Oxidation-reduction responsive carrier protein and its use in the preparation of vaccines

Abstract

The application discloses a redox response carrier protein and application thereof in preparation of vaccines, wherein at least one hapten structure is chemically coupled and modified on the carrier protein through a connecting arm containing a disulfide bond structure, and the hapten structure is selected from one of dinitrobenzene, galactose-alpha-(1,3)-galactose, rhamnose and phosphorylcholine. The redox response disulfide bond connecting arm can modify the hapten to the carrier protein, and can be recognized and combined by the naturally existing antibody in the body to form a complex, promote phagocytosis, processing and presentation of antigen presenting cells, thereby effectively improving the immunogenicity of the vaccine molecule and improving the antigen related immune response. After the redox disulfide bond connecting arm enters the lysosome, the disulfide bond is broken through response to the reducing property in the cell, lysosome escape occurs, cross-layer delivery of the antigen is realized, and the immunogenicity of the antigen is greatly improved.

Description

Technical Field

[0001] This invention relates to a redox-responsive carrier protein and its application in vaccines, belonging to the field of vaccine development technology. Background Technology

[0002] Glycoantigens are highly expressed on the surface of various pathogenic bacteria and tumor cells, possessing specific chemical structures. Using these specific glycans as vaccine components can induce a specific immune response in the human body against these glycans, thereby preventing or treating diseases and making them important targets for vaccine design. Currently, the US FDA has approved 10 glycoantigen vaccine products, primarily for the prevention of diseases caused by bacterial infections. Developing more safe and effective glycoantigen vaccines targeting a wider range of diseases is a key focus of current glycoantigen vaccine research.

[0003] However, the immunogenicity of glycoantigens is weak; they are generally T-cell-independent antigens, posing a significant obstacle to the development of glycoantigen vaccines. For pathogens such as bacteria, fungi, and parasites, their glycan structures differ considerably from those in the human body, usually evoking a stronger immune response. Therefore, research on these types of glycoantigen vaccines faces relatively fewer challenges. The development of tumor glycoantigen vaccines is more difficult because tumor-associated carbohydrate antigens (TACAs) are human autoantigens with weak immunogenicity, failing to induce a sufficiently strong immune response against TACAs in the human body. Currently, there are no tumor glycoantigen vaccines on the market. Viruses themselves are produced by human expression systems, and their glycan structures are not significantly different from those in human cells. The main research approach for viral glycoantigen vaccines is to design vaccines that mimic the antigenic epitopes recognized by broadly neutralizing antibodies, aiming to induce antibodies similar to broadly neutralizing antibodies in the human body, thereby achieving viral prevention or therapeutic effects.

[0004] To improve the immunogenicity of glycoprotein vaccines, the traditional vaccine design strategy involves conjugating them with carrier proteins to create glycoprotein conjugate vaccines. However, this type of vaccine design still has some shortcomings. First, traditional carrier proteins, such as keyhole limpet hemocyanin (KLH) and diphtheria toxin CRM197, contain a large number of carrier protein epitopes, competing for a significant amount of immune system resources, causing unnecessary immune responses, and even inhibiting the immune response to the glycoprotein antigen itself, resulting in low selectivity of the generated serum antibodies. Second, the number of protein carriers currently available for clinical use is very limited. Repeated use of the same vaccine carrier may interfere with the immune response process and cause instability in immunization efficiency. Therefore, there is an urgent need to develop new vaccine protein carrier strategies.

[0005] In recent years, redox-responsive linkers have been used in the construction of pharmaceutical molecules and vaccines. The team of Chen Yongming / Liu Zhijia at Sun Yat-sen University and their collaborators developed a library of biocompatible redox-responsive cationic peptide polymer materials based on Ugi multicomponent chemistry. The screened polymer carriers, when co-assembled with protein drugs, can form small-sized nanocomposites with high drug encapsulation efficiency. These nanocomposites enter cells via macropinocytosis and / or caveolin-mediated endocytosis, escape via lysosomes, and are effectively released into the cell while maintaining their biological activity due to the rapid degradation of disulfide bonds in the polymer backbone triggered by intracellular glutathione. This demonstrates that multicomponent reactions can be used as a material discovery tool to screen biofunctional polymer carrier materials. Professor Tang Li's research group designed a redox-responsive condensation polymer epitope (PNE) by polycondensing a peptide neoantigen with an adjuvant using redox-responsive linker monomers. After internalization, the PNE rapidly releases the epitope under intracellular reducing conditions, promoting lysosomal escape and cytoplasmic delivery of the peptide antigen.

[0006] Leveraging the advantages of redox-responsive linkers, this invention designs and synthesizes a redox-responsive linker to couple a hapten to a glycoprotein. The hapten on this linker recruits endogenous antibodies to enhance the immune response. Unlike traditional highly immunogenic carrier proteins, this invention significantly improves the immune activation of low-immunogenic carrier proteins while simultaneously reducing the immune system's response to the carrier protein itself. This enhances the specific immune response against the coupled exogenous antigens (e.g., tumor-associated glycoantigens, fungal glycoantigens, etc.). Furthermore, after entering the lysosome, this redox-responsive linker responds to intracellular reducing properties, enabling lysosomal escape and inducing cross-presentation, thereby greatly enhancing the immune response to the glycoantigen. Summary of the Invention

[0007] Technical Problem: The purpose of this invention is to utilize a redox-responsive linker to modify haptens onto glycoproteins to prepare glycoprotein vaccines. The modified hapten on the carrier protein recruits naturally occurring antibodies to form immune complexes, thereby enhancing the phagocytosis, processing, and presentation of antigens by antigen-presenting cells, thus improving the effectiveness of the immune response. Furthermore, the redox-responsive linker enables lysosomal escape, inducing cross-delivery of glycoantigens. This invention can enhance the immune system's immune response to exogenous antigens while reducing the response to carrier proteins, avoiding interference between identical carrier proteins, and significantly improving the immune response to glycoantigens through lysosomal escape, thus solving the problem of weak immunogenicity of glycoantigens.

[0008] Technical solution: The technical solution adopted in this invention is as follows.

[0009] The first objective of this invention is to provide a method for preparing a redox-responsive carrier protein, wherein the carrier protein is modified with at least one hapten structure by means of a redox-responsive linker arm, wherein the redox-responsive linker arm has a disulfide bond chemical structure and the linker arm can be broken in response to redox conditions.

[0010] Furthermore, as a preferred embodiment, the hapten structure is coupled to the amino or carboxyl group of the carrier protein via an amide condensation reaction.

[0011] Furthermore, as a preferred embodiment, the hapten is a chemical group capable of recruiting corresponding specific natural antibodies, including but not limited to any one of the following groups: dinitrobenzene (DNP), galactose-α-(1,3)-galactose (α-Gal), rhamnose (Rha), and phosphorocholine (PC).

[0012] Furthermore, as a preferred embodiment, the carrier protein is selected from one or more of the following fusion proteins: bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin (OVA), diphtheria toxoid, the non-toxic mutant of diphtheria toxoid CRM197, tetanus toxoid, keyhole hemocyanin (KLH), ferritin, superantigen, and bacterial expressed proteins.

[0013] A second objective of this invention is to provide a redox-responsive carrier protein, wherein at least one hapten structure is modified onto the carrier protein via a redox-responsive disulfide linker arm, the linker arm being cleavable in response to redox conditions, and the redox-responsive carrier protein has the following structural formula:

[0014]

[0015] In the formula, Linker represents the connecting arm and has the following structure:

[0016] CP stands for carrier protein.

[0017] Furthermore, as one embodiment of this application, Rha is used as an example for illustration. However, it should be noted that when the hapten is other substances such as dinitrobenzene or phosphorycholine, the same principle applies as when Rha is used, and all fall within the scope of protection of this invention, which will not be elaborated further here. When the hapten is Rha, a redox-responsive carrier protein is provided, wherein at least one hapten structure is modified on the carrier protein through a redox-responsive disulfide bond linker arm, the linker arm of which can break in response to redox conditions. The redox-responsive carrier protein has the following structural formula:

[0018]

[0019] Where n and m are natural numbers, n represents the number of repeating structural units, i.e., the length of the PEG chain; m represents the number of haptens Rha.

[0020] The connecting arm has the following structural formula:

[0021] In this invention, the connecting arm is used as a switch for redox reactions, and is activated in the intracellular environment to enable lysosomal escape.

[0022] A third objective of this invention is to provide a redox-responsive conjugate comprising a redox-responsive carrier protein and a target antigen, wherein the redox-responsive carrier protein is modified with the target antigen, and wherein the redox-responsive carrier protein is a redox-responsive carrier protein prepared by the method described above, or is a redox-responsive carrier protein described above.

[0023] Furthermore, as one embodiment of this application, an example is provided using Rha as the hapten, which is not intended to limit the scope of protection of this invention. Other similar cases will not be described in detail here. When the hapten is Rha, the conjugate has the following structural formula:

[0024]

[0025] Where n', n, and m are natural numbers, representing the number of glycoantigens, the number of structural repeating units (i.e., the length of the PEG chain), and the number of hapten Rha, respectively.

[0026] The connecting arm has the following structural formula:

[0027]

[0028] Furthermore, as one embodiment of this application, β-glucan is used as an example for illustration. This example is not intended to limit the scope of protection of the present invention, and other similar cases will not be repeated here. When the glycoantigen is β-glucan, the glycoantigen has the following structural formula:

[0029]

[0030] A fourth objective of this invention is to provide a method for preparing a redox-responsive conjugate, wherein the conjugate is prepared by coupling a redox-responsive carrier protein with a target antigen. The redox-responsive carrier protein is either a redox-responsive carrier protein prepared by the method described above, or a redox-responsive carrier protein as described above; or it is composed of a carrier protein modified with the target antigen modified with at least one hapten structure via a redox-responsive disulfide linker arm. The conjugate obtained by coupling a hapten with a target antigen-modified carrier protein achieves lysosomal escape in response to intracellular reducing properties, greatly enhancing the immunogenicity of the glycoantigen. Further, as a preferred embodiment, the conjugate (also referred to as a conjugate) is a conjugate obtained by coupling a hapten with a carrier protein modified with a redox-responsive disulfide linker arm and a target antigen, wherein the target antigen includes any one of tumor antigens, bacterial antigens, viral antigens, and fungal antigens.

[0031] Furthermore, as a preferred embodiment, the target antigen includes tumor antigen, bacterial antigen, viral antigen, and fungal antigen.

[0032] Furthermore, as a preferred embodiment, the target glycoantigen is a tumor antigen or a fungal antigen.

[0033] Furthermore, as a preferred embodiment, the target antigen is selected from tumor-associated glycoantigens and fungal-associated glycoantigens, including: TF, Tn, sTn, Globo H, GM2, GM3, GD2, GD3, MUC1 and derivatives of MUC1, β-glucan, β-mannan, β-1,3-glucan, β-1,6-glucan, β-1,2-mannan, etc.

[0034] A fifth object of the present invention is to provide an immunogenic composition comprising a conjugate of the hapten-modified carrier protein or a pharmaceutically acceptable excipient, carrier, or diluent thereof.

[0035] When the target antigen is a glycoprotein, this invention provides a redox-responsive linker based on common carrier proteins, which is used to couple the hapten to the glycoprotein to form a redox-responsive conjugate, i.e., a glycoprotein vaccine.

[0036] Furthermore, as a preferred embodiment, Rha-OVA pre-immunization is performed before using the conjugate for immunization.

[0037] Another object of the present invention is to provide the use of conjugates of the hapten-modified redox-responsive carrier protein, or immunogenic compositions comprising conjugates of the hapten-modified redox-responsive carrier protein, in the preparation of vaccines.

[0038] Another object of the present invention is to provide the use of conjugates of the hapten-modified redox-responsive carrier protein in the preparation of medicaments for the prevention or treatment of cancer. The cancers described in this aspect include tumors and other types of cancer.

[0039] Beneficial effects:

[0040] (1) Compared with the rigid linker arm of glutaric acid disuccinate (DSG), the redox-responsive linker arm used in this invention breaks the disulfide bond by responding to the reducing properties in the cell, realizing lysosomal escape and antigen cross-delivery, which greatly improves the immunogenicity of glycoantigens; and compared with traditional carrier proteins, the hapten-modified carrier protein provided by this invention can promote the uptake, processing and presentation of antigen-presenting cells by interacting with naturally occurring antibodies in the human body, thereby significantly improving the immune response of the immune system to exogenous antigens conjugated on the carrier protein, while reducing the immune response of the immune system to the carrier protein.

[0041] (2) The application of this invention in vaccine construction by modifying haptens onto glycoproteins using a redox-responsive linker not only greatly solves the problem of weak immunogenicity of glycoantigens, but also provides a new approach to vaccine construction. This strategy provides a simple and effective method to enhance the effectiveness of carrier proteins in vaccine construction, and allows for the rational modification of commonly used carrier proteins to obtain more carrier proteins that can be used for vaccine preparation. Furthermore, it avoids the problem of mutual interference between the same carrier proteins.

[0042] (3) Considering the wide application of carbohydrate antigens in vaccine research and industry, this invention can be used to construct new carbohydrate complexes for vaccine development. The conjugates of hapten-modified glycoproteins of this invention can also be used in the preparation of drugs for the prevention or treatment of cancer. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the structure of the redox-responsive carrier protein of the present invention.

[0044] Figure 2The design and structure of the β-glucan-BSA-Rha vaccine constructs are described below. CP represents the carrier protein, and n' and m are natural numbers representing the number of glycoantigens and hapten Rha, respectively. The constructs are: Compound 1 (CP = BSA, β-glucan-BSA, containing 7.9% w / w β-glucan and 0% w / w Rha); Compound 2 (CP = HSA, β-glucan-HSA, containing 3.7% w / w β-glucan and 0% w / w Rha); Compound 3 (CP = BSA, β-glucan-BSA-L1-Rha, containing 7.9% w / w β-glucan and 2.2% w / w Rha); and Compound 4 (CP = BSA, β-glucan-BSA-L2-Rha, containing 7.9% w / w β-glucan and 2.4% w / w Rha).

[0045] Figure 3 The synthetic methods for compounds 1-4 are as follows: (a) CH3CN, 12 h, rt; (b) saturated NH4HCO3, 45 °C, 4 Days; (c) glutaric acid disuccinate, DMF:PBS (4:1), rt; (d) BSA / HSA, PBS, rt; (e) Ac2O, pyridine, rt; (f) triethylene glycol azide, BF3·Et2O, CH2Cl2, rt, 68%; (g) MeONa, MeOH; (h) 10% Pd / c, H2, MeOH, rt; (i) glutaric acid disuccinate, DMF:PBS (4:1), rt; (j) 4, PBS, rt.

[0046] Figure 4 This is the NMR spectrum of the redox linker.

[0047] Figure 5 The molecular weight of β-glucan was determined using MALDI-TOF-MS.

[0048] Figure 6 To determine the molecular weight of β-glucan-NH2 using MALDI-TOF-MS.

[0049] Figure 7 The molecular weight of β-glucan-BSA was determined using MALDI-TOF-MS.

[0050] Figure 8 The molecular weight of β-glucan-HSA was determined using the MALDI-TOF-MS method.

[0051] Figure 9 This is the NMR spectrum of compound 14.

[0052] Figure 10The molecular weight of β-glucan-BSA-L1-Rha was determined using MALDI-TOF-MS.

[0053] Figure 11 The molecular weight of β-glucan-BSA-L2-Rha was determined using MALDI-TOF-MS.

[0054] Figure 12 To collect the mean titers of Rha-specific antibodies in the antiserum of mice in groups 5 and 6 on day 21 after Rha-OVA inoculation, the mean antibody titers from three parallel experiments are shown for each sample, and the error bars show the standard deviations of the three parallel experiments.

[0055] Figure 13 For the immunological evaluation of the synthetic vaccine: (A) Antibody titers in each group on day 28, with compound 4 used as the coating antigen to detect β-glucan-specific antibodies; (B) Mean titers of total β-glucan-specific antibodies collected in the serum of mice in groups 1–6 on days 7, 14, 21, and 28; (C) Evaluation of BSA-specific and β-glucan-specific antibodies in the mixed antiserum of groups 3–6; (D) Antibody isotypes and subtypes of the conjugate vaccine. The mean antibody titers from three parallel experiments are shown for each sample. Error bars represent the standard error of the three replicates (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.005).

[0056] Figure 14 Assessment of IFN-γ and IL-6 in the serum of immunized mice: (A) IFN-γ; (B) IL-6. The mean antibody titer for each sample is shown in three parallel experiments, and the error bars show the standard deviation of the three parallel experiments.

[0057] Figure 15 For the immunological evaluation of the synthetic vaccine: the binding of antiserum from groups 1-6 to heat-inactivated HKCA was analyzed by laser confocal microscopy. (A) BSA; (B) β-glucan-BSA; (C) β-glucan-BSA-L1-Rha; (D) β-glucan-BSA-L2-Rha; (E) Pre-immunized β-glucan-BSA-L1-Rha; (F) Pre-immunized β-glucan-BSA-L2-Rha.

[0058] Figure 16 For the in vitro kinetic study of the redox connecting arm.

[0059] Figure 17The results of the immunogenicity of the vaccine conjugate obtained in Example 2 are as follows: (B) Antibody titers. (A) Average titers of total STn-specific antibodies in the serum of mice in groups 1-5 were collected on days 7, 14, 21 and 28, respectively; (B) Antibody isotypes and subtypes of the conjugate vaccine. Detailed Implementation

[0060] The present invention will be further described below with reference to the accompanying drawings and embodiments. The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the present invention as described in detail in the claims.

[0061] For the following embodiments, it should be noted in advance that the method of the present invention first modifies at least one hapten structure onto the carrier protein to form a hapten structure-modified carrier protein, such as BSA+Rha→BSA-Rha, etc. Figure 1 As shown; the carrier protein modified with the obtained hapten structure is then coupled with the target antigen to obtain conjugates, such as BSA-Rha+β-glucan→BSA-Rha-β-glucan, etc. Figure 2 As shown. However, in the synthesis process, this experiment was designed to first conjugate the carrier protein with the target antigen, such as β-glucan, to form conjugates with the same loading, and then divide them into two parts, each modified with a different linker arm to modify a hapten, such as Rha, to ensure that the amount of the target antigen β-glucan is consistent. This facilitates the comparison of the effects of haptens modified with different linker arms on the conjugates.

[0062] Example 1: Preparation of redox-responsive L-rhamnose Rha-modified carrier protein BSA and its conjugated antigen β-glucan conjugate

[0063] The carrier protein is bovine serum albumin (BSA), the hapten structure is L-rhamnose 10 (Rha), and the target antigen is β-1,3-glucan fungal antigen with a degree of polymerization of 5-20.

[0064] 1.1: Synthesis of β-glucan and Rha derivatives and their coupling reaction with BSA

[0065] like Figure 3As shown, in this embodiment, the carrier proteins BSA and HAS are first conjugated with the target antigen β-glucan to prepare compound 1 (CP = BSA, β-glucan-BSA, containing 7.9% w / w β-glucan and 0% w / w Rha) and compound 2 (CP = HSA, β-glucan-HSA, containing 3.7% w / w β-glucan and 0% w / w Rha). Then, based on compound 1, the hapten Rha is further modified onto the carrier protein of compound 1 using different linker arms L1 and L2 to prepare redox-responsive conjugates, namely compound 3 (CP = BSA, β-glucan-BSA-L1-Rha, containing 7.9% w / w β-glucan and 2.2% w / w Rha) and compound 4 (CP = BSA, β-glucan-BSA-L2-Rha, containing 7.9% w / w β-glucan and 0% w / w Rha). 2.4% w / w), where, as a control group, L1 is a rigid connecting arm with the following structural formula: As the experimental group, the structural formula of L2 is:

[0066] like Figure 3 The following is a description of the synthetic process for compounds 1-4:

[0067] (1) Composite connecting arm

[0068] (a) In accordance with the method described in the literature (Org. Bioorg. Chem., 2017, 5, 1227, DOI: 10.1016 / j.bmc.2016.12.033), the redox linker L2 (compound 6) was synthesized, as follows: Figure 4 As shown.

[0069] (2) The carrier proteins BSA and HSA were conjugated with the target antigen β-glucan to prepare compounds 1 and 2.

[0070] (g)MeONa,MeOH; (h)10%Pd / c,H2,MeOH,rt; (i)Glutaric acid disuccinate,DMF:PBS (4:1),rt; (j)4,PBS,rt.

[0071] (b) β-glucan antigens (compound 7) with different degrees of polymerization were synthesized by acid hydrolysis. Figure 5 ), and modified the hydroxyl group at position 1 to an amino group (compound 8, Figure 6 ).

[0072] Then, the β-glucan antigen was bound to bovine serum albumin (BSA) via a bifunctional glutarate method. This is a well-established method used in our previous project (Org. Biomol. Chem., 2014, 12, 3238, DOI: 10.1039 / c4ob00390j) and does not affect the conjugate. Specifically, the steps (c) and (d) are as follows:

[0073] (c) Compound 8 was reacted with a large excess (15 equivalents) of glutaric acid disuccinate (DSG) in N,N-dimethylformamide (DMF) to give the corresponding monoester 9, which was then purified by repeated precipitation to remove excess DSG.

[0074] (d) Mix monoester 9 with BSA or HSA in PBS buffer to generate conjugated β-glucan-BSA conjugate 1 (compound 1) or β-glucan-HSA conjugate 2 (compound 2). Purify using a centrifugal filtration device (MW cutoff: 10 kDa) to remove excess monoester 9 and other small molecules (reaction process as follows). Figure 3 As shown in the figure). The β-glucan antigen loading in conjugate 1 and conjugate 2 was determined by MALDI-TOF-MS to be 7.9% and 3.7%, respectively. Figure 7 , Figure 8 This indicates that the coupling reaction was successful and the antigen loading level was suitable for biological research.

[0075] (3) Synthesize the Rha hapten and couple it to the linker arm.

[0076] In this embodiment, the synthesis of Rha hapten starting from L-rhamnose 10 specifically includes the following steps:

[0077] (e) First, rhamnose 10 was peracetylated to obtain compound 11.

[0078] (f) Compound 12 was obtained by glycosylation of triglyceride with BF3·Et2O and CH2Cl2.

[0079] (g) Under Zemplén transesterification conditions, the acetyl group was removed to give compound 13, which was then hydrogenated to reduce the azide group, yielding compound 14 with an amine group at the reduced end. Figure 9 ).

[0080] (h) Monoester 14 is reacted with a large excess (15 equivalents) of DSG, namely L1 or the redox linker L2 obtained in step (1), to obtain monoester 15 and monoester 16.

[0081] (4) Compound 1 was conjugated with the Rha hapten to prepare compounds 3 and 4.

[0082] (i) β-glucan-BSA conjugate 1 was incubated with monoester 15 and monoester 16 in an equivalent ratio of 1:15 in PBS buffer (pH=7.4) to obtain β-glucan-BSA-Rha conjugates 3 and 4.

[0083] The loading of Rha hapten in conjugated conjugate 3 and conjugate 4 was determined by MALDI-TOF-MS, and was 2.2% and 2.4%, respectively. Figure 10 , Figure 11 Therefore, in subsequent experiments, these two conjugates were named β-glucan-BSA-L1-Rha and β-glucan-BSA-L2-Rha. The different linker arms in β-glucan-BSA-Rha (conjugate 3 and conjugate 4) that connect Rha to the carrier protein help to reveal the ability of redox linker arms to mediate anti-β-glucan-BSA vaccination.

[0084] The structural formula of the compound is as follows Figure 2 As shown, the results are characterized as follows:

[0085] Compound 1CP=BSAβ-glucan-BSA (β-glucan loading=7.9%, Rha loading=0%)

[0086] Compound 2CP=HSAβ-glucan-HSA (β-glucan loading=3.7%, Rha loading=0%)

[0087] Compound 3CP=BSAβ-glucan-BSA-L1-Rha (β-glucan loading=7.9%, Rha loading=2.2%)

[0088] Compound 4CP=BSAβ-glucan-BSA-L2-Rha (β-glucan loading=7.9%, Rha loading=2.4%)

[0089] 1.2: Immunological activity of vaccine conjugates

[0090] Six groups of mice, C57BL / 6J (female, 6 weeks old) (groups 1-6), were used in these studies. The experimental setup was as follows:

[0091] Group 1: Immunized with BSA

[0092] Group 2: Immunized with β-glucan-BSA conjugate 1 that does not contain Rha;

[0093] Groups 3 and 4: Immunized with β-glucan-BSA-Rha (conjugate 3 and conjugate 4), respectively;

[0094] Groups 5-6: Pre-immunized with Rha-OVA, then immunized with β-glucan-BSA-Rha (3, 4) respectively.

[0095] Previous studies have demonstrated (J Am Chem Soc. 2010; 132(48): 17236-46. doi: 10.1021 / ja107029z) that laboratory mice do not contain significant titers of natural anti-Rha antibodies. Therefore, we pre-immunized mice in groups 5–6 with Rha-OVA to establish high levels of anti-Rha antibodies, which were considered to be endogenous antibodies. The pre-immunization method was as follows: Rha-OVA (3 μg sugar per mouse per dose) was dissolved in 0.3 ml of 10× phosphate-buffered saline (PBS) buffer and then diluted to 1.25 ml of 1× PBS solution. The solution was mixed with 1.25 ml of Freund's complete adjuvant (1:1, v / v) and an emulsion was formed according to the manufacturer's protocol. Five female C57BL / 6 mice in each group were immunized on day 1 by intramuscular injection of 0.1 ml of the above-prepared conjugate vaccine and Freund's complete adjuvant emulsion. Following the initial immunization, mice were boosted three times by subcutaneous injection of the same conjugate emulsion on days 7, 14, and 21. Blood samples were collected from each mouse via a leg vein on day 0 before the initial immunization and on days 7, 14, 21, and 28 post-immunization. Antiserum extracted from the blood samples was stored at -80°C before use. Antibody titers were displayed ( Figure 12 After immunization with Rha-OVA, Rha-specific antibodies showed high titers.

[0096] Antibodies (approximately 200,000) were detected in the serum of mice in groups 5 and 6. Subsequently, mice in groups 5 and 6 were immunized with β-glucan-BSA-Rha conjugates 3 and 4 on day 1, with booster immunizations on days 7, 14, and 21. Blood samples were collected from mice on days 7, 14, 21, and 28 after the first immunization. Antibodies were detected using enzyme-linked immunosorbent assay (ELISA). Regression analysis of the OD value versus serum dilution curves determined the antibody titer to be the dilution factor at which the optical density (OD) at 415 nm reached 0.2.

[0097] Enzyme-linked immunosorbent assay (ELISA) protocol: Dissolve 100 μl of β-glucan-HSA or Rha-HSA (2 μg / ml) in coating buffer (0.1 M bicarbonate, pH 9.6) and treat each well of the ELISA plate at 4°C, then incubate at 37°C for 1 h. Wash three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST), followed by three washes with PBST. Incubate for 1 h with blocking buffer (gelatin blocking buffer) at rt. Add mixed or single mouse antiserum (serial semi-logarithmic dilutions in PBS from 1:300 to 1:656100) to the coated ELISA plates (100 μL / well) and incubate at 37°C for 2 h. Then wash the plates with PBST and incubate at rt for 1 h with goat anti-mouse IgG or IgM antibody (100 μL / well). Finally, the plates were washed with PBST and developed with 100 μL of p-nitrophenyl phosphate (PNPP) solution (1.67 mg / mL in buffer) at room temperature for 30 minutes. Colorimetric readings were then performed at 415 nm using an iMark microplate reader. Optical density (OD) values ​​were plotted on a logarithmic scale based on the antiserum dilution values ​​to obtain the best-fit line. The dilution values ​​were calculated using a linear equation, where the OD was 0.2, and the antibody titer was calculated as the reciprocal of the dilution value.

[0098] Figure 13 Table 1 summarizes the ELISA results of the antiserum from the 7 groups on day 28.

[0099] Group 1, immunized with BSA, produced the lowest level of β-glucan-specific antibodies (approximately 3771).

[0100] Group 2 was immunized with Rha-free β-glucan-BSA conjugate 1, and the resulting β-glucan-specific antibody level was 16775.

[0101] Groups 3 and 4 were immunized with β-glucan-BSA-Rha (3, 4), and significantly high-titer anti-β-glucan specific antibodies were obtained, namely 34501 and 38762, respectively. Compared with Group 1, the induced antibody levels were at least about 9 times higher and up to about 10 times higher.

[0102] Groups 5 and 6 were pre-immunized with Rha-OVA and then immunized with β-glucan-BSA-Rha (1-3). The production of anti-β-glucan specific antibodies was significantly increased, at 123,443 and 186,445 respectively. Compared with Group 1, the production of anti-β-glucan antibodies increased by 32 and 49 times respectively.

[0103] Table 1 Comparison of Immunological Activity Results of Vaccine Conjugates

[0104]

[0105]

[0106] These results indicate that pre-existing endogenous anti-Rha antibodies in mouse serum can target APC cells to deliver glycoconjugates (3, 4), thereby achieving more efficient transport and internalization, as well as a more effective overall immune response. Notably, anti-β-glucan titers were closely correlated with the Rha loading level in the glycoconjugates (groups 3-4 and 5-6). Binding of the Rha hapten to the vaccine did not inhibit the immunogenicity of β-glucan. In contrast, in the presence of anti-Rha antibodies, a high Rha loading in the vaccine generally led to better uptake of β-glucan precursors and the generation of more anti-β-glucan-specific antibodies. Furthermore, glycoconjugate 4 (groups 4 and 6), containing a redox linker, generated more β-glucan-specific antibodies compared to conjugate 3 (groups 3 and 5), which had a rigid linker.

[0107] We also analyzed the progression of anti-β-glucan-specific immune responses in groups 2-4 and 5-6. For example... Figure 13 As shown in Figure B, mice in groups 2-4 produced low titers of anti-β-glucan specific antibodies on day 21. However, anti-β-glucan levels significantly increased on day 28. Notably, pre-immunized groups 5-6 produced higher levels of anti-β-glucan specific antibodies on day 28 compared to non-pre-immunized groups 3-4, and groups 4 and 6, which used redox linkers, produced significantly more anti-β-glucan specific antibodies than groups 3 and 5. This interesting finding suggests that redox linkers enable lysosomal escape of the vaccine, thereby achieving cross-delivery of antigens and greatly improving immunization efficiency. Furthermore, the β-glucan-BSA-Rha vaccine may have a self-adjuvant effect in the later stages of immunization, possibly due to an antigen uptake mechanism mediated by anti-Rha antibodies. Therefore, with the help of endogenous anti-Rha antibodies, a faster anti-β-glucan immune response can be elicited. Figure 13 B).

[0108] Anti-BSA and anti-β-glucan specific antibodies in groups 5 and 6 were detected by ELISA. The results showed that the anti-β-glucan specific antibody production in groups 5 and 6, which underwent pre-immunization with OVA-Rha, was significantly higher than that of BSA specific antibodies. Figure 13(C) This result indicates that BSA has weak immunogenicity, and with the help of endogenous Rha antibodies, the immune response against β-glucan is enhanced without suppression. This result inspires us to believe that carrier proteins with low immunostimulatory capacity, utilizing hapten modification strategies, may be applied in future carbohydrate vaccines and cancer treatments.

[0109] ELISA was used to detect antibody isotypes and isotypes in groups 1-6. Clearly, IgG2b and IgG2c were the major isotypes in groups 3-6, indicating that the Rha-modified BSA carrier protein successfully stimulated T-cell immunity. Figure 13 D).

[0110] The expression levels of IFN-γ and IL-6 in the serum of groups 1-6 were detected by ELISA. IFN-γ and IL-6 were produced in the serum of all groups (1-6), indicating that both Th1 and Th2 immune responses occurred. Figure 14 A, Figure 14 B).

[0111] 1.3: Immunological evaluation of synthetic vaccines

[0112] Antiserum collected from mice immunized in groups 1-6 was co-incubated with heat-inactivated HKCA. After fluorescent dual antibody labeling, the binding ability of serum to fungi was analyzed using laser confocal microscopy. It was clearly observed that the serum from groups 2-6 (BF group) could bind to heat-inactivated HKCA. Figure 15 Compared with the negative control group 1 (group A), the fungal particles and hyphae cells in the experimental group were stained evenly, indicating that the antiserum had a strong binding force with HKCA cells.

[0113] 1.4: In vitro kinetic study of the redox connecting arm

[0114] To verify whether the redox linker L2 could break under reducing conditions, Rha-L2 was reacted with DTT at 37°C. The concentration of p-nitrophenol in the reaction solution was measured at OD 405 nm. Readings were taken at 0 min, 1 min, 1 h, 3 h, and 6 h after the reaction. It was found that under DTT reducing agent conditions, the concentration of p-nitrophenol almost reached its maximum at 1 h, while the concentration continued to increase slowly without DTT. Figure 16 This indicates that the redox connecting arm can break quickly under reducing conditions.

[0115] Example 2: Synthesis of STn and Rha derivatives and their coupling reaction with BSA

[0116] The target antigen was STn. Using the same method as in Example 1, the antigen was conjugated to the carrier protein bovine serum albumin (BSA) via an amide condensation reaction to form a glycoprotein conjugate. Using the same method as in Example 1, different linker arms were used to conjugate with the hapten Rha, resulting in two different STn-BSA-Rha conjugate vaccines.

[0117] The immunogenicity of the vaccine conjugate was determined using the method described in 1.2 above, and the results are as follows: Figure 17 As shown in Figure A, the group containing the redox linker in the STn-BSA-Rha conjugate vaccine produced higher anti-STn antibody titers, indicating that the redox linker enables lysosomal escape of the antigen and cross-delivery, greatly improving the antigen's immunogenicity. It also elicited high levels of Ig2b and IgG2c antibody titers, as well as a relatively high level of IgG1 antibody titer. Figure 17 B). This demonstrates that the BSA carrier protein modified with Rha successfully stimulated T-cell immunity.

[0118] In summary, this invention proposes a vaccine, its preparation method, and its application based on a redox-responsive linker arm. By chemically coupling at least one rhamnose hapten (Rha) or a hapten to a carrier protein via a redox linker arm, the hapten-modified carrier protein can be recognized and bound by naturally occurring antibodies in vivo, forming a complex. This promotes phagocytosis, processing, and presentation by antigen-presenting cells, thereby effectively improving the immunogenicity of the vaccine molecule and enhancing antigen-related immune responses. Furthermore, after entering the lysosome, the redox linker arm can break disulfide bonds in response to intracellular reducing properties, allowing the antigen to escape from the lysosome, achieving cross-delivery of the antigen and greatly improving immunization efficiency. Similarly, the conjugate of this invention, which modifies a carrier protein with a hapten via a redox linker arm, can also be applied to the preparation of drugs for the prevention or treatment of cancer.

[0119] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A redox-responsive carrier protein, characterized in that, At least one hapten structure is modified onto a carrier protein via a redox-responsive disulfide linker arm, said linker arm being cleavable in response to redox conditions. The redox-responsive carrier protein has the following structural formula: ， The structural formula of the connecting arm is: ; The hapten is rhamnose; The carrier protein is bovine serum albumin.

2. The redox-responsive carrier protein according to claim 1, characterized in that, The hapten structure is coupled to the amino or carboxyl group of the carrier protein via a linker arm having a disulfide bond structure.

3. The use of the redox-responsive carrier protein according to claim 1 or 2 in the preparation of vaccines.

4. A redox-responsive conjugate, characterized in that, The conjugate comprises a redox-responsive carrier protein and a target antigen, wherein the redox-responsive carrier protein is modified with the target antigen. The redox-responsive carrier protein is the redox-responsive carrier protein as described in claim 1 or 2; The target antigen is β-glucan.

5. The use of the redox-responsive conjugate according to claim 4 in the preparation of vaccines.

6. An immunogenic composition, characterized in that, Contains the redox-responsive conjugate of claim 4 or a pharmaceutically acceptable carrier thereof.

7. The use of the immunogenic composition as described in claim 6 in the preparation of a vaccine.

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