Toll-like receptor 4 immune agonist and application thereof, antiviral vaccine molecule and preparation method and application thereof

Abstract

The present invention relates to the interdisciplinary field of pharmaceutical chemistry and immunology, and discloses a Toll-like receptor 4 immunostimulator and its application, an antiviral vaccine molecule, and its preparation method and application. The Toll-like receptor 4 immunostimulator is a compound represented by formula (1) and a pharmaceutically acceptable salt thereof, wherein m is an integer of 0-21, n is 0 or 1, o is an integer of 0-10, and p is an integer of 1-25; X and Y are each independently an oxygen element, a nitrogen element, or a carbon element; D is an oxygen element or a sulfur element; and any two of A, B, and C are ‑CH2OH, and the remaining one is ‑CH2NH2, ‑COOH, ‑CH2OH, ‑CH2SH, ‑CF3, ‑CHF2, ‑CH2F, ‑CH2OCH3, ‑CH2NH‑, ‑CH2N3‑, ‑CH2O‑, ‑CH2S‑, or ‑C(O)‑. The Toll-like receptor 4 immunostimulator of the present invention can be used as a vaccine adjuvant, and the antiviral vaccine molecule prepared has a simple structure and is easy to prepare and store.

Description

Technical Field

[0001] The present invention relates to the interdisciplinary field of medicinal chemistry and immunology, and specifically to a Toll-like receptor 4 immune agonist and application thereof, an antiviral vaccine molecule and a preparation method and application thereof. Background Art

[0002] Adjuvants are an almost indispensable component of modern vaccines. Their rational use is an important factor in improving the immunogenicity of antigens, reducing the amount of antigens used, reducing the number of immunizations, and inducing efficient and long-term specific immunity. Lipopolysaccharide (LPS) is a component of the Gram-negative cell wall and a natural ligand for the TLR4 receptor. Its main active ingredient is lipid A (Lipid A). Lipid A can induce a strong immune response, but its high toxicity limits its use as an adjuvant. Chemically modified monophosphoryl lipid A (MPLA) retains the immune activity of the parent molecule, and preliminary toxicological analysis has demonstrated its low toxicity. Currently, MPLA has been approved for use in a variety of vaccines.

[0003]

[0004] Lipid A binds to Toll-like receptor 4 (TLR4), inducing antigen-presenting cells (APCs) to produce cytokines such as interferon-γ (IFN-γ), interleukin-6 (IL-6), and interleukin-12 (IL-12), promoting a Th1-biased immune response. This response plays a crucial role in killing endogenous pathogens, including tumor cells. The complex synthesis of lipid A and its low yield limit its large-scale application in vaccines and related drugs. To address this, researchers have developed simpler lipid A derivatives, such as PET-A and CRX-527. Therefore, the development of simpler synthetic routes or derivative molecules, while maintaining the adjuvant's effectiveness, is highly urgent and necessary.

[0005]

[0006] Previous patents disclosed PET-A and its derivatives, including a series of derivatives with modified side chains, but did not include glucosamine aminoalkyl 4-phosphate (GAP) compounds and their salt derivatives and their use in medicine.

[0007] At the same time, because immune adjuvants can act as "danger signals" to stimulate a strong immune response in the body, they are often mixed or coupled with different types of antigens to enhance the immunogenicity of the antigens. Many studies have shown that covalent coupling of adjuvants with different types of antigens can significantly enhance the immune response. This is because, compared to the physical mixing of adjuvants and antigens, covalent coupling allows the adjuvant molecules and antigens to be delivered together to the same antigen-presenting cell, thereby enhancing the efficiency of the immune response.

[0008] However, for many antigenic proteins, when linked to lipid molecule adjuvants such as lipid A, the solubility of the resulting conjugate decreases sharply as the number of coupled lipid molecules increases, thus limiting the preparation and application of "lipid molecule-protein" vaccines. Some "lipid molecule-protein" related studies have used methods such as linking short peptides or hydrophilic polyethylene glycol chains to aid solubility, but this also increases the difficulty of synthesizing vaccine molecules. Therefore, it is very necessary to explore a vaccine molecule design strategy with a simple structure and efficient immune performance. Summary of the Invention

[0009] The purpose of the present invention is to overcome the shortcomings of the existing lipid A in preparation, the poor antigen immunogenicity of the existing technology, and the difficulty in preparing "lipid molecule-protein" vaccines, and to provide Toll-like receptor 4 immune agonists and their applications, antiviral vaccine molecules and their preparation methods and applications.

[0010] In order to achieve the above-mentioned object, the first aspect of the present invention provides a Toll-like receptor 4 immunoagonist, wherein the Toll-like receptor 4 immunoagonist is a compound represented by formula (1) and a pharmaceutically acceptable salt thereof.

[0011]

[0012] Wherein, the structure of R in formula (1) is as follows,

[0013]

[0014] wherein m is an integer from 0 to 21, n is 0 or 1, o is an integer from 0 to 10, and p is an integer from 1 to 25;

[0015] X and Y are each independently oxygen, nitrogen or carbon;

[0016] D is oxygen or sulfur;

[0017] Any two of A, B, and C are -CH2OH, and the other one is -CH2NH2, -COOH, -CH2OH, -CH2SH, -CF3, -CHF2, -CH2F, -CH2OCH3, -CH2NH-, -CH2N3-, -CH2O-, -CH2S-, or -C(O)-.

[0018] The second aspect of the present invention provides an application of the above-mentioned Toll-like receptor 4 immune agonist in the preparation of vaccines, cell activators for in vitro activation, expansion and differentiation of immune cells, immune targeted drugs, pharmaceutical preparations combined or coupled with chemotherapy drugs, pharmaceutical preparations for antagonists of autoimmune diseases and immunomodulators for allergic diseases.

[0019] The third aspect of the present invention provides an antiviral vaccine molecule, which is a two-component conjugate comprising a Toll-like receptor 4 immune agonist and a protein antigen, wherein the Toll-like receptor 4 immune agonist is covalently linked to the N-terminal amino acid of the protein antigen.

[0020] Or the Toll-like receptor 4 immune agonist is covalently linked to the N-terminal amino acid of the protein antigen via a covalent linker arm;

[0021] Wherein, the Toll-like receptor 4 immune agonist is a compound represented by formula (1) and a pharmaceutically acceptable salt thereof,

[0022]

[0023] Wherein, the structure of R in formula (1) is as follows,

[0024]

[0025] wherein m is an integer from 0 to 21, n is 0 or 1, o is an integer from 0 to 10, and p is an integer from 1 to 25;

[0026] X and Y are each independently oxygen, nitrogen or carbon;

[0027] D is oxygen or sulfur;

[0028] Any two of A, B, and C are -CH2OH, and the remaining one serves as a connection site, and the structure of the connection site is -CH2NH-, -CH2N3-, -CH2O-, -CH2S-, or -C(O)-.

[0029] A fourth aspect of the present invention provides a method for preparing the above-mentioned antiviral vaccine molecule, the method comprising:

[0030] The α-NH2 at the N-terminus of the protein antigen is converted into an α-ketoamide through a transamination reaction, and the α-ketoamide is reacted with an alkoxyamine reagent to generate an oxime, and then the Toll-like receptor 4 immunoagonist is linked to the oxime; or

[0031] Converting the α-NH2 at the N-terminus of the protein antigen into α-ketoamide through a transamination reaction, and reacting the α-ketoamide with a Toll-like receptor 4 immunoagonist derivative modified with an alkoxyamine reagent to generate an oxime; or

[0032] The α-NH2 at the N-terminus of the protein antigen is converted to an α-ketoamide by a transamination reaction; the α-ketoamide is reacted with an alkoxyamine reagent to generate an oxime, and then the oxime is reduced to a secondary amine using sodium cyanoborohydride, and then the Toll-like receptor 4 immunoagonist is linked to the secondary amine; or

[0033] The α-NH2 at the N-terminus of the protein antigen is converted into an α-ketoamide through a transamination reaction; the α-ketoamide is reacted with a hydrazine reagent to generate a hydrazone, and then a Toll-like receptor 4 immunoagonist is linked to the hydrazone; or

[0034] The α-NH2 at the N-terminus of the protein antigen is converted into α-ketoamide through a transamination reaction, and the α-ketoamide is reacted with a Toll-like receptor 4 immunoagonist derivative modified with a hydrazine reagent to generate a hydrazone;

[0035] Wherein, the transamination reaction of the N-terminal amino acid of the protein antigen is completed by mediating pyridoxal phosphate.

[0036] The fifth aspect of the present invention provides a use of the above-mentioned antiviral vaccine molecule in the preparation of an antiviral vaccine.

[0037] The present invention is the first in the vaccine field to use pyridoxal phosphate (PLP)-mediated protein N-terminal amino acid transamination reaction to prepare a site-specific covalently coupled "lipid molecule-protein" subunit vaccine. This reaction can specifically oxidize the protein N-terminal amino acid to an aldehyde or ketone, and then react with an alkoxyamine to generate the corresponding oxime product, thereby site-specifically coupling the alkoxyamine to the terminal amino acid of the protein. Therefore, when the N-terminus of the protein is an amino acid that can be efficiently converted into an oxime, this reaction can be used to obtain the corresponding protein oxime product under mild conditions, thereby achieving site-specific covalent modification of the protein. After the adjuvant molecule is modified, the present invention uses this transamination reaction to site-specifically covalently link it to the antigen protein. While controlling the connection of only one lipid adjuvant molecule to ensure water solubility, it will not affect the structure and function of the immunogenic epitope on the antigen protein. This vaccine design strategy is applicable to antigen proteins from various sources and various types of lipid adjuvants. The vaccine molecule has a simple structure and is easy to prepare and store. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the cytokine level in the serum of mice in Test Example 1;

[0039] Figure 2 is the IgG antibody titer in the mouse serum in Test Example 1;

[0040] Figure 3 is the IgG subtype antibody titer of mouse serum in Test Example 1;

[0041] Figure 4 is the number of interferon-γ spots produced by T cells in the spleen of the mouse in Test Example 1;

[0042] Figure 5 is the proportion of CD4+ T cells producing interferon-γ and tumor necrosis factor-α cytokines in the spleen of the mouse in Test Example 1;

[0043] Figure 6 is the proportion of CD8+ T cells producing interferon-γ and tumor necrosis factor-α cytokines in the spleen of the mouse in Test Example 1;

[0044] Figure 7 is the level of interleukin-6 and α-tumor necrosis factor cytokine secretion by bone marrow dendritic cells before and after TAK242 treatment in Test Example 1;

[0045] Figure 8 is the matrix-assisted laser desorption ionization time-of-flight mass spectrometry data of the conjugate vaccine in Example 2;

[0046] Figure 9 is the ratio of dendritic cells activated in the spleen and lymph nodes of mice in Test Example 2;

[0047] Figure 10 is the activation ratio of mouse bone marrow-derived dendritic cells and macrophages in Test Example 2;

[0048] Figure 11 is the IgG antibody titer in the mouse serum in Test Example 2;

[0049] Figure 12 is the IgG antibody subtype titer in the mouse serum in Test Example 2;

[0050] Figure 13 The titer of antibodies against SARS-CoV-2 pseudovirus and variants in mouse serum in Test Example 2;

[0051] Figure 14 is the number of interferon-γ spots produced by T cells in the spleen of the mouse in Test Example 2;

[0052] Figure 15 is the ratio of CD8+ and CD4+ T cells producing interferon-γ and tumor necrosis factor-α cytokines in the spleen of the mouse in Test Example 2;

[0053] Figure 16The ratio of CD8+ and CD4+ T cells to central memory T cells in the spleen of the mouse in Test Example 2;

[0054] Figure 17 is the activation level of central memory T cells of CD8+ and CD4+ T cells in the lymph nodes of mice in Test Example 2;

[0055] Figure 18 is the content of glutamate transaminase, aspartate transaminase, alkaline phosphatase and urea in the serum of the mice in Test Example 2;

[0056] Figure 19 This is a tissue section of the important organ of the mouse in Test Example 2. DETAILED DESCRIPTION

[0057] The following describes the specific embodiments of the present invention in detail with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0058] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0059] The first aspect of the present invention provides a Toll-like receptor 4 immunoagonist, wherein the Toll-like receptor 4 immunoagonist is a compound represented by formula (1) and a pharmaceutically acceptable salt thereof.

[0060]

[0061] Wherein, the structure of R in formula (1) is as follows,

[0062]

[0063] wherein m is an integer from 0 to 21, n is 0 or 1, o is an integer from 0 to 10, and p is an integer from 1 to 25;

[0064] X and Y are each independently oxygen, nitrogen or carbon;

[0065] D is oxygen or sulfur;

[0066] Any two of A, B, and C are -CH2OH, and the other one is -CH2OH, -CH2NH2, -COOH, -CH2SH, -CF3, -CHF2, -CH2F, -CH2OCH3, -CH2NH-, -CH2N3-, -CH2O-, -CH2S-, or -C(O)-.

[0067] In the Toll-like receptor 4 immunoagonist described in the present invention, preferably, B and C are both -CH2OH, A is -CH2OH, -CH2NH2, -COOH, -CH2SH, -CF3, -CHF2, -CH2F, -CH2NH-, -CH2N3-, -CH2O-, -CH2S- or -C(O)-, and more preferably, A, B and C are all -CH2OH.

[0068] The Toll-like receptor 4 immunoagonist of the present invention may be a compound represented by formula (1) and a pharmaceutically acceptable salt thereof.

[0069] The Toll-like receptor 4 immune agonist of the present invention belongs to GAP (Glucosaminide Aminoalkyl4-Phosphates, glucosamine aminoalkyl 4-phosphate).

[0070] In a preferred embodiment, the structural formula of the Toll-like receptor 4 immune agonist is:

[0071]

[0072] Further preferably, the structural formula of the Toll-like receptor 4 immune agonist is:

[0073]

[0074] The Toll-like receptor 4 immune agonist of the present invention can improve the immune capacity of humans or animals.

[0075] The second aspect of the present invention provides an application of the above-mentioned Toll-like receptor 4 immune agonist in the preparation of vaccines, cell activators for in vitro activation, expansion and differentiation of immune cells, immune targeted drugs, pharmaceutical preparations combined or coupled with chemotherapy drugs, pharmaceutical preparations for antagonists of autoimmune diseases and immunomodulators for allergic diseases.

[0076] Preferably, the vaccine is a semi-synthetic vaccine, a fully synthetic sugar vaccine, a peptide vaccine, a glycopeptide vaccine, a subunit vaccine, a nucleic acid vaccine, a virus-like particle vaccine, an inactivated vaccine or an attenuated vaccine.

[0077] When the Toll-like receptor 4 immunoagonist of the present invention is used to prepare a vaccine, the vaccine may contain a single component or a combined component of the Toll-like receptor 4 immunoagonist and its derivatives (such as an aluminum salt, zinc salt or manganese salt adjuvant adsorption preparation, a combined or coupled preparation with other immunoagonists, or the molecule and derivative contained in a carrier such as a liposome).

[0078] Preferably, the immune cells are DC cells, NK cells, NKT cells, macrophages or CAR-T cells.

[0079] When the Toll-like receptor 4 immunoagonist of the present invention is used to prepare an immune-targeted drug, the Toll-like receptor 4 immunoagonist is coupled with a targeted conjugated drug to obtain the immune-targeted drug.

[0080] In a preferred embodiment, the targeted conjugated drug is a monoclonal antibody or an immune agonist.

[0081] In a preferred embodiment, the monoclonal antibody is (Glaxo-Welcome), (IDEC / Genentech / Hoffman la Roche)、 (Wyeth), (Millennium), (IDEC and Schering AG), (Corixa / GSK), (Imclone / BMS), (Genentech) or (Genentech / Hoffman la Roche).

[0082] In a preferred embodiment, the immune agonist is a Toll-like receptor 1-10 agonist, a NOD receptor agonist, a lectin receptor agonist or a STING agonist.

[0083] Preferably, the chemotherapy drugs are gemcitabine, methotrexate, vincristine doxorubicin, cisplatin, non-sugar-containing chloroethyl nitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, paclitaxel, fragillin, meglumine GLA, valrubicin, nitrosocarb and polyphenylpropion, RAS farnesyl transferase inhibitors, farnesyl transferase inhibitors, melphalan and prednisone (MP), pemetrexed disodium (MTA / LY231514), lometrexol (LY264618), lometrexol, glatiramer, acetaminophen ... Acyldinaline (CI-994), methionine aminopeptidase-2 inhibitor (TNP-470), midostaurin (PKC412), valsputa (PSC833), mitoxantrone hydrochloride (Novantrone), batimastat, E7070, troxacitabine (BCH-4556), sapacitabine (CS-682), 9-anthracenecarboxylic acid (9-AC), prinomastat (AG3340), biricodar (VX-710), tegafur (UFT™), levamisole (Ergamisol), irinotecan Tumod / Relite, Cladribine Paclitaxel (Paxex), liposomal doxorubicin Kailai / doxorubicin liposome, fludarabine Epirubicin / Epirubicin, Cytarabine Bis-naphthalimide (LU79553), dolastatin (LU103793), liposomal ketidase / doxorubicin, ifosfamide Mesna Niposide Carbon Platinum Plantinol / cisplatin, Vepeside / Etoposide, Docetaxel Guanine arabinoside prodrugs, taxane analogs, nitrosoureas, alkylating agents such as phenylalanine mustard and cyclophosphamide, aminoglutethimide, asparaginase, busulfan, carboplatin, chlorambucil, cytarabine hydrochloride, dactinomycin, daunorubicin hydrochloride, estramustine phosphate, etoposide (VP16-213), floxuridine, fluorouracil (5-FU), flutamide, hydroxyurea (hydroxyurea), ifosfamide, lomustine (CCNU), mechlorethamine hydrochloride (nitrogen mustard), mercaptopurine, mesna, mitotane (o- DDD (o, p'-DDD), mitoxantrone hydrochloride, octreotide, plicamycin, procarbazine hydrochloride, streptozocin, tamoxifen citrate, thioguanine thiotepa, vinblastine, amsacrine (m-AMSA), azacitidine erythropoietin, hexamethylmelamine (HMM), interleukin-2, mitoguanidine (methyl-GAG), methylglycyrrhizin (MGBG), pentostatin (2'-deoxycoformycin), semustine (methyl-CCNU), teniposide (VM-26), or vindesine sulfate.

[0084] Preferably, the autoimmune disease is chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis or ulcerative colitis.

[0085] Preferably, the allergic disease is allergic rhinitis, allergic asthma, allergic dermatitis or allergic urticaria.

[0086] Preferably, when the Toll-like receptor 4 immunoagonist of the present invention is used to prepare a pharmaceutical preparation, the pharmaceutical preparation is a solid preparation, a liquid preparation, a powder preparation or an inhalation preparation.

[0087] The Toll-like receptor 4 immune agonist of the present invention can activate antigen-presenting cells and stimulate the production of cytokines, thereby increasing the antibody level in the immunized animal.

[0088] The third aspect of the present invention provides an antiviral vaccine molecule, which is a two-component conjugate comprising a Toll-like receptor 4 immune agonist and a protein antigen, wherein the Toll-like receptor 4 immune agonist is covalently linked to the N-terminal amino acid of the protein antigen.

[0089] Or the Toll-like receptor 4 immune agonist is covalently linked to the N-terminal amino acid of the protein antigen via a covalent linker arm;

[0090] Wherein, the Toll-like receptor 4 immune agonist is a compound represented by formula (1) and a pharmaceutically acceptable salt thereof,

[0091]

[0092] Wherein, the structure of R in formula (1) is as follows,

[0093]

[0094] wherein m is an integer from 0 to 21, n is 0 or 1, o is an integer from 0 to 10, and p is an integer from 1 to 25;

[0095] X and Y are each independently oxygen, nitrogen or carbon;

[0096] D is oxygen or sulfur;

[0097] Any two of A, B, and C are -CH2OH, and the remaining one serves as a connection site, and the structure of the connection site is -CH2O-, -CH2NH-, -CH2N3-, -CH2S-, or -C(O)-.

[0098] In the antiviral vaccine molecule of the present invention, in a preferred embodiment, B and C are both -CH2OH, and the structure of A is -CH2O-.

[0099] In the antiviral vaccine molecule of the present invention, the Toll-like receptor 4 immunoagonist contained therein may be a compound represented by formula (1) and a pharmaceutically acceptable salt thereof. In the antiviral vaccine molecule of the present invention, in a preferred case, the structural formula of the Toll-like receptor 4 immunoagonist is:

[0100]

[0101] The antiviral vaccine molecule described in the present invention is a site-specific covalently coupled "lipid molecule-protein" subunit vaccine molecule, which is easy to prepare and has a strong immune effect.

[0102] In the antiviral vaccine molecule of the present invention, the protein antigen is bovine serum albumin, tetanus toxoid, diphtheria toxoid, group B meningococcal outer membrane protein complex, pertussis toxoid, diphtheria toxin mutant, respiratory syncytial virus adhesion protein, fusion protein, influenza A virus membrane protein, rotavirus structural protein, filovirus structural protein and coronavirus structural protein; or

[0103] Bovine serum albumin, tetanus toxoid, diphtheria toxoid, serogroup B meningococcal outer membrane protein complex, pertussis toxoid, diphtheria toxin mutant, respiratory syncytial virus adhesion protein, fusion protein, influenza A virus membrane protein, rotavirus structural protein, filovirus structural protein, subunit of coronavirus structural protein; or

[0104] Modified derivatives of bovine serum albumin, tetanus toxoid, diphtheria toxoid, group B meningococcal outer membrane protein complex, pertussis toxoid, diphtheria toxin mutants, respiratory syncytial virus adhesion protein, fusion protein, influenza A virus membrane protein, rotavirus structural protein, filovirus structural protein and coronavirus structural protein.

[0105] Preferably, the coronavirus structural protein is Middle East Respiratory Syndrome Coronavirus, SARS or 2019 Novel Coronavirus structural protein.

[0106] Preferably, the 2019 novel coronavirus structural protein is a nucleocapsid protein, a spike protein, or a subunit of a nucleocapsid protein or a spike protein.

[0107] Preferably, the structure of the covalent linker arm is

[0108]

[0109]

[0110]

[0111] -CO-, -O-CO-, -NH-CO-, -NH(C=NH)-, -SO2-, -O-SO2-, -NH-, -NH-CO-CH2-, -CH2-, -C2H4-, -C3H6-, -C4H8-, -C5H 10 -、-C6H 12 -、-C7H 14 -、-C8H 16 -、-C9H 18 -、-C 10 H 20 -, -CH(CH3)-, -C[(CH3)2]-, -CH2-CH(CH3)-, -CH(CH3)-CH2-, -CH(CH3)-C2H4-, -CH2-CH(CH3)-CH2-, -C2H4-CH(C H3)-, -CH2-C[(CH3)2]-, -C[(CH3)2]-CH2-, -CH(CH3)-CH(CH3)-, -C[(C2H5)(CH3)]-, -CH(C3H7)-, -(CH2-CH2-O) q-CH2-CH2-, -CO-CH2-, -CO-C2H4-, -CO-C3H6-, -CO-C4H8-, -CO-C5H 10 -、-CO-C6H 12 -、-CO-C7H 14 -、-CO-C8H 16 -、-CO-C9H 18 -、-CO-C 10 H 20 -, -CO-CH(CH3)-, -CO-C[(CH3)2]-, -CO-CH2-CH(CH3)-, -CO-CH(CH3)-CH2-, -CO-CH(CH3)-C2H4-, -CO-CH2-CH(CH3)-CH2-, -CO-C2H4-CH (CH3)-, -CO-CH2-C[(CH3)2]-, -CO-C[(CH3)2]-CH2-, -CO-CH(CH3)-CH(CH3)-, -CO-C[(C2H5)(CH3)]-, -CO-CH(C3H7)-, and -CO-(CH2-CH2-O) q -CH2-CH2-;

[0112] Wherein, in the structure of the covalently linked arm,

[0113] Each q is independently selected from an integer of 1-60;

[0114] Each G is independently selected from at least one of -NH-, -O-, -S- and -SS-;

[0115] A fourth aspect of the present invention provides a method for preparing the above-mentioned antiviral vaccine molecule, the method comprising:

[0116] The α-NH2 at the N-terminus of the protein antigen is converted into an α-ketoamide through a transamination reaction, and the α-ketoamide is reacted with an alkoxyamine reagent to generate an oxime, and then the Toll-like receptor 4 immunoagonist is linked to the oxime; or

[0117] Converting the α-NH2 at the N-terminus of the protein antigen into α-ketoamide through a transamination reaction, and reacting the α-ketoamide with a Toll-like receptor 4 immunoagonist derivative modified with an alkoxyamine reagent to generate an oxime; or

[0118] The α-NH2 at the N-terminus of the protein antigen is converted to an α-ketoamide by a transamination reaction; the α-ketoamide is reacted with an alkoxyamine reagent to generate an oxime, and then the oxime is reduced to a secondary amine using sodium cyanoborohydride, and then the Toll-like receptor 4 immunoagonist is linked to the secondary amine; or

[0119] The α-NH2 at the N-terminus of the protein antigen is converted into an α-ketoamide through a transamination reaction; the α-ketoamide is reacted with a hydrazine reagent to generate a hydrazone, and then a Toll-like receptor 4 immunoagonist is linked to the hydrazone; or

[0120] The α-NH2 at the N-terminus of the protein antigen is converted into α-ketoamide through a transamination reaction, and the α-ketoamide is reacted with a Toll-like receptor 4 immunoagonist derivative modified with a hydrazine reagent to generate a hydrazone;

[0121] Wherein, the transamination reaction of the N-terminal amino acid of the protein antigen is mediated by pyridoxal phosphate (PLP).

[0122] The present invention is the first in the vaccine field to use pyridoxal phosphate (PLP)-mediated protein N-terminal amino acid transamination reaction to prepare a site-specific covalently coupled "lipid molecule-protein" subunit vaccine. This reaction can specifically oxidize the protein N-terminal amino acid to an aldehyde or ketone, and then react with an alkoxyamine to generate the corresponding oxime product, thereby site-specifically coupling the alkoxyamine to the terminal amino acid of the protein. Therefore, when the N-terminus of the protein is an amino acid that can be efficiently converted into an oxime, this reaction can be used to obtain the corresponding protein oxime product under mild conditions, thereby achieving site-specific covalent modification of the protein. After the adjuvant molecule is modified, the present invention uses this transamination reaction to site-specifically covalently link it to the antigen protein. While controlling the connection of only one lipid adjuvant molecule to ensure water solubility, it will not affect the structure and function of the immunogenic epitope on the antigen protein. This vaccine design strategy is applicable to antigen proteins from various sources and various types of lipid adjuvants. The vaccine molecule has a simple structure, is easy to prepare and store, and has stable properties and good immune effects. It can better activate dendritic cells and macrophages, while triggering strong humoral immunity and cellular immunity.

[0123] In one embodiment of the present invention, RBD is used as a protein antigen, and the process and structure of preparing the antiviral vaccine molecule of the present invention by combining a Toll-like receptor 4 immune agonist (GAP) and a protein antigen are as follows:

[0124]

[0125] The fifth aspect of the present invention provides a use of the above-mentioned antiviral vaccine molecule in the preparation of an antiviral vaccine.

[0126] The Toll-like receptor 4 immune agonist of the present invention can be used as a vaccine adjuvant for the treatment or adjuvant treatment of viral diseases such as hepatitis B and C, HIV, coronaviruses (including SARS, MERS, and SARS-CoV-2), monkeypox virus, Langya virus, human influenza, and avian influenza, as well as various cancers. Antigens can include attenuated or inactivated antigens, protein structures, polysaccharide structures, nucleic acid structures, and recombinant subunit structures.

[0127] The present invention will be described in detail below through examples, but the scope of the present invention is not limited thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0128] The normal temperature in the following examples refers to 25°C.

[0129] Example 1: Preparation of Toll-like receptor 4 immune agonist, the preparation process is as follows:

[0130]

[0131] The structures of the Toll-like receptor 4 immune agonists (Compounds 7a, 7b, and 7c) synthesized by the following method are shown below:

[0132]

[0133] Where D is oxygen, A, B, and C are all -CH2OH;

[0134] The structure of R in compound 7a is shown below:

[0135]

[0136] Where n is 1, p is 3, o is 1, m is 10, and X and Y are both carbon elements;

[0137] The structure of R in compound 7b is shown below:

[0138]

[0139] Where n is 1, p is 1, o is 2, m is 10, and X and Y are both carbon elements;

[0140] The structure of R in compound 7c is shown below:

[0141]

[0142] Where n is 1, p is 7, o is 1, m is 10, and X and Y are both carbon elements;

[0143] The preparation method is as follows:

[0144] Synthesis of compound 2a: Compound 1 (1.5 g, 1.8 mmol) was weighed and added to dichloromethane (5 mL) to dissolve, followed by adding 1 mol / L trimethylphosphine solution (3 mL), stirring at room temperature under argon atmosphere for 2 h, then removing the liquid by rotary evaporation, adding ethanol (5 mL) under Ar atmosphere to dissolve the residue, then adding hydrazine hydrate (2 mL), and then heating under reflux for 3 hours, then removing the liquid by rotary evaporation, and separating and purifying by column chromatography to obtain the intermediate compound (881 mg, yield 89%); under argon atmosphere, The obtained intermediate compound (1 equivalent) and the fatty chain module (aliphatic chain with a secondary acyl chain having 10 carbon atoms) (4.5 equivalents) were dissolved in dichloromethane (DCM) (30 mL) under a carbon atmosphere. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (4.5 equivalents) and 4-dimethylaminopyridine (DMAP) (0.03 equivalents) were then added. The reaction was stirred for 4 h, and the liquid was then removed by rotary evaporation. Compound 2a (55 mg, 39% yield) was obtained after purification by column chromatography.

[0145] The synthesis method and principle of compound 2b are similar to those of compound 2a, except that the fatty chain module used is a fatty chain with a secondary acyl chain carbon number of 12, to obtain compound 2b (46 mg, yield 49%).

[0146] The synthesis method and principle of compound 2c are similar to those of compound 2a, except that the fatty chain module used is a fatty chain with a secondary acyl chain carbon number of 14, to obtain compound 2c (150 mg, yield 73%).

[0147] Compound 2a: 1 H NMR (600MHz, CDCl3) δ7.49–7.30(m,12H),6.19(s,1H),6.05(d,J=8.9Hz,1H),5.49(d,J=5.6Hz,1H),5. 15(q,J=10.4Hz,2H),5.11–4.98(m,2H),4.47(q,J=15.3,11.3Hz,5H),4.33(dd,J=10.6,5.1Hz,1H),4. 08(d,J=15.3Hz,2H),4.03–3.92(m,1H),3.75(ddt,J=28.0,17.7,9.1Hz,5H),3.64(t,J=9.5Hz,1H),3. 51–3.38(m,1H),2.67–1.99(m,12H),1.68–1.43(m,16H),1.40–1.04(m,114H),0.88(t,J=7.1Hz,24H). 1313C NMR (100 MHz, CDCl3) δ 173.54, 173.18, 170.18, 170.06, 169.68, 157.90, 148.91, 139.58, 138.28, 129.05, 128.32, 128.19, 128.16, 127.61, 127.54, 127.49, 126.13, 118.62, 102.32, 101.41, 77.20, 76.35, 73.32, 71.49, 71.23, 69.87, 68.85, 59.75, 41.40, 39.02, 35.04, 34.50, 34.31, 33.85, 31.92, 31.89, 29.72, 29.69, 29.66, 29.60, 29.51, 29.48, 29.39, 29.37, 29.33, 29.30, 29.22, 29.12, 28.13, 25.38, 25.01, 24.97, 22.69, 14.11.

[0148] Compound 2b: 1 1H NMR (600 MHz, CDCl3) δ 7.47 (dd, J = 6.4, 3.0 Hz, 2H), 7.41–7.30 (m, 16H), 6.24 (d, J = 4.8 Hz, 1H), 6.14 (d, J = 9.0 Hz, 1H), 5.53 (d, J = 3.3 Hz, 1H), 5.21 (dd, J = 11.3, 8.0 Hz, 2H), 5.16–4.99 (m, 2H), 4.52 (t, J = 5.9 Hz, 5H), 4.37 (dd, J = 10.6, 4.9 Hz, 1H), 4.21–4.15 (m, 1H), 4.12 (d, J = 10.1 Hz, 2H), 4.01 (q, J = 9.0 Hz, 1H), 3.93–3.74 (m, 5H), 3.69 (td, J = 9.4, 2.9 Hz, 1H), 3.49 (td, J = 9.7, 4.9 Hz, 1H), 2.71–2.13 (m, 12H), 1.74–1.43 (m, 12H), 1.29 (d, J = 5.3 Hz, 125H), 0.92 (t, J = 6.7 Hz, 23H). 13C NMR (100MHz, CDCl3) δ177.54,173.53,170.14,138.25,136.89,129.03,128.30,128.15,127.65,127.59,127.54,12 7.52,127.49,127.47,126.11,126.03,102.48,101.39,101.27,77.20,73.32,71.47,71.19,70.97,69.86,68.83,6 8.57,66.32,59.77,42.04,41.39,39.01,34.45,34.30,34.24,34.11,33.83,31.91,29.72,29.67,29.64,29.59,29.55,29.46,29.38,29.36,29.32,29.26,29.22,29.11,29.04,25.36,25.01,24.96,24.77,22.67,14.10. Mass spectrum: [M+Na] + Theoretical composition C 109 H 182 N2O 16 Na + : 1798.3387, measured value: 1798.3391.

[0149] Compound 2c: 1 H NMR(600MHz, CDCl3)δ7.45(d,J=5.7Hz,2H),7.42–7.29(m,13H),6.22(s,1H),6.11(d,J=8.7H z,1H),5.51(s,1H),5.17(d,J=9.2Hz,2H),5.07(d,J=37.4Hz,2H),4.49(d,J=8.1Hz,5H),4.4 4–4.33(m,1H),4.09(s,2H),3.99(d,J=9.4Hz,1H),3.87–3.72(m,6H),3.67(t,J=9.3Hz,1H), 3.48(d,J=11.5Hz,1H),2.66–2.13(m,12H),1.26(d,J=7.6Hz,130H),0.90(t,J=6.9Hz,20H). 13C NMR (151MHz, CDCl3) δ170.15,138.19,136.83,129.00,128.27,128.12,127.5 7,127.50,127.44,126.07,101.34,73.26,71.47,70.95,69.82,68.75,68.53 ,68.40,66.20,59.72,38.97,34.45,34.40,34.26,33.81,31.89,29.69,29.65,29.55,29.42,29.35,29.19,29.09,25.34,24.97,22.66,14.09. Mass spectrum: [M+Na] + Theoretical composition C 115 H 194 N2O 16 Na: 1882.4326, measured value: 1882.4345.

[0150] Synthesis of compound 3a: Compound 2a (55 mg, 32 μmol) was weighed and added to dichloromethane (3 mL) and trifluoroacetic acid (0.5 mL). The reaction was allowed to proceed for 30 min, followed by the addition of toluene (5 mL × 3). The mixture was frozen and then evaporated to remove the liquid. Compound 3a (35 mg, 69% yield) was obtained by purification by column chromatography.

[0151] The synthesis method and principle of compound 3b are similar to those of compound 3a, except that compound 2b is used to obtain compound 3b (38 mg, yield 88%).

[0152] The synthesis method and principle of compound 3c are similar to those of compound 3a, except that compound 2c was used to obtain compound 3c (45 mg, yield 96%).

[0153] Compound 3a: 1 H NMR (400MHz, CDCl3) δ7.54–7.31(m,12H),6.19(s,1H),5.99(d,J=8.9Hz,1H),5.21– 5.01(m,3H),4.90(t,J=9.8Hz,1H),4.50(p,J=9.0Hz,4H),4.40(d,J=8.1Hz,1H),4.2 2(d,J=10.1Hz,1H),4.05–3.85(m,4H),3.75(d,J=18.9Hz,5H),3.62(t,J=8.9Hz,1H) ,3.56–3.37(m,2H),2.68–2.13(m,15H),1.75–1.49(m,17H),0.92(t,J=6.6Hz,30H). 13C NMR (150MHz, CDCl3) δ174.65,173.37,171.80,170.28,169.63,138.11,128.30 ,127.60,127.50,101.51,75.47,73.28,71.38,71.27,70.93,69.34,68.50,67 .88,62.28,59.76,53.39,42.26,41.42,40.30,34.86,34.43,34.15,31.89,29.66,29.52,29.34,29.17,25.32,25.14,24.95,22.67,15.28,14.11. Mass spectrum: [M+Na] + Theoretical composition C 96 H 166 N2O 16 Na: 1626.2135, measured value: 1626.2120.

[0154] Compound 3b: 1 H NMR (400MHz, CDCl3) δ7.54–7.31(m,12H),6.19(s,1H),5.99(d,J=8.9Hz,1H),5.21– 5.01(m,3H),4.90(t,J=9.8Hz,1H),4.50(p,J=9.0Hz,4H),4.40(d,J=8.1Hz,1H),4.2 2(d,J=10.1Hz,1H),4.05–3.85(m,4H),3.75(d,J=18.9Hz,5H),3.62(t,J=8.9Hz,1H) ,3.56–3.37(m,2H),2.68–2.13(m,15H),1.75–1.49(m,17H),0.92(t,J=6.6Hz,30H). 13 C NMR (150MHz, CDCl3) δ174.65,173.37,171.80,170.28,169.63,138.11,128.30 ,127.60,127.50,101.51,75.47,73.28,71.38,71.27,70.93,69.34,68.50,67 .88,62.28,59.76,53.39,42.26,41.42,40.30,34.86,34.43,34.15,31.89,29.66,29.52,29.34,29.17,25.32,25.14,24.95,22.67,15.28,14.11. Mass spectrum: [M+Na] + Theoretical composition C 102 H178 N2O 16 Na: 1710.3074, measured value: 1710.3067.

[0155] Compound 3c: 1 H NMR (600MHz, CDCl3) δ7.34–7.21(m,11H),6.15(s,1H),5.99(d,J=9.1Hz,1H),5.04(dq,J=34.9,6.2Hz,3H),4.84( q,J=10.2Hz,1H),4.46(qd,J=12.0,5.0Hz,4H),4.35(t,J=11.1Hz,1H),4.17(d,J=10.3Hz,1H),3.96(d,J=10.2Hz ,1H),3.93–3.87(m,2H),3.83(d,J=9.3Hz,1H),3.71(d,J=10.1Hz,3H),3.67(d,J=9.4Hz,1H),3.57(d,J=9.8Hz,1 H),3.45–3.34(m,2H),2.63–2.09(m,16H),1.81–1.40(m,26H),1.24(d,J=10.1Hz,140H),0.87(t,J=7.0Hz,25H). 13 C NMR (150MHz, CDCl3) δ209.37,174.60,173.49,173.34,171.76,170.26,169.60,138.14,128.29,127. 58,127.51,127.47,101.53,75.44,73.27,71.38,71.24,70.92,69.40,68.53,67.90,62.32,59.77,5 3.37,42.27,41.42,40.28,34.85,34.45,34.41,34.17,31.90,29.69,29.66,29.58,29.54,29.51,29.35,29.27,29.21,29.16,29.11,25.34,25.30,25.14,25.00,24.95,24.91,22.67,14.10. Mass spectrum: [M+Na] + Theoretical composition C 108 H 190 N2O 16 Na: 1794.4013, measured value: 1794.4005.

[0156] Synthesis of compound 4a: Compound 3a (1.0 equiv) was weighed and dissolved in dichloromethane (8.0 mL). Tert-butyldimethylsilyl chloride (TBSCl) (1.5 equiv) and imidazole (2 equiv) were added, and the mixture was reacted at room temperature for 12 h. After completion of the reaction, the liquid was removed by rotary evaporation and purified by column chromatography to obtain compound 4a (36 mg, 95% yield).

[0157] The synthesis method and principle of compound 4b are similar to those of compound 4a, except that compound 3b is used to obtain compound 4b (34 mg, yield 83%).

[0158] The synthesis method and principle of compound 4c are similar to those of compound 4a, except that compound 3c is used to obtain compound 4c (47 mg, yield 96%).

[0159] Compound 4a: 1 H NMR (600MHz, CDCl3) δ7.30(q,J=12.3,9.8Hz,10H),6.19(s,1H),6.00(d,J=9.0Hz,1H),5.21–4.97(m,3H),4.88(t,J=9.9Hz,1H),4.46(dt,J=9. 0,5.5Hz,4H),4.31(d,J=8.3Hz,1H),4.09–3.96(m,2H),3.95–3.85(m,2H),3.81(dd,J=16.7,7.2Hz,2H),3.78–3.72(m,2H),3.70(d,J=9.2Hz,1 H),3.65(t,J=9.2Hz,1H),3.60(s,1H),3.33(dt,J=9.9,5.2Hz,1H),2.60(dd,J=15.3,8.1Hz,1H),2.52(dd,J=15.4,4.1Hz,1H),2.43(dd,J=14. 8,5.9Hz,1H),2.39–2.12(m,10H),1.56(qt,J=26.5,15.8Hz,14H),1.26 (q,J=10.6,9.2Hz,108H),0.87(d,J=7.6Hz,32H),0.07(d,J=3.0Hz,7H). 13C NMR (150MHz, CDCl3) δ186.12,173.96,173.40,170.01,169.60,138.23,128.27,127.54, 127.46,109.19,101.74,75.67,74.14,73.24,72.03,71.40,70.94,70.66,68.86,68.52 ,68.25,64.21,59.68,53.35,41.95,41.31,39.75,34.44,34.15,31.89,29.66,29.59,29.49,29.35,29.18,25.81,25.36,25.14,24.97,22.67,18.21,14.12,-5.46. Mass spectrum: [M+Na] + Theoretical composition C 102 H 180 N2O 16 SiNa: 1740.3000, measured value: 1740.2998.

[0160] Compound 4b: 1 H NMR(600MHz, CDCl3)δ7.45–7.26(m,10H),6.19(s,1H),6.01(d,J=8.9Hz,1H),5.18–4.98(m,3H),4 .88(t,J=9.9Hz,1H),4.46(dt,J=9.1,5.5Hz,4H),4.31(d,J=8.3Hz,1H),4.08–3.97(m,2H),3.95–3 .86(m,2H),3.87–3.78(m,2H),3.77–3.53(m,5H),3.33(dt,J=9.8,5.1Hz,1H),2.68–2.12(m,13H) ,1.72–1.42(m,15H),1.25(q,J=12.9,9.1Hz,118H),0.88(d,J=6.5Hz,31H),0.06(d,J=3.1Hz,7H). 13C NMR (150MHz, CDCl3) δ271.60,173.96,173.42,170.01,169.60,138.23,128.27 ,127.54,127.46,101.75,75.66,74.15,73.24,70.95,70.65,68.85,68.53,68 .25,64.21,59.68,53.35,41.95,41.31,39.75,34.45,34.16,31.91,29.66,29.56,29.37,29.19,25.81,25.37,25.13,24.97,22.68,14.12,-5.45. Mass spectrum: [M+Na] + Theoretical composition C 108 H 192 N2O 16 SiNa: 1824.3939, measured value: 1824.3940.

[0161] Compound 4c: 1 H NMR (600MHz, CDCl3) δ7.31(q,J=12.5,9.9Hz,10H),6.18(s,1H),5.98(d,J=8.7Hz,1H),5.18–4.97(m,3H),4.88(t,J=9. 9Hz,1H),4.46(p,J=4.7,3.6Hz,5H),4.32(d,J=8.2Hz,1H),4.07–3.97(m,2H),3.95–3.85(m,3H),3.82(dd,J=17.2,6.9H z,2H),3.34(dt,J=10.7,5.4Hz,1H),2.61(dd,J=15.4,8.1Hz,1H),2.52(dd,J=15.3,3.9Hz,1H),2.43(dd,J=14.8,5.9Hz ,1H),2.27(dddd,J=62.0,35.4,12.8,5.2Hz,12H),1.37–1.16(m,173H),0.87(d,J=7.2Hz,35H),0.07(d,J=3.1Hz,10H). 13C NMR (151MHz, CDCl3) δ173.94,171.35,169.59,138.27,128.28,127.54,127.50,127.45 ,101.78,100.16,74.16,73.46,73.26,70.93,70.64,68.87,68.54,64.23,59.70,53.39 ,41.97,39.76,34.45,34.18,31.91,29.71,29.67,29.64,29.59,29.56,29.52,29.36,29.20,29.16,25.81,25.36,25.14,24.98,22.67,18.20,14.11,-5.44,-5.50. Mass spectrum: [M+Na] + Theoretical composition C 114 H 204 N2O 16 SiNa: 1908.4878, measured value: 1908.4880.

[0162] Synthesis of compound 5a: Compound 4a (36 mg, 21 mmol) was weighed and dissolved in dichloromethane (5 mL) from which water had been removed. Activated 4 molecular sieves (100 mg) were added and stirred under an Ar atmosphere for 30 min. 20 mg (3 equivalents) of dibenzyl N,N-diisopropylphosphoramidite was added at 0°C, followed by a solution of 8 mg (6 equivalents) of 1H-tetrazole dissolved in 1 mL of acetonitrile. The mixture was stirred for 12 h. When the reaction was completed as monitored by thin layer chromatography (TLC), 16 μL (9 equivalents) of tert-butyl hydroperoxide was added and stirred for 30 min. The liquid was removed by rotary evaporation, and the crude product was purified by column chromatography to afford compound 5a (26 mg, 62% yield) as a white solid.

[0163] The synthesis method and principle of compound 5b are similar to those of compound 5a, except that compound 4b is used to obtain compound 5b (27 mg, yield 68%).

[0164] The synthesis method and principle of compound 5c are similar to those of compound 5a, except that compound 4c is used to obtain compound 5c (38 mg, yield 90%).

[0165] Compound 5a: 1H NMR (600MHz, CDCl3) δ7.40–7.26(m,20H),6.18(d,J=15.0Hz,2H),5.18(dt,J=26.3,8.2Hz,2H),5.08( dd,J=16.2,9.5Hz,2H),4.95(dd,J=28.0,7.9Hz,4H),4.54(d,J=8.3Hz,1H),4.46(d,J=7.4Hz,4H),4. 38(q,J=9.5Hz,1H),4.03(s,2H),3.91(d,J=11.7Hz,1H),3.75(td,J=27.7,25.8,9.6Hz,6H),3.43(d, J=7.8Hz,1H),2.55–2.09(m,12H),1.61(d,J=13.7Hz,14H),1.39–1.01(m,101H),0.99–0.72(m,32H). 13 C NMR (100MHz, CDCl3) δ173.50,169.85,138.28,135.58,128.51,128.26,127.99,127.84,127.44,73.71,73.2 4,72.89,69.47,68.54,61.89,59.71,34.43,31.92,29.68,29.38,25.82,25.39,25.01,22.69,14.12,-5.12. 31 P NMR (162MHz,CDCl3)δ-4.00.Mass spectrum: [M+Na] + Theoretical composition C 116 H 193 N2O 19 PSiNa: 2000.3602, measured value: 2000.3566.

[0166] Compound 5b: 1H NMR (600MHz, CDCl3) δ7.32 (q, J=15.6, 9.9Hz, 28H), 6.19 (d, J=20.5Hz, 2H), 5.21 (dd, J=24.7, 14.6Hz ,2H),5.08(d,J=20.2Hz,2H),4.97(dd,J=28.3,7.8Hz,4H),4.56(d,J=8.1Hz,1H),4.48(d,J=7.4Hz,4 H),4.39(q,J=9.6Hz,1H),4.05(s,2H),3.92(d,J=11.7Hz,1H),3.77(td,J=26.6,24.1,9.7Hz,6H),3. 44(s,1H),2.56–2.11(m,12H),1.68(d,J=48.2Hz,18H),1.25(d,J=9.8Hz,119H),0.93–0.79(m,32H). 13 C NMR (100MHz, CDCl3) δ173.50,169.85,138.28,135.58,128.51,128.26,127.99,127.84,127.44,73.71,73.2 4,72.89,69.47,68.54,61.89,59.71,34.43,31.92,29.68,29.38,25.82,25.39,25.01,22.69,14.12,-5.12. 31 P NMR (161MHz,CDCl3)δ-4.12.Mass spectrum: [M+Na] + Theoretical composition C 122 H 205 N2O 19 PSiNa: 2084.4541, measured value: 2084.4537.

[0167] Compound 5c: 1H NMR (600MHz, CDCl3) δ7.41–7.29(m,18H),6.22(s,1H),6.18(d,J=8.4Hz,1H),5.24(t,J=9.9Hz,1H),5.19(t,J=6.0Hz ,1H),5.13–5.05(m,3H),5.01(t,J=9.0Hz,3H),4.95(d,J=7.8Hz,2H),4.57(d,J=8.3Hz,1H),4.49(d,J=7.0Hz,4H),4 .40(q,J=9.3Hz,1H),4.29–4.20(m,1H),4.06(s,2H),3.93(d,J=11.7Hz,1H),3.85–3.67(m,6H),3.48(ddt,J=29.3,9 .3,5.7Hz,2H),2.63–2.14(m,12H),1.41–1.14(m,148H),0.90(t,J=7.0Hz,23H),0.86(s,10H),0.02(d,J=3.5Hz,9H). 13 C NMR (101MHz, CDCl3) δ173.50,169.85,138.28,135.58,128.51,128.26,127.99,127.84,127.44,73.71,73.2 4,72.89,69.47,68.54,61.89,59.71,34.43,31.92,29.68,29.38,25.82,25.39,25.01,22.69,14.12,-5.12. 31 P NMR (161 MHz, CDCl3) δ-2.06. Mass spectrum: [M+Na] + Theoretical composition C 128 H 217 N2O 19 PSiNa: 2168.5480, measured value: 2168.5478.

[0168] Synthesis of compound 6a: Compound 5a (26 mg, 13 μmol) was weighed, dichloromethane (5 mL) and trifluoroacetic acid (0.5 mL) were added, and the mixture was reacted for 30 min. Dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and compound 6a (20 mg, yield 91%) was obtained by purification by column chromatography.

[0169] The synthesis method and principle of compound 6b are similar to those of compound 6a, except that compound 5b is used to obtain compound 6b (22 mg, yield 95%).

[0170] The synthesis method and principle of compound 6c are similar to those of compound 6a, except that compound 5c is used to obtain compound 6c (35 mg, yield 91%).

[0171] Compound 6a: 1 H NMR (600MHz, CDCl3) δ7.40–7.28(m,15H),6.18(d,J=21.7Hz,2H),5.23(t,J=9.7Hz,1H),5.15 –4.89(m,8H),4.58(d,J=7.9Hz,1H),4.47(t,J=4.8Hz,5H),4.40(d,J=9.6Hz,1H),4.13(d,J=1 0.2Hz,1H),4.02(d,J=10.3Hz,1H),3.85(d,J=9.3Hz,1H),3.76(d,J=17.2Hz,6H),3.69(d,J= 9.3Hz,1H),3.37(d,J=9.1Hz,1H),2.50–2.14(m,14H),1.24(s,137H),0.88(t,J=6.8Hz,18H). 13 C NMR (100MHz, CDCl3) δ173.49,173.28,170.19,159.39,138.25,128.80,128.73,128.64,128.61,128.33,12 8.29,127.97,127.53,127.48,127.45,101.06,74.72,73.30,72.51,71.42,70.89,70.03,69.84,68.68,68. 58,60.68,59.80,54.64,42.19,41.31,38.93,34.47,34.42,34.26,31.92,30.00,29.71,29.67,29.61,29.59,29.57,29.54,29.47,29.45,29.41,29.38,29.36,29.25,29.20,25.32,25.10,25.00,22.68,14.10,8.29. 31 P NMR (162MHz,CDCl3)δ-2.85.Mass spectrum: [M+Na] + Theoretical composition C 110 H 179 N2O 19 PNa: 1886.2737, measured value: 1886.2729.

[0172] Compound 6b: 1H NMR (600MHz, CDCl3) δ7.31(dq,J=24.8,11.0,8.5Hz,21H),6.21(d,J=8.0Hz,2H),5.22(t,J=9.8Hz,1H),5.15–5.07(m ,2H),5.07–5.01(m,2H),5.00–4.88(m,4H),4.57(d,J=8.3Hz,1H),4.47(q,J=6.4,5.2Hz,4H),4.42–4.36(m,1H),4.30 (t,J=6.7Hz,2H),4.13(d,J=10.3Hz,1H),4.01(d,J=10.3Hz,1H),3.84(d,J=9.3Hz,1H),3.82–3.72(m,6H),3.69(d,J =9.3Hz,1H),3.37(d,J=9.7Hz,1H),2.51–2.04(m,16H),1.85–1.63(m,10H),1.62–1.45(m,15H),1.46–1.05(m,143H). 13 C NMR (100MHz, CDCl3) δ302.80,231.51,186.86,173.51,173.31,170.23,169.86,138.19 ,135.18,128.76,128.62,128.30,127.96,127.54,127.46,113.91,101.04,74.68,73. 28,72.46,71.39,70.88,70.01,68.59,68.29,60.65,59.77,54.54,42.15,41.26,38.88,37.87,34.43,34.24,31.91,29.67,29.50,29.36,29.18,25.33,24.99,22.68,14.12. 31 P NMR (162MHz,CDCl3)δ-2.92.Mass spectrum: [M+Na] + Theoretical composition C 116 H 191 N2O 19 PNa: 1970.3676, measured value: 1970.3684.

[0173] Compound 6c: 1H NMR (600MHz, CDCl3) δ7.40–7.28(m,15H),6.18(d,J=21.7Hz,2H),5.23(t,J=9.7Hz,1H),5.15 –4.89(m,8H),4.58(d,J=7.9Hz,1H),4.47(t,J=4.8Hz,5H),4.40(d,J=9.6Hz,1H),4.13(d,J=1 0.2Hz,1H),4.02(d,J=10.3Hz,1H),3.85(d,J=9.3Hz,1H),3.76(d,J=17.2Hz,6H),3.69(d,J= 9.3Hz,1H),3.37(d,J=9.1Hz,1H),2.50–2.14(m,14H),1.24(s,137H),0.88(t,J=6.8Hz,20H). 13 C NMR (100MHz, CDCl3) δ173.54,173.33,170.26,169.89,138.19,135.19,132.25,130.90,128 .81,128.74,128.63,128.35,128.29,127.97,127.55,127.48,101.04,74.68,73.29,71.40, 70.89,70.02,69.82,68.53,68.28,65.56,60.63,59.77,54.52,42.14,41.25,38.88,34.44,34.23,31.91,30.53,29.67,29.58,29.36,29.18,25.32,24.99,22.68,19.16,14.11,13.72. 31 P NMR (162MHz,CDCl3)δ-0.33.Mass spectrum: [M+K] + Theoretical composition C 122 H 203 N2O 19 PK: 2070.4355, measured value: 2070.4897.

[0174] Synthesis of compound 7a (GAP110): Compound 6a (20 mg, 12 μmol) was weighed and dissolved in 5 mL of a mixture of dichloromethane and methanol (volume ratio of dichloromethane to methanol was 1:1). Pd / C (20 mg) was added, and the mixture was stirred under 1 atm H2 atmosphere for 3 h. The liquid was removed by rotary evaporation to obtain compound 7a (18 mg, quantitative).

[0175] The synthesis method and principle of compound 7b (GAP112) are similar to those of compound 7a, except that compound 6b is used to obtain compound 7b (19 mg, quantitative).

[0176] The synthesis method and principle of compound 7c (GAP114) are similar to those of compound 7a, except that compound 6c is used to obtain compound 7c (32 mg, quantitative).

[0177] Compound 7a: 1 H NMR (600MHz, CDCl3 and CD3OD 1:1)δ5.30–4.98(m,4H),4.42(d,J=11.1Hz,1H),4.31–4.16(m,1H),4.02–3.70(m,4H),3.70–3.53(m,4H),3.46(d,J =9.9Hz,1H),3.36–3.34(m,1H),2.69–2.22(m,12H),1.79–1.53(m,12H),1.49–1.13(m,90H),0.85(t,J=6.9Hz,18H). 31 P NMR (162MHz, CDCl3 and CD3OD 1:1) δ-3.23. Mass spectrum: [M+Na] + Theoretical composition C 82 H 155 N2O 19 PNa: 1526.0859, measured value: 1526.0834.

[0178] Compound 7b: 1 H NMR (600MHz, CDCl3 and CD3OD 1:1)δ5.31–4.93(m,4H),4.41(d,J=10.0Hz,1H),4.32–4.15(m,1H),3.97–3.71(m,4H),3.69–3.53(m,4H),3.44(d,J=11.6Hz,1 H),3.35–3.34(m,1H),2.69–2.17(m,12H),1.62–1.50(dt,J=21.1,10.7Hz,12H),1.30–1.23(m,102H),0.85(t,J=6.9Hz,18H). 31 P NMR (162 MHz, CDCl3 and CD3OD 1:1) δ-3.24. Mass spectrum: [M+H] + Theoretical composition C 88 H 168 N2O 19P: 1588.1979, measured value: 1588.1976.

[0179] Compound 7c: 1 H NMR (400MHz, CDCl3 and CD3OD 1:1)δ5.32–5.08(m,4H),4.48(d,J=8.4Hz,1H),4.33–4.19(m,1H),4.04–3.73(m,4H),3.70–3.63(m,4H),3.52(d,J=1 1.7Hz,1H),3.45–3.37(m,1H),2.73–2.26(m,12H),1.63–1.60(m,12H),1.35–1.26(m,114H),0.90(t,J=6.6Hz,18H). 13 C NMR (100MHz, CDCl3 and CD3OD 1:1)δ173.42,173.40,173.11,171.67,170.71,170.02,100.70,74.32,72.60,70.66,70.19,69.46,66.48,60.88,60.73,53.01,40.86, 40.18,38.15,34.12,33.72,33.68,33.65,33.56,31.18,31.15,28.91,28.81,28.64,28.58,24.62,24.44,24.33,24.28,21.86,12.99. 31 P NMR (162MHz, CDCl3 andCD3OD 1:1)δ-0.20. Mass spectrum: [M+Na] + Theoretical composition C 94 H 179 N2O 19 PNa: 1694.2737, measured value: 1694.4765.

[0180] Test Example 1 Biological Activity Test

[0181] This test example is used to illustrate the activity detection of the Toll-like receptor 4 immune agonist obtained in Example 1. GAP110, GAP112, and GAP114 are used as adjuvants and are respectively prepared into vaccines in the form of liposome encapsulation with protein antigens. The immune effect of the vaccine on mice is tested to evaluate its activity.

[0182] Liposomes were prepared by the reverse evaporation method, and distearoylphosphatidylcholine (DSPC), cholesterol (Chol), chicken ovalbumin (OVA) and adjuvant were configured in a molar ratio of 5:4:1:1, and comparative examples 1-4 were added. The specific dosages of the vaccines in Example 1 and Comparative Examples 1-4 are shown in Table 1, wherein Comparative Example 1 is a phosphate buffered saline (PBS) solution, and the adjuvants in Comparative Examples 2 and 3 are replaced by MPLA (CAS: 1246298-63-4) and PET-A (Publication No.: US7820627B2), respectively. No adjuvant was used in Comparative Example 4, and the adjuvants in Example 1 were GAP110, GAP112 and GAP114, respectively. Comparative Examples 2-4 and Example 1 were prepared into vaccines for use;

[0183] The vaccine preparation method is as follows: distearoylphosphatidylcholine, cholesterol and adjuvant are first dissolved in a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane and methanol is 1:1), and then the organic solvent is evaporated under reduced pressure to remove it. A phosphate buffered saline (PBS) solution containing chicken egg albumin is then added, and ultrasonicated for 20 minutes before injection (the comparative example 1 group is directly injected with PBS solution, and is also ultrasonicated for 20 minutes before injection).

[0184] Table 1

[0185]

[0186] Immunization method: intraperitoneal injection was performed on days 1, 15, and 29. Blood was collected by tail cutting at 2 hours after injection on day 1, day 14, day 28, and day 42. The serum collected 2 hours after injection on day 1 was used for the determination of cytokines (interleukin-6, interleukin-12, and tumor necrosis factor-α). The results are shown in Table 1. Figure 1 The serum collected on days 14, 28, and 42 was used for antibody (IgG and its subtype) testing, and the results were as follows: Figure 2 、 3 As shown, Figure 2 These are the test results of serum antibody IgG collected on days 14, 28, and 42. Figure 3 The results of serum antibody IgG subtype test on day 42; the spleen of mice was taken on day 42 for enzyme-linked immunosorbent assay and intracellular cytokine staining test, and the results were as follows: Figure 4-6 As shown, Figure 5 is the proportion of CD4+T cells producing IFN-γ and TNF-α cytokines in the spleen of mice, Figure 6 The ratio of CD8+T cells producing IFN-γ and TNF-α cytokines in the spleen of mice; Toll-like receptor 4 inhibition experiment was performed on the bone marrow dendritic cells of mice. The results are as follows Figure 7 shown.

[0187] Cytokine assay

[0188] (1) Antigen coating: The capture antibody is used as the coating antigen. Dilute the capture antibody 200-fold with coating buffer (containing 0.1 M Na2HCO3 and 0.03 M Na2CO3, pH 9.5) and add 100 μL per well to a high-adsorption 96-well plate. Coat overnight at 4°C.

[0189] (2) Washing: Pour out the solution in the 96-well plate, then add 200 μL of PBST solution (phosphate buffered saline solution PBS containing 0.05 wt% Tween-20) to each well, place it on a constant temperature oscillator (special for microplates, maintained at 22-25°C), and oscillate for 2-3 minutes. Pour out the solution in the plate, then turn the 96-well plate upside down on absorbent paper and gently press it to allow the absorbent paper to absorb the remaining liquid. Repeat the washing operation 3 times.

[0190] (3) Blocking: Add 200 μL of phosphate buffered saline solution containing 1 wt% bovine serum albumin (BSA) to each well, place on a constant temperature shaker and shake for 1 hour, then wash: the operation is the same as step (2).

[0191] (4) Adding standards and serum: a) According to the instructions of the cytokine kit, add the corresponding gradient dilution of cytokine standards (phosphate buffered saline containing 1 wt% bovine serum albumin as the diluent) to each well in sequence; b) Dilute the serum to the corresponding concentration with phosphate buffered saline containing 1 wt% bovine serum albumin, add 100 μL to each well, place on a constant temperature oscillator and oscillate for 2 h, and then wash: the operation is the same as step (2).

[0192] (5) Adding detection antibody: dilute the detection antibody 200-fold with phosphate buffered saline containing 1 wt% bovine serum albumin, add 100 μL to each well, place on a constant temperature oscillator and shake for 1 hour, then wash: the operation is the same as step (2).

[0193] (6) Adding enzyme-labeled secondary antibody: dilute the enzyme-labeled secondary antibody 1000 times with phosphate buffered saline containing 1 wt% bovine serum albumin, add 100 μL to each well, seal with plastic wrap, place on a constant temperature oscillator and oscillate for 30 minutes, then wash: same as step (2).

[0194] (7) Color development and reading: Add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) color development solution to each well, place the 96-well plate on a constant temperature oscillator in the dark for 20-30 minutes, then add 50 μL of H2SO4 (2M) stop solution to each well, finally, place the 96-well plate in a microplate reader and measure the absorbance at 450 nm. Finally, according to the instructions of the cytokine kit, plot the absorbance values ​​of the standard sample into a standard curve. Then, substitute the measured serum absorbance value into the standard curve to determine the concentration of cytokines in the serum.

[0195] The results are as follows Figure 1 As shown, GAP110 and GAP112 can induce high concentrations of tumor necrosis factor, interleukin-12, and interleukin-6, with efficacy comparable to that of the widely used MPLA adjuvant, while GAP114 has a moderate efficacy.

[0196] Antibody testing

[0197] A 96-well ELISA plate was coated with 100 μL of carbonate buffer solution (pH 9.0-9.6) containing chicken ovalbumin (concentration 1 μg / mL), then incubated at 4°C overnight and washed three times with PBST (phosphate buffered saline containing 0.05% by volume of Tween-20). Then, the plate was blocked with 1.5% by weight bovine serum albumin solution, incubated at 37°C for 1 hour, and washed three times with PBST. Serum diluted to a specific concentration with 10% by weight phosphate buffer containing bovine serum albumin was added. The plate was incubated at 37°C for 1 hour and washed three times with PBST. Horseradish peroxidase-labeled goat anti-mouse secondary antibodies IgG, IgM, IgG1, IgG2a, IgG2b, and IgG3 diluted 4000:1 in phosphate buffer were added. The plate was incubated at 37°C for 1 hour, and washed three times with PBST. Freshly prepared 3,3',5,5'-tetramethylbenzidine colorimetric solution was then added and incubated for 15 minutes. The plate was terminated by adding 2M H2SO4 solution, and the absorbance was measured by a microplate reader at a wavelength of 450 nm.

[0198] The results are as follows Figure 2 、 Figure 3 As shown, GAP110 and GAP112 can induce higher titers of IgG antibodies, while GAP114 is lower. All three agonists can induce multiple IgG subtypes, which can provide more comprehensive protection.

[0199] ELISpot test

[0200] (1) Cell culture: Add 200 μL of activation solution to the coated plate and let it stand at room temperature for 5-10 minutes. Add 100 μL of cell suspension and stimulate with 10 μL of chicken ovalbumin in phosphate-buffered saline (1 μg / mL) per well. Incubate in a CO2 incubator for 16-24 hours.

[0201] (2) Cell lysis: Pour out the cells and culture medium in the wells, add 100 μL of ice-cold deionized water to each well and lyse for 10 min;

[0202] (3) Washing: Add 50-fold diluted washing buffer to each well and wash 6 times;

[0203] (4) Add detection antibody: Add 100 μL of detection antibody diluent prepared according to the instructions to each well and incubate at 37°C for 1 h;

[0204] (5) Washing: Wash 6 times with washing buffer;

[0205] (6) Add HRP enzyme: Add 100 μL of HRP enzyme diluent prepared according to the instructions to each well and incubate at 37°C for 1 h;

[0206] (7) Washing: Wash 5 times with washing buffer;

[0207] (8) Color development: Add 100 μL of freshly prepared AEC color development solution to each well. Air-dry at room temperature in the dark. Count the number of spots after air-drying.

[0208] The results are as follows Figure 4 As shown, the results showed that GAP110 and GAP112 could stimulate more T cells to produce interferon-γ spots, reflecting their stronger T cell-dependent immune response, while GAP114 produced less.

[0209] Intracellular cytokine staining test

[0210] (1) Mouse spleen cell extraction: The mouse was killed by dislocating the neck and soaked in alcohol for 3-5 minutes, and the mouse spleen was dissected out. 3 mL of lymph separation fluid was added to the glass dish, and the cell suspension was obtained by grinding and transferred to a 15 mL centrifuge tube. 800 μL of serum-free 1640 culture medium was added along the wall of the centrifuge tube, and a clear interface was ensured. Centrifuge in a high-speed centrifuge at room temperature (800 g, 30 min). After centrifugation, the cell suspension was aspirated and washed with 4 mL of serum-free 1640 culture medium, and centrifuged at room temperature (350 g, 10 min). The supernatant was discarded and the cells were resuspended in 1640 complete medium containing 10% by volume of fetal bovine serum and 1% by volume of double antibody to obtain 1 mL of cell suspension and count.

[0211] (2) Flow cytometry preparation: Add 400 μL of 1640 complete medium to a 24-well plate, then add 300 μL of cell suspension. Stimulate with 10 μL of chicken ovalbumin in phosphate buffer (1 μg / mL) per well. Incubate in a CO2 incubator for 3 h, and then add 2 μL of the inhibitor brefeldin.

[0212] (3) Cell staining: Prepare phosphate buffer (staining buffer) containing 1 wt% bovine serum albumin and 1 volume% fetal bovine serum. Combine the cells of each mouse, wash with 1 mL of staining buffer, and centrifuge to retain the cells at the bottom. Add 100 μL of cell staining solution (1 μL CD3, 1 μL CD4, 1 μL CD8 diluted in 100 μL staining buffer) and 400 μL of staining buffer to each group of cells, mix well, and stain in an ice bath for 30 min. Then wash twice with staining buffer, add 250 μL of fixative, and place in the dark at room temperature for 20 min.

[0213] (4) Cytokine staining: Wash with permeabilization solution for 1 min, centrifuge and add 250 μL of cytokine staining solution (1 μL of interferon-γ, 0.4 μL of tumor necrosis factor-α added to 250 μL of 10-fold diluted permeabilization solution), place in the dark at 4°C for 30 min, then wash once with 500 μL of permeabilization solution, and centrifuge to retain the cells at the bottom.

[0214] (5) Sample loading test: Resuspend the cells in phosphate buffered saline with 0.1% bovine serum albumin by volume, pass through a cell sieve, transfer to a flow cytometry tube, and load the cells for testing.

[0215] The results are as follows Figure 5 and Figure 6 As shown, the results showed that GAP110 and GAP112 could stimulate more T cells to produce gamma interferon and alpha tumor necrosis factor, reflecting their stronger T cell-dependent immune response, while GAP114 produced less.

[0216] Toll-like receptor 4 inhibition assay

[0217] Single-cell suspensions of whole bone marrow cells were isolated from the femurs and tibias of unimmunized female BALB / c mice. The cells were washed with phosphate-buffered saline and then treated with red blood cell lysis buffer for 7 minutes. The collected cells were centrifuged, washed, and then lysed at 2 × 10 6 The cells were resuspended in RPMI 1640 complete medium (containing 10% by weight fetal bovine serum, 100 μg / mL penicillin, and 100 μg / mL streptomycin) at a density of 10 cells / mL and cultured in a 37°C, 5% CO2 incubator. The culture medium was supplemented with mouse interleukin-4 (10 ng / mL) and mouse granulocyte-macrophage colony-stimulating factor (20 ng / mL). After 6 days, bone marrow dendritic cells were collected and transferred to 96-well plates at a density of 2 × 10 cells / well. 5Cells were incubated for 12 hours in a GAP112+TAK-242 group, a GAP112 group, and a phosphate-buffered saline (PBS) group. TAK-242 (DMSO solution) was added to the GAP112+TAK-242 group at a final concentration of 5 μM and incubated for 1 hour. The phosphate-buffered saline (PBS) and GAP112 groups were left untreated. The GAP112+TAK-242 and GAP112 groups were then incubated for 12 hours with 0.5 μg of GAP112. The phosphate-buffered saline group was then added with 0.5 μg of phosphate-buffered saline. The cultures were centrifuged, and the supernatants were collected. Cytokine concentrations (interleukin-6 and tumor necrosis factor-α) in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA).

[0218] The results are as follows Figure 7 As shown, GAP112 can stimulate bone marrow-derived dendritic cells to produce interleukin-6 and α-TNF. However, after TAK-242 inhibits the TLR4 signaling pathway, GAP112 no longer stimulates bone marrow-derived dendritic cells to produce interleukin-6 and α-TNF. This suggests that GAP112 activates bone marrow dendritic cells through the TLR4 pathway.

[0219] Example 2

[0220] This example is used to illustrate the preparation method of the antiviral vaccine molecule of the present invention, and the process is as follows.

[0221]

[0222] Synthesis of compound 9: Tetraethylene glycol (17.1 g, 88 mmol, 1 equivalent), p-toluenesulfonyl chloride (42.3 g, 220 mmol, 2.5 equivalents) and triethylamine (22.3 g, 220 mmol, 2.5 equivalents) were dissolved in dichloromethane (150 mL) from which water had been removed, and the mixture was reacted for 2 hours. After the reaction was completed by thin layer chromatography, the resulting mixture was washed with saturated brine (50 mL) and dichloromethane (the number of dichloromethane washes was 3 times, each time with an amount of 50 mL). The washed dichloromethane was collected, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography to obtain a colorless liquid product 8; the product 8 (38.83 g, 77.26 mmol, 1 equivalent) was dissolved in N,N-dimethylformamide (DMF) (40 mL) from which water had been removed, and then sodium azide (15.1 g, 232 mmol, 3 equivalents) was added under an argon atmosphere and reacted for 12 hours. The resulting mixture was heated to 65° C. for 1.5 hours and then cooled to room temperature. After the reaction was completed by thin layer chromatography, the resulting mixture was washed with saturated brine (50 mL) and dichloromethane (the number of dichloromethane washes was 3 times, each time with an amount of 50 mL). The washed dichloromethane was collected and dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography to obtain a colorless liquid product 9 (16.98 g, 69.54 mmol, yield 90%): 1 H NMR (600MHz, CDCl3) δ3.68 (d, J = 3.8Hz, 12H), 3.40 (t, J = 5.1Hz, 4H). 13 C NMR (151 MHz, CDCl3) δ 70.63, 69.97, 50.61. Mass spectrum: [M+Na] + Theoretical composition C8H 16 N6O3Na: 267.11816, measured value 267.11656.

[0223] Synthesis of compound 10: Compound 9 (1.2963 g, 5.31 mmol, 1.0 equivalent) was dissolved in methanol (8 mL), and then Pd / C (130 mg) was added under H2 atmosphere and reacted for 3 hours. When thin layer chromatography analysis showed that the raw material was completely converted, the resulting mixture was filtered and concentrated in vacuo to obtain a light yellow liquid product 10 (0.74 g, 3.82 mmol, yield was 72%).

[0224] Synthesis of compound 11: tert-Butoxycarbonylaminooxyacetic acid (200 mg, 1.046 mol, 1 equivalent), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (220 mg, 1.151 mmol, 1.1 equivalent), and pentafluorophenol (212 mg, 1.151 mol, 1.1 equivalent) were added to dichloromethane (10 mL) from which water had been removed and reacted for 15 hours. After completion of the reaction, silica gel (2.65 g) was added, stirred for 10 minutes, filtered, and the residue was thoroughly washed with dichloromethane. The filtrate was collected and dried to give the product 11 as a white solid (334 mg, 0.935 mmol, yield 89%): 1 H NMR (400 MHz, CDCl3) δ7.68 (s, 1H), 4.80 (s, 2H), 1.50 (s, 9H). Mass spectrum: [M+Na] + Theoretical composition C 13 H 12 F5NO5Na: 380.05333, measured value 380.05249.

[0225] Synthesis of compound 12: Under Ar atmosphere, compound 10 (162.7 mg, 0.847 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (1.1 g, 1.4 mL, 8.47 mmol, 10 equivalents) were dissolved in dichloromethane (5 mL) from which water was removed. Then, compound 11 (42 mL, 0.34 mol, 2.0 equivalents) was slowly added and stirred for 3 hours. The crude product was concentrated in vacuo and purified by column chromatography to give compound 12 (105.2 mg, 0.288 mmol, 34% yield) as a light yellow oily liquid.

[0226] Synthesis of compound 13: Compound 6 (64.3 mg, 0.033 mol, 1 equivalent) and carbonyldiimidazole (160.5 mg, 0.99 mol, 30 equivalents) were dissolved in dichloromethane (3 mL) from which water had been removed. Triethylamine (100.2 mg, 58 μL, 0.99 mol, 30 equivalents) was then added and reacted under an argon atmosphere for 2.5 hours. After completion of the reaction, the crude product was concentrated in vacuo and purified by column chromatography to obtain a white intermediate. The obtained intermediate (64.0 mg, 0.031 mmol, 1 equivalent) and compound 12 (91.59 mg, 0.251 mol, 8 equivalents) were dissolved in dichloromethane (3 mL) after removal of moisture, and then triethylamine (253.61 mg, 147 μL, 2.51 mmol, 80 equivalents) was added and reacted under Ar atmosphere for 14 hours. After the reaction was completed by thin layer chromatography, the crude product was concentrated in vacuo and purified by column chromatography to obtain a white solid 13 (56.45 mg, 0.024 mmol, yield 77%):1 H NMR(400MHz,CDCl3)δ8.46(s,1H:),8.00-7.85(m,1H),7.31(d,J=3.0Hz,11H),7.27(s,4H),7.25(d,J=3.5Hz,5H),6.31(d,J=8.4Hz,1H),6.26(s,1H),5.73(t,J=5.8Hz,1H),5.23(t,J=9.8Hz,1H),5.18-5.03(m,3H),4.99(d,J=8.9Hz,2H),4.90(d,J=7.8Hz,2H),4.59(d,J=8.2Hz,1H),4.47(d,J=3.2Hz,4H),4.45-4.38(m,2H),4.33(s,2H),4.16(t,J=14.0,9.4,8.9Hz,1H),4.05(s,2H),3.84-3.73(m,4H),3.71(d,J=9.2Hz,1H),3.66-3.54(m,12H),3.49(t,J=6.2Hz,4H),3.30(d,J=6.2Hz,1H),2.48-2.17(m,12H),1.57(td,J=14.3,7.2Hz,12H),1.46(s,9H),1.24(d,J=5.6Hz,102H),0.88(t,J=6.7Hz,18H). 13C NMR (101MHz, CDCl3) δ173.50,173.43,173.24,170.23,170.08,169.81,169.08 ,157.41,155.96,138.15,135.42,135.36,135.29,128.47,128.42,128.40,128 .19,127.96,127.90,127.45,127.40,127.36,101.19,82.17,75.66,73.62,73.56,73.19,73.16,72.49,72.38,71.37,70.80,70.36,70.32,70.14,69.87,69. 77,69.62,69.57,69.52,69.37,68.63,68.37,68.31,62.55,59.75,54.39,53.34,41.88,41.08,40.74,38.96,38.73,34.41,34.37,34.34,34.26,34.21,31.8 3,29.64,29.63,29.59,29.57,29.52,29.47,29.40,29.37,29.33,29.30,29.28,29.16,29.11,29.10,28.05,25.30,25.27,25.03,24.93,24.91,22.59,14.03. 31 P NMR (162MHz,CDCl3)δ-1.89.Mass spectrum: [M+Na] + Theoretical composition C 132 H 220 N5O 27 PNa: 2361.56310, measured value 2361.55976.

[0227] Synthesis of compound 14: Compound 13 (40 mg, 0.020 mmol, 1.0 equivalent) was dissolved in 2 mL of a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol was 1:1), and then Pd / C (40 mg) was added under a H2 atmosphere and stirred for 3 hours. After the reaction was completed by thin layer chromatography, the mixture was concentrated in vacuo to obtain the intermediate product as a white solid. The intermediate product (32 mg, 0.016 mol, 1.0 equivalent) was dissolved in 2 mL of a mixed solution of dichloromethane and methanol (the volume ratio of dichloromethane to methanol was 1:1), and then trifluoroacetic acid was added to react for 3 hours. After the reaction was complete by thin layer chromatography, compound 14 (30 mg, 0.016 mmol, quantitative) was obtained as a white solid: 1H NMR (600MHz, CDCl3) δ5.18(s,4H),4.57(s,1H),4.16(s,3H),3.91(s,3H),3.55(d,J=79.0Hz,18H) ,3.32(s,4H),2.73-2.15(m,12H),1.57(s,12H),1.26(d,J=16.7Hz,102H),0.87(d,J=7.1Hz,18H). 13 C NMR (151MHz, CDCl3) δ175.15l,174.98,174.76,174.53,173.38,171.57,170.24,160.7 9,156.73,100.25,72.44,72.40,71.61,71.28,71.13,71.07,70.95,70.05,69.94,69. 67,69.63,69.39,65.36,59.64,57.70,41.55,40.58,39.48,39.09,35.01,34.51,34.39,31.91,29.66,29.57,29.36,29.15,28.54,25.12,24.95,22.68,15.24,14.08,12.93. 31 P NMR (162 MHz, CDCl3) δ-2.96. Mass spectrum: [MH] - Theoretical composition C 99 H 187 N5O 25 P:1877.3258, measured value:1877.3221.

[0228] Phosphate buffered saline (pH 6.5) was used as the solvent to prepare 10 mmol / L pyridoxal phosphate solution and 10 μmol / L RBD solution. After mixing equal volumes of the two, the mixture was reacted at 37°C for 24 h. The mixture was centrifuged three times at 6000 rpm using a 0.5 mL ultrafiltration tube (Millipore, 10 kD) to obtain a purified protein solution. The purified protein solution was mixed with compound 14 (200 equivalents) and 800 μL of phosphate buffered saline was added. The protein was purified by centrifugation at 6000 rpm for 3 times using a 0.5 mL ultrafiltration tube (Millipore, 10 kD) to obtain a purified protein solution. The protein solution was lyophilized and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results are as follows: Figure 8As shown, it shows that one RBD molecule is covalently linked to one GAP112 derivative to form a conjugate vaccine (GAP112-RBD).

[0229] Test Example 2 Biological Activity Test

[0230] In order to better reflect the effect of the antiviral vaccine obtained in Example 2, Comparative Examples 5-8 are also provided. The vaccine components in Example 2 and Comparative Examples 5-8 are shown in Table 2. Comparative Example 8 and Example 2 are both prepared in the form of liposomes for testing and use. The preparation method is as follows: the vaccine components of Comparative Example 8 and Example 2 are respectively mixed with distearoylphosphatidylcholine and cholesterol in a molar ratio of 1:50:40 and dissolved in a mixture of 2 mL of dichloromethane and methanol (the volume ratio of dichloromethane and methanol is 1:1). After the solvent is removed by rotary evaporation, a thin layer of lipid film is left on the wall of the bottle; 10 mL of phosphate buffered saline is added thereto and shaken under argon protection at 55°C for 1 hour; finally, the resulting emulsion is ultrasonicated for 15 minutes to obtain the desired liposomes;

[0231] Comparative Example 5 is a phosphate buffered saline solution, Comparative Example 6 is a phosphate buffered saline solution containing 10 μg of RBD protein, and Comparative Example 7 is a phosphate buffered saline solution containing 10 μg of RBD protein and 4 mg of Alum adjuvant.

[0232] Table 2

[0233]

[0234]

[0235] Example 2 and Comparative Examples 5-8 were used as five groups of vaccines and were subcutaneously injected into BALB / C mice twice, on the 1st day and the 21st day respectively.

[0236] On day 23, the spleen and lymph nodes of mice were aseptically isolated for in vivo dendritic cell activation test. Figure 9 As shown;

[0237] Bone marrow dendritic cells and RAW264.7 were used for in vitro cell activation test. The results were as follows: Figure 10 shown.

[0238] On the 42nd day of immunization, blood was collected from the eye sockets and centrifuged to separate the serum. The serum collected on the 42nd day was used for antibody testing (IgG and its subtypes) and pseudovirus neutralization experiments. The results are as follows: Figure 11 、 12 and 13;

[0239] On day 42, the spleens of mice were aseptically separated and ground to obtain lymphocytes for enzyme-linked immunosorbent assay, intracellular cytokine staining test, and central memory T cell test. The results were as follows: Figure 14 、 15 , 16 and 17;

[0240] On the 42nd day, the important organs and serum of the mice were used for tissue section staining and biochemical index testing. Figure 18 and 19 shown.

[0241] In vivo determination of activation levels of lymph node / spleen-derived dendritic cells

[0242] 48 hours after the second vaccination, single-cell suspensions were isolated from the spleen or lymph of female BALB / c mice, the cells were washed with phosphate-buffered saline, and the red blood cells in the spleen were lysed at room temperature for 7 minutes. The cells were then incubated with anti-CD11c, anti-CD80, and anti-CD86 antibodies at 4°C for 30 minutes. The cells were centrifuged, washed, filtered, and resuspended in phosphate-buffered saline containing 0.1% by weight bovine serum albumin and stored in an ice bath. All antibody-labeled cells were tested on a flow cytometer.

[0243] The results are as follows Figure 9 As shown, the results showed that the conjugate vaccine (GAP112-RBD) could activate a higher proportion of dendritic cells.

[0244] Determination of activation level of bone marrow-derived dendritic cells (BMDCs) in vitro

[0245] Single-cell suspensions of whole bone marrow cells were isolated from the femurs and tibias of female BALB / c mice. The cells were washed with phosphate-buffered saline, and then erythrocytes were lysed at room temperature for 7 minutes. The collected cells were centrifuged, washed, and then centrifuged at 2 × 10 6 The cells were resuspended at 100 cells / ml and cultured in RPMI 1640 complete medium (containing 10% FBS, 100 μg / ml penicillin and 100 μg / ml streptomycin) supplemented with mouse IL-4 (10 ng / mL) and mouse GM-CSF (20 ng / mL). After 6 days, bone marrow-derived dendritic cells were collected and seeded into 24-well plates. After 24 hours of culture, 2×10 cells / well were added. 5Cells were transferred to 96-well plates and treated with PBS, RBD (PBS and RBD were 10 μg / well), GAP112 / RBD (GAP112 was 10 μg / well, RBD was 0.5 μg / well), and GAP112-RBD (10.5 μg / well) at 37°C for 24 hours. After stimulation, the cells were incubated with anti-CD11c, anti-CD80, and anti-CD86 antibodies for 30 minutes, followed by filtration. The filtered cells were resuspended in phosphate-buffered saline (containing 0.1% by weight bovine serum albumin) and stored in an ice bath. All antibody-labeled cells were tested on a flow cytometer.

[0246] In vitro RAW264.7 cell activation level determination

[0247] RAW264.7 cells were maintained in RPMI 1640 complete medium and seeded in 24-well culture plates (3 × 10 cells per well) at 37°C in a 5% carbon dioxide atmosphere. 5 Cells were cultured with PBS, RBD (10 μg / well of PBS and RBD), GAP112 / RBD (10 μg / well of GAP112 and 0.5 μg / well of RBD), and GAP112-RBD (10.5 μg / well) for 20 hours, followed by incubation with anti-CD80 and anti-CD86 antibodies at 4°C for 30 minutes. The cells were centrifuged, washed, filtered, resuspended in phosphate-buffered saline (containing 0.1% by weight bovine serum albumin), and stored on ice. All labeled cells were analyzed on a flow cytometer.

[0248] The results are as follows Figure 10 As shown, the results indicate that the conjugate vaccine (GAP112-RBD) of the present invention can activate a higher proportion of dendritic cells and macrophages in vitro.

[0249] Determination of antibody content in sera of immunized mice

[0250] In a 96-well ELISA plate (purchased from Corning 3590), each well was coated with 100 μL of antigen (RBD was dissolved in a carbonate buffer solution with a pH of 9.4-9.6 at a concentration of 1 μg / ml) and incubated at 4°C overnight; the plate was washed three times with PBST (phosphate buffered saline containing 0.05% by volume Tween-20), and then blocked with phosphate buffered saline containing 3% by weight casein (RBD-coated ELISA plates were blocked with phosphate buffered saline containing 3% by weight bovine serum albumin), and incubated at 37°C for 1 hour; the plate was washed again with PBST three times, and 0.1% by weight bovine serum albumin was added. The serum was diluted with phosphate-buffered saline and incubated at 37°C for 1 h; the plate was washed three times with PBST, and HRP-labeled goat anti-mouse secondary antibodies IgG, IgM, IgG1, IgG2a, IgG2b, and IgG3 diluted 4000:1 with PBS solution were added and incubated at 37°C for 1 h; the plate was washed twice with phosphate-buffered saline and three times with PBST, and freshly prepared 3,3',5,5'-tetramethylbenzidine colorimetric solution was added and incubated in the dark for 5 min. 2 M sulfuric acid was added to stop the color development, and the absorbance at a wavelength of 450 nm was measured by a microplate reader.

[0251] The results are as follows Figure 11 、 12 As shown, it is shown that the conjugate vaccine of the present invention (GAP112-RBD) can produce high-titer IgG antibodies, which is 2 orders of magnitude higher than the traditional GAP112 / RBD vaccine, fully demonstrating the advantages of the agonist-antigen coupling strategy of the present invention.

[0252] Determination of pseudovirus neutralizing antibody titers in sera of immunized mice

[0253] The sera of immunized mice from different groups were heat-inactivated (56°C water bath, 15-30 min). The treated serum samples were diluted with Opti-MEM according to different concentration gradients, pipetted evenly, and added to a 96-well plate. The pseudovirus (SARS-CoV-2 wild type and its variants) was then taken out from -80°C and reconstituted at 4°C. The reconstituted pseudovirus was diluted with Opti-MEM (0.15 μL pseudovirus / 25 μL diluent) and added to the above 96-well plate and incubated with the serum sample at room temperature for 1 hour. During the co-incubation period, ACE2-HEK293T cells were prepared, ACE2-HEK293T cells were counted, and the cell concentration was adjusted to 300,000 cells / mL with complete culture medium (without double antibody). They were placed in a cell culture incubator for use. After the serum sample and pseudovirus incubation is complete, the prepared ACE2-HEK293T cells are removed and pipetted evenly. 50 μL of the cell suspension is added to each well, and the edges are sealed with phosphate-buffered saline. The 96-well plate is then placed in a cell culture incubator and incubated. After 24 hours, 50 μL of complete culture medium is added to each well and incubated for another 24 hours. After incubation, the culture medium is carefully removed from the wells, and the plates are washed once with phosphate-buffered saline. 100 μL of cell lysis buffer is added to each well, and the plates are shaken at room temperature for 15 minutes. Then, 80 μL of luciferase detection reagent is added, and the plates are protected from light and mixed thoroughly. The absorbance at 450 nm is measured using a microplate reader.

[0254] The results are as follows Figure 13 As shown, the neutralization of the variant virus is the detection result of the conjugate vaccine (GAP112-RBD), which shows that the neutralizing antibodies produced by the conjugate vaccine (GAP112-RBD) of the present invention can better neutralize SARS-CoV-2 pseudovirus and its variants.

[0255] Determination of the number of antigen-specific IFN-γ secreting cells in the spleen of immunized mice

[0256] The whole process must be carried out in a clean bench to maintain aseptic operation; the spleens of blank mice and immunized mice were taken, ground into 5 mL of cold phosphate buffered saline solution through a cell sieve, and the cells were collected by centrifugation. 2 mL of red blood cell lysis buffer was added, and the cells were lysed for 3 minutes and then centrifuged again. After the cells were collected, they were washed with cold phosphate buffered saline solution and culture medium 1640 respectively, and centrifuged; the cells were collected and resuspended in 3 mL of complete culture medium. In the clean bench, the ELISPOT plate pre-coated with capture antibody was activated with 200 μL of serum-free culture medium for 10 minutes, the culture medium was poured out, and 100 μL of the cell suspension of the corresponding group (10 6cell / well), add 2 μL peptide library for stimulation, and incubate in a cell culture incubator for 18-24 hours (movement or shaking is strictly prohibited during incubation). After the incubation is completed, pour out the liquid, add 200 μL cold deionized water, and place at 4 ° C for 10 minutes to lyse the cells; shake out the liquid, wash 6 times with Washing Buffer, blot dry, add 100 μL diluted biotin-labeled antibody, and incubate at 37 ° C for 1 hour; shake out the liquid, wash 6 times with Washing Buffer, blot dry, add 100 μL diluted HRP-labeled avidin, and incubate at 37 ° C for 1 hour; shake out the liquid, wash 5 times with Washing Buffer, wash 2 times with deionized water, and blot dry; add 100 μL freshly prepared AEC color development solution to each well, and incubate at 37 ° C for 30 minutes; shake out the liquid, wash 5 times with deionized water, stop color development, place the ELISPOT plate in a cool place to dry naturally, and count with an ELISPOT analyzer.

[0257] The results are as follows Figure 14 As shown, it shows that the conjugate vaccine of the present invention (GAP112-RBD) can produce more IFN-γ spots, reflecting its stronger T cell-dependent immune response.

[0258] Determination of antigen-specific CD4+ and CD8+ T cell levels in the spleen of immunized mice

[0259] The collection of splenic lymphocytes was the same as that described in the above-mentioned determination of the number of antigen-specific IFN-γ secreting cells; the resuspended cell solution was added to a 24-well plate in a clean bench (400 μL complete medium and 200 μL cell suspension per well), 2 μL peptide library was added for stimulation, and the cells were incubated in a cell culture incubator; after 3 hours, the cells were taken out and 4 μL protein transport inhibitors (2 μL monensin and 2 μL brefeldin A) were added to each well, and the cells were continued to be incubated in the cell culture incubator for 12 hours; the cell solution was transferred to a 2 mL centrifuge tube, centrifuged, and the supernatant was discarded; the cells were washed with Stain Buffer (phosphate buffered saline containing 1 wt% bovine serum albumin, 1 wt% fetal bovine serum, and 0.1 wt% NaN3), centrifuged, and the supernatant was discarded; 500 μL of diluted CD3+, CD4+, and CD8+ cell staining solution was added to each tube, mixed, and stained on ice for 30 minutes; centrifuged, the supernatant was discarded, and Stain Wash twice with Washing Buffer; add 250 μL of fixation and permeabilization buffer to each tube, fix at 4°C for 10 minutes, and wash twice with Washing Buffer; add 250 μL of diluted IFN-γ and TNF-α cytokine staining solution to each tube, mix well, and stain at 4°C in the dark for 30 minutes; centrifuge, discard the supernatant, wash twice with Washing Buffer, resuspend the cells in 400 μL of 0.1% by weight bovine serum albumin in PBS, filter the cells, and test on a flow cytometer.

[0260] The results are as follows Figure 15 As shown, the left figure represents the proportion of CD8+T cells in the mouse spleen that can produce interferon γ and α-tumor necrosis factor cytokines, and the right figure represents the proportion of CD4+T cells in the mouse spleen that can produce interferon γ and α-tumor necrosis factor cytokines. The results show that the conjugate vaccine (GAP112-RBD) can produce higher levels of antigen-specific CD4+ and CD8+T cells, reflecting its stronger T cell-dependent immune response.

[0261] Determination of spleen / lymph memory T cell levels in immunized mice

[0262] Single-cell suspensions were isolated from the spleen and lymph of female BALB / c mice on day 42. The cells were washed with phosphate-buffered saline (PBS), and then lysed from the spleen's erythrocytes for 7 minutes at room temperature. The cells were then incubated with anti-CD3, anti-CD4, anti-CD8, anti-CD44, and anti-CD62 antibodies for 30 minutes at 4°C. The cells were centrifuged, washed, filtered, resuspended in phosphate-buffered saline (containing 0.1% by weight bovine serum albumin), and stored on ice. All labeled cells were analyzed by flow cytometry.

[0263] The results are as follows Figure 16 、 17 As shown, Figure 16 The left picture in the middle shows the ratio of CD8+ T cells to central memory T cells in the spleen of mice, and the right picture shows the ratio of CD4+ T cells to central memory T cells in the spleen of mice;

[0264] Figure 17 The left image in the middle shows the activation level of central memory T cells of CD8+ T cells in the lymph nodes of mice, and the right image shows the activation level of central memory T cells of CD4+ T cells in the lymph nodes of mice;

[0265] The results showed that the conjugate vaccine (GAP112-RBD) can produce high levels of central memory T cells, reflecting its good long-term T cell immunity.

[0266] Vaccine safety testing

[0267] On day 42, mice were killed by cervical dislocation and serum levels of glutamate transaminase, aspartate transaminase, alkaline phosphatase and urea were determined. Figure 18 As shown in Figure 2, all groups showed no difference in glutamate transaminase, aspartate transaminase, alkaline phosphatase, and urea levels. Vital organs (heart, liver, spleen, lung, kidney, and brain) were removed and preserved in formalin for tissue section testing. Figure 19As shown, it can be seen that the tissues of all groups showed normal behavior, with no abnormal cell death, tissue reaction or inflammatory infiltration immunity. This reflects that the conjugate vaccine (GAP112-RBD) of the present invention has good safety.

[0268] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A Toll-like receptor 4 immune agonist, characterized in that The Toll-like receptor 4 immune agonist is a compound represented by formula (1) and a pharmaceutically acceptable salt thereof. Formula (1) The structure of R in formula (1) is as follows: wherein m is an integer from 0 to 21, n is 0 or 1, o is an integer from 0 to 10, and p is an integer from 1 to 25; X and Y are both carbon elements; D is oxygen or sulfur; Any two of A, B, and C are -CH2OH, and the remaining one is -COOH or -CH2OH.

2. Use of the Toll-like receptor 4 immune agonist according to claim 1 in the preparation of vaccines, cell activators for in vitro activation, expansion and differentiation of immune cells, immune targeted drugs, pharmaceutical preparations combined or coupled with chemotherapy drugs, pharmaceutical preparations for antagonists of autoimmune diseases and immunomodulators for allergic diseases.

3. The use according to claim 2, characterized in that The vaccine is a semi-synthetic vaccine, a fully synthetic sugar vaccine, a peptide vaccine, a glycopeptide vaccine, a subunit vaccine, a nucleic acid vaccine, a virus-like particle vaccine, an inactivated vaccine or an attenuated vaccine.

4. The use according to claim 2, characterized in that The immune cells are DC cells, NK cells, NKT cells, macrophages or CAR-T cells.

5. The use according to claim 2, characterized in that The autoimmune disease is chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis or ulcerative colitis.

6. The use according to claim 2, characterized in that The allergic disease is allergic rhinitis, allergic asthma, allergic dermatitis or allergic urticaria.

7. An antiviral vaccine molecule, characterized in that The antiviral vaccine molecule is a two-component conjugate comprising a Toll-like receptor 4 immune agonist and a protein antigen, wherein the Toll-like receptor 4 immune agonist is covalently linked to the N-terminal amino acid of the protein antigen. Or the Toll-like receptor 4 immune agonist is covalently linked to the N-terminal amino acid of the protein antigen via a covalent linker arm; Wherein, the Toll-like receptor 4 immune agonist is a compound represented by formula (1) and a pharmaceutically acceptable salt thereof, Formula (1) The structure of R in formula (1) is as follows: wherein m is an integer from 0 to 21, n is 0 or 1, o is an integer from 0 to 10, and p is an integer from 1 to 25; X and Y are both carbon elements; D is oxygen or sulfur; Any two of A, B, and C are -CH2OH, and the remaining one serves as a connection site, and the structure of the connection site is -CH2O-.

8. The antiviral vaccine molecule according to claim 7, characterized in that The protein antigen is bovine serum albumin, tetanus toxoid, diphtheria toxoid, group B meningococcal outer membrane protein complex, pertussis toxoid, diphtheria toxin mutant, respiratory syncytial virus adhesion protein, fusion protein, influenza A virus membrane protein, rotavirus structural protein, filovirus structural protein and coronavirus structural protein; or Bovine serum albumin, tetanus toxoid, diphtheria toxoid, serogroup B meningococcal outer membrane protein complex, pertussis toxoid, diphtheria toxin mutant, respiratory syncytial virus adhesion protein, fusion protein, influenza A virus membrane protein, rotavirus structural protein, filovirus structural protein, subunit of coronavirus structural protein; or Modified derivatives of bovine serum albumin, tetanus toxoid, diphtheria toxoid, group B meningococcal outer membrane protein complex, pertussis toxoid, diphtheria toxin mutants, respiratory syncytial virus adhesion protein, fusion protein, influenza A virus membrane protein, rotavirus structural protein, filovirus structural protein and coronavirus structural protein.

9. The antiviral vaccine molecule according to claim 8, characterized in that The coronavirus structural protein is the Middle East Respiratory Syndrome Coronavirus, SARS or 2019 novel coronavirus structural protein.

10. The antiviral vaccine molecule according to claim 9, characterized in that The 2019 novel coronavirus structural protein is a nucleocapsid protein, a spike protein, or a subunit of a nucleocapsid protein or a spike protein.

11. The antiviral vaccine molecule according to claim 7, characterized in that The structure of the covalent linker arm is: -CO-, -O-CO-, -NH-CO-, -NH(C=NH)-, -SO2-, -O-SO2-, -NH-, -NH-CO-CH2-, -CH2-, -C2H4-, -C3H6-, -C4H8-, -C5H 10 -, -C6H 12 -, -C7H 14 -, -C8H 16 -, -C9H 18 -, -C 10 H 20 -, -CH(CH3)-, -C[(CH3)2]-, -CH2-CH(CH3)-, -CH(CH3)-CH2-, -CH(CH3)-C2H4-, -CH2-CH(CH3)-CH2-, -C2H4-CH(CH3)-, -CH2-C[(CH3)2]-, -C[(CH3)2]-CH2-, -CH(CH3)-CH(CH3)-, -C[(C2H5)(CH3)]-, -CH(C3H7)-, -(CH2-CH2-O) q -CH2-CH2-, -CO-CH2-, -CO-C2H4-, -CO-C3H6-, -CO-C4H8-, -CO-C5H 10 -, -CO-C6H 12 -, -CO-C7H 14 -, -CO-C8H 16 -, -CO-C9H 18 -, -CO-C 10 H 20 -, -CO-CH(CH3)-, -CO-C[(CH3)2]-, -CO-CH2-CH(CH3)-, -CO-CH(CH3)-CH2-, -CO-CH(CH3)-C2H4-, -CO-CH2-CH(CH3)-CH2-, -CO-C2H4-CH(CH3)-, -CO-CH2-C[(CH3)2]-, -CO-C[(CH3)2]-CH2-, -CO-CH(CH3)-CH(CH3)-, -CO-C[(C2H5)(CH3)]-, -CO-CH(C3H7)-, and -CO-(CH2-CH2-O) q -CH2-CH2-; Wherein, in the structure of the covalently linked arm, Each q is independently selected from an integer of 1-60; Each G is independently selected from at least one of -NH-, -O-, -S- and -SS-.

12. A method for preparing the antiviral vaccine molecule according to any one of claims 7 to 11, characterized in that: The method includes: The α-NH2 at the N-terminus of the protein antigen is converted into an α-ketoamide through a transamination reaction, and the α-ketoamide is reacted with an alkoxyamine reagent to generate an oxime, and then the Toll-like receptor 4 immunoagonist is linked to the oxime; or Converting the α-NH2 at the N-terminus of the protein antigen into α-ketoamide through a transamination reaction, and reacting the α-ketoamide with a Toll-like receptor 4 immunoagonist derivative modified with an alkoxyamine reagent to generate an oxime; or The α-NH2 at the N-terminus of the protein antigen is converted to an α-ketoamide by a transamination reaction; the α-ketoamide is reacted with an alkoxyamine reagent to generate an oxime, and then the oxime is reduced to a secondary amine using sodium cyanoborohydride, and then the Toll-like receptor 4 immunoagonist is linked to the secondary amine; or The α-NH2 at the N-terminus of the protein antigen is converted into an α-ketoamide through a transamination reaction; the α-ketoamide is reacted with a hydrazine reagent to generate a hydrazone, and then a Toll-like receptor 4 immunoagonist is linked to the hydrazone; or The α-NH2 at the N-terminus of the protein antigen is converted into α-ketoamide through a transamination reaction, and the α-ketoamide is reacted with a Toll-like receptor 4 immunoagonist derivative modified with a hydrazine reagent to generate a hydrazone; Wherein, the transamination reaction of the N-terminal amino acid of the protein antigen is completed by mediating pyridoxal phosphate.

13. Use of the antiviral vaccine molecule according to any one of claims 7 to 11 in the preparation of an antiviral vaccine.

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