tumor growth conditions of mice in each group in the breast cancer therapeutic model. P < 0.05; p < 0.01; p < 0.001.

FIG. 6 shows the detection of humoral immune response elicited by NACD tumor vaccine (prepared from NY-ESO-1 protein and aluminum hydroxide gel/CpG oligonucleotide// immunomodulatory active peptide mixture). and a, detecting the IgG titer of the NY-ESO-1 specific antibody. b, detecting the titer of subtype IgG1 and IgG2c of the NY-ESO-1 specific antibody. P < 0.05; p < 0.01.

FIG. 7 shows the detection of cellular immune response induced by NACD tumor vaccine immunization. a and b flow cytometry detection of IFN-gamma secreting CD4 ⁺ (a) Or CD8 ⁺ T (b) cells. ELISPOT detects T cells secreting IL-4(c) and IFN- γ (d). A, p<0.05；**,p<0.01；***,p<0.001。

FIG. 8 is an immunological memory T cell assay in groups of mice following NACD tumor vaccine immunization.

FIG. 9 shows weights (a) and HE staining of important organs (b) of various groups of mice immunized with the NACD tumor vaccine.

FIG. 10 blood routine analysis of groups of mice after immunization with NACD tumor vaccine. Wherein WBC: number of leukocytes; RBC: the number of red blood cells; PLT: number of platelets; HGB: (ii) hemoglobin; HCT: hematocrit; MPV: mean platelet volume; MCH: mean corpuscular hemoglobin content; MCHC: mean corpuscular hemoglobin concentration; MCV: mean red blood cell volume. FIG. 11. Biochemical analysis of blood of groups of mice after immunization with NACD tumor vaccine. TP: total protein; ALB: albumin; ALT: glutamic-pyruvic transaminase; AST: glutamic-oxalacetic transaminase; ALP: alkaline phosphatase; and (3) CREA: creatinine; UREA: urea; UA: uric acid; GLU: glucose; LDH: a lactate dehydrogenase; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol.

FIG. 12 in vitro RT-PCR detection of transcription factor expression. P < 0.05; p < 0.001.

FIG. 13 is involved in the DP7 activity signaling pathway study. P < 0.05; p < 0.01; p < 0.001.

FIG. 14 GPR35 interaction study with DP 7. Dp7 induces GPR35 receptor internalization; RT-PCR to detect the effect of GPR35 silencing on transcription factor expression; DP7 upregulation of Erk1/2 phosphorylation was dependent on GPR 35. P < 0.05; p < 0.001.

Detailed Description

In order to develop an ideal immunologic adjuvant, the invention researches a plurality of single and compound formulas of the prior immunologic adjuvant in the early period, and accidentally discovers that a specific compound is formed by using aluminum hydroxide, CpG oligonucleotide (CpG ODN, CpG for short) and certain active polypeptide, and when the compound is used as the immunologic adjuvant, the compound is favorable for assisting in exciting the immune reaction of an organism and improving the immune effect of an antigen.

On this basis, a variety of aluminum hydroxide/CpG ODN/polypeptide formulations were further investigated. The invention screens a plurality of immunoregulation active polypeptides to obtain the formula components of the composite immunologic adjuvant which is formed by the immunoregulation active polypeptides and the invention. Meanwhile, in order to obtain better effect, the dosage of the aluminum hydroxide, the CpG ODN and the immunoregulation active peptide with different proportions is screened, and the optimization of the formula among the aluminum hydroxide, the CpG ODN and the polypeptide is completed. And finally, the optimized formula ratio is 1-25: 1-4 of aluminum adjuvant: CpG oligonucleotide: polypeptide. The preferable ratio is aluminum adjuvant: CpG oligonucleotide: polypeptide: 1-25: 1: 2. CpG ODN are used in the art specifically, with or without modification, with or without a thio modification treatment. If the modification is carried out, the stability can be improved, and the modification of the whole chain skeleton is generally carried out.

The invention compares the addition of the aluminum hydroxide gel, the CpG ODN, the immunoregulation active peptide and the like which are used as the adjuvant singly or jointly, and finds that the aluminum hydroxide gel/the CpG ODN/the immunoregulation active peptide are compounded as the adjuvant, so that the organism can be effectively stimulated to generate antigen specific immunoreaction, and the immunological memory can be prolonged.

The three components adopted by the composite adjuvant have good advantages: wherein the aluminum hydroxide gel adjuvant is a kind of Al-containing gel adjuvant ³⁺ The inorganic salt has good adsorption effect and can adsorb soluble antigen on the surface of an alumina gel molecule; is also a good precipitator, can concentrate antigen and reduce injection dosage. The aluminum hydroxide gel has low cost, convenient use and no toxicity, is an adjuvant with the widest application, and is the only adjuvant which is approved by FDA to be used in human vaccines. However, a large number of CpG ODN adjuvant vaccines related clinical studies show that CpG ODN can enhance the humoral immunity and cellular immune response of organisms to pathogens, allergens and tumor antigens. Immunomodulatory active peptides can induce the body to produce cytokines and chemokines, recruit DC cells and monocytes directly or indirectly to the site of infection, promote phagocytosis of antigens by antigen presenting cells, and it canSo as to stimulate the innate immunity of the body and further enhance the acquired immune response.

The composite immune adjuvant can be added with antigen to form immune adjuvant-antigen composite, namely vaccine. The antigen used for preparing the vaccine may be various antigens commonly used in the art. Such as full-length antigens with a proteinaceous component, such as proteins or peptides, may also be antigens that are further modified post-translationally, for example, certain glycoproteins or lipoproteins. The antigen may also be a tumor antigen, including a specific protein or polypeptide expressed by a tumor cell. Such as WT1, MUC1, EGFRvIII, HER-2, MAGE-A3, NY-ESO-1, PSMA, GD2 or MART1, or individualized mutant neo-antigen combinations based on patient tumor sequence determination. The antigen may also be a viral antigen, such as a protein or polypeptide that makes up a viral portion, or a specific protein or polypeptide that is expressed in a cell infected with a virus that is under the control of the viral expression mechanism. Such as EBV, LMP2, HPV E6E 7, adenovirus 5Hexon or HCMV pp 65. The antigen may also be a bacterial antigen, including a protein or polypeptide expressed by a bacterium. Such as Pseudomonas aeruginosa antigen, tetanus bacillus antigen, Streptococcus pneumoniae, Salmonella, etc.

In the process of preparing the vaccine in the development process, the invention finds that the weight of the added antigen and the weight of the polypeptide in the composite immunologic adjuvant are closely related to obtain good vaccine effect. Through a large number of researches, the antigen weight and the weight ratio of the immunoregulation active polypeptide to the immunoregulation active polypeptide of 1: 1-400 are determined to obtain better effects. The tumor antigen weight and the immunoregulation active peptide weight are 1: 1-10, and the virus antigen weight and the immunoregulation active peptide weight are 1: 200-400, which are preferred. Alternatively, the ratio may be selected within the above range according to the self-characteristics of the antigen and other specific conditions. The verification is carried out on the hepatitis antigen HBsAg and the tumor antigen NY-ESO-1, and the result shows that the prepared vaccine has good effect. In the hepatitis vaccine model, the body of the immunized mouse generates high-titer HBsAg specific antibody; in a tumor vaccine model, the vaccine can stimulate the specific humoral immunity and cellular immune response of the antigen of an organism and further can effectively inhibit the growth of tumors.

Based on the above-disclosed technical schemes, such as the idea of the present invention, the ratio range of the aluminum hydroxide/CpG ODN/polypeptide composite adjuvant and the ratio range of the aluminum hydroxide/CpG ODN/polypeptide composite adjuvant with the antigen, it is obvious to those skilled in the art that the obtained formula range can be further optimized according to different specific antigens. It is not excluded, where necessary, to add other adjuvant ingredients acceptable in vaccine preparation to the basic formulation above for the purpose of specific dosage form requirements. It is clear that these embodiments are within the scope of the invention.

The process of the present invention is further illustrated below with reference to examples.

The main reagents used in the examples were:

aluminum hydroxide adjuvant (Alhydrogel) was purchased from brentag Biosector, denmark.

HBsAg was obtained from ARP Products, USA.

CpG ODN (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3', SEQ ID No.1) was purchased from invitrogen.

Horse radish peroxidase-labeled goat anti-mouse IgG and subtype detection kit were purchased from southern Biotech.

C57BL/6, Balb/C mice were purchased from Wintolite, Beijing.

MCP-1 cytokine detection kit is purchased from R & D.

The cytokine chip assay kit HCYTOMAG-60K was purchased from Millipox corporation.

Goat anti-mouse, goat anti-rabbit secondary antibodies and related immunohistochemical kits were obtained from Abcam corporation, antibodies anti-CD4, anti-CD8, anti-CD56, F4/80, Gr1, CD11b, and the like.

The polypeptides DP 2-DP 11 used were all synthesized by Shanghai peptide biology, Inc., and the specific sequences are shown in Table 1.

Other reagents are imported or domestic analytical pure products.

EXAMPLE A preliminary assay for CpG/active polypeptide ratios and toxicity

First, CpG/immune regulation active peptide ratio determination

CpG is mixed with alternative active polypeptide (see Table I) in the ratio of 4:1(wt/wt), 2:1(wt/wt), 1:2(wt/wt) and 1:4(wt/wt), respectively, and the mixture is kept still for 10-15min at 37 ℃, wherein the CpG amount is 5 mug. To each CpG and immunomodulatory peptide mixture was added 10. mu.l of 6 × loading buffer and mixed. 1% agarose gels were prepared, 10. mu.l of each sample was spotted and electrophoresed using a 1% agarose gel at 90V for 20min and imaged using a gel imaging system.

Table i amino acid sequences of active polypeptides used in the examples

Numbering	Amino acid sequence
		DP2	VQWRIRVCVIRA(SEQ ID No.2)
DP3	VQWRIRIAVIRA(SEQ ID No.3)
		DP4	VCWRIRVAVIRA(SEQ ID No.4)
DP5	VQLRIRVCVIRR(SEQ ID No.5)
		DP6	KQWRIRVAVIRA(SEQ ID No.6)
DP7	VQWRIRVAVIRK(SEQ ID No.7)
		DP8	VQLRIRVCVIRK(SEQ ID No.8)
DP9	KQWRIRVCVIRA(SEQ ID No.9)
		DP10	VQLRCRVCVIRK(SEQ ID No.10)
DP11	VQWRIRIAVIRK(SEQ ID No.11)

The results show (fig. 1 a): with the increase of defense short peptides, the content of free CpG ODN is less, and when the mixing ratio of the CpG ODN and the candidate polypeptide is 1:2(wt/wt), the CpG ODN and the candidate polypeptide can completely form a stable complex.

Second, toxicity test of the Complex as an adjuvant

1. LDH Release test

Human peripheral blood PBMC lymphocytes were isolated and PBMC were diluted to 1X 10 with RPMI1640+ 5% FBS medium ⁷ cells/ml, 500. mu.l/well in 24-well plates, 37 ℃ and 5% CO2 incubator for 1 hour. 10 μ g of CpG and immunomodulatory active peptide were prepared in a ratio of 4:1(wt/wt), 2:1(wt/wt), 1:2(wt/wt) and 1:4(wt/wt), respectively, and allowed to stand at room temperature for 10-15 minutes, after which the CpG/immunomodulatory active peptide mixture was added to the upper lymphocyte culture medium and incubated at 37 ℃ with 5% CO2 for 24 hours. The supernatant was collected, 250g, and centrifuged at 4 ℃ for 5 min. Positive, negative and blank controls were set according to the LDH kit instructions and their LDH values were determined.

2. Erythrocyte Heme Release assay

Peripheral red blood cells were collected and washed 3 times with 0.85% physiological saline. Erythrocytes were diluted 4-fold with physiological saline, 100. mu.l/well plated in 96-well plates, and 50. mu.l of different concentrations of CpG/immunomodulatory active peptide were added, and the final working concentrations of CpG and immunomodulatory peptide were CpG 20. mu.g/ml, immunomodulatory peptide 40. mu.g/ml, incubated overnight at 37 ℃ with 5% CO2, using Triton-100 (1%) as a positive control and physiological saline as a negative control. Centrifugation is carried out at 4000rpm for 10min, the supernatant is collected, and the toxicity of the adjuvant to the erythrocytes is calculated by using the readings of an enzyme-labeling instrument A450.

The experimental results show (fig. 1 b): the CpG/immunomodulatory active peptide mixture has no detectable level of toxic effect on both peripheral blood mononuclear cells and erythrocytes.

EXAMPLE two CpG/immunomodulatory active peptide complexes screening

First, CpG/immune regulation active peptide compound stimulates human PBMC to secrete MCP-1 detection

Separating human peripheral mononuclear lymphocytes (PBMC) with lymphocyte secretion, and diluting the separated lymphocytes into 1 × 10 in 1640 complete medium ⁶ cells/ml, 24-well plates, 500. mu.l/well, 37 ℃ C., 5% CO ₂ Incubate for 1 h. Adding adjuvant or adjuvant complex CpG, DP2-11, and CpG-DP2-11 dropwise into lymphocyte culture solution at 37 deg.C and 5% CO ₂ Incubation for 24h, where CpG: 20. mu.g/ml, DP 2-11: 40. mu.g/ml. 16000g, centrifuged at 4 ℃ for 5min, the supernatant was collected and MCP-1 release was detected using an ELISA kit.

The experimental results show (fig. 2 a): MCP-1 is a monocyte chemotactic factor capable of chemotactic monocytes/macrophages, T cells, NK cells and neutrophils. After PBMC is stimulated by the double composite adjuvants of CpG/DP7, CpG/DP8 and CpG/DP10, the secretion amount of MCP-1 is 4572pg/ml, 4826pg/ml and 3534pg/ml respectively. Compared with other groups, the three groups secreted MCP-1 in a higher amount than other groups (P < 0.05). The synergy coefficient of CpG and DP7, DP8 and DP10 is more than 2, and the CpG, DP7, DP8 and DP10 can synergistically promote MCP-1 secretion of PBMC.

Second, detection of immune response elicited by HbsAg/aluminum hydroxide gel/CpG/DP 2-11

Protein vaccines were prepared according to the following protocol, with a total dose of 100. mu.l per mouse supplemented with less than 100. mu.l of PBS. Each group of 10 mice was dosed as follows:

(ii) NS group of 100. mu.l PBS

② alum group 0.1. mu.g HBsAg + 25. mu.g alum

③alum/CpG:0.1μg HBsAg+25μg alum+20μg CpG

alum/DP 2-11 groups, 0.1 μ g HBsAg +25 μ g alum +40 μ g DP 2-DP 11

0.1 μ g HBsAg +25 μ g alum +20 μ g CpG +40 μ g DP 2-DP 11 of the group of (alum/CpG/DP 2-11)

The preparation scheme of the vaccine is as follows:

a. adding any one of the needed polypeptides DP 2-DP 11 and CpG ODN, mixing uniformly, and incubating for 15min at 37 ℃;

b. adding desired aluminum hydroxide gel (Alhydrogel, referred to as alum in the embodiment) of Brenntag Biosector, Denmark, mixing, and incubating at 37 deg.C for 10 min;

c. finally 0.1. mu.g of HBsAg is added and made up to a volume of 100. mu.l with sterile PBS.

Mice were immunized using a prophylactic immunization protocol three times at weeks 0, 2, and 4, and blood was taken at week 5 to detect HbsAg-specific antibodies.

The results show (FIG. 2b) that at week 5 of immunization, the median anti-HBsAg total antibody titer of the alum/CpG group was 64000, whereas the median anti-HBsAg total antibody titer of the alum/CpG/DP7, alum/CpG/DP8 and alum/CpG/DP10 groups was 512000, with statistical differences compared to the alum/CpG group.

Tri, DP7, DP8, and DP10 recruit lymphocytes in vivo

The C57BL/6 mice were injected intraperitoneally with 200. mu.g of polypeptides DP7, DP8 and DP10, respectively, and the control group was injected with the same volume of sterile saline. 24h after injection, mice were harvested for peritoneal lavage and flow analyzed for chemotactic lymphocytes. F4/80 ⁺ CD11b ⁺ Double positive is macrophage, F4/80 ⁺ Gr1 ⁺ Double positive is a monocyte, F4/80 ^- Gr1 ⁺ The marker was neutrophil.

The flow results show (fig. 2c) that the proportion of mononuclear cells in the total cells of the peritoneal lavage fluid of the DP7 and DP8 injected groups was significantly increased, and the proportion of mononuclear cells in the total cells of the peritoneal lavage fluid of the DP10 injected group was also increased, compared to the control group (saline, sterile saline). The proportion of the neutrophils in the total cells of the peritoneal lavage fluid in the DP7 injection group is obviously increased, and the proportion of the neutrophils in the total cells of the peritoneal lavage fluid in the DP8 and DP10 injection groups is also increased. The proportion of macrophages in the total cells of the peritoneal lavage fluid in the DP8 injection group is obviously increased, and the proportion of macrophages in the total cells of the peritoneal lavage fluid in the DP7 and DP10 injection groups is also increased. It was shown that DP7, DP8 and DP10 have immunomodulatory effects and are capable of recruiting lymphocytes in vivo.

Example three CpG/immunomodulatory active peptide complexes promote human peripheral blood mononuclear cytokine production

Human peripheral blood mononuclear cells were isolated as described above. PBMC were diluted to 4X 10 with 1640+ 10% FBS ⁶ A48-well plate was placed in 125. mu.l/well. 125. mu.l of 2 Xadjuvant stimulator stock (DP7, CpG/DP7) was added, mixed well and incubated in a 37 ℃ incubator for 48 h. Wherein the stimulation concentration of the defense oligopeptide is 40 mug/ml, and the stimulation concentration of CpG is 20 mug/ml. PHA (3 μ g/ml) stimulation was used as a positive control, medium alone as a blank control, and unstimulated human PBMC medium supernatant as a negative control. Centrifuging at 250g for 5min, collecting culture medium supernatant, and detecting with cytokine chip detection kit (IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(P70), IL-17A, TNF-alpha and IFN-gamma).

As a result, compared with a single adjuvant (figure 3), the double compound adjuvant obviously promotes the secretion of immune positive regulation cytokines IL-1 beta, TNF-alpha and IFN-gamma. Cytokine secretion in the supernatant of the CpG/DP7 group media was as follows: the secretion amount of IFN-gamma after stimulation by the double adjuvant is 542 pg/ml; the IL-1 beta secretion amount is 8045 pg/ml; the secreted TNF-alpha is: 6704 pg/ml; the IL-10 secretion was 6260 pg/ml. Compared with DP7 and CpG groups, the dual compound adjuvant CpG/DP7 can obviously promote the secretion of immune positive regulation cell factors IL-1 beta, TNF-alpha and IFN-gamma and inhibit the secretion of immune negative regulation cell factors IL-10. Example four CpG/immunomodulatory active peptide complexes activate BMDCs Activity

CpG/immune-modulating active peptide complexes to promote antigen uptake

1. Isolation and culture of BMDCs cells

The mice are killed by neck fracture in 6-8 weeks, and bone marrow cells of tibia and fibula are taken; the medium containing bone marrow cells was collected in 50ml BD tubes, centrifuged at 1500rpm for 3min, resuspended in erythrocyte lysate (1:10), allowed to stand at room temperature for 5min, centrifuged at 1500rpm for 3min, and resuspended in RPMI1640+ FBS. Cells were filtered through a 70 μm cell screen, debris removed, and cultured with fresh medium and 10ng/ml GM CSF and 10ng/ml IL 4 added, replacing medium half every other day. Subsequent experiments were performed on day 7 of culture.

2. Alexa using protein red fluorescence labeling kit

594Microscale Protein Labeling Kit labeled NY ESO1 Protein with red fluorescence. In vitro fluorescence labeled NY ESO1 mixed with adjuvant or adjuvant complex DP7, CpG/DP7) respectively, and incubated at room temperature for 10min, wherein NY ESO 1:10 μ g/ml, CpG:20 μ g/ml, DP 740 μ g/ml). The adjuvant or adjuvant complex was added to BMDCs cell cultures and mixed well and incubated for 1 h. Washed with PBS 3 times, and fixed with 4% paraformaldehyde in dark at room temperature for 10 min. After 3 washes with PBS, DAPI was stained for 1 min. After 3 washes with PBS, the slides were quenched and observed under a confocal microscope.

The results show (FIG. 4a), the mean fluorescence intensity of the red fluorescence of BMDC cells in CpG/DP7 group is significantly higher than that of other control groups, and it can be seen that CpG/DP7 adjuvant mixture group can promote the intake of NY-ESO-1 antigen by BMDC cells.

Di, CpG/immune-modulating active peptide compound for promoting BMDCs maturation

The adjuvant or adjuvant complex was mixed in vitro with DP7, CpG/DP7(CpG: 20. mu.g/ml, DP 740. mu.g/ml). Adding BMDCs cell culture, mixing evenly, and incubating for 16 h. The antibodies APC-anti-Mouse CD11c, FITC-anti-Mouse-CD40, PerCP-Cy5.5-anti-Mouse-CD8 or FITC-anti-Mouse-CD86 stained BMDCs prior to detection by flow cytometry.

The results show (fig. 4b, 4c and 4d) that CpG/DP7 significantly increased the expression of CD40, CD80 and CD86 compared to Control, DP7 and CpG, indicating that the CpG/DP7 adjuvant mixture group can promote the maturation of BMDC cells.

Thirdly, CpG/immune-modulating active peptide compound promotes the expression of pERK1/2 and p-p65

The adjuvant or adjuvant complex was mixed in vitro with DP7, CpG/DP7(CpG: 20. mu.g/ml, DP 740. mu.g/ml). Adding into BMDCs cell culture, mixing, and incubating for 30 or 60 min. Cell samples were collected, subjected to 12% SDS-PAGE to separate proteins in the samples and transferred to PVDF membrane. After blocking for 1h at room temperature, primary antibody incubation: PVDF membranes were incubated overnight at 4 ℃ with shaking in a medium containing anti-NF-. kappa. B p65, Phospho-NF-. kappa. B p65, Erk1/2 and Phospho-Erk1/2 antibodies (dilution ratio 1:2000), respectively. The next day after incubation for 1h with the corresponding secondary antibody, exposure was performed.

The results show (FIG. 4e) that CpG/DP7 significantly increased the expression of pERK1/2 and p-p65 compared to Control, DP7, CpG.

EXAMPLE V study of antitumor Effect of the present invention Using aluminum hydroxide gel/CpG/active Polypeptides as Complex adjuvant for tumor vaccine

First, preventive immune model

(1) Preparation of NY-ESO-1 tumor protein vaccine

Protein vaccines were prepared according to the following protocol, with a total dose of 100. mu.l per mouse supplemented with less than 100. mu.l of PBS. 10 mice per group were dosed as follows:

1. NS group of 100. mu.l PBS

2. The alum group comprises 5 mu g NY-ESO-1 or 10 mu g OVA +125 mu g alum

3. Alum + CpG group 5 μ g NY-ESO-1 or 10 μ g OVA +125 μ g alum +20 μ g CpG

4. The alum + DP7 group is 5 μ g NY-ESO-1 or 10 μ g OVA +125 μ g alum +40 μ g DP7

5. Group alum + CpG + DP7 5. mu.g NY-ESO-1 or 10. mu.g OVA + 125. mu.g alum + 20. mu.g CpG + 40. mu.g DP7

The preparation scheme of the vaccine is as follows:

a. adding the required DP7 and CpG ODN, mixing uniformly, and incubating for 15min at 37 ℃;

b. adding the required aluminum hydroxide gel, mixing uniformly, and incubating for 10min at 37 ℃;

c. finally 5. mu.g NY-ESO-1 protein or 10. mu.g OVA were added and the volume was made up to 100. mu.l with sterile PBS.

(2) Immunization protocols

Female mice (C57 BL/6J mice selected by a B16-NY-ESO-1 model and Balb/C mice selected by a 4T1-NY-ESO-1 model) of about 18g, which are 5-7 weeks old, are selected, and are randomly grouped after being fed in an animal room of the laboratory for one week. 0. Multiple subcutaneous immunizations were performed at 2, 4 weeks, and tumor cells were subcutaneously inoculated into the back of each mouse at 5 weeks (B16-NY-ESO-1: 2X 10) ⁵ ；EG7.OVA:2×10 ⁶ ；4T1-NY-ESO-1:2×10 ⁵ ) Tumors were measured once every 3 days after they grew out and observed for survival. The tumor volume is calculated by the formula of 0.52X length X width ² 。

The experimental results show (fig. 5 a): in the B16 melanoma model, tumor growth was somewhat inhibited in mice of the alum group, the alum/DP7 group, and the alum/CpG group. The mean tumor volume was 955. + -. 298mm on day 22 after tumor inoculation ³ 、906±623mm ³ And 642 +/-236 mm ³ . The growth of the tumor of the mice in the alum/CpG/DP7 group is obviously inhibited, and the average tumor volume is 224 +/-126 mm ³ There were significant statistical differences compared to the NS group (p < 0.05). The tumor suppression rates were 62.6%, 64.5% and 74.9% for the alum group, the alum/DP7 group and the alum/CpG group, respectively, at day 22 after tumor inoculation. While the swelling inhibition rate of the alum/CpG/DP7 group was 91.2%.

Tumor growth was also inhibited in mice of the alum group, the alum/DP7 group and the alum/CpG group in the T lymphoma model (EG7.OVA) (FIG. 5 c). The mean tumor volume was 3725 + -478 mm on day 26 after tumor inoculation ³ 、2180±454mm ³ And 2446 + -576 mm ³ . The growth of the tumor of the mice in the alum/CpG/DP7 group is obviously inhibited, and the average tumor volume is 1232 +/-543 mm ³ There was a significant statistical difference (p <0.05) compared to the NS and alum groups. On day 26 after tumor inoculation, the tumor suppression rate of the alum/CpG/DP7 group was 74.9%.

In the breast cancer model (fig. 5e), mice began to appear frizzled, poorly conditioned 28 days after tumor inoculation, so mice were sacrificed at this time point. The experimental result shows that the NS group mice have rapid tumor growth, and the average tumor volume can reach 1382 +/-86 mm ³ . The mean tumor volumes of mice in the alum group, the alum/DP7 group, the alum/CpG group and the alum/CpG/DP7 group were 1232 + -87 mm, respectively ³ 、1116±91mm ³ 、1104±65mm ³ And 755. + -. 78mm ³ . It can be seen that the tumor growth was significantly inhibited in mice of the alum/CpG/DP7 group, with a significant statistical difference compared to the other control groups (p < 0.05). On day 28 after tumor inoculation, the tumor suppression rate of the alum/CpG/DP7 group was 45.4%.

Second, therapeutic immune model

The grouping and mouse administration dose was the same as the prophylactic immunization dose. The immunization protocol was as follows: each mouse was inoculated subcutaneously into the back of the mouse on day 0 with tumor cells (B16-NY-ESO-1: 2X 10) ⁵ ；EG7.OVA:2×10 ⁶ ；4T1-NY-ESO-1:2×10 ⁵ ) The volume of the tumor to be treated is about 50mm ³ In this case, the immunization was performed 1 time per week for 3 times.

In the melanoma model (FIG. 5b), the mean tumor volumes of mice in the NS, alum/DP7, alum/CpG and alum/CpG/DP7 groups were 2081. + -. 201mm, respectively, at day 24 post-tumor inoculation ³ 、2447±692mm ³ 、1507±539mm ³ 、2144±707mm ³ And 1111 + -201 mm ³ . Tumor growth was significantly slowed in the alum/CpG/DP7 group mice compared to the NS group (p < 0.05).

In the T lymphoma model (FIG. 5d), the average tumor volumes of mice in NS, alum/DP7, alum/CpG and alum/CpG/DP7 groups were 2913. + -. 407mm, respectively, at day 28 post-tumor inoculation ³ 、2880±402mm ³ 、2333±722mm ³ 、2677±614mm ³ And 1073. + -. 284mm ³ . Tumor growth was significantly slowed in the alum/CpG/DP7 group mice compared to the NS group (p < 0.05).

In the breast cancer model (FIG. 5f), the average tumor volumes of mice of NS, alum group, alum/DP7 group, alum/CpG group and alum/CpG/DP7 group were 1727 + -97 mm, respectively, at day 32 post-tumor inoculation (FIG. 5f) ³ 、1703±107mm ³ 、1703±192mm ³ 、1646±215mm ³ And 1071. + -. 244mm ³ . Tumor growth was significantly slowed in mice of the alum/CpG/DP7 group compared to the other 4 groups (p < 0.05). .

Test example detection of immune response elicited by six tumor vaccine NY-ESO-1/alum/CpG/DP7

First, detection of humoral immune response

The immune response elicited by NACD was tested in the preventive model in example four.

1. Elisa detection of NY-ESO-1 specific antibody

Detecting NY-ESO-1 specific antibodies and antibody subtypes by an ELISA method. The detection method comprises the following steps: the NY-ESO-1 protein was diluted to 1. mu.g/ml with a coating buffer (0.05M carbonate buffer, pH9.6), and 100. mu.l was added to each well of a 96-well plate overnight at 4 ℃. PBST (PBS + 0.5% Tween20) was washed 3 times. 5% skimmed milk powder was blocked at 37 ℃ for 1 hour. PBST was washed 5 times. Serum was diluted in a gradient and 100. mu.l was added to each well and incubated at 37 ℃ for 1 hour. PBST wash plate 5 times. Thereafter, 100. mu.L of HRP-labeled goat anti-mouse IgG (1:4000 dilution) was added to each well, and incubated at 37 ℃ for 1 hour. PBST was washed 5 times. To each reaction well, 100. mu.l of a temporarily prepared TMB solution (KPL Co.) was added, and after development at room temperature for 20 minutes, 0.5MH was added ₂ SO ₄ The reaction was stopped at 100. mu.l and read at 450nm wavelength.

The experimental results showed (fig. 6a) that the median values of IgG antibody titers of the alum group, alum/DP7 group, alum/CpG group and alum/CpG/DP7 group were 640000, 960000, 640000 and 1280000, respectively. The NY-ESO-1 specific antibody in the serum of the mice in the alum/CpG/DP7 group is obviously higher than that of other groups, and has statistical difference with the alum group (p is less than 0.05). Thus, the alum/CpG/DP7 composite adjuvant can obviously enhance the specific humoral immune response of NY-ESO-1.

2. Detection of antibody subtypes

The mixed serum is diluted by 10 times from 1:100, the diluted serum is used as primary antibody for Elisa detection, the appropriate initial dilution ratio is selected to determine the antibody subtype range, 8 gradients are diluted by 2 times as the primary antibody, and different antibody subtype specific antibodies are respectively used as secondary antibodies (IgG1, IgG2 c; diluted by 1: 4000), and the subtype titer is determined according to the ELISA scheme.

We tested subtypes of NY-ESO-1 specific antibodies (IgG1 and IgG2 c). By calculation (FIG. 6b), the IgG1/IgG2a ratios of the alum group, the alum/DP7 group, the alum/CpG group, and the alum/CpG/DP7 group were: 21.3, 6, 25.6 and 2. Compared with the alum/CpG group, the serum of mice in the alum/CpG/DP7 group has obviously increased NY-ESO-1 specific IgG2c titer and decreased IgG 1.

Second, cellular immune response detection

The cellular immune response elicited by NACD was tested in the preventive model in example four.

1. IFN-gamma intracellular staining

Spleen lymphocytes were isolated, counted in a resuspension, 3X 10 ⁶ Individual splenic lymphocytes/well were plated in 24-well plates. 10. mu.g/ml NY-ESO-1 (DMSO for negative control, conA 5. mu.g/ml for positive control) was added and stimulated at 37 ℃ for 1h followed by incubation with Golgiplug (1. mu.l added to 1ml cell culture) for 6-12 h. The cells were then harvested and blocked with CD16/CD 324 ℃ for 15 min. After washing cells with the stabilizing buffer, the cells were resuspended, FITC-anti-mouse-CD3 ε, PE-Cy7-anti-mouse CD8 α or PE-Cy7-anti-mouse CD4 was added, and incubated at 4 ℃ for 30min in the absence of light. Subsequently using stating buffer washing 2 times, adding 250 u l of the fusion/permability vortex fully suspended, room temperature and light from incubation fixed cells for 20 min. After the cells are washed by BD Perm/wash buffer, the cells are resuspended, intracellular antigen antibody (PE-anti-mouse IFN-gamma) is added, and the cells are incubated for 30min at room temperature in the dark, and after being washed for 2 times by PBS, the flow analysis can be carried out.

The results showed (FIGS. 7a and 7b) IFN-. gamma.secreting CD4 in spleen lymphocytes from mice of the alum group, the alum/DP7 group and the alum/CpG group ⁺ CD3 occupancy by T cells ⁺ The T cell ratios are 0.44%, 0.36% and 0.47%, respectively, while IFN-gamma-secreting CD4 in spleen lymphocytes of mice in the alum/CpG/DP7 group ⁺ CD3 occupancy by T cells ⁺ The T cell proportion can reach 0.88%, compared with NS and alum/CpG groups, the statistical difference (p)<0.05). For IFN-gamma-secreting CD8 ⁺ Analysis of T cells found: the double positive cells of the alum group and the alum/DP7 group account for CD3 ⁺ The proportion of T cells is 0.52 percent and 0.79 percent respectively, and the double positive cells of the alum/CpG group and the alum/CpG/DP7 group account for CD3 ⁺ The proportion of T cells was 0.88% and 1.28%, respectively, which was significantly higher than that of each of the other control groups, and was statistically different (p) from NS<0.05). Thus, the IFN-gamma-secreting CD4 cells in spleen lymphocytes of mice in the alum/CpG/DP7 group are shown ⁺ T cells and CD8 ⁺ T cells were all significantly increased.

2. ELISPOT detection

5×10 ⁵ Individual spleen lymphocytes/well were plated into 96-well plates precoated with IFN-. gamma.or IL-4, 10. mu.g/ml NY-ESO-1 was added, incubated at 37 ℃ for 24h, and positive spots were detected as per the instructions.

The results showed (FIGS. 7c and 7d) that the positive cell spots secreting IFN-. gamma.in spleen lymphocytes of mice in NS, alum/DP7 and alum/CpG groups were 31, 117, 252 and 133, respectively, while the positive cell spot secreting IFN-. gamma.in spleen lymphocytes of mice in alum/CpG/DP7 group was 379, which is statistically different from the other groups (p < 0.05); while the spots of IL-4-secreting positive cells in spleen lymphocytes of mice in NS group, alum/DP7 group and alum/CpG group were 0.3, 41, 65 and 33, respectively, while the spot of IL-4-secreting positive cells in spleen lymphocytes of mice in alum/CpG/DP7 group was 116, which was statistically different from the other groups (p <0.05)

Third, detection of immunological memory T cells

Using the prophylactic immunization protocol of the prophylactic model in example four, 2 weeks after 3 immunizations, mouse spleen lymphocytes were isolated, labeled with fluorescent antibodies CD44 and CD62L, and the status of effector and central memory T cells after vaccine immunization was examined by flow.

The results show (fig. 8): CD4 in spleen cells of alum/CpG/DP7 group mice ⁺ CD44 ⁺ CD62L ^- And CD8 ⁺ CD44 ⁺ CD62L ^- The proportion of T lymphocytes is 23.56% and 28.15%, respectively, which are higher than that of other groups (p)<0.05). And CD4 ⁺ CD44 ⁺ CD62L ⁺ And CD8 ⁺ CD44 ⁺ CD62L ⁺ T lymphocytes did not change significantly, and this result may be related to the time point we chose. Effective memory CD4 in spleen cells of alum/CpG/DP7 group of mice 2 weeks after 3 immunizations ⁺ And CD8 ⁺ T lymphocytes are increased obviously. As can be seen, the NY-ESO-1/alum/CpG/DP7 vaccine can obviously prolong the immunological memory after being immunized.

Test example seven vaccines NY-ESO-1/alum/CpG/DP7 safety preliminary evaluation

First, body weight detection and H & E staining

(1) Groups were divided according to the prophylactic immunization protocol of example four, and groups of mice were immunized using the prophylactic immunization protocol.

(2) The weight of the mice was weighed throughout the experiment and observed for abnormalities in the state and daily behavior of the mice.

(3) After 3 times of immunization, important organs such as heart, liver, spleen, lung and kidney of the mice are taken for H & E staining.

In the experiment, whether the vaccine can generate toxic or side effect on organisms or not is detected. In the safety detection test of the vaccine, a preventive immunization scheme is adopted to immunize mice, the mice are killed two weeks after immunization, and vital organs such as heart, liver, spleen, lung and kidney are taken for HE detection. In the whole experiment process, the mice have no phenomena such as weight loss, piloerection, appetite reduction, behavior abnormality and the like, and the important organ HE staining detection has no obvious abnormality (figure 9a and 9 b).

Second, routine blood detection

(1) Groups of mice were immunized using a prophylactic immunization protocol.

(2) After 1 week of 3 rd immunization, orbital blood from mice was taken for blood routine analysis.

In the experiment, whether the vaccine can generate toxic or side effect on organisms or not is detected. In the safety detection test of the vaccine, a preventive immunization scheme is adopted for immunizing the mice, and after 1 week after 3 rd immunization, the mice are subjected to blood detection routine. The red blood cell number, platelet number, hemoglobin, hematocrit, average platelet volume, average red blood cell hemoglobin content, average red blood cell hemoglobin concentration and average red blood cell volume of the five groups of mice were all within the normal range (fig. 10).

Biochemical detection of blood

(1) Groups of mice were immunized using a prophylactic immunization protocol.

In the experiment, whether the vaccine can generate toxic or side effect on organisms or not is detected. In the safety detection test of the vaccine, a preventive immunization scheme is adopted to immunize the mouse, and the blood of the mouse is biochemically detected after immunization. Total protein of five groups of mice; albumin, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, alkaline phosphatase, creatinine, urea, uric acid, glucose, lactate dehydrogenase, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol were all within normal range, and there was no difference in blood biochemistry between five groups of mice (fig. 11).

Experimental example eight DP7 activates Gi/PKC or PI3k/Erk1/2 signal pathway

1. RNA-seq analysis of DP7 Effect on transcription of BMDCs

On the fifth day of induction of BMDCs with GM-CSF and IL-4, cells were harvested and sorted with CD11c magnetic beads. After stimulating sorted BMDCs for 30min with 40. mu.g/ml DP7 (control in equal amounts of DMSO), cells were harvested, total RNA extracted, and second generation high-throughput sequencing was performed.

The results showed that bioinformatics analysis showed a total detection of 22954 genes in the Control and DP7 treatment groups. Compared with the control group, the DP 7-treated group has 118 genes with differential expression (expression fold is more than or equal to 2 times). With online String: protein-protein interaction network software protein interaction network analysis was performed on 118 genes, and was shown to be divided into two major networks. The most important network is further shown in the whole interaction network, namely the transcription factor interaction network taking Egr1 as the core, wherein the network has 11 proteins: zfp36, Btg2, Atf3, Dusp1, JunB, Ier2, Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a 1. We picked 6 transcription factors with larger change fold and more important in the network for further research, including Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1, with the upper modulation fold being 16.63, 2.40, 15.70, 6.34, 114.79 and 19.81, and these 6 transcription factors are also genes with larger change in RNA-seq analysis.

2. DP7 upregulated expression of Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1

JAWSII cells were divided into 6-well plates, 2 × 10 per well ⁵ And (4) cells. Cells were stimulated with 40. mu.g/ml DP7 for 30min, and controls with equal amounts of DMSO. Removing the supernatant culture solution, adding 1ml of Trizol reagent into each hole, extracting the total RNA of the sample and performing reverse transcription to obtain cDNA, and performing quantitative detection on the target gene by an RT-PCR method.

The results show (FIG. 12), considering the expression level of transcription factor in Control as 1, it can be seen that Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1 are up-regulated by 22.05, 2.79, 15.36, 19.51, 143.80 and 5.63, respectively, and basically match the results of RNA-seq, indicating that the up-regulated 6 transcription factors play an important role in DP7 action.

3. DP7 activates the Gi/PKC or PI3k/Erk1/2 signaling pathways

JAWSII cells were divided into 6-well plates, 2 × 10 per well ⁵ (ii) individual cells; cells were stimulated with 40. mu.g/ml DP7 for 30min after pre-treatment with small molecule inhibitors at 37 ℃ with control groups stimulated with equal amounts of DMSO. And (3) carrying out quantitative detection on the target gene by an RT-PCR method. The inhibitors and their receptors used were: the small molecule inhibitors of Erk1/2 are Selumetinib, Pertussis Toxin (PTX) is a specific inhibitor of Gi-like proteins, AS-605240 is a selective inhibitor of PI3K gamma, and Sotrastaurin is an inhibitor of PKC θ, respectively.

The results show (FIG. 13a) that DP7 up-regulated the expression of Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1 statistically significantly (p <0.05) after pre-treatment of cells with small molecule inhibitors.

4. DP7 activation Erk1/2 phosphorylation is inhibited by PTX

Divide BMDCs into 6-well plates, 5 × 10 per well ⁵ And (4) cells. After 3h of PTX pre-treatment of the cells, the cells were stimulated with 40. mu.g/ml DP7 for 30min, and the control group was stimulated with the same amount of DMSO. The supernatant medium was removed and 100. mu.l of RIPA lysate was added to each well and the cells were lysed thoroughly on ice for 30 min. After collection of cell lysates, the cells were centrifuged at 13000rpm, 10min and 4 ℃. Western blots were used to detect the expression of Erk1/2 and p-Erk 1/2.

From the results (FIG. 13b), DP7 showed a marked up-regulation of Erk1/2 phosphorylation 30min after exposure to cells, while Erk1/2 phosphorylation decreased 60min after exposure. Similarly, the upregulation of Erk1/2 phosphorylation by DP7 was reduced to varying degrees after PTX pretreatment.

5. DP7 is capable of upregulating intracellular levels of Ga2 +.

Collecting JAWSII cells at 1 × 10 per well ⁴ One was plated on a black 96-well plate for two days. The supernatant medium was discarded and the cells were washed once with 100. mu.l Hank' buffer. Mu.l of Fluo 3-AM (diluted in Hank' buffer) at a final concentration of 5. mu.M was added to each well and incubated at 37 ℃ for 30 min. Remove supernatant, 100. mu.l Hank' buffer washes the cells once more. Fluorescence was measured by ceigo after stimulation with 200. mu.l of medium or medium containing 40. mu.g/ml DP7 per well.

From the results (FIG. 13c), the mean fluorescence intensity of the individual cells was significantly increased (p <0.05vs Control) after DP7 stimulated the cells for 15min, and continued to increase (p <0.01vs Control) after DP7 stimulated the cells for 30 min.

Test example nine GPR35 is a potential target for DP7

1. DP7 induced internalization of GPR35 receptor

After 60min of 40 μ g/ml DP7 stimulation of mouse BMDCs, cells were fixed and blocked, incubated overnight with the primary GPR35 antibody, and stained with FITC-labeled rabbit anti-goat secondary antibody, and finally observed by confocal microscopy.

The results show (fig. 14a) that GPR35 is predominantly located on the cell membrane in control, whereas GPR35 has a phenomenon of cellular internalization following DP7 stimulation.

2. DP7 upregulation of transcription factor expression dependent on GPR35

After stimulating GPR35 shRNA cells and a control group of scrambled shRNA cells for 30min by 40 mu g/ml DP7, collecting samples, extracting total RNA, and detecting the expression of beta-actin, Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1 by an RT-PCR method.

The results show (figure 14b) that relative fold changes of Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1 were significantly reduced in GPR 35-silenced cells relative to control scrambled shRNA cells (p < 0.05).

3. DP7 upregulation of Erk1/2 phosphorylation dependent on GPR35

After stimulating GPR35 shRNA cells and a control group of scrambled shRNA cells for 30min by 40 mu g/ml DP7, samples were collected and subjected to western blot detection Erk1/2 phosphorylation.

The results show (FIG. 14c) that DP7 was acting in scrambled shRNA cells, and Erk1/2 phosphorylation was enhanced compared to Control. In GPR35 shRNA cells, after DP7 acts for 30min, Erk1/2 phosphorylation is almost unchanged.

4. Mechanism of action of aluminum hydroxide/CpG/DP 7 adjuvant in BMDCs or macrophages

The DP7 polypeptide interacts with GPR35 on the cell surface, activating PI3K γ, PKC θ and ERK1/2 signaling pathways, inducing the production of transcription factors, ultimately leading to the production of chemokines or cytokines. In addition, DP7 activation of NF-. kappa.B may also have an effect on chemokine or cytokine production. CpG combined with TLR9 activates ERK1/2 or NF-kappa B, and promotes the release of inflammatory factors such as IL-6 and TNF-alpha. Aluminum hydroxide activates the NALP3 inflammatory complex, which regulates the secretion of the proinflammatory cytokines IL-1 β and IL-18. Chemokines and cytokines induced by aluminum hydroxide, CpG and DP7 contribute to enhancing the immune effect of the composite adjuvant.

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Claims (18)

1. The composite immunologic adjuvant mainly comprises an aluminum adjuvant, CpG oligonucleotide and polypeptide.

2. The composite immunologic adjuvant according to claim 1, wherein: the polypeptide is at least one selected from polypeptides shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No. 10.

3. The composite immunoadjuvant of claim 1, characterized in that: the weight ratio of the immunologic adjuvant is aluminum adjuvant: CpG oligonucleotide: polypeptide: 1-25: 1-4.

4. The composite immunoadjuvant of claim 1, characterized in that: the weight ratio of the immunologic adjuvant is aluminum adjuvant: CpG oligonucleotide: polypeptide: 1-25: 1: 2.

5. The composite immunoadjuvant of claim 1, characterized in that: the aluminum adjuvant is aluminum hydroxide gel.

6. The composite immunologic adjuvant according to any one of claims 1-5, wherein: the CpG oligonucleotide sequence is 5'-TCGTCGTTTTGTCGTTTTTGTCGTT-3' (SEQ ID No. 1).

7. Use of the composite immunoadjuvant of any one of claims 1 to 5 for the preparation of a vaccine.

8. Use according to claim 7, characterized in that: the vaccine is a therapeutic vaccine or a prophylactic vaccine.

9. An immunoadjuvant-antigen complex characterized by: the compound immunoadjuvant according to any one of claims 1 to 5, wherein the compound immunoadjuvant is prepared from the antigen as a main raw material.

10. The immunoadjuvant-antigen complex according to claim 9, characterized in that: the weight ratio of the antigen to the composite immunologic adjuvant in the immunologic adjuvant-antigen composite is that the antigen to the polypeptide is 1: 1-400.

11. The immunoadjuvant-antigen complex according to claim 10, characterized in that it is a therapeutic vaccine or a prophylactic vaccine.

12. An immunoadjuvant-antigen complex according to any one of claims 9 to 11, wherein the antigen is at least one of a tumor antigen, a viral antigen or a bacterial antigen.

13. The immunoadjuvant-antigen complex of claim 12, wherein the viral antigen is at least one of EBV, LMP2, HPV E6E 7, adenovirus 5Hexon, HCMV pp65, hepatitis b virus HBsAg.

14. The immunoadjuvant-antigen complex of claim 12, wherein said tumor antigen is at least one of WT1, MUC1, EGFRvIII, HER-2, MAGE-A3, NY-ESO-1, PSMA, GD2, or MART 1.

15. The immunoadjuvant-antigen complex of claim 12, wherein said bacterial antigen is at least one of a pseudomonas aeruginosa antigen, a tetanus antigen, a streptococcus pneumoniae antigen, a salmonella antigen.

16. An immunoadjuvant-antigen complex according to any one of claims 9 to 15, characterized in that: when the antigen in the vaccine is a tumor antigen, the weight ratio of the antigen to the composite immunologic adjuvant is that the tumor antigen is 1: 1-10;

or when the antigen is a virus antigen, the weight ratio of the antigen to the composite immunologic adjuvant is 1: 200-400 of the virus antigen to the immunoregulation active peptide.

17. A method for preparing the composite immunoadjuvant according to any one of claims 1 to 6, characterized by comprising the steps of:

a. taking the polypeptide and the CpG oligonucleotide according to the proportion, mixing uniformly, and incubating for 10-20min at room temperature;

b. and adding the aluminum hydroxide gel according to the proportion, and uniformly mixing to obtain the composite immunologic adjuvant.

18. A method of preparing an immunoadjuvant-antigen complex according to any one of claims 9 to 15, characterized by comprising the steps of:

a. taking the polypeptide and the CpG oligonucleotide according to the proportion, mixing uniformly, and incubating for 10-20min at room temperature;

b. adding aluminum hydroxide gel according to the proportion, and uniformly mixing to obtain the composite immunologic adjuvant;

c. then adding the antigen according to the proportion, and mixing evenly to obtain the immune adjuvant-antigen compound.

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