阅读说明：本技术 含有至少2个硫原子的杂环化合物在制备纳米疫苗中的应用及制得的纳米疫苗 (Application of heterocyclic compound containing at least 2 sulfur atoms in preparation of nano vaccine and prepared nano vaccine ) 是由 杨黄浩 李娟� 张达 刘小龙 于 2020-04-24 设计创作，主要内容包括：本发明涉及免疫治疗或疫苗防治技术领域,尤其涉及含有两个或两个以上硫原子的杂环化合物及其制备纳米疫苗中的应用。本发明提供了含有至少两个硫原子且能够与多肽以共价或非共价连接的杂环化合物在制备纳米疫苗中的应用。该化合物与抗原自组装制得的纳米颗粒能够通过跨膜方式进入树突状细胞质内,提高对抗原及免疫佐剂的摄取效率。由于在进入细胞的过程中,能有效躲避或降低溶酶体内酶体对抗原或核酸佐剂生物降解,故而本发明的纳米疫苗能够高效激活树突状细胞,并提高对抗原的交叉呈递作用,进而有效激活CD8+T细胞,并促进T细胞增殖。因此,本发明的纳米疫苗能够利用高效的免疫激活与免疫调节作用预防肿瘤细胞增殖与病毒感染。(The invention relates to the technical field of immunotherapy or vaccine prevention and treatment, in particular to a heterocyclic compound containing two or more than two sulfur atoms and application thereof in preparation of a nano vaccine. The invention provides application of a heterocyclic compound which contains at least two sulfur atoms and can be connected with a polypeptide in a covalent or non-covalent mode in preparation of a nano vaccine. Nanoparticles prepared by self-assembly of the compound and antigen can enter dendritic cytoplasm in a transmembrane mode, so that the uptake efficiency of the antigen and immune adjuvant is improved. In the process of entering cells, the antigen or nucleic acid adjuvant biodegradation of a lysosome in vivo can be effectively avoided or reduced, so that the nano vaccine disclosed by the invention can efficiently activate dendritic cells and improve the cross presentation effect on the antigen, thereby effectively activating CD8+ T cells and promoting the proliferation of the T cells. Therefore, the nano vaccine can prevent tumor cell proliferation and virus infection by utilizing efficient immune activation and immune regulation.)

1. The application of the compound of the formula I in preparing nano vaccines;

A-L-F

formula I

Wherein A is a heterocyclic group containing two or more S atoms;

l is nothing or an intermediate linking group;

f is a group capable of covalent or non-covalent attachment to the polypeptide.

2. The use according to claim 1, wherein the heterocyclic group in A is a 4-to 8-membered ring.

3. Use according to claim 1 or 2,

l is- (CH)₂)_n-、

Wherein n is 1-5; r is-H, C_1～5Alkyl group of (A) or (B), m＝1～50。

4. Use according to any one of claims 1 to 3, wherein F is:

5. the use according to claim 1,

the A is as follows:

the F is as follows:

l is:

6. the use according to claim 1, wherein the compound of formula I is:

7. a nanoparticle whose starting materials include a compound of formula I, an antigenic peptide;

A-L-F

formula I

Wherein A is a heterocyclic group containing two or more S atoms;

l is nothing or an intermediate linking group;

f is a group capable of covalent or non-covalent attachment to the polypeptide.

8. The nanoparticle according to claim 7, wherein the antigenic peptide is selected from a tumor antigen, a bacterial antigen or a viral antigen.

9. The nanoparticle according to claim 8, wherein the tumor antigen is a tumor neoantigen selected from neoantigens of bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, genitourinary tract cancer, head cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, muscle tissue cancer, neck cancer, oral or nasal mucosa cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer and/or thyroid cancer;

the virus antigen is an influenza virus antigen, an Ebola virus antigen, a coronavirus antigen, a hepatitis virus antigen, a mumps virus antigen, a varicella-zoster virus antigen, a measles virus antigen, a rubella virus antigen, an influenza virus antigen and/or an avian influenza virus antigen;

the bacterial antigen is a combined mycobacterial antigen, a staphylococcal antigen, a streptococcal antigen, a pneumococcal antigen, a bacillus anthracis antigen, a diphtheria antigen, a proteus antigen, a bordetella pertussis antigen, a vibrio cholerae antigen, an meningococcal antigen and/or a typhoid bacillus antigen.

10. The nanoparticle of claim 7, further comprising a nucleic acid adjuvant, wherein the nucleic acid adjuvant is CpG-ODN or Poly I: C.

11. The nanoparticle of any one of claims 7 to 11, wherein the molar ratio of the compound of formula I, the nucleic acid adjuvant and the antigenic peptide is (50-100): (0-1): (20-50).

12. A method for preparing nanoparticles as claimed in any one of claims 7 to 12, comprising: the preparation method of the nano vaccine comprises the following steps: mixing the compound of formula I, the nucleic acid adjuvant and the polypeptide antigen with a TM buffer solution, stirring for 15min at 37 ℃, and then dialyzing in deionized water to prepare a solution containing the nanoparticles.

13. A nano-vaccine comprising the nanoparticle of any one of claims 6 to 11 and an adjuvant acceptable in a vaccine.

14. Use of the nano-vaccine of claim 13 for the preparation of a medicament for the prevention and treatment of tumors, bacterial infections and/or viral infections.

Technical Field

The invention relates to the technical field of immunotherapy or vaccine prevention and treatment, in particular to application of a heterocyclic compound containing two or more than two sulfur atoms in preparation of a nano vaccine and the prepared nano vaccine.

Background

In recent years, the development and development of immune vaccines have made remarkable progress in the comprehensive prevention and treatment of cancer and virus infection, and have higher clinical application value in the aspect of anti-tumor or anti-virus infection. Currently, various anti-tumor or anti-viral vaccines have been developed successively in various countries for disease prevention or treatment and have entered the clinical stage I, II, and some have been approved for clinical use in the united states and europe. However, such vaccines still face some challenges in stimulating antigen presenting cell maturation and enhancing cross presentation, for example, simple mixed adjuvants and antigen preparations are not efficiently taken up by dendritic cells, and the taken-up antigen or adjuvant is more easily degraded by enzyme after entering intracellular lysosome and other organelles, so as to reduce or lose activation of dendritic cells, further weaken the antigen cross presentation effect, and fail to effectively activate the immune system of the body to cope with external or internal tumor or virus infection, and even fail to cope with specific types of tumor or virus infection.

Efficient antigen presentation and activation of the immune system and immunoregulation are mainly dependent on whether antigen presenting cells can efficiently take in antigen or can be effectively activated to perform cross presentation on antigen. The existing nano vaccine mainly enters dendritic cells by an endocytosis-lysosome way, namely after being endocytosed by the dendritic cells, endocytosomes or vesicles are formed and then fused with the intracellular lysosomes, nuclease or proteolytic enzyme in the lysosome can shear or hydrolyze nucleic acid adjuvant or antigen peptide released by the nano vaccine, so that the stimulation and activation of the nucleic acid adjuvant on the dendritic cells are reduced, the cross presentation effect of the dendritic cells on the antigen peptide is influenced, the activation and proliferation promotion effect on immature T lymphocytes are realized, the cellular immune response of an organism to the antigen peptide is weakened, and the tumor immunotherapy effect is poor.

In recent years, liposome mRNA or polypeptide vaccine developed based on liposome embedding technology can reduce the degradation of active ingredients of lysosome in vivo to a certain extent, but still depends on endocytosis-lysosome pathway to be taken into lymphocytes, and can not avoid the degradation effect of lysosome enzyme; and because the polyethylene glycol on the surface of the liposome can effectively resist or block the endocytosis of lymphocytes such as macrophages and the like, the antigen uptake efficiency and the lymphocyte activation ratio are reduced, and the cross presentation effect on the antigen is reduced. Therefore, there is an urgent need to develop a new antigen uptake and presentation mode for efficient lymphocyte activation and cross-presentation efficiency improvement.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide an application of a heterocyclic compound containing a disulfide bond in the preparation of a nano vaccine and the prepared nano vaccine, wherein a thiol compound and a tumor neogenesis polypeptide antigen or a virus polypeptide antigen are prepared and synthesized by a nucleic acid adjuvant through electrostatic force, hydrogen bond and van der waals force, and the nano vaccine can efficiently enter the inside of a dendritic cell cytoplasm through a transmembrane pathway to reduce the degradation of the polypeptide antigen and nucleic acid inside the dendritic cell by protease or nuclease in an lysosome.

The invention provides application of a compound shown in a formula I in preparation of a nano vaccine;

A-L-F

formula I

Wherein A is a heterocyclic group containing two or more S atoms;

l is nothing or an intermediate linking group;

f is a group capable of covalent or non-covalent attachment to the polypeptide.

The compound of the formula I contains at least two sulfur atoms, and can be self-assembled with a nucleic acid adjuvant and a neoantigen peptide to form a nano vaccine. Wherein the F group is connected with the polypeptide, and the A group is subjected to mercaptan exchange with a cell membrane and mediates the direct transmembrane entry of the nano vaccine into dendritic cytoplasm. The mode of entering the cell does not pass through an endocytosis-lysosome route, and after entering the cell, the cell is disintegrated under the action of an electrolyte buffer solution in dendritic cytoplasm to release the nucleic acid adjuvant and the neoantigen peptide in the cell. The released nucleic acid adjuvant can more effectively stimulate dendritic cells to mature, enhance the cross presentation effect of the dendritic cells on the neoantigen peptide, enhance the activation and proliferation promoting capability on immature T lymphocytes, enhance the specific cell immune response of an organism to tumor cells expressed by the neoantigen peptide and enhance the T cell-dependent tumor specific cell immunotherapy.

In the present invention, the heterocyclic group in A is a 4-to 8-membered ring.

In some embodiments, the a is:

in the present invention, L is- (CH)₂)_n-、Wherein n is 1-5; r is-H, C_1～5Alkyl group of (A) or (B),m＝1～50。

In some embodiments, the L is:

in the present invention, the covalent attachment means may be selected from: click chemical reaction and crosslinking reaction; the non-covalent attachment means include: electrostatic interactions, hydrophobic interactions, affinity interactions.

Wherein, the F group that can be attached to the polypeptide by a click chemistry reaction comprises:

f groups that can be attached to a polypeptide by a crosslinking reaction include:

f groups that can form covalent linkages with polypeptides by other reaction means include:

f groups that can be attached to polypeptides by electrostatic interaction include:

f groups that can be attached to a polypeptide by hydrophobic interactions include:

f groups that can be attached to a polypeptide by affinity include:

in some embodiments, F is:

in some embodiments, the A isL is F is

In some embodiments, the compound of formula I is

The compound of formula I provided by the invention can be used for preparing nanoparticles with polypeptides, but experiments show that the compound of formula I with different structures needs to be selected according to different polypeptides, otherwise, the particle size of the formed particles is too large to be taken up by cells.

The invention also provides a nanoparticle, the raw materials of which comprise the compound shown in the formula I, a nucleic acid adjuvant and an antigen peptide;

A-L-F

formula I

Wherein A is a heterocyclic group containing two or more S atoms;

l is nothing or an intermediate linking group;

f is a group capable of covalent or non-covalent attachment to the polypeptide.

In the present invention, the antigenic peptide is selected from a tumor antigen, a bacterial antigen or a viral antigen.

In the present invention, the tumor antigen is a tumor neoantigen selected from neoantigens of bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal tract cancer, genitourinary tract cancer, head cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, muscle tissue cancer, neck cancer, oral or nasal mucosa cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer and/or thyroid cancer;

in the invention, the virus antigen is influenza virus antigen, Ebola virus antigen, coronavirus antigen, hepatitis virus antigen, mumps virus antigen, varicella-zoster virus antigen, measles virus antigen, rubella virus antigen, influenza virus antigen and/or avian influenza virus antigen;

the coronavirus is novel coronavirus COVID-19 and SARS virus; the hepatitis virus is hepatitis A virus, hepatitis B virus or hepatitis C virus; the influenza virus is influenza A virus, influenza B virus or influenza C virus, wherein the type of hemagglutinin protrusion of the influenza A virus comprises H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14 or H15, and the type of neuraminidase protrusion is N1, N2, N3, N4, N5, N6, N7, N8 or N9.

In the present invention, the bacterial antigen is a binding mycobacterial antigen, a staphylococcal antigen, a streptococcal antigen, a pneumococcal antigen, a bacillus anthracis antigen, a diphtheria antigen, a proteus antigen, a bordetella pertussis antigen, a vibrio cholerae antigen, a meningococcal antigen and/or a typhoid bacillus antigen.

In some embodiments, the tumor antigen is a liver cancer antigen, specifically a liver cancer H22 polypeptide antigen, more specifically, its antigenic sequence is HTDAHAQAFAALFDSMH. The virus antigen is a coronavirus antigen, in particular to a novel coronavirus antigen; more specifically, the antigenic sequence is SYYSLLMPI, RYVLMDGSI, AYANSVFNI, TYASALWEI or GYLKLTDNV.

In the examples of the present invention, the antigen has an isoelectric point of < 6.5.

In the embodiment of the invention, the nucleic acid adjuvant is CpG-ODN and Poly I: C.

In the present invention, a Cy3 fluorophore is added to the N-terminus of a polypeptide antigen, and a FAM fluorophore is labeled to the 3' -terminus of a nucleic acid adjuvant. The fluorescent group is used for positioning the nano vaccine in the dendritic cell. And experiments show that whether the fluorescent group is added or not has no obvious influence on the activation effect.

In the embodiment of the invention, in the nanoparticles, the molar ratio of the compound shown in the formula I, the nucleic acid adjuvant and the antigen peptide is (50-100): (0.5-1): 20-50). In some embodiments, the nanoparticle has a molar ratio of the compound of formula I, the nucleic acid adjuvant, and the antigenic peptide of 50:0.5: 20.

The nano-particles are prepared and synthesized by self-assembly through interaction of intermolecular electrostatic force between guanidino in a positively charged compound in the formula I and phosphate radical in a negatively charged nucleic acid adjuvant. Wherein the nucleic acid adjuvant may be CpG-ODN (murine or human) or Poly I: C (murine or human); the isoelectric point (pI) of the neoantigenic peptide is less than 6.

In some embodiments, the method of preparing the nanoparticle comprises: the preparation method of the nano vaccine comprises the following steps: the compound of formula I, nucleic acid adjuvant and polypeptide antigen are mixed with TM buffer (pH 7.4), stirred at 37 deg.C and 100rpm for 15min, and dialyzed in deionized water to obtain solution containing nanoparticles.

The volume ratio of the total volume of the compound shown in the formula I, the nucleic acid adjuvant and the polypeptide antigen solution to the TM buffer solution is 1: 60-70.

In a specific embodiment, a 146.2mM methanol solution of the compound of formula I, a 100mM nucleic acid adjuvant solution, and a 2.5mg/mL polypeptide antigen solution are mixed at a volume ratio of 1:1:1, and then mixed with a TM buffer (pH 7.4) having a volume 60-70 times the total volume of the mixture.

The invention also provides a nano vaccine which comprises the nano particles and acceptable auxiliary materials in the vaccine.

Acceptable auxiliary materials in the vaccine comprise: solvent, osmotic pressure regulator. Preferably, the nano vaccine prepared by the invention comprises the nano particles and sodium chloride injection.

The nano vaccine is absorbed into dendritic cytoplasm in a thiol exchange mediated transmembrane mode, and is disintegrated to release polypeptide antigen and nucleic acid adjuvant due to an intracytoplasmic buffer solution, so that dendritic cells are assisted and efficiently activated, the antigen cross presentation effect of the dendritic cells is improved, and CD8+ T cells are effectively activated.

The invention also provides an anti-tumor method, which is to administer the nano vaccine.

The invention also provides a method for resisting virus and/or cell infection, which is to administer the nano vaccine.

The invention relates to the technical field of immunotherapy or vaccine prevention and treatment, in particular to a heterocyclic compound containing two or more than two sulfur atoms and application thereof in preparation of a nano vaccine. The invention provides application of a heterocyclic compound which contains at least two sulfur atoms and can be connected with a polypeptide in a covalent or non-covalent mode in preparation of a nano vaccine. The compound, nucleic acid adjuvant and antigen are self-assembled to prepare the nano-particles. The nano-particles can enter dendritic cytoplasm through a transmembrane mode, so that the uptake efficiency of the antigen and the immunologic adjuvant is improved. In the process of entering cells, the antigen or nucleic acid adjuvant biodegradation of a lysosome in vivo can be effectively avoided or reduced, so that the nano vaccine disclosed by the invention can efficiently activate dendritic cells and improve the cross presentation effect on the antigen, thereby effectively activating CD8+ T cells and promoting the proliferation of the T cells. Therefore, the nano vaccine can prevent tumor cell proliferation and virus infection by utilizing efficient immune activation and immune regulation.

Drawings

FIG. 1 shows a schematic diagram of the preparation and anti-tumor of a nano vaccine;

FIG. 2 shows the SEM analysis of the nano-vaccine of example 3;

FIG. 3 shows the particle size analysis of the Nanoprotein and control Nanoprotein of example 3

FIG. 4 shows confocal fluorescence imaging images of transmembrane nano-vaccine uptake into dendritic cytoplasm after co-culture of nano-vaccine and dendritic cells for 30min in example 3, wherein red light is polypeptide antigen labeled fluorescence, green light is nucleic acid adjuvant labeled fluorescence, and the scale is 20 μm;

FIG. 5 shows confocal fluorescence imaging images of the uptake of the nano-vaccine into the dendritic cytoplasm across the membrane in example 3, where green light is the lysosome indicator, red light is the polypeptide antigen-labeled fluorescence, and the scale is 20 μm;

FIG. 6 shows confocal fluorescence imaging images of transmembrane nano-vaccine uptake into dendritic cytoplasm after co-culture of nano-vaccine and dendritic cells for 30min in example 4, wherein red light is polypeptide antigen labeled fluorescence, green light is nucleic acid adjuvant labeled fluorescence, and the scale is 10 μm;

FIG. 7 shows confocal fluorescence imaging of the uptake of the nano-vaccine into the dendritic cytoplasm across the membrane in example 4, where green light is the lysosome indicator, red light is the polypeptide antigen-labeled fluorescence, and the scale is 10 μm;

FIG. 8 shows confocal fluorescence imaging images of transmembrane nano-vaccine uptake into dendritic cytoplasm after co-culture of nano-vaccine and dendritic cells for 30min in example 5, wherein red light is polypeptide antigen labeled fluorescence, green light is nucleic acid adjuvant labeled fluorescence, and the scale is 10 μm;

FIG. 9 shows confocal fluorescence imaging of the uptake of the nano-vaccine into the dendritic cytoplasm across the membrane in example 5, where green light is the lysosome indicator, red light is the polypeptide antigen-labeled fluorescence, and the scale is 10 μm;

FIG. 10 shows the activation of dendritic cells after co-culturing the nano-vaccine prepared in example 3 with dendritic cells for 3 d;

FIG. 11 illustrates proliferation of activated dendritic cells after 2d co-culture with CD8+ T cells in the nano-vaccine prepared in example 3;

FIG. 12 shows the mouse tumor growth curve of the nano-vaccine prepared in example 3 for H22 mouse liver cancer anti-tumor effect.

Detailed Description

The invention provides application of a heterocyclic compound containing at least two S atoms in preparation of a nano vaccine and the prepared nano vaccine, and a person skilled in the art can realize the preparation by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

(1) Chemical reagents: synthesis of all nucleic acid adjuvant sequences (see Table 1 for sequence) were prepared by Shanghai Bioengineering Co., Ltd and synthesized using HPLC purification method. LysoTracker and Hoechst 33342 are available from Sammer Feishel technologies, USA. Ultrapure water was prepared by a Milli-Q water purification system (18.2 M.OMEGA.).

(2) Cell lines and cell cultures: murine hepatoma cells H22 were purchased from American Type Culture Collection (ATCC). H22 cells were cultured in DMEM medium (HyClone) supplemented with 10% fetal bovine serum (Gibco) at final concentration and penicillin-streptomycin at 100IU/mL at 37 ℃ with 5% CO₂The culture is performed in the atmosphere of (2).

(3) The required murine nucleic acid adjuvant CpG-ODN is used as an adjuvant for activating dendritic cells, and the base sequence is as follows:

TABLE 1 CpG-ODN base sequence Listing used in the examples

Name (R)	Base sequence (5 '→ 3')
		Fluorescent labeling of CpG-ODN by FAM	TCCATGACGTTCCTGACGTT-FAM
Label-free CpG-ODN	TCCATGACGTTCCTGACGTT

A compound:

compound 1:synthesized in example 1;

compound 2:synthesized in example 2;

compound 3:available from mclin biochemistry technologies, ltd.

The invention is further illustrated by the following examples:

example 1

The dichloromethane solution containing N, N' -carbonyl diimidazole and lipoic acid is gradually dropped into dichloromethane of ethylenediamine at 0 ℃, stirred for 1h at 0 ℃ and then stirred for 1h at room temperature, and water is removed by anhydrous sodium sulfate, and then the mixture is concentrated under reduced pressure to obtain oily matter. The oily substance was dissolved in a dichloromethane solution containing 1H-pyrazole-1-carboxamidine hydrochloride, and after stirring, distillation was performed under reduced pressure, and the obtained precipitate was dissolved in methanol and washed with diethyl ether to finally obtain compound 1.

Example 2

Anhydrous CH of molecule 1₂Cl₂Adding 1, 1-Carbonyl Diimidazole (CDI) into the solution for activation, and then adding molecule 2 for condensation reaction to obtain molecule 3. In CH of molecule 3₂Cl₂Trifluoroacetic acid (TFA) was added to the solution to remove the tert-butoxycarbonyl protecting group to give molecule 4. In the absence of water CH₂Cl₂To this solution, molecule 4, levulinic acid, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N, N-Diisopropylethylamine (DIPEA) were added to obtain molecule 5 by condensation reaction.

Example 3

The preparation method of the nano vaccine comprises the following steps: mixing the compound 1 (the concentration is 146.2mM) prepared in example 1, a 100mM nucleic acid adjuvant (CpG-ODN) and a 2.5mg/mL polypeptide antigen (mouse hepatoma cell H22 polypeptide antigen, the amino acid sequence is HTDAHAQAFAALFDSMH, the N end is connected with a Cy3 label, and the isoelectric point is 5.71) according to the volume ratio of 1:1:1, putting the mixture into a TM buffer solution (pH 7.4) with the total volume 60-70 times of the mixture, mixing and stirring the mixture at 37 ℃ for 15min, dialyzing the mixture in deionized water for 24H, and removing the nucleic acid adjuvant or the polypeptide antigen which is not loaded, thereby finally obtaining the nano vaccine.

After the nano vaccine is prepared by concussion dialysis, the particle size of the nano vaccine is measured, and a scanning electron microscope represents that the nano vaccine is successfully prepared. The results show that the CpG-ODN, the polypeptide antigen and the compound 1 can form spherical nano-vaccines in a TM buffer (pH 7.5) buffer solution, the particle size of the nano-vaccines is about 100nm (shown in figure 2), the concentration of the effective component CpG-ODN is 8.8 mu g/mL, and the concentration of the polypeptide antigen is 6.11 mu g/mL.

Comparative example

The compound 1 (concentration of 146.2mM) prepared in example 1, a 100mM nucleic acid adjuvant (CpG-ODN), and a 2.5mg/mL comparative polypeptide antigen (amino acid sequence of YKYRYLRHGKLR, N-terminal connected with Cy3 label, isoelectric point of 10.55) were mixed according to a volume ratio of 1:1:1, and put into a TM buffer solution (pH 7.4) 60-70 times the total volume, mixed and stirred at 37 ℃ for 15min, and dialyzed in deionized water for 24h, and the unloaded nucleic acid adjuvant or polypeptide antigen was removed, thereby obtaining a comparative nano-vaccine. After the concussion dialysis preparation, the nano vaccine and the contrast vaccine are represented by a nano particle size analyzer, and the result shows that the particle size of the nano vaccine is about 100nm, and the particle size of the contrast nano vaccine is about 600 nm. The difference of the isoelectric points of the polypeptides shows that the size of the formed control nano vaccine is too large, and efficient cell uptake is difficult to realize.

Example 4

The preparation method of the nano vaccine comprises the following steps: mixing the compound 2 (the concentration is 146.2mM) prepared in the example 2, 100mM nucleic acid adjuvant (CpG-ODN), and 2.5mg polypeptide antigen (neocoronavirus antigen polypeptide 1, the amino acid sequence is FYYVWKSYV, the N-terminal is connected with Cy3 marker, and the isoelectric point is 8.43) according to the volume ratio of 1:1:1, putting the mixture into TM buffer solution (pH 7.4) with the total volume of 60-70 times of the mixture, mixing and stirring the mixture at 37 ℃ for 15min, dialyzing the mixture in deionized water for 24h, and removing the nucleic acid adjuvant or the polypeptide antigen which is not loaded, thereby finally obtaining the nano vaccine.

After the nano vaccine is prepared by concussion dialysis, the particle size of the nano vaccine is measured, and a scanning electron microscope represents that the nano vaccine is successfully prepared. Wherein the concentration of the CpG-ODN which is an effective component is 8.8 mug/mL, and the concentration of the polypeptide antigen is 6.11 mug/mL.

Example 5

The preparation method of the nano vaccine comprises the following steps: mixing a compound 3 (the concentration is 146.2mM), a 100mM nucleic acid adjuvant (CpG-ODN) and 2.5mg polypeptide antigen (a new coronavirus polypeptide antigen 2, the amino acid sequence is KYTQLCQYL, the N end is connected with a Cy3 label, and the isoelectric point is 8.5) according to the volume ratio of 1:1:1, putting the mixture into a TM buffer solution (pH 7.4) with the total volume 60-70 times of that of the mixture, mixing and stirring the mixture at 37 ℃ for 15min, dialyzing the mixture in deionized water for 24h, and removing the nucleic acid adjuvant or the polypeptide antigen which is not loaded, thereby finally obtaining the nano vaccine.

After the nano vaccine is prepared by concussion dialysis, the particle size of the nano vaccine is measured, and a scanning electron microscope represents that the nano vaccine is successfully prepared. Wherein the concentration of the CpG-ODN which is an effective component is 8.8 mug/mL, and the concentration of the polypeptide antigen is 6.11 mug/mL.

Example 6

The preparation method of the nano vaccine comprises the following steps: mixing the compound 1 (the concentration is 146.2mM) prepared in the example 1, 100mM nucleic acid adjuvant (CpG-ODN), and 2.5mg polypeptide antigen (neocoronavirus polypeptide antigen 3, the amino acid sequence is SYYSLLMPI, and the isoelectric point is 5.55) according to the volume ratio of 1:1:1, putting the mixture into TM buffer solution (pH 7.4) with the total volume of 60-70 times of the mixture, mixing and stirring the mixture at 37 ℃ for 15min, dialyzing the mixture in deionized water for 24h, and removing the nucleic acid adjuvant or the polypeptide antigen which is not loaded, thereby finally obtaining the nano vaccine.

After the nano vaccine is prepared by concussion dialysis, the particle size of the nano vaccine is measured, and a scanning electron microscope represents that the nano vaccine is successfully prepared. Wherein the concentration of the CpG-ODN which is an effective component is 7.8 mu g/mL, and the concentration of the polypeptide antigen is 7.41 mu g/mL.

Example 7

The preparation method of the nano vaccine comprises the following steps: mixing the compound 1 (the concentration is 146.2mM) prepared in the example 1, 100mM nucleic acid adjuvant (CpG-ODN), and 2.5mg polypeptide antigen (the polypeptide antigen 4 of the new coronavirus, the amino acid sequence is RYVLMDGSI, and the isoelectric point is 6.21) according to the volume ratio of 1:1:1, putting the mixture into TM buffer solution (pH 7.4) with the total volume of 60-70 times of the mixture, mixing and stirring the mixture at 37 ℃ for 15min, dialyzing the mixture in deionized water for 24h, and removing the nucleic acid adjuvant or the polypeptide antigen which is not loaded, thereby finally obtaining the nano vaccine.

After the nano vaccine is prepared by concussion dialysis, the particle size of the nano vaccine is measured, and a scanning electron microscope represents that the nano vaccine is successfully prepared. Wherein the concentration of the CpG-ODN which is an effective component is 7.1 mu g/mL, and the concentration of the polypeptide antigen is 5.82 mu g/mL.

Example 8

The preparation method of the nano vaccine comprises the following steps: mixing the compound 1 (the concentration is 146.2mM) prepared in the example 1, 100mM nucleic acid adjuvant (CpG-ODN), and 2.5mg polypeptide antigen (neocoronavirus polypeptide antigen 5, the amino acid sequence is AYANSVFNI, and the isoelectric point is 5.55) according to the volume ratio of 1:1:1, putting the mixture into TM buffer solution (pH 7.4) with the total volume of 60-70 times of the mixture, mixing and stirring the mixture at 37 ℃ for 15min, dialyzing the mixture in deionized water for 24h, and removing the nucleic acid adjuvant or the polypeptide antigen which is not loaded, thereby finally obtaining the nano vaccine.

After the nano vaccine is prepared by concussion dialysis, the particle size of the nano vaccine is measured, and a scanning electron microscope represents that the nano vaccine is successfully prepared. The concentration of the effective component CpG-ODN is 9.1 mug/mL, and the concentration of the polypeptide antigen is 7.21 mug/mL.

Example 9

The preparation method of the nano vaccine comprises the following steps: mixing the compound 1 (the concentration is 146.2mM) prepared in the example 1, 100mM nucleic acid adjuvant (CpG-ODN), and 2.5mg polypeptide antigen (the polypeptide antigen 6 of the new coronavirus, the amino acid sequence is TYASALWEI, and the isoelectric point is 3.75) according to the volume ratio of 1:1:1, putting the mixture into TM buffer solution (pH 7.4) with the total volume of 60-70 times of the mixture, mixing and stirring the mixture at 37 ℃ for 15min, dialyzing the mixture in deionized water for 24h, and removing the nucleic acid adjuvant or the polypeptide antigen which is not loaded, thereby finally obtaining the nano vaccine.

After the nano vaccine is prepared by concussion dialysis, the particle size of the nano vaccine is measured, and a scanning electron microscope represents that the nano vaccine is successfully prepared. Wherein the concentration of the CpG-ODN which is an effective component is 7.1 mu g/mL, and the concentration of the polypeptide antigen is 7.67 mu g/mL.

Example 10

The preparation method of the nano vaccine comprises the following steps: mixing the compound 1 (the concentration is 146.2mM) prepared in the example 1, 100mM nucleic acid adjuvant (CpG-ODN), and 2.5mg polypeptide antigen (neocoronavirus polypeptide antigen 7, the amino acid sequence is GYLKLTDNV, and the isoelectric point is 6.21) according to the volume ratio of 1:1:1, putting the mixture into TM buffer solution (pH 7.4) with the total volume of 60-70 times of the mixture, mixing and stirring the mixture at 37 ℃ for 15min, dialyzing the mixture in deionized water for 24h, and removing the nucleic acid adjuvant or the polypeptide antigen which is not loaded, thereby finally obtaining the nano vaccine.

After the nano vaccine is prepared by concussion dialysis, the particle size of the nano vaccine is measured, and a scanning electron microscope represents that the nano vaccine is successfully prepared. . Wherein the concentration of the CpG-ODN which is an effective component is 9.12 mu g/mL, and the concentration of the polypeptide antigen is 7.14 mu g/mL.

Effect verification

A. Laser confocal fluorescence imaging analysis of transmembrane entry of nano vaccine into dendritic cells:

the nano vaccine (example 3-10) prepared by using CpG-ODN labeled with FAM and Cy3 respectively and polypeptide antigen and immature dendritic cells are co-cultured in a confocal culture dish for 30min, and then Hoechst 33342 is used for dyeing and laser confocal microscope detection to observe the subcellular localization.

The nano vaccine and the dendritic cells are co-cultured for 30min, and the nano vaccine can rapidly enter the dendritic cells, wherein the effect of the nano vaccine entering the cells in example 3 is shown in figure 3. In order to further verify that the pathway of the vaccine entering the cell is a thiol exchange-mediated transmembrane mode rather than an endocytosis pathway, the LysoTracker lysosome green dye is used for effectively distinguishing the red fluorescent nano vaccine from the LysoTracker lysosome dye, so that the nano vaccine can enter the cell through the thiol exchange transmembrane and non-endocytosis pathway, and the degradation risk of the lysosome on CpG-ODN and polypeptide antigen is obviously reduced (figure 4).

In addition, the effect of the nano vaccine in the example 4 entering the cells is shown in figures 5-6; the effect of the nano vaccine entering cells in example 5 is shown in FIGS. 7-8.

B. The nano vaccine can efficiently activate dendritic cells to mature:

dendritic cells were seeded in 6-well plates and co-cultured with PBS buffer, CpG-ODN alone, polypeptide antigen, CpG-ODN and polypeptide antigen mixed solution, nano-vaccine (example 3) at 37 ℃ for 3d, with LPS as positive control. Then stained with CD11c, CD80, CD86 fluorescent-labeled antibodies and phenotyped using flow cytometry. Analysis shows that the nano vaccine treatment group can effectively activate DC cell maturation, the maturation degree of the nano vaccine treatment group is obviously higher than that of other treatment groups (PBS, single CpG-ODN, polypeptide antigen, mixed solution of CpG-ODN and polypeptide antigen), and LPS is a positive control group (as shown in figure 9).

C. The dendritic cells after the high-efficiency activation of the nano vaccine can effectively activate T cells to mature and proliferate:

to demonstrate that the nano-vaccine (example 3) can efficiently activate T cell-dependent immune response, enhance T cell maturation and cell proliferation, dendritic cells from different treatment groups in experiment B above were co-cultured with immature mouse primary T cells inoculated in 6-well plates for 2d, and T cells stimulated by dendritic cells were stained with CD8 fluorescent-labeled antibody and phenotyped by flow cytometry. In addition, the proliferation of CD8+ T cells was evaluated by stimulating T cells via dendritic cells using primarily CFSE dye labeling, and co-culturing was continued for 2d using flow cytometry for quantitative fluorescence analysis. The results show that the dendritic cells treated by the nano vaccine can effectively stimulate the maturation of T cells and effectively promote the proliferation of the T cells, and the left fluorescence intensity is enhanced (P2) (as shown in figure 10).

D. The mice immunized by the nano vaccine can effectively prevent tumors

To prove that the mice immunized by the nano vaccine synthesized in example 3 can prevent tumors, the PBS buffer solution, the single CpG-ODN, the polypeptide antigen, the mixed solution of the CpG-ODN and the polypeptide antigen and the nano vaccine are injected subcutaneously into a BCLB/c male mouse (5 weeks old), the immunization is performed once every 5 days, after 5 times of total immunization, the H22 tumor is transplanted into the mice immunized by the PBS or the nano vaccine, the tumor size is measured once every 3 days, and a tumor growth curve is drawn. As shown in fig. 11, the tumor growth of the mice treated with the nano-vaccine was significantly transplanted, and the transplantation efficiency was higher than that of the other treatment groups. The nano vaccine is proved to be capable of inhibiting the growth of H22 liver cancer tumor more efficiently, and provides research basis for the prevention and treatment of tumor immunity.

Therefore, the nano vaccine has good anti-tumor activity and good clinical transformation and application prospects in the aspect of tumor immunotherapy.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

<110> Fuzhou university

<120> application of heterocyclic compound containing at least 2 sulfur atoms in preparation of nano vaccine and prepared nano vaccine

<130> MP2006389

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

His Thr Asp Ala His Ala Gln Ala Phe Ala Ala Leu Phe Asp Ser Met

1 5 10 15

His

<210> 2

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu Arg

1 5 10

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Phe Tyr Tyr Val Trp Lys Ser Tyr Val

1 5

<210> 4

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Lys Tyr Thr Gln Leu Cys Gln Tyr Leu

1 5

<210> 5

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ser Tyr Tyr Ser Leu Leu Met Pro Ile

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Arg Tyr Val Leu Met Asp Gly Ser Ile

1 5

<210> 7

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ala Tyr Ala Asn Ser Val Phe Asn Ile

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Thr Tyr Ala Ser Ala Leu Trp Glu Ile

1 5

<210> 9

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gly Tyr Leu Lys Leu Thr Asp Asn Val

1 5

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tccatgacgt tcctgacgtt 20