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 particularly relates to an aluminum hydroxide-CpG oligonucleotide-polypeptide composite adjuvant, a vaccine, a preparation method and application thereof. 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 composite immunologic adjuvant mainly containing an aluminum adjuvant, CpG oligonucleotide and 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-polypeptide composite 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.

Description

Aluminum hydroxide-CpG oligonucleotide-polypeptide complex adjuvant, vaccine and preparation method thereof use

technical field

The invention belongs to the field of vaccine preparation, and in particular relates to a composite immune adjuvant with aluminum hydroxide, CpG oligonucleotides and polypeptides as main components, a prepared vaccine, and a preparation method and application thereof.

Background technique

Adjuvants play an important role in the efficacy of vaccines and are one of the important factors in determining vaccine development. The purpose of adding adjuvant to the vaccine is to enhance the immunogenicity and immune protection effect of the antigen, which is manifested as enhancing the specific humoral immunity and/or cellular immune response of the specific antigen, thereby improving the production or/and specificity of the specific antibody. Cellular immune function, reducing the amount of antigen required for effective immunization, reducing the frequency of need for booster immunization, improving early immune response and the response success rate of immunocompromised patients, etc. At present, the commonly used vaccine adjuvants on the market are mainly aluminum adjuvants (the main type is the main component of aluminum hydroxide gel AL(OH)3 or ALPO3, referred to as aluminum salts), and aluminum hydroxide is the only one approved by the FDA for clinical use. Vaccine adjuvants. The main mechanism of aluminum hydroxide as an adjuvant to stimulate the immune response is: after the antigen is wrapped in its gel-like emulsion, the antigen is slowly released at the injection site of the body to increase the "half-life" of the antigen in the body, so as to improve the vaccine. It mainly induces the body's Th2 cells to act on B cells to induce the body's humoral immunity. Another advantage of aluminum adjuvant is good safety; however, aluminum adjuvant can induce IgE antibodies produced by the body, which can induce allergic reactions, and such adjuvants cannot induce Th1 immune responses, which The response is involved in the response to clear intracellular microbial infection, namely the killer T lymphocyte response (CTL). These all limit the application of aluminum salts as adjuvants.

Therefore, for those vaccines that have been successfully developed for human or veterinary use, the development of a new adjuvant or adjuvant complex that can effectively enhance or modulate antigen-specific immune responses is a major advance. The natural immune system of vertebrates has developed different mechanisms to recognize antigens based on various antigen-specific signaling molecules, including toll-like receptors (TLRs), NODs, and the like. In the emergency immune response of the body to clear foreign microbial infection, a series of signal transduction based on ligand recognition of TLR receptors play an important role; moreover, TLR receptors can also induce the maturation of DC cells - this is The necessary and most important step in initiating adaptive immunity. Among the TLR receptor recognition molecules, the most studied and mature is the unmethylated CpG oligonucleotide (CpGoligodeoxynucleotide, CpG ODN, CpG for short) derived from Escherichia coli that recognizes TLR-9. CpG ODN can bind to TLR9 in the Toll-like receptor family to activate DC cells and induce Th1 cytokines such as IL-12, IFN-γ, TNF-α; it can also activate NK cells and enhance the cytotoxicity of macrophages and NK cells to promote antigen-specific Th1-type immune responses.

However, CpG ODNs lack cell specificity and are negatively charged, so they are not easily taken up by cells, and the CpG ODN receptor TLR9 is located in cells, so its utilization as an adjuvant alone is low. However, some studies have reported that high-dose CpG ODN can also cause tissue edema and may trigger autoimmune diseases. How to improve the use efficiency and security is a major difficulty on the road of CpG ODN application.

Some polypeptides have immunomodulatory function and direct antibacterial ability. These immunomodulatory functions are mainly reflected in that they can induce enhanced expression of cytokines and chemokines, directly or indirectly recruit granulocytes to the site of infection, stimulate mast cells to release histamine, dendritic cell (DC) maturation and wound healing, while It is difficult to obtain polypeptides with good immunomodulatory ability.

In recent years, tumor immunotherapy has attracted more and more attention as a new possible way to treat tumors, and tumor vaccines for the prevention and treatment of tumors have also become a research hotspot of tumor immunotherapy. Among them, the search for specific tumor antigens and effective auxiliary means for antigen uptake and presentation is one of the key issues in the development of tumor vaccines. However, since tumor antigens are often weak in immunogenicity, when used alone, the stimulated immune response is not strong enough, and an effective anti-tumor immune response must be induced in the body with the assistance of immune adjuvants. However, the existing conventional adjuvants are not effective when used in conjunction with tumor antigens, and new antigen adjuvants suitable for tumor immunotherapy must be developed.

There is an urgent need in the art to develop an immune adjuvant that can stimulate more signaling pathways to synergistically increase the immune response, thereby stimulating a stronger specific immune response, and is more suitable for the preparation of tumor vaccines. R&D provides new and effective options.

SUMMARY OF THE INVENTION

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 compound immune adjuvant. The composite immune adjuvant mainly contains aluminum adjuvant, CpG oligonucleotide and at least one polypeptide selected from the amino acid sequence of SEQ ID No.7, SEQ ID No.8 and SEQ ID No.10.

Wherein, the weight ratio of each main component in the above-mentioned composite immune adjuvant is aluminum adjuvant:CpG oligonucleotide:polypeptide=1～25:1:1～4.

Wherein, the preferred weight ratio of each main component in the above-mentioned immune adjuvant is aluminum adjuvant: CpG oligonucleotide: polypeptide=1～25:1:2.

Further, the above-mentioned aluminum adjuvant is aluminum hydroxide gel.

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

Wherein, the nucleotide sequence of the CpG oligonucleotide described in the above-mentioned composite immune adjuvant is:

5'-TCGTCGTTTGTCGTTTTTGTCGTT-3' (SEQ ID No. 1).

Wherein, the amino acid sequence of the polypeptide SEQ ID No.7 in the above-mentioned immune adjuvant is: VQWRIRVAVIRK (named DP7); the amino acid sequence of SEQ ID No.8 is: VQLRIRVCVIRK (named DP8); the amino acid sequence of SEQ ID No.2 The sequence is: VQLRCRVCVIRK (designated DP10).

According to another aspect of the present invention, the present invention also provides the use of the above-mentioned composite immune adjuvant in preparing a vaccine.

Wherein, the above-mentioned vaccines are therapeutic vaccines or prophylactic vaccines.

Meanwhile, the present invention also provides an immune adjuvant-antigen complex formed by adding an antigen to the above-mentioned composite immune adjuvant.

Wherein, the weight ratio between the antigen in the immune adjuvant-antigen complex and the complex immune adjuvant is the weight of the antigen: the weight of the immunomodulatory active peptide=1:1～400.

Wherein tumor antigen weight: immunomodulatory active peptide weight=1:1-10, virus antigen weight: immunomodulatory active peptide weight=1:200-400.

Further, the above-mentioned immune adjuvant-antigen complex can be prepared as a therapeutic vaccine or a preventive vaccine.

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

Further, the tumor antigens include specific proteins or polypeptides expressed by tumor cells. Such as WT1, MUC1, EGFRvIII, HER-2, MAGE-A3, NY-ESO-1, PSMA, GD2 or MART1 and other tumor antigens that have been reported or a combination of individualized mutant neoantigens based on patient tumor sequence determination. The viral antigen may be a protein or polypeptide constituting a part of the virus, or a specific protein or polypeptide expressed in virus-infected cells controlled by the virus expression mechanism. Such as EBV, LMP2, HPV E6 E7, adenovirus 5Hexon, HCMV pp65, HBsAg protein and other virus-related antigens.

The bacterial antigens include proteins or polypeptides expressed by bacteria. Such as Pseudomonas aeruginosa antigen, tetanus antigen, Streptococcus pneumoniae, Salmonella and other bacterial antigens.

On the basis of the above technical solutions, the present invention also provides a method for preparing the above composite immune adjuvant. The method includes the following steps:

a. Take the peptide and CpG oligonucleotide according to the ratio, mix well, and incubate at room temperature for 10-20min;

b. Add aluminum hydroxide gel according to the proportion, and mix well to obtain a composite immune adjuvant.

The present invention also provides a method for preparing the above-mentioned immune adjuvant-antigen complex.

It is characterized by comprising the following steps:

a. Take the peptide according to the ratio and mix it with CpG oligonucleotide, mix well, and incubate at room temperature for 10-20min;

b. Add aluminum hydroxide gel according to the proportion, and mix to obtain a composite immune adjuvant;

c. Then add the antigen according to the proportion, and mix well to obtain the immune adjuvant-antigen complex.

In step a of the above method, it is generally sufficient to incubate at room temperature for about 15 min. The immune adjuvant-antigen complex obtained in step c can be filled with sterile PBS to make up the required volume for subsequent use.

The beneficial effects of the present invention are as follows: the present invention creatively obtains a new compound immune adjuvant formula composed of aluminum hydroxide/CpG oligonucleotide/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 have shown that the various vaccines prepared by the aluminum hydroxide/CpG oligonucleotide/polypeptide adjuvant of the present invention have higher immune and therapeutic effects, can effectively stimulate the body to produce antigen-specific immune responses, and Extend immune memory. More importantly, the anti-tumor vaccine prepared by the immune adjuvant of the present invention has a very good effect, which provides a new choice for the development and application of vaccines in the field.

Description of drawings

Fig. 1 Determination of the ratio of CpG/immunomodulatory active peptide complexes and detection of toxicity. a: Gel retardation experiment Lane 1: CpG (5μg)+DP7 (20μg); Lane 2: CpG (5μg)+DP7 (10μg); Lane 3: CpG (5μg)+DP7 (5μg); Lane 4: CpG (5μg)+DP7 (2.5μg); Lane 5: CpG (5μg)+DP7 (1.25μg); Lane 6: CpG (5μg); b: Erythrocytes treated with CpG/DP (2-11) Heme release after complex stimulation and LDH release from PBMCs stimulated by CpG/DP(2-11) complex.

Figure 2. Screening of CpG/immunomodulatory active peptide complexes. a: Detection of MCP-1 release after human PBMCs were stimulated by CpG ODN/defense peptide DP2-DP11 complexes. b: Detection of immune responses stimulated by HbsAg/alum/CpG/DP2～DP11. c: In vivo chemotaxis of DP7, DP8, and DP10, saline, DP7, DP8, and DP10, respectively, from left to right in each group. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 3. Detection of CpG/DP7 complex adjuvant stimulated human peripheral blood mononuclear cells to produce cytokines IL-1β, IFN-γ, TNF-α and IL-10. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 4 CpG/DP7 activates BMDC activity. a. CpG/DP7 promotes antigen uptake by BMDCs; b-d. CpG/DP7 promotes BMDCs maturation; c. CpG/DP7 upregulates the expression of pERK1/2 and p-p65.

Figure 5 shows the anti-tumor effect of tumor vaccine prepared by NY-ESO-1 protein or OVA protein and aluminum hydroxide gel/CpG oligonucleotide//immunomodulatory active peptide mixture. a: Tumor growth of mice in each group in the melanoma preventive model. b: Tumor growth of mice in each group in a therapeutic model of melanoma. c: Tumor growth of each group of mice in the T lymphoma preventive model. d: Tumor growth of each group of mice in the T lymphoma therapeutic model. e: Tumor growth in each group of mice in a breast cancer preventive model. f: Tumor growth of each group of mice in a breast cancer therapeutic model. *, p<0.05; **, p<0.01; ***, p<0.001.

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

Figure 7 shows the detection of cellular immune responses stimulated by NACD tumor vaccine immunization. a and b: CD4 ⁺ (a) or CD8 ⁺ T (b) cells secreting IFN-γ were detected by flow cytometry. c and d: ELISPOT detection of T cells secreting IL-4 (c) and IFN-γ (d). *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 8 shows the detection of immune memory T cells of mice in each group after immunization with NACD tumor vaccine.

Figure 9 shows the body weight (a) and HE staining of important organs (b) of mice in each group after immunization with NACD tumor vaccine.

Figure 10 Blood routine analysis of mice in each group after immunization with NACD tumor vaccine. where WBC: number of white blood cells; RBC: number of red blood cells; PLT: number of platelets; HGB: hemoglobin; HCT: hematocrit; MPV: mean platelet volume; MCH: mean red blood cell hemoglobin content; MCHC: mean red blood cell hemoglobin concentration; MCV: mean red blood cell volume. Figure 11. Blood biochemical analysis of mice in each group after NACD tumor vaccine immunization. TP: total protein; ALB: albumin; ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; CREA: creatinine; UREA: urea; UA: uric acid; GLU: glucose; LDH: lactate dehydrogenase ; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol.

Figure 12 Detection of transcription factor expression by RT-PCR in vitro. *, p<0.05; ***, p<0.001.

Figure 13. Involvement in DP7 activity signaling pathway studies. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 14. GPR35 and DP7 interaction studies. a. DP7 induces GPR35 receptor internalization; b. RT-PCR detects the effect of GPR35 silencing on transcription factor expression; c. DP7 up-regulates Erk1/2 phosphorylation dependent on GPR35. *, p<0.05; ***, p<0.001.

Detailed ways

In order to develop an ideal immune adjuvant, the present invention investigated a variety of existing immune adjuvant single and composite formulations in the early stage, and accidentally found that the use of aluminum hydroxide, CpG oligonucleotide (CpG ODN, CpG for short) and some Active polypeptides form a specific complex, and when used as an immune adjuvant, it is beneficial to help stimulate the immune response of the body and improve the immune effect of the antigen.

On this basis, various aluminum hydroxide/CpG ODN/peptide formulations were further investigated. In the present invention, various immunomodulatory active polypeptides are screened, and the formula components of the compound immune adjuvant composed of the active polypeptide and the present invention are obtained. At the same time, in order to achieve better results, the dosages of aluminum hydroxide, CpG ODN, and immunomodulatory active peptides with different ratios were screened, and the optimization of the formula between aluminum hydroxide, CpG ODN and peptides was completed. The final optimized formula ratio is aluminum adjuvant:CpG oligonucleotide:polypeptide=1～25:1:1～4. The preferred ratio is aluminum adjuvant:CpG oligonucleotide:polypeptide=1～25:1:2. In the specific use of CpG ODN in the art, thio-modification treatment may be performed, or no modification may be performed. For example, thiomodification can increase its stability, and thiomodification of the entire chain backbone is generally performed.

The present invention compares aluminum hydroxide gel, CpG ODN, immunomodulatory active peptide, etc. alone or in combination as adjuvants to add antigens, and finds that aluminum hydroxide gel/CpG ODN/immunomodulatory active peptide compound as adjuvant can effectively Stimulate the body to produce antigen-specific immune responses and prolong immune memory.

The three components used in this composite adjuvant all have good advantages: among them, aluminum hydroxide gel adjuvant is a kind of inorganic salt containing Al ³⁺ , which not only has good adsorption effect, but also can convert soluble antigens into soluble antigens. It is adsorbed on the surface of aluminum glue molecules; it is also a good precipitant, which can concentrate the antigen and reduce the injection dose. Aluminum hydroxide gel is the most widely used adjuvant and the only adjuvant approved by the FDA for human vaccines so far. At present, a large number of clinical studies related to CpG ODN adjuvant vaccines have shown that CpG ODN can enhance the body's humoral and cellular immune responses to pathogens, allergens and tumor antigens. Immunomodulatory active peptides can induce the body to produce cytokines and chemokines, directly or indirectly recruit DC cells and monocytes to the site of infection, promote the phagocytosis of antigens by antigen-presenting cells, and stimulate the body's innate immunity, thereby enhancing Acquired immune response.

The above-mentioned compound immune adjuvant of the present invention can be added with antigen to form an immune adjuvant-antigen complex, that is, a vaccine. Antigens used for preparing vaccines can be various antigens commonly used in the art. For example, full-length antigens with proteinaceous components, such as proteins or peptides, may also be antigens that are further modified after translation, such as certain glycoproteins or lipoproteins. Antigens can also be tumor antigens, including specific proteins or polypeptides expressed by tumor cells. For example, WT1, MUC1, EGFRvIII, HER-2, MAGE-A3, NY-ESO-1, PSMA, GD2 or MART1 have been reported tumor antigens or individualized combination of mutant neoantigens based on patient tumor sequence determination. Antigens can also be viral antigens, such as proteins or polypeptides that form part of the virus, or specific proteins or polypeptides that are expressed in virus-infected cells controlled by the viral expression machinery. For example, virus-associated antigens such as EBV, LMP2, HPV E6 E7, adenovirus 5Hexon or HCMV pp65. Antigens can also be bacterial antigens, including proteins or polypeptides expressed by bacteria. Such as Pseudomonas aeruginosa antigen, tetanus antigen, Streptococcus pneumoniae, Salmonella and other bacterial antigens.

In the process of preparing the vaccine in the development process, the present invention finds that in order to obtain a good vaccine effect, the weight of the added antigen and the weight of the polypeptide in the compound immune adjuvant are closely related. After a lot of research, it is determined that the weight ratio of antigen weight: immunomodulatory active polypeptide=1:1～400 can achieve better results. Among them, tumor antigen weight: immunomodulatory active peptide weight=1:1-10, virus antigen weight: immunomodulatory active peptide weight=1:200-400 is a better choice. Alternatively, another appropriate ratio can be selected within the above-mentioned range according to the characteristics of the antigen itself and other specific conditions. Validated on hepatitis antigen HBsAg and tumor antigen NY-ESO-1, the results show that the prepared vaccine has a good effect. In the hepatitis vaccine model, the immunized mice produced high titers of HBsAg-specific antibodies; in the tumor vaccine model, it could stimulate the body's antigen-specific humoral and cellular immune responses, and then effectively inhibit tumor growth.

Those skilled in the art, on the basis of knowing the idea of the present invention, as well as the ratio range of aluminum hydroxide/CpG ODN/polypeptide composite adjuvant and the ratio range of antigens and other technical solutions disclosed above, can clearly A further optimization was carried out in the range of formulations obtained above. Under necessary conditions, for the requirements of a specific dosage form, it is not excluded to add other acceptable auxiliary components in the preparation of vaccines on the basis of the above-mentioned basic formula to complete the preparation of vaccines. Obviously, these embodiments fall within the scope of the present invention.

The method of the present invention will be further described below in conjunction with the examples.

The main reagents used in the examples are:

Aluminum hydroxide adjuvant (Alhydrogel) was purchased from Brenntag Biosector, Denmark.

HBsAg was purchased from American Research Products.

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

Horseradish peroxidase-labeled goat anti-mouse IgG and isotype detection kit were purchased from SouthernBiotech.

C57BL/6 and Balb/c mice were purchased from Beijing Weitong Lihua Company.

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

Cytokine chip detection kit HCYTOMAG-60K was purchased from Milliplex.

Goat anti-mouse, goat anti-rabbit secondary antibodies and related immunohistochemical kits were purchased from Fuzhou Maixin Biotechnology Company, and anti-CD4, anti-CD8, anti-CD56, F4/80, Gr1, CD11b and other antibodies were purchased from Abcam company.

The used polypeptides DP2 to DP11 were all synthesized by Shanghai Kepeptide Biological Co., Ltd., and the specific sequences are shown in Table 1.

All other reagents are imported or domestic analytically pure products.

Example 1 CpG/active polypeptide ratio and preliminary detection of toxicity

1. Determination of CpG/immunomodulatory active peptide ratio

CpG was mixed in the ratios of 4:1(wt/wt), 2:1(wt/wt), 1:1(wt/wt), 1:2(wt/wt), 1:4(wt/wt), respectively. Mixed with alternative active polypeptides (see Table 1), and allowed to stand at 37°C for 10-15min, wherein the amount of CpG was 5 μg. Add 10 μl of 6× loading buffer to each CpG and immunomodulatory active peptide mixture and mix well. Prepare a 1% agarose gel, take 10 μl of each sample for spotting, use 1% agarose gel for electrophoresis at 90V for 20 min, and use a gel imaging system to image.

Table 1. 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 showed (Fig. 1a): with the increase of defense peptides, the free CpG ODNs became less and less. When the mixing ratio of CpG ODNs and alternative peptides was 1:2 (wt/wt), CpG ODNs and alternative The polypeptides can fully form stable complexes.

2. Toxicity test of the complex as an adjuvant

1. LDH release experiment

Isolate human peripheral blood PBMC lymphocytes, use RPMI 1640+5% FBS medium, dilute PBMC to 1×10 ⁷ cells/ml, 500 μl/well, spread on 24-well plate, and culture in 37°C, 5% CO2 incubator for 1 hour . 10 μg of CpG and immunomodulatory active peptides were prepared at 4:1 (wt/wt), 2:1 (wt/wt), 1:1 (wt/wt), 1:2 (wt/wt), 1:4 (wt, respectively The CpG/immunomodulatory peptide mixture was prepared at a ratio of /wt), and the mixture was allowed to stand at room temperature for 10-15 minutes, then the CpG/immunomodulatory peptide mixture was added to the upper lymphocyte culture medium, and incubated at 37°C with 5% CO2 for 24 hours. The supernatant was collected and centrifuged at 250 g for 5 min at 4°C. According to the instructions of the LDH kit, set positive, negative and blank controls to determine the LDH value.

2. Erythrocyte heme release assay

Peripheral blood red blood cells were collected and washed three times by adding 0.85% normal saline. The red blood cells were diluted 4 times with normal saline, 100 μl/well was placed in a 96-well plate, and 50 μl of different concentrations of CpG/immunomodulatory active peptides were added. The final working concentrations of CpG and immunomodulatory peptides were CpG 20μg/ml and immunomodulatory peptides 40μg/ml , 37 ℃, 5% CO2 incubated overnight, which used Triton-100 (1%) as a positive control, normal saline as a negative control. 4000rpm, 10min centrifugation, collect the supernatant, use the microplate reader A450 to read, and calculate the toxicity of the adjuvant to erythrocytes.

The experimental results showed (Fig. 1b): the CpG/immunomodulatory active peptide mixture had no detectable level of toxicity on peripheral blood mononuclear cells and erythrocytes.

Example 2 Screening of CpG/immunomodulatory active peptide complexes

1. Detection of MCP-1 secretion from human PBMCs stimulated by CpG/immunomodulatory active peptide complexes

Isolate human peripheral mononuclear lymphocytes (PBMCs) with lymphocyte secretion fluid, dilute the above-isolated lymphocytes into 1×10 ⁶ cells/ml in 1640 complete medium, spread 24-well plates, 500 μl per well, 37°C, 5 % _CO2 for 1 h. Add the adjuvant or adjuvant complex CpG, DP2-11, CpG-DP2-11 dropwise to the lymphocyte culture medium at 37°C and incubate for 24h in 5% _CO2 , wherein CpG: 20μg/ml, DP2-11: 40μg/ml . Centrifuge at 16000g for 5 min at 4°C, collect the supernatant, and use an ELISA kit to detect the release of MCP-1.

The experimental results show (Fig. 2a): MCP-1 is a monocyte chemokine that can chemotactic monocytes/macrophages, T cells, NK cells and neutrophils. After the double compound adjuvant CpG/DP7, CpG/DP8 and CpG/DP10 stimulated PBMC, the secretion of MCP-1 was 4572pg/ml, 4826pg/ml and 3534pg/ml, respectively. Compared with other groups, the secretion of MCP-1 in these three groups was significantly higher than that in other groups (P<0.05). Moreover, the synergy coefficients between CpG and DP7, DP8, and DP10 were all greater than 2, indicating that CpG and DP7, DP8, and DP10 could synergistically promote the secretion of MCP-1 from PBMCs.

2. Detection of immune responses stimulated by HbsAg/aluminum hydroxide gel/CpG/DP2-11

The protein vaccines were prepared according to the following schemes. The total dose of each mouse was 100 μl, and PBS was added to make up for the less than 100 μl. There were 10 mice in each group, and each mouse was administered the following doses:

①NS group: 100μl PBS

②alum group 0.1μg HBsAg+25μg alum

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

④alum/DP2～11 group: 0.1μg HBsAg+25μg alum+40μg DP2～DP 11

⑤alum/CpG/DP2～11 group 0.1μg HBsAg+25μg alum+20μg CpG+40μg DP2～DP 11

The above vaccine preparation scheme is:

a. Add any of the desired polypeptides DP2 to DP 11 and CpG ODN, mix well, and incubate at 37°C for 15 minutes;

b. Add the required aluminum hydroxide gel (Alhydrogel from Brenntag Biosector, Denmark, referred to as alum in the examples), mix well, and incubate at 37°C for 10 minutes;

c. Finally, add 0.1 μg of HbsAg and make up to a volume of 100 μl with sterile PBS.

The mice were immunized with a preventive immunization scheme, three times at the 0th, 2nd, and 4th weeks, and the blood was collected at the 5th week to detect HbsAg-specific antibodies.

The results showed (Fig. 2b), at week 5 of immunization, the median anti-HbsAg total antibody titer in the alum/CpG group was 64,000, while the alum/CpG/DP7, alum/CpG/DP8 and alum/CpG/DP10 groups had a median total antibody titer of 64,000. The median value of HbsAg total antibody titer was 512000, which was significantly different from the alum/CpG group.

3. DP7, DP8 and DP10 recruit lymphocytes in vivo

C57BL/6 mice were injected intraperitoneally with 200 μg of polypeptides DP7, DP8 and DP10, respectively, and the control group was injected with the same volume of sterile normal saline. Twenty-four hours after injection, the mouse peritoneal lavage fluid was collected and the chemotactic lymphocytes were analyzed by flow cytometry. F4/80 ⁺ CD11b ⁺ double positive is macrophages, F4/80 ⁺ Gr1 ⁺ double positive is monocytes, and F4/80 ^- Gr1 ⁺ is labeled as neutrophils.

The flow cytometry results showed (Fig. 2c), compared with the control group (saline, sterile saline), the proportion of monocytes in the total cells of the peritoneal lavage fluid in the DP7 and DP8 injection groups was significantly increased, and the peritoneal lavage in the DP10 injection group was significantly increased. The proportion of monocytes in the total cells of the washes was also increased. The proportion of neutrophils in the total cells of the peritoneal lavage fluid in the DP7 injection group was significantly increased, and the proportion of neutrophils in the total cells in the peritoneal lavage fluid of the DP8 and DP10 injection groups also increased. The proportion of macrophages in the total cells of the peritoneal lavage fluid in the DP8 injection group was significantly increased, and the proportion of macrophages in the total cells in the peritoneal lavage fluid of the DP7 and DP10 injection groups also increased. It was shown that DP7, DP8 and DP10 have immunomodulatory effects and are able to recruit lymphocytes in vivo.

Example 3 CpG/immunomodulatory active peptide complexes promote the production of cytokines in human peripheral blood mononuclear cells

Isolation of human peripheral blood mononuclear cells, the method is the same as above. PBMCs were diluted to 4×10 ⁶ /ml with 1640+10% FBS, 125 μl/well were plated in 48-well plates. Add 125 μl of 2× adjuvant stimulus stock solution (DP7, CpG, CpG/DP7), mix well, and incubate in a 37° C. incubator for 48 hours. The concentration of defense peptide stimulation was 40μg/ml, and the stimulation concentration of CpG was 20μg/ml. PHA (3 μg/ml) stimulation was used as positive control, simple medium was used as blank control, and unstimulated human PBMC medium supernatant was used as negative control. Centrifuge at 250 g for 5 min, collect the supernatant of the medium and use the cytokine chip detection kit to detect (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (P70), IL -17A, TNF-α and IFN-γ).

The results showed that compared with single adjuvant (Fig. 3), dual adjuvant significantly promoted the secretion of immune positive regulator cytokines IL-1β, TNF-α and IFN-γ. The secretion of cytokines in the supernatant of the CpG/DP7 group medium was as follows: after stimulation with double adjuvant, the secretion of IFN-γ was 542 pg/ml; the secretion of IL-1β was 8045 pg/ml; the secretion of TNF-α was: 6704 pg /ml; IL-10 secretion was 6260pg/ml. It can be seen that compared with DP7 and CpG groups, the dual adjuvant CpG/DP7 can significantly promote the secretion of immune positive regulatory cytokines IL-1β, TNF-α and IFN-γ, and inhibit the secretion of immune negative regulatory cytokine IL-10. . Example 4 CpG/immunomodulatory active peptide complex activates the activity of BMDCs

1. CpG/immunomodulatory active peptide complexes promote antigen uptake

1. Isolation and culture of BMDCs

6-8 week old mice were sacrificed by neck fracture, and bone marrow cells from tibia and fibula were collected; the culture medium containing bone marrow cells was collected into a 50ml BD tube, centrifuged at 1500rpm for 3min, resuspended with red blood cell lysate (1:10), and allowed to stand at room temperature Centrifuge at 1500rpm for 5min for 3min, and resuspend with RPMI1640+FBS. Cells were filtered through a 70 μm cell mesh to remove debris, incubated with fresh medium and added with 10 ng/ml GM CSF and 10 ng/ml IL 4, and the medium was replaced by half every other day. Subsequent experiments were performed on day 7 of culture.

594Microscale Protein LabelingKit将NY ESO1蛋白标记红色荧光。体外将荧光标记的NY ESO 1分别与佐剂或佐剂复合物混合DP7、CpG、CpG/DP7)，室温孵育10min，其中NY ESO 1:10μg/ml,CpG:20μg/ml,DP7 40μg/ml)。将佐剂或佐剂复合物加入BMDCs细胞培养物中混匀，孵育1h。先用PBS洗3次，再用4％多聚甲醛,室温避光固定10min。PBS清洗3次后，DAPI染色1min。PBS清洗3次后，用防淬灭封片，在共聚焦显微镜下观察。2. Use protein red fluorescent labeling kit Alexa

594Microscale Protein LabelingKit labels NY ESO1 protein with red fluorescence. In vitro, fluorescently labeled NY ESO 1 was mixed with adjuvant or adjuvant complex (DP7, CpG, CpG/DP7), and incubated at room temperature for 10 min, wherein NY ESO 1: 10 μg/ml, CpG: 20 μg/ml, DP7 40 μg/ml ). The adjuvant or adjuvant complex was added to the BMDCs cell culture, mixed, and incubated for 1 h. Washed 3 times with PBS, then fixed with 4% paraformaldehyde for 10 min at room temperature in the dark. After washing 3 times with PBS, DAPI staining was performed for 1 min. After washing 3 times with PBS, the slides were mounted with anti-quenching and observed under a confocal microscope.

The results showed (Fig. 4a) that the mean fluorescence intensity of red fluorescence of BMDC cells in the CpG/DP7 group was significantly higher than that in the other control groups, indicating that the CpG/DP7 adjuvant mixture group could promote the uptake of NY-ESO-1 antigen by BMDC cells.

2. CpG/immunomodulatory active peptide complexes promote the maturation of BMDCs

Adjuvant or adjuvant complex was mixed in vitro with DP7, CpG, CpG/DP7 (CpG: 20 μg/ml, DP7 40 μg/ml). Add to BMDCs cell culture, mix well, and incubate for 16h. BMDCs were stained with antibodies APC-anti-Mouse CD11c, FITC-anti-mouse-CD40, PerCP-Cy5.5-anti-mouse-CD8 or FITC-anti-mouse-CD86 and detected by flow cytometry.

The results showed (Figures 4b, 4c and 4d) that compared with Control, DP7 and CpG, CpG/DP7 significantly increased the expressions of CD40, CD80 and CD86. It can be seen that the CpG/DP7 adjuvant mixture group can promote the maturation of BMDC cells.

3. CpG/immunomodulatory active peptide complexes promote the expression of pERK1/2 and p-p65

Adjuvant or adjuvant complex was mixed in vitro with DP7, CpG, CpG/DP7 (CpG: 20 μg/ml, DP7 40 μg/ml). Add to BMDCs cell culture, mix well, and incubate for 30 or 60 min. Cell samples were collected, subjected to 12% SDS-PAGE electrophoresis to separate proteins in the samples and transferred to PVDF membranes. After blocking at room temperature for 1 h, the primary antibody was incubated: PVDF membranes were placed in anti-NF-κB p65, Phospho-NF-κB p65, Erk1/2 and Phospho-Erk1/2 antibodies (dilution ratio of 1:2000), 4 Incubate overnight with shaking. The next day, the corresponding secondary antibody was incubated for 1 h, and then exposed.

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

Example 5 Study on the anti-tumor effect of the present invention using aluminum hydroxide gel/CpG/active polypeptide as a composite adjuvant for tumor vaccines

1. Preventive immune model

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

The protein vaccines were prepared according to the following schemes. The total dose of each mouse was 100 μl, and PBS was added to make up for the less than 100 μl. There were 10 mice in each group, and each mouse was administered the following doses:

1. NS group: 100 μl PBS

2. Alum group: 5μg NY-ESO-1 or 10μg OVA+125μg alum

3. alum+CpG group: 5μg NY-ESO-1 or 10μg OVA+125μg alum+20μg CpG

4. alum+DP7 group: 5μg NY-ESO-1 or 10μg OVA+125μg alum+40μg DP7

5. alum+CpG+DP7 group: 5μg NY-ESO-1 or 10μg OVA+125μg alum+20μg CpG+40μg DP7

The above vaccine preparation scheme is:

a. Add the required DP7 and CpG ODN, mix well, and incubate at 37°C for 15min;

b. Add the required aluminum hydroxide gel and mix, incubate at 37°C for 10min;

c. Finally, add 5 μg NY-ESO-1 protein or 10 μg OVA, and make up to a volume of 100 μl with sterile PBS.

(2) Immunization program

Select 5-7 weeks old female mice of about 18g (C57BL/6J mice for the B16-NY-ESO-1 model; Balb/c mice for the 4T1-NY-ESO-1 model). After one week of housekeeping, they were randomly divided into groups. Multi-point subcutaneous immunization at 0, 2, and 4 weeks, and tumor cells (B16-NY-ESO-1: 2×10 ⁵ ; EG7.OVA: 2×10 ⁶ ; 4T1-NY- ESO-1: 2×10 ⁵ ), the tumor was measured after the tumor grew out, once every 3 days, and the survival period was observed. The tumor volume was calculated as 0.52×length×width ² .

The experimental results showed (Fig. 5a): in the B16 melanoma model, the tumor growth of mice in the alum group, alum/DP7 group and alum/CpG group was inhibited to a certain extent. On day 22 after tumor inoculation, the mean tumor volumes were 955 ± 298 mm ³ , 906 ± 623 mm ³ and 642 ± 236 mm ³ , respectively. The tumor growth of the alum/CpG/DP7 group was significantly inhibited, and the average tumor volume was 224±126 mm ³ , which was significantly different from that of the NS group (p﹤0.05). On the 22nd day after tumor inoculation, the tumor suppression rates of the alum group, alum/DP7 group and alum/CpG group were 62.6%, 64.5% and 74.9%, respectively. The tumor suppression rate in alum/CpG/DP7 group was 91.2%.

In the T lymphoma model (EG7.OVA) (Fig. 5c), the tumor growth of mice in the alum group, alum/DP7 group and alum/CpG group was also inhibited to a certain extent. On day 26 after tumor inoculation, the mean tumor volumes were 3725 ± 478 mm ³ , 2180 ± 454 mm ³ and 2446 ± 576 mm ³ , respectively. The tumor growth of the alum/CpG/DP7 group was significantly inhibited, and the average tumor volume was 1232±543 mm ³ , which was significantly different from the NS group and the alum group (p﹤0.05). On the 26th day after tumor inoculation, the tumor inhibition rate of alum/CpG/DP7 group was 74.9%.

In the breast cancer model (Fig. 5e), on the 28th day after tumor inoculation, the mice began to have frizzy hair and were in poor condition, so the mice were sacrificed at this time point. The experimental results showed that the tumors of the mice in the NS group grew rapidly, and the average tumor volume could reach 1382±86 mm ³ . The mean tumor volumes of mice in alum group, alum/DP7 group, alum/CpG group and alum/CpG/DP7 group were 1232±87 mm ³ , 1116±91 mm ³ , 1104±65 mm ³ and 755±78 mm ³ , respectively. It can be seen that the tumor growth of mice in alum/CpG/DP7 group was significantly inhibited, and compared with other control groups, there was a significant statistical difference (p﹤0.05). On the 28th day after tumor inoculation, the tumor inhibition rate of the alum/CpG/DP7 group was 45.4%.

2. Therapeutic immune model

The doses of groups and mice administered were the same as the preventive immunization doses. The immunization protocol was as follows: tumor cells (B16-NY-ESO-1: 2×10 ⁵ ; EG7.OVA: 2×10 ⁶ ; 4T1-NY-ESO-1: 2×) were inoculated subcutaneously on the back of each mouse on day 0 10 ⁵ ), and when the tumor volume was about 50 mm ³ , the patients were immunized once a week for a total of 3 times.

In the melanoma model (Fig. 5b), on day 24 after tumor inoculation, the mean tumor volume of mice in NS, alum group, alum/DP7 group, alum/CpG group and alum/CpG/DP7 group was 2081±201 mm, respectively ³ , 2447±692mm ³ , 1507±539mm ³ , 2144±707mm ³ and 1111±201mm ³ . Compared with the NS group, the tumor growth of the alum/CpG/DP7 group was significantly slowed down (p﹤0.05).

In the T lymphoma model (Fig. 5d), on day 28 after tumor inoculation, the mean tumor volume of mice in NS, alum group, alum/DP7 group, alum/CpG group and alum/CpG/DP7 group was 2913± 407mm ³ , 2880±402mm ³ , 2333±722mm ³ , 2677±614mm ³ and 1073±284mm ³ . Compared with the NS group, the tumor growth of the alum/CpG/DP7 group was significantly slowed down (p﹤0.05).

In the breast cancer model (Fig. 5f), the mean tumor volume of mice in the NS, alum group, alum/DP7 group, alum/CpG group and alum/CpG/DP7 group was 1727 ± 97 mm ^on day 32 after tumor inoculation, respectively. , 1703±107mm ³ , 1703±192mm ³ , 1646±215mm ³ and 1071±244mm ³ . Compared with the other 4 groups, the tumor growth of the mice in the alum/CpG/DP7 group was significantly slowed down (p﹤0.05). .

Test Example 6 Detection of immune responses stimulated by tumor vaccine NY-ESO-1/alum/CpG/DP7

1. Detection of humoral immune response

In the preventive model in Example 4, the immune response stimulated by NACD was detected.

1. Elisa detects NY-ESO-1 specific antibody

NY-ESO-1 specific antibodies and antibody subtypes were detected by ELISA. The detection method is as follows: NY-ESO-1 protein was diluted to 1 μg/ml with coating buffer (0.05M carbonate buffer, pH 9.6), and 100 μl was added to each well of a 96-well plate, and the temperature was 4°C overnight. Washed 3 times with PBST (PBS+0.5% Tween20). Block with 5% nonfat dry milk at 37°C for 1 hour. Washed 5 times with PBST. Serum was serially diluted, 100 μl was added to each well, and incubated at 37°C for 1 hour. Plates were washed 5 times with PBST. After that, 100 μL of HRP-labeled goat anti-mouse IgG (1:4000 dilution) was added to each well, and incubated at 37° C. for 1 hour. Washed 5 times with PBST. 100 μl of temporarily prepared TMB solution (KPL company) was added to each reaction well, and after 20 minutes of color development at room temperature, 100 μl of 0.5MH ₂ SO ₄ was added to stop the reaction, and the reaction was read at 450 nm.

The experimental results showed (Fig. 6a) that the median values of IgG antibody titers in alum group, alum/DP7 group, alum/CpG group and alum/CpG/DP7 group were 640,000, 960,000, 640,000 and 1,280,000, respectively. The NY-ESO-1-specific antibody in the serum of mice in alum/CpG/DP7 group was significantly higher than that in other groups, and there was a statistical difference with the alum group (p﹤0.05). It can be seen that alum/CpG/DP7 adjuvant can significantly enhance the specific humoral immune response of NY-ESO-1.

2. Detection of antibody subtypes

Start with mixed serum starting from 1:100, and wait for 10-fold dilution. The diluted serum is used as the primary antibody for Elisa detection. To determine the range of antibody subtypes, select the appropriate initial dilution ratio and 2-fold dilution for 8 gradients as a primary antibody. Antibodies of different subtypes were used as secondary antibodies (IgG1, IgG2c; both were diluted 1:4000), and the titers of each subtype were determined according to the above ELISA protocol.

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

2. Detection of cellular immune response

In the preventive model in Example 4, the cellular immune response stimulated by NACD was detected.

1. Intracellular staining of IFN-γ

Spleen lymphocytes were isolated, resuspended and counted, and 3×10 ⁶ spleen lymphocytes/well were plated in 24-well plates. Add 10 μg/ml NY-ESO-1 (DMSO for negative control, 5 μg/ml conA for positive control) to stimulate for 1 h at 37°C, then add Golgiplug (1 μl to 1 ml of cell culture) and incubate for 6-12 h. Cells were then harvested and blocked with CD16/CD32 for 15 min at 4°C. After washing the cells with staining buffer, resuspend the cells, add FITC-anti-mouse-CD3ε, PE-Cy7-anti-mouse CD8α or PE-Cy7-anti-mouse CD4, and incubate at 4°C for 30 min in the dark. Then wash twice with staining buffer, add 250 μl fixation/permeabilization, vortex to suspend well, and incubate at room temperature for 20 min in the dark to fix the permeabilized cells. After washing the cells with BDPerm/wash buffer, resuspend the cells, add intracellular antigen antibody (PE-anti-mouse IFN-γ) and incubate at room temperature for 30 min in the dark. After washing twice with PBS, flow analysis can be performed.

The results showed (Figures 7a and 7b) that the proportion of CD4 ⁺ T cells secreting IFN-γ in the spleen lymphocytes of mice in alum group, alum/DP7 group and alum/CpG group accounted for 0.44% and 0.36% of CD3 ⁺ T cells, respectively. % and 0.47%, while the proportion of CD4 ⁺ T cells secreting IFN-γ in the spleen lymphocytes of mice in the alum/CpG/DP7 group can reach 0.88%, and the proportion of CD3 ⁺ T cells can reach 0.88%. Statistical difference (p<0.05). Analysis of CD8 ⁺ T cells secreting IFN-γ found that the proportion of double positive cells in alum group and alum/DP7 group accounted for 0.52% and 0.79% of CD3 ⁺ T cells, respectively, while the alum/CpG group and alum/CpG/DP7 group accounted for 0.52% and 0.79% of CD3 + T cells, respectively. The proportion of double positive cells in the group was 0.88% and 1.28% of CD3 ⁺ T cells, which were significantly higher than those in other control groups, and compared with NS, there was a statistical difference (p<0.05). It can be seen that CD4 ⁺ T cells and CD8 ⁺ T cells secreting IFN-γ in the spleen lymphocytes of mice in alum/CpG/DP7 group were significantly increased.

2. ELISPOT detection

5×10 ⁵ spleen lymphocytes/well were plated into 96-well plates pre-coated with IFN-γ or IL-4, 10 μg/ml NY-ESO-1 was added, incubated at 37°C for 24 hours, and positive spots were detected according to the instructions.

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

3. Detection of immune memory T cells

The preventive immunization scheme of the preventive model in Example 4 was used. After 3 times of immunization for 2 weeks, the spleen lymphocytes of the mice were isolated, labeled with fluorescent antibodies CD44 and CD62L, and the effector memory T cells and central nervous system were detected by flow cytometry after vaccine immunization. The case of sexual memory T cells.

The results showed (Fig. 8): the proportions of CD4 ⁺ CD44 ⁺ CD62L ^- and CD8 ⁺ CD44 ⁺ CD62L ^- T lymphocytes in the spleen cells of mice in the alum/CpG/DP7 group were 23.56% and 28.15%, respectively, which were higher than those of other groups. group (p<0.05). However, there was no significant change in CD4 ⁺ CD44 ⁺ CD62L ⁺ and CD8 ⁺ CD44 ⁺ CD62L ⁺ T lymphocytes, which may be related to the time point we selected. Effector memory CD4 ⁺ and CD8 ⁺ T lymphocytes were significantly increased in the spleen cells of mice in the alum/CpG/DP7 group after 3 immunizations for 2 weeks. It can be seen that NY-ESO-1/alum/CpG/DP7 vaccine can significantly prolong the immune memory after immunization.

Test Example 7 Preliminary evaluation of the safety of vaccine NY-ESO-1/alum/CpG/DP7

1. Body weight detection and H&E staining

(1) Grouping according to the preventive immunization scheme of Example 4, each group of mice was immunized with the preventive immunization scheme.

(2) During the whole experiment, the weight of the mice was weighed, and the state and daily behavior of the mice were observed for abnormality.

(3) One week after the three immunizations, the heart, liver, spleen, lung, kidney and other important organs of the mice were taken for H&E staining.

In this experiment, it was tested whether the vaccine immunization would have toxic and side effects on the body. In the safety test of the vaccine, the mice were immunized with a preventive immunization scheme, and the mice were sacrificed two weeks after immunization, and the heart, liver, spleen, lung, kidney and other important organs were taken for HE detection. During the whole experiment, the mice did not experience weight loss, standing hair, loss of appetite, abnormal behavior, etc., and there were no obvious abnormalities in the HE staining of important organs (Figures 9a and 9b).

2. Routine blood test

(1) Mice in each group were immunized with a preventive immunization program.

(2) One week after the third immunization, the orbital blood of the mice was collected for blood routine analysis.

In this experiment, it was tested whether the vaccine immunization would have toxic and side effects on the body. In the safety detection test of the vaccine, the mice were immunized with a preventive immunization scheme, and the blood routine of the mice was tested 1 week after the third immunization. The erythrocyte count, platelet count, hemoglobin, hematocrit, mean platelet volume, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, and mean corpuscular volume of the five groups of mice were within the normal range (Figure 10).

3. Blood biochemical test

(1) The mice in each group were immunized with a preventive immunization program.

In this experiment, it was tested whether the vaccine immunization would have toxic and side effects on the body. In the safety detection test of the vaccine, the mice were immunized with a preventive immunization scheme, and the blood biochemistry of the mice was detected after immunization. The total protein of five groups of mice; albumin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatinine, urea, uric acid, glucose, lactate dehydrogenase, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol were all normal value range, and there were no differences in blood biochemistry between the five groups of mice (Figure 11).

Test Example 8 DP7 activates Gi/PKC or PI3k/Erk1/2 signaling pathway

1. RNA-seq analysis of the effect of DP7 on the transcription of BMDCs

On the fifth day after BMDCs were induced with GM-CSF and IL-4, cells were collected and sorted by CD11c magnetic beads. After stimulating the sorted BMDCs with 40 μg/ml DP7 for 30 min (the control group was the same amount of DMSO), the cells were collected, and total RNA was extracted for second-generation high-throughput sequencing.

RESULTS: Bioinformatics analysis revealed a total of 22,954 genes detected in the Control and DP7-treated groups. Compared with the control group, 118 genes were differentially expressed in the DP7 treatment group (expression fold ≥ 2 times). Using the online String: Protein-Protein Interaction Network software, 118 genes were subjected to protein interaction network analysis and showed that they were divided into two main networks. In the whole interaction network, the most important network is further demonstrated-the transcription factor interaction network with Egr1 as the core. There are 11 proteins in the network: Zfp36, Btg2, Atf3, Dusp1, JunB, Ier2, Egr1, Egr2, Egr3 , c-Fos, Fosb and Nr4a1. We selected 6 transcription factors with larger and more important fold changes in the network for further research, including Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1, with up-regulated fold changes of 16.63, 2.40, 15.70, and 6.34, respectively. , 114.79 and 19.81, these six transcription factors are also genes with relatively large changes in RNA-seq analysis.

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

Divide JAWSII cells into 6-well plates, ² *105 cells per well. The cells were stimulated with 40 μg/ml DP7 for 30 min, and the control group was stimulated with the same amount of DMSO. The supernatant medium was removed, and 1 ml of Trizol reagent was added to each well to extract the total RNA of the sample and reverse-transcribe it into cDNA, and quantitatively detect the target gene by RT-PCR.

The results showed (Fig. 12) that the expression of transcription factors in Control was regarded as 1, and the expressions of Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1 were up-regulated by 22.05, 2.79, 15.36, 19.51, 143.80 and 5.63, respectively. Basically consistent with the results of RNA-seq, indicating that the up-regulated six transcription factors play an important role in the role of DP7.

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

Divide JAWSII cells into 6-well plates, with 2*10 ⁵ cells per well; pre-treated with small molecule inhibitors at 37°C for 1-3 h, 40 μg/ml DP7 stimulated cells for 30 min, and the control group was stimulated with the same amount of DMSO. The target gene was quantitatively detected by RT-PCR. The inhibitors and their receptors used are: Selumetinib is a small molecule inhibitor of Erk1/2, pertussis toxin (PTX) is a specific inhibitor of class Gi proteins, AS-605240 is a selective inhibitor of PI3Kγ, and Sotrastaurin is PKCθ inhibitor.

The results showed (Fig. 13a) that after pre-treatment of cells with small molecule inhibitors, the up-regulated expressions of Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1 by DP7 were inhibited, and the difference was statistically significant (p<0.05).

4. DP7 activates Erk1/2 phosphorylation and is inhibited by PTX

Divide BMDCs into 6-well plates with ⁵ *105 cells per well. After pre-treatment of cells with PTX for 3h, cells were stimulated with 40μg/ml DP7 for 30min, and the control group was stimulated with the same amount of DMSO. Remove the supernatant medium, add 100 μl of RIPA lysis buffer to each well, and place on ice for 30 min to fully lyse the cells. After collecting the cell lysate, centrifuge at 13000rpm*10min*4℃. The expressions of Erk1/2 and p-Erk1/2 were detected by western blot.

From the results (Fig. 13b), the phosphorylation of Erk1/2 was significantly up-regulated after DP7 was exposed to cells for 30 min, while the phosphorylation of Erk1/2 was decreased after 60 min of exposure to DP7. Likewise, the upregulation of Erk1/2 phosphorylation by DP7 was attenuated to varying degrees after PTX pretreatment.

5. DP7 can up-regulate the level of intracellular Ga2+.

JAWSII cells were collected, and 1*10 ⁴ cells per well were plated in black 96-well plates and cultured for two days. The supernatant medium was discarded, and the cells were washed once with 100 μl Hank'buffer. 100 μl of Fluo 3-AM (diluted in Hank'buffer) at a final concentration of 5 μM was added to each well and incubated at 37° C. for 30 min. The supernatant was removed and the cells were washed once more with 100 μl of Hank'buffer. Fluorescence was detected by celigo after stimulation with 200 μl medium or medium containing 40 μg/ml DP7 per well.

From the results (Fig. 13c), the mean fluorescence intensity of a single cell was significantly enhanced after 15 min of DP7 stimulation (p<0.05 vs Control), and the mean fluorescence intensity of a single cell continued to increase after 30 min of DP7 stimulation (p < 0.01 vs Control).

Test Example 9 GPR35 is a potential target of DP7

1. DP7 induces GPR35 receptor internalization

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

The results showed (Fig. 14a) that in control, GPR35 was mainly located on the cell membrane, while after DP7 stimulation, GPR35 was internalized into cells.

2. DP7 up-regulates transcription factor expression dependent on GPR35

GPR35 shRNA cells and their control group scrambled shRNA cells were stimulated with 40 μg/ml DP7 for 30 min, samples were collected, total RNA was extracted, and the expressions of β-actin, Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1 were detected by RT-PCR.

The results showed (Fig. 14b) that the relative fold changes of Egr1, Egr2, Egr3, c-Fos, Fosb and Nr4a1 were significantly reduced (p<0.05) in GPR35 silenced cells compared to control scrambled shRNA cells.

3. DP7 up-regulation of Erk1/2 phosphorylation depends on GPR35

GPR35 shRNA cells and their control group scrambled shRNA cells were stimulated with 40 μg/ml DP7 for 30 min, and then the samples were collected for western blot detection of Erk1/2 phosphorylation.

The results showed (Fig. 14c) that, in scrambled shRNA cells, phosphorylation of Erk1/2 was enhanced after the action of DP7 compared with Control. In GPR35 shRNA cells, the phosphorylation of Erk1/2 was almost unchanged after DP7 was treated for 30 min.

4. The mechanism of action of aluminum hydroxide/CpG/DP7 adjuvant in BMDCs or macrophages

The polypeptide DP7 interacts with GPR35 on the cell surface, activates the PI3Kγ, PKCθ and ERK1/2 signaling pathways, induces the production of transcription factors, and ultimately leads to the production of chemokines or cytokines. In addition, the activation of NF-κB by DP7 may also affect the production of chemokines or cytokines. Binding of CpG to TLR9 activates ERK1/2 or NF-κB and promotes the release of inflammatory factors such as IL-6 and TNF-α. Aluminum hydroxide activates the NALP3 inflammasome, which regulates the secretion of proinflammatory cytokines IL-1β and IL-18. Chemokines and cytokines induced by aluminum hydroxide, CpG and DP7 contributed to enhancing the immune effect of complex adjuvants.

sequence listing

<110> Sichuan University

<120> Aluminum hydroxide-CpG oligonucleotide-polypeptide complex adjuvant, vaccine and preparation method and use

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 1

tcgtcgtttt gtcgtttttg tcgtt 25

<210> 3

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 3

Val Gln Trp Arg Ile Arg Val Cys Val Ile Arg Ala

1 5 10

<210> 3

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 3

Val Gln Trp Arg Ile Arg Ile Ala Val Ile Arg Ala

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 4

Val Cys Trp Arg Ile Arg Val Ala Val Ile Arg Ala

1 5 10

<210> 5

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 5

Val Gln Leu Arg Ile Arg Val Cys Val Ile Arg Arg

1 5 10

<210> 6

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 6

Lys Gln Trp Arg Ile Arg Val Ala Val Ile Arg Ala

1 5 10

<210> 7

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 7

Val Gln Trp Arg Ile Arg Val Ala Val Ile Arg Lys

1 5 10

<210> 8

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 8

Val Gln Leu Arg Ile Arg Val Cys Val Ile Arg Lys

1 5 10

<210> 9

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 9

Lys Gln Trp Arg Ile Arg Val Cys Val Ile Arg Ala

1 5 10

<210> 10

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 10

Val Gln Leu Arg Cys Arg Val Cys Val Ile Arg Lys

1 5 10

<210> 11

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 11

Val Gln Trp Arg Ile Arg Ile Ala Val Ile Arg Lys

1 5 10

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.

Images