3D-MLA and QS-21 composite adjuvant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of vaccine adjuvants and drug delivery, and discloses a 3D-MLA and QS-21 composite adjuvant as well as a preparation method and application of the 3D-MLA and QS-21 composite adjuvant. A saponin QS-21; polyethylene glycol sodium alginate; embedding particles with pH-sensitive calcium chloride; the preparation method of the vaccine antigen comprises the following steps: performing pegylation modification on sodium alginate; embedding calcium chloride with a pH sensitive polymer, and carrying out spray drying to prepare cross-linking agent particles; dissolving the synthetic monophosphoryl lipid A and saponin QS-21 to prepare a composite adjuvant solution; mixing the adjuvant solution with the pegylated sodium alginate solution to prepare a precursor preparation; and adding antigen and cross-linking agent particles into the precursor preparation, and uniformly mixing to obtain a final product. According to the invention, the composite adjuvant is delivered through the pH responsive in-situ gel, an immune reservoir is formed, and a powerful and lasting comprehensive immune response biased to the Th1 type is safely induced.

Description

A 3D-MLA and QS-21 composite adjuvant, its preparation method and application Technical Field

[0001] This invention relates to the field of vaccine adjuvants and drug delivery technology, and in particular to a 3D-MLA and QS-21 composite adjuvant, its preparation method and application. Background Technology

[0002] To effectively prevent and control major infectious diseases caused by viruses, intracellular bacteria, and other pathogens, it is crucial to develop novel vaccines capable of inducing a strong and durable protective immune response. The efficacy of a vaccine depends not only on the antigen itself but also, and more importantly, on the selection and application of the adjuvant. An ideal adjuvant should enhance the strength and duration of the immune response and direct it to the correct type (such as cellular or humoral immunity) to address the challenges posed by different pathogens. Therefore, designing novel adjuvant delivery systems that combine high efficiency and safety is a core research focus and urgent need in the field of modern vaccinology.

[0003] In existing technologies, the application of vaccine adjuvants mainly involves several technical solutions. Traditional aluminum adjuvants, represented by aluminum hydroxide or aluminum phosphate, exert a reservoir effect and induce local inflammatory responses by forming micron-sized particles that adsorb antigens at the injection site, making them the most widely used adjuvants. In addition, to obtain stronger immune activation signals, various novel adjuvants have been developed, such as monophospholipid A (MPLA), which activates the Toll-like receptor 4 (TLR4) signaling pathway, and the saponin adjuvant QS-21, which can activate multiple innate immune pathways. At the delivery system level, carriers such as liposomes and emulsions are also used to co-deliver antigens and adjuvants to enhance immune efficacy.

[0004] While existing technologies have improved vaccine efficacy to some extent, several shortcomings remain: First, traditional liquid formulations or free adjuvants are rapidly diluted and cleared by body fluids at the injection site after injection, resulting in a short window of action between the antigen and adjuvant, making it difficult to form lasting immune memory. Second, the mechanism of action of traditional aluminum adjuvants mainly induces Th2 humoral immune responses, while their protective effect against intracellular pathogens such as viruses that require strong Th1 cellular immunity for effective clearance is limited. Finally, the application of some novel and highly effective adjuvants is constrained by their safety profile. For example, saponin QS-21, whose molecular basis as an adjuvant lies in its ability to disrupt cell membrane stability, also exhibits significant hemolytic and local reactivity when applied in its free state, limiting its clinical application potential. Summary of the Invention

[0005] The purpose of this invention is to provide a 3D-MLA and QS-21 composite adjuvant, its preparation method and application, which solves the problems of short immune action time, insufficient ability to induce Th1 cell immunity and the limitation of application by the self-toxicity of high-efficiency adjuvants in existing vaccine adjuvant technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a 3D-MLA and QS-21 composite adjuvant, employing the following technical solution:

[0008] A 3D-MLA and QS-21 compound adjuvant comprises the following components in parts by weight:

[0009] Synthetic monophospholipid A: 0.1-1.0 parts;

[0010] Saponin QS-21: 0.2-10.0 parts;

[0011] Polyethylene glycolated sodium alginate: 15.0-220.0 parts;

[0012] pH-sensitive calcium chloride encapsulated particles: 0.3-22.0 parts;

[0013] Vaccine antigen: 0.1-5.5 doses.

[0014] By employing the above technical solution, this invention constructs a multi-component synergistic composite adjuvant system. This system uses polyethylene glycol-modified sodium alginate as a cross-linkable biocompatible framework, and synthetic monophospholipid A (TLR4 agonist) and saponin QS-21, which have synergistic immune-enhancing effects, as the core of the composite immune-enhancing effect. Furthermore, it innovatively introduces pH-sensitive calcium chloride-encapsulated particles as a smart, responsive multivalent ionic cross-linking agent precursor. Its function is as follows:

[0015] Liquid before injection: Before storage and injection, the adjuvant system is a flowable liquid formulation in which the components are stably dispersed, ensuring the stability and injectability of the product.

[0016] Post-injection in situ gel: After injection into the body, triggered by the physiological microenvironment (pH value of approximately 7.4) at the injection site, the pH-sensitive polymer shell of the pH-sensitive calcium chloride embedded particles undergoes a conformational change or swelling, responsively releasing the embedded calcium ions.

[0017] Formation of an immune reservoir: The released calcium ions undergo ionic cross-linking with the surrounding polyethylene glycol-modified sodium alginate molecular chains, rapidly transforming the liquid formulation into a three-dimensional hydrogel network structure, thereby forming an immune reservoir in situ at the injection site.

[0018] This in-situ formed immune reservoir encapsulates vaccine antigens, synthetic monophospholipid A, and saponin QS-21 within the same physical space, thereby achieving the following beneficial effects:

[0019] Colocalization and codelivery: This ensures that antigen-presenting cells can simultaneously capture antigens and two adjuvant molecules, achieving synergistic amplification of immune signals;

[0020] Long-term sustained release: Through the slow degradation of the hydrogel network, antigens and adjuvants are continuously released, prolonging the time the immune system is stimulated and helping to induce more durable immune memory;

[0021] Reduced toxic side effects: By confining saponin QS-21, which has potential hemolytic and other toxic properties, to the injection site, its rapid entry into the systemic circulation is avoided, thus improving the overall safety of the adjuvant.

[0022] In summary, this invention effectively solves the problems of inconsistent spatiotemporal distribution of adjuvants and antigens and toxic side effects through an in-situ dynamic cross-linking mechanism, synergistically enhancing the immunogenicity of the vaccine and inducing stronger and more durable humoral and cellular immune responses.

[0023] Preferably, the PEGylated sodium alginate is obtained by grafting sodium alginate with a molecular weight of 100-300 kDa onto polyethylene glycol with a molecular weight of 5,000-20,000 Da, and the grafting rate of polyethylene glycol is 5%-20% w / w.

[0024] By adopting the above technical solution, the immunogenicity of the polymer backbone itself is effectively reduced by grafting polyethylene glycol onto sodium alginate, giving it immune inertness or stealth properties, prolonging its retention time in vivo, and improving its solubility and biocompatibility.

[0025] Preferably, the pH-sensitive calcium chloride encapsulated particles are formed by encapsulating calcium chloride as a multivalent ion source in a pH-sensitive polymer carboxymethyl cellulose-acrylic acid copolymer. The average particle size distribution of the particles is 100-500 nanometers, and the calcium chloride loading rate is 10%-30% w / w.

[0026] By adopting the above technical solution, the particle size and loading rate of pH-sensitive calcium chloride encapsulated particles, which serve as the crosslinking trigger core, are limited, ensuring that the particles are stably dispersed in liquid formulations without premature sedimentation, and guaranteeing that they can efficiently and controllably release sufficient calcium ions to complete the gelation process at physiological pH.

[0027] Preferably, the pH-sensitive polymer carboxymethyl cellulose-acrylic acid copolymer is obtained by copolymerizing sodium carboxymethyl cellulose and acrylic acid in a molar ratio of 1:0.1-0.5, wherein the sodium carboxymethyl cellulose has a molecular weight of 90-250 kDa and a degree of substitution of 0.6-0.9.

[0028] By adopting the above technical solution and limiting the raw material molar ratio of the pH-sensitive polymer, the pH response threshold of the polymer is precisely controlled, so that it remains stable in acidic or neutral storage environments without releasing calcium ions, while responding rapidly in the physiological pH environment (about 7.4) in vivo, thus ensuring the intelligence and controllability of the in-situ gelation process.

[0029] Secondly, the present invention provides a method for preparing a composite adjuvant of 3D-MLA and QS-21, using the following technical solution:

[0030] A method for preparing a 3D-MLA and QS-21 composite adjuvant, for preparing the 3D-MLA and QS-21 composite adjuvant as described in any one of the preceding claims, comprising the following steps:

[0031] S1. Preparation of polyethylene glycolated sodium alginate;

[0032] S2. Preparation of pH-sensitive calcium chloride encapsulated particles;

[0033] S3. Prepare a solution of the adjuvant active ingredient;

[0034] S4. Preparation of adjuvant-modified polymeric composite precursor formulation;

[0035] S5. Prepare the final adjuvant formulation.

[0036] By employing the above technical solution, this method prepares the core functional components (polyethylene glycol-modified sodium alginate and pH-sensitive calcium chloride encapsulated particles) in steps, followed by orderly mixing. The process steps are clear, the conditions are mild, and it is easy to implement and scale up for production. The key to this preparation method is the addition of the pH-sensitive calcium chloride encapsulated particles, which serve as a crosslinking agent precursor, in the final step. This ensures that the system does not gel during preparation and storage, ultimately yielding a stable, homogeneous, injectable liquid compound adjuvant formulation, guaranteeing product quality and the feasibility of clinical application.

[0037] Preferably, in step S1, the step of preparing polyethylene glycol-modified sodium alginate includes: dissolving sodium alginate, adding an activator and stirring at pH 5.0-6.0 for 2-4 hours; subsequently, adding methoxy polyethylene glycol-N-hydroxysuccinimide ester and reacting at pH 6.0-7.0 for 12-24 hours; finally, purifying the reaction product by dialysis and freeze-drying to obtain the polyethylene glycol-modified sodium alginate.

[0038] By adopting the above technical solution, the specific process parameters of the polyethylene glycolation reaction were defined, ensuring the efficient grafting of polyethylene glycol and the purity of the product, thus providing reliable raw materials for the subsequent construction of a stable hydrogel framework.

[0039] Preferably, in step S2, the step of preparing pH-sensitive calcium chloride encapsulated particles includes: reacting sodium carboxymethyl cellulose, acrylic acid, and an initiator at 60-80°C for 3-6 hours to obtain the pH-sensitive polymer carboxymethyl cellulose-acrylic acid copolymer; subsequently, preparing the pH-sensitive calcium chloride encapsulated particles by spray drying, wherein the inlet temperature of the spray drying is 120-150°C and the outlet temperature is 70-90°C.

[0040] By adopting the above technical solution, a specific method for obtaining pH-sensitive calcium chloride encapsulated particles with stable structure and performance is disclosed, including two steps: polymer synthesis and particle forming, which ensures the reproducibility of this key intelligent response component.

[0041] Preferably, in step S3, the amount of phosphate buffered saline used in the preparation of the adjuvant active ingredient solution is controlled within the range of 0.1-6.0 mg / mL.

[0042] By adopting the above technical solution, the concentration of the adjuvant active ingredient is limited, ensuring the accuracy of the distribution ratio of each group in subsequent steps, which is an important guarantee for achieving uniform and controllable final product quality.

[0043] Preferably, in step S4, the step of preparing the adjuvant-modified polymeric composite precursor formulation includes: preparing the polyethylene glycolated sodium alginate into a solution with a concentration of 0.5%-2.0% w / v; subsequently, slowly adding the adjuvant active ingredient solution at a stirring rate of 50-200 μL / min at a stirring rate of 100-300 rpm, and continuing to stir for 30-60 minutes.

[0044] By employing the above technical solution, the concentration of polyethylene glycol-modified sodium alginate, which serves as the gel backbone, and the mixing method and rate of the two solutions are limited. The gentle and slow mixing method avoids adjuvant molecule aggregation that could result from excessively high local concentrations, ensuring uniform dispersion of the adjuvant active ingredient within the polymer backbone.

[0045] Preferably, in step S5, the step of preparing the final adjuvant formulation includes: adding the vaccine antigen to the adjuvant-modified polymeric composite precursor formulation and mixing for 15-30 minutes at a temperature of 4-25°C and a stirring rate of 50-250 rpm; subsequently, adding the pH-sensitive calcium chloride encapsulated particles and continuing to mix for 5-15 minutes.

[0046] By adopting the above technical solution, the mild conditions of the final mixing step, especially the temperature and stirring rate, are limited, which effectively protects the biological activity and spatial conformation of vaccine antigens (especially protein antigens) from being destroyed during the preparation process, thus ensuring the effectiveness of the final vaccine product.

[0047] Thirdly, this invention provides an application of the 3D-MLA and QS-21 composite adjuvant, employing the following technical solution:

[0048] The use of a 3D-MLA and QS-21 combined adjuvant as described in any of the preceding claims in the preparation of a vaccine adjuvant or vaccine composition for the prevention or treatment of viral infectious diseases.

[0049] By adopting the above-mentioned technical solutions, and addressing the technical challenge that recombinant protein antigens typically have weak immunogenicity, the composite adjuvant provided by this invention, through its in-situ gelation, co-delivery, and sustained-release mechanisms, can effectively enhance the strength and durability of the immune response to these recombinant protein vaccines, and has significant clinical application value.

[0050] In summary, the present invention has at least one of the following beneficial technical effects:

[0051] 1. This invention enhances and prolongs the intensity and persistence of the body's immune response by forming a three-dimensional hydrogel reservoir in situ at the injection site. Its key technical feature lies in the fact that pH-sensitive calcium chloride-embedded particles trigger polyethylene glycol-modified sodium alginate cross-linking under physiological conditions. This reservoir co-encapsulates antigens and adjuvants, achieving synergistic delivery and sustained release, thereby prolonging the effective interaction time between the immune system and antigens, leading to more efficient antibody production and the formation of immune memory.

[0052] 2. This invention utilizes the synergistic effect of a Toll-like receptor 4 agonist (synthetic monophospholipid A) and saponin QS-21 in inducing a Th1-type immune response. The in-situ gel delivery system ensures the spatiotemporal consistency of the two adjuvants and the antigen in the immune microenvironment, maximizing the synergistic effect and efficiently activating IFN-γ-producing T cells, which is crucial for clearing intracellular pathogens such as viruses.

[0053] 3. This invention improves the biocompatibility and application safety of adjuvant formulations. The core lies in the in-situ formed hydrogel network structure, which physically shields and encapsulates components with cell membrane-disrupting activity, such as saponin QS-21. This design reduces the direct contact concentration of adjuvant molecules with body tissues and cells, thereby mitigating potential hemolytic activity and adverse reactions such as injection site reactions, making the application of highly effective adjuvants safer. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to embodiments, comparative examples and test examples.

[0055] Example 1:

[0056] This embodiment provides a method for preparing a 3D-MLA and QS-21 composite adjuvant, the final product of which contains the following components in parts by weight: synthetic monophospholipid A: 0.5 parts, saponin QS-21: 2.5 parts, polyethylene glycolated sodium alginate: 100.0 parts, pH-sensitive calcium chloride embedded particles: 5.0 parts, and recombinant herpes zoster virus glycoprotein E (gE antigen): 1.0 part.

[0057] The specific preparation steps are as follows:

[0058] S1. Preparation of polyethylene glycol-modified sodium alginate: 150 kDa sodium alginate was dissolved in deionized water, and activators EDC and NHS were added. The mixture was stirred and activated at pH 6.5 for 3 hours. Subsequently, 5,000 Da mPEG-NHS was added, the pH was adjusted to 6.8, and the reaction was carried out for 18 hours. The reaction product was purified by dialysis and then freeze-dried to obtain polyethylene glycol-modified sodium alginate.

[0059] S2. Preparation of pH-sensitive calcium chloride encapsulated particles: Sodium carboxymethyl cellulose and acrylic acid were reacted at a molar ratio of 1:0.2 at 70°C in the presence of initiator APS for 4 hours to obtain a pH-sensitive polymer, a carboxymethyl cellulose-acrylic acid copolymer. This copolymer was mixed with calcium chloride, and particles were prepared by spray drying. The inlet temperature was set at 130°C and the outlet temperature at 80°C to obtain pH-sensitive calcium chloride encapsulated particles.

[0060] S3. Preparation of adjuvant active ingredient solution: 0.5 parts by weight of synthetic monophospholipid A and 2.5 parts by weight of saponin QS-21 were added to an appropriate amount of phosphate buffered saline (PBS, pH 7.4) and sonicated until fully dissolved to obtain a homogeneous solution with a total adjuvant active ingredient concentration of 1.0 mg / mL.

[0061] S4. Preparation of adjuvant-modified polymeric composite precursor formulation: Dissolve 100.0 parts by weight of the polyethylene glycolated sodium alginate obtained in step S1 in phosphate buffered saline to prepare a solution with a concentration of 1.0% w / v. Then, under a stirring speed of 200 rpm, slowly add the adjuvant active ingredient solution obtained in step S3 to this solution at a rate of 100 μL / min. After the addition is complete, continue stirring for 45 minutes to obtain the adjuvant-modified polymeric composite precursor formulation.

[0062] S5. Preparation of the final adjuvant formulation: Add 1.0 part by weight of gE antigen to the prodrug formulation and mix for 20 minutes at 20°C and 150 rpm. Then, add 5.0 parts by weight of the pH-sensitive calcium chloride encapsulated particles obtained in step S2 and continue to mix gently for 10 minutes under the same conditions until homogeneous.

[0063] Example 2:

[0064] This embodiment provides a method for preparing a 3D-MLA and QS-21 composite adjuvant, the final product of which comprises the following components in parts by weight: synthetic monophospholipid A: 1.0 part, saponin QS-21: 10.0 part, polyethylene glycol-modified sodium alginate: 220.0 part, pH-sensitive calcium chloride embedded particles: 22.0 part, and recombinant respiratory syncytial virus pre-fusion F protein (Pre-F antigen): 5.5 parts. The specific preparation steps are as follows:

[0065] S1. Preparation of polyethylene glycol-modified sodium alginate: 250 kDa sodium alginate was dissolved in deionized water, and activators EDC and NHS were added. The mixture was stirred and activated at pH 6.0 for 4 hours. Subsequently, 10,000 Da mPEG-NHS was added, the pH was adjusted to 6.5, and the reaction was carried out for 24 hours. The reaction product was purified by dialysis and then freeze-dried to obtain polyethylene glycol-modified sodium alginate.

[0066] S2. Preparation of pH-sensitive calcium chloride encapsulated particles: Sodium carboxymethyl cellulose and acrylic acid were reacted at a molar ratio of 1:0.4 at 60℃ in the presence of initiator APS for 6 hours to obtain a pH-sensitive polymer, a carboxymethyl cellulose-acrylic acid copolymer. This copolymer was mixed with calcium chloride, and particles were prepared by spray drying. The inlet temperature was set at 150℃ and the outlet temperature at 90℃ to obtain pH-sensitive calcium chloride encapsulated particles.

[0067] S3. Preparation of adjuvant active ingredient solution: 1.0 parts by weight of synthetic monophospholipid A and 10.0 parts by weight of saponin QS-21 were added to an appropriate amount of phosphate buffered saline and sonicated until fully dissolved to obtain a homogeneous solution with a total adjuvant active ingredient concentration of 6.0 mg / mL.

[0068] S4. Preparation of adjuvant-modified polymeric composite precursor formulation: Dissolve 220.0 parts by weight of the polyethylene glycolated sodium alginate obtained in step S1 in phosphate buffered saline to prepare a solution with a concentration of 2.0% w / v.

[0069] Subsequently, the adjuvant active ingredient solution prepared in step S3 was slowly added dropwise to the solution at a stirring rate of 200 μL / min under a stirring rate of 300 rpm. After the addition was completed, stirring was continued for 60 minutes to obtain the adjuvant-modified polymeric composite precursor formulation.

[0070] S5. Preparation of the final adjuvant formulation: Add 5.5 parts by weight of Pre-F antigen to the prodrug formulation and mix for 30 minutes at 25°C and 250 rpm. Then, add 22.0 parts by weight of the pH-sensitive calcium chloride encapsulated particles obtained in step S2, and continue to mix gently for 15 minutes under the same conditions until homogeneous.

[0071] Example 3:

[0072] This embodiment provides a method for preparing a 3D-MLA and QS-21 composite adjuvant, the final product of which contains the following components in parts by weight: synthetic monophospholipid A: 0.1 parts, saponin QS-21: 0.2 parts, polyethylene glycolated sodium alginate: 15.0 parts, pH-sensitive calcium chloride embedded particles: 0.3 parts, and recombinant herpes zoster virus glycoprotein E (gE antigen): 0.1 parts.

[0073] The specific preparation steps are as follows:

[0074] S1. Preparation of polyethylene glycol-modified sodium alginate: 150 kDa sodium alginate was dissolved in deionized water, and activators EDC and NHS were added. The mixture was stirred and activated at pH 5.0 for 2 hours. Subsequently, 5,000 Da mPEG-NHS was added, the pH was adjusted to 7.0, and the reaction was carried out for 12 hours. The reaction product was purified by dialysis and then freeze-dried to obtain polyethylene glycol-modified sodium alginate.

[0075] S2. Preparation of pH-sensitive calcium chloride encapsulated particles: Sodium carboxymethyl cellulose and acrylic acid were reacted at a molar ratio of 1:0.1 in the presence of initiator APS for 3 hours at 80℃ to obtain a pH-sensitive polymer, a carboxymethyl cellulose-acrylic acid copolymer. This copolymer was mixed with calcium chloride, and particles were prepared by spray drying. The inlet temperature was set at 120℃ and the outlet temperature at 70℃ to obtain pH-sensitive calcium chloride encapsulated particles.

[0076] S3. Preparation of adjuvant active ingredient solution: 0.1 parts by weight of synthetic monophospholipid A and 0.2 parts by weight of saponin QS-21 were added to an appropriate amount of phosphate buffered saline and sonicated until fully dissolved to obtain a homogeneous solution with a total adjuvant active ingredient concentration of 0.1 mg / mL.

[0077] S4. Preparation of adjuvant-modified polymeric composite precursor formulation: Dissolve 15.0 parts by weight of the polyethylene glycolated sodium alginate obtained in step S1 in phosphate buffered saline to prepare a solution with a concentration of 0.5% w / v.

[0078] Subsequently, the adjuvant active ingredient solution prepared in step S3 was slowly added dropwise to the solution at a stirring rate of 50 μL / min under a stirring rate of 100 rpm. After the addition was completed, stirring was continued for 30 minutes to obtain the adjuvant-modified polymeric composite precursor formulation.

[0079] S5. Preparation of the final adjuvant formulation: Add 0.1 parts by weight of gE antigen to the prodrug formulation and mix for 15 minutes at 4°C and 50 rpm. Then, add 0.3 parts by weight of the pH-sensitive calcium chloride encapsulated particles obtained in step S2 and continue to mix gently for 5 minutes under the same conditions until homogeneous.

[0080] Example 4:

[0081] This embodiment provides a method for preparing a 3D-MLA and QS-21 composite adjuvant, the final product of which comprises the following components in parts by weight: synthetic monophospholipid A: 0.5 parts, saponin QS-21: 2.5 parts, polyethylene glycol-modified sodium alginate: 100.0 parts, pH-sensitive calcium chloride embedded particles: 5.0 parts, and recombinant herpes zoster virus glycoprotein E (gE antigen): 1.0 part. The specific preparation steps are as follows:

[0082] S1. Preparation of polyethylene glycol-modified sodium alginate: 250 kDa sodium alginate was dissolved in deionized water, and activators EDC and NHS were added. The mixture was stirred and activated at pH 6.0 for 4 hours. Subsequently, 10,000 Da mPEG-NHS was added, the pH was adjusted to 6.5, and the reaction was carried out for 24 hours. The reaction product was purified by dialysis and then freeze-dried to obtain polyethylene glycol-modified sodium alginate.

[0083] S2. Preparation of pH-sensitive calcium chloride encapsulated particles: Sodium carboxymethyl cellulose and acrylic acid were reacted at a molar ratio of 1:0.2 at 70°C in the presence of initiator APS for 4 hours to obtain a pH-sensitive polymer, a carboxymethyl cellulose-acrylic acid copolymer. This copolymer was mixed with calcium chloride, and particles were prepared by spray drying. The inlet temperature was set at 130°C and the outlet temperature at 80°C to obtain pH-sensitive calcium chloride encapsulated particles.

[0084] S3. Preparation of adjuvant active ingredient solution: 0.5 parts by weight of synthetic monophospholipid A and 2.5 parts by weight of saponin QS-21 were added to an appropriate amount of phosphate buffered saline and sonicated until fully dissolved to obtain a homogeneous solution with a total adjuvant active ingredient concentration of 1.0 mg / mL.

[0085] S4. Preparation of adjuvant-modified polymeric composite precursor formulation: Dissolve 100.0 parts by weight of the polyethylene glycolated sodium alginate obtained in step S1 in phosphate buffered saline to prepare a solution with a concentration of 1.0% w / v.

[0086] Subsequently, the adjuvant active ingredient solution prepared in step S3 was slowly added dropwise to the solution at a stirring rate of 100 μL / min under a stirring rate of 200 rpm. After the addition was completed, stirring was continued for 45 minutes to obtain the adjuvant-modified polymeric composite precursor formulation.

[0087] S5. Preparation of the final adjuvant formulation: Add 1.0 part by weight of gE antigen to the prodrug formulation and mix for 20 minutes at 20°C and 150 rpm. Then, add 5.0 parts by weight of the pH-sensitive calcium chloride encapsulated particles obtained in step S2 and continue to mix gently for 10 minutes under the same conditions until homogeneous.

[0088] Example 5:

[0089] This embodiment provides a method for preparing a 3D-MLA and QS-21 composite adjuvant, the final product of which contains the following components in parts by weight: synthetic monophospholipid A: 0.5 parts, saponin QS-21: 2.5 parts, polyethylene glycolated sodium alginate: 100.0 parts, pH-sensitive calcium chloride embedded particles: 5.0 parts, and recombinant herpes zoster virus glycoprotein E (gE antigen): 1.0 part.

[0090] The specific preparation steps are as follows:

[0091] S1. Preparation of polyethylene glycol-modified sodium alginate: 150 kDa sodium alginate was dissolved in deionized water, and activators EDC and NHS were added. The mixture was stirred and activated at pH 6.5 for 3 hours. Subsequently, 5,000 Da mPEG-NHS was added, the pH was adjusted to 6.8, and the reaction was carried out for 18 hours. The reaction product was purified by dialysis and then freeze-dried to obtain polyethylene glycol-modified sodium alginate.

[0092] S2. Preparation of pH-sensitive calcium chloride encapsulated particles: Sodium carboxymethyl cellulose and acrylic acid were reacted at a molar ratio of 1:0.1 in the presence of initiator APS for 3 hours at 80℃ to obtain a pH-sensitive polymer, a carboxymethyl cellulose-acrylic acid copolymer. This copolymer was mixed with calcium chloride, and particles were prepared by spray drying. The inlet temperature was set at 120℃ and the outlet temperature at 70℃ to obtain pH-sensitive calcium chloride encapsulated particles.

[0093] S3. Preparation of adjuvant active ingredient solution: 0.5 parts by weight of synthetic monophospholipid A and 2.5 parts by weight of saponin QS-21 were added to an appropriate amount of phosphate buffered saline and sonicated until fully dissolved to obtain a homogeneous solution with a total adjuvant active ingredient concentration of 1.0 mg / mL.

[0094] S4. Preparation of adjuvant-modified polymeric composite precursor formulation: Dissolve 100.0 parts by weight of the polyethylene glycolated sodium alginate obtained in step S1 in phosphate buffered saline to prepare a solution with a concentration of 1.0% w / v.

[0095] Subsequently, the adjuvant active ingredient solution prepared in step S3 was slowly added dropwise to the solution at a stirring rate of 100 μL / min under a stirring rate of 200 rpm. After the addition was completed, stirring was continued for 45 minutes to obtain the adjuvant-modified polymeric composite precursor formulation.

[0096] S5. Preparation of the final adjuvant formulation: Add 1.0 part by weight of gE antigen to the prodrug formulation and mix for 20 minutes at 20°C and 150 rpm. Then, add 5.0 parts by weight of the pH-sensitive calcium chloride encapsulated particles obtained in step S2 and continue to mix gently for 10 minutes under the same conditions until homogeneous.

[0097] Example 6:

[0098] This embodiment provides a method for preparing a 3D-MLA and QS-21 composite adjuvant, the final product of which contains the following components in parts by weight: synthetic monophospholipid A: 0.5 parts, saponin QS-21: 2.5 parts, polyethylene glycolated sodium alginate: 100.0 parts, pH-sensitive calcium chloride embedded particles: 5.0 parts, and recombinant herpes zoster virus glycoprotein E (gE antigen): 1.0 part.

[0099] The specific preparation steps are as follows:

[0100] S1. Preparation of polyethylene glycol-modified sodium alginate: 150 kDa sodium alginate was dissolved in deionized water, and activators EDC and NHS were added. The mixture was stirred and activated at pH 6.5 for 3 hours. Subsequently, 5,000 Da mPEG-NHS was added, the pH was adjusted to 6.8, and the reaction was carried out for 18 hours. The reaction product was purified by dialysis and then freeze-dried to obtain polyethylene glycol-modified sodium alginate.

[0101] S2. Preparation of pH-sensitive calcium chloride encapsulated particles: Sodium carboxymethyl cellulose and acrylic acid were reacted at a molar ratio of 1:0.2 at 70°C in the presence of initiator APS for 4 hours to obtain a pH-sensitive polymer, a carboxymethyl cellulose-acrylic acid copolymer. This copolymer was mixed with calcium chloride, and particles were prepared by spray drying. The inlet temperature was set at 130°C and the outlet temperature at 80°C to obtain pH-sensitive calcium chloride encapsulated particles.

[0102] S3. Preparation of adjuvant active ingredient solution: 0.5 parts by weight of synthetic monophospholipid A and 2.5 parts by weight of saponin QS-21 were added to an appropriate amount of phosphate buffered saline and sonicated until fully dissolved to obtain a homogeneous solution with a total adjuvant active ingredient concentration of 1.0 mg / mL.

[0103] S4. Preparation of adjuvant-modified polymeric composite precursor formulation: Dissolve 100.0 parts by weight of the polyethylene glycolated sodium alginate obtained in step S1 in phosphate buffered saline to prepare a solution with a concentration of 1.0% w / v.

[0104] Subsequently, the adjuvant active ingredient solution prepared in step S3 was slowly added dropwise to the solution at a stirring rate of 150 μL / min under a stirring rate of 250 rpm. After the addition was completed, stirring was continued for 45 minutes to obtain the adjuvant-modified polymeric composite precursor formulation.

[0105] S5. Preparation of the final adjuvant formulation: Add 1.0 part by weight of gE antigen to the prodrug formulation and mix for 20 minutes at 10°C and 200 rpm. Then, add 5.0 parts by weight of the pH-sensitive calcium chloride encapsulated particles obtained in step S2, and continue to mix gently for 10 minutes under the same conditions until homogeneous.

[0106] Comparative Example 1:

[0107] The difference from Example 1 is that, in step S5, pH-sensitive calcium chloride encapsulated particles are not added; instead, an aqueous solution of calcium chloride in an equimolar amount of the calcium chloride contained in the particles is directly added. All other steps are the same.

[0108] Comparative Example 2:

[0109] The difference from Example 1 is that its components do not contain saponin QS-21, and only synthetic monophospholipid A is dissolved in step S3. All other aspects are the same.

[0110] Comparative Example 3:

[0111] The difference from Example 1 is that its components do not contain synthetic monophospholipid A, and only saponin QS-21 is dissolved in step S3. All other aspects are the same.

[0112] Comparative Example 4:

[0113] Compared with Example 1, the difference is that the formulation of this comparative example is a free adjuvant vaccine, and its components do not contain polyethylene glycol sodium alginate and pH-sensitive calcium chloride embedded particles. Instead, the same amount of synthetic monophospholipid A, saponin QS-21 and gE antigen as in Example 1 are directly dissolved in phosphate buffered saline.

[0114] Comparative Example 5:

[0115] The difference from Example 1 is that, in step S1, the polyethylene glycolation reaction is not performed; instead, unmodified sodium alginate with a molecular weight of 150 kDa is used directly as the gel matrix material. All other aspects are the same.

[0116] Comparative Example 6:

[0117] Compared with Example 1, the difference is that this comparative formulation does not use the in-situ gel adjuvant system of the present invention. Instead, it mixes an equal amount of gE antigen and synthetic monophospholipid A with aluminum hydroxide adjuvant as in Example 1 to prepare a conventional adsorbent vaccine formulation.

[0118] Test Example 1:

[0119] Experimental materials and equipment:

[0120] Samples to be tested: Formulations of Examples 1-6 and Comparative Example 1.

[0121] Buffers: acetate buffer (pH 5.5, 0.1M), phosphate buffered saline (PBS, pH 7.4, 0.01M).

[0122] Equipment: 1.5mL centrifuge tubes, pipettes, vortex mixer, constant temperature water bath.

[0123] Sample grouping and processing: Take clean 1.5 mL centrifuge tubes and label them as groups. Accurately transfer 1.0 mL of the corresponding reagent to each centrifuge tube labeled pH 5.5, and then add 1.0 mL of pH 5.5 acetate buffer. Accurately transfer 1.0 mL of the corresponding reagent to each centrifuge tube labeled pH 7.4, and then add 1.0 mL of pH 7.4 PBS.

[0124] Mixing and Incubation: Vortex the liquid in each tube for 5 seconds to ensure thorough mixing. Then, immediately place all centrifuge tubes labeled pH 7.4 in a 37°C water bath for incubation. Centrifuge tubes labeled pH 5.5 were left to stand at room temperature (25°C).

[0125] Status Observation and Judgment: At the set time points (0, 2, 5, 10, 30 minutes), remove the centrifuge tubes of the pH 7.4 group from the water bath, invert them 180 degrees and hold for 1 minute, and observe the flow state of the substances inside the tubes. Perform the same operation on the pH 5.5 group at 30 minutes. The status judgment criteria are as follows:

[0126] Liquid: Macroscopic flow occurs within 1 minute after the sample is inverted.

[0127] Gel formation: No macroscopic flow occurred in the bulk of the sample within 1 minute after inversion.

[0128] Precipitation / flocculation: Solid-liquid separation occurred in the sample, forming non-uniform flocculent or particulate aggregates. The test results are shown in Table 1.

[0129] Table 1: State changes of each test group under different pH conditions

[0130] Test Group pH 5.5 (30 min) pH 7.4 (2 min) pH 7.4 (5 min) pH 7.4 (10 min) pH 7.4 (30 min) Example 1 Liquid-Liquid Gel Formation Gel Formation Gel Formation Example 2 Liquid Gel Formation Gel Formation Gel Formation Example 3 Liquid-Liquid-Liquid-Liquid Gel Formation Example 4 Liquid-Liquid Gel Formation Gel Formation Example 5 Liquid-Liquid-Liquid Gel Formation Gel Formation Example 6 Liquid-Liquid Gel Formation Gel Formation Comparative Example 1 Liquid Precipitation / Flocculation Precipitation / Flocculation Precipitation / Flocculation Precipitation / Flocculation Precipitation / Flocculation surface

[0131] From Table 1, we can obtain:

[0132] Test results showed that the formulations in Examples 1-6 remained in a liquid state at pH 5.5, but all underwent a sol-gel transition at pH 7.4. The mechanism of this phenomenon is as follows: in a neutral pH (7.4) environment, the pH-sensitive calcium chloride encapsulated particles and their polymer backbone (carboxymethyl cellulose-acrylic acid copolymer) in the formulation swell or dissolve, thereby releasing the internal divalent calcium ions. These released calcium ions undergo ionic cross-linking with the guluronic acid (G) units on the polyethylene glycol-modified sodium alginate molecular chain, forming a three-dimensional network structure, ultimately manifesting macroscopically as the formation of a homogeneous hydrogel.

[0133] The results of Comparative Example 1 validated the necessity of this mechanism. Due to the direct addition of free calcium ions, the sodium alginate molecular chains underwent transient and uneven cross-linking, leading to rapid polymer aggregation and precipitation, rather than the formation of a homogeneous gel reservoir suitable for drug delivery.

[0134] Therefore, the data from this test case confirm that the adjuvant formulation system has precise pH responsiveness and controllable in-situ gel formation capability, providing feasibility verification for its use as a technical solution for in-situ formation of an immune reservoir after injection.

[0135] Test Example 2:

[0136] Experimental materials and equipment:

[0137] Sample to be tested: Formulation of Example 1.

[0138] Release medium: phosphate-buffered saline (PBS, pH 7.4, 0.01M).

[0139] Equipment: Dialysis bags (molecular weight cutoff 3.5 kDa), constant temperature shaking water bath, high performance liquid chromatography system (HPLC), volumetric flasks, pipettes.

[0140] Sample preparation and gelation: Accurately measure 1.0 mL of the formulation from Example 1 and mix it with 1.0 mL of pH 7.4 PBS. Incubate at 37°C for 10 minutes to allow it to completely transform into a gel state. Transfer the adjuvant-loaded gel sample completely into a pre-treated dialysis bag and seal it tightly.

[0141] Dialysis apparatus setup: Immerse the dialysis bag containing the gel sample completely into a container containing 50 mL of PBS release medium preheated to 37°C.

[0142] Release experiment: The container was placed in a constant temperature shaking water bath, the temperature was set at 37℃ and the shaking rate was 100rpm.

[0143] Sample collection: At preset time points (1, 4, 8, 12, 24, 48, 72, 96 hours), 1.0 mL of sample was drawn from the release medium outside the dialysis bag for analysis, and 1.0 mL of fresh, isothermal PBS release medium was immediately added to the container to maintain a constant total release volume.

[0144] Content determination and calculation: After appropriate dilution, the collected samples were analyzed by HPLC. The concentrations of synthetic monophospholipid A (3D-MLA) and saponin QS-21 in the samples were calculated according to the standard curve. The cumulative release rate was calculated using the following formula:

[0145] Cumulative release rate In the formula, For; cumulative release rate; To release the total volume of the medium, For each sampling volume, For the first The concentration of the drug in the release medium during the second sampling. For the first The concentration of the drug in the release medium during the second sampling. The initial total amount of drug in the gel is shown in Table 2.

[0146] Table 2: Cumulative in vitro release rates of 3D-MLA and QS-21 in the gel system of Example 1

[0147] Time (hours) 3D-MLA Cumulative Release Rate (%) QS-21 Cumulative Release Rate (%) 15.2±0.8 7.1±1.14 12.8±1.3 16.5±1.98 20.1±1.92 4.8±2.31 226.4±2.53 2.0±2.82 434.6±2.14 1.2±3.44 852.1±3.66 0.9±4.072 63.8±4.17 5.4±4.59 670.3±4.58 3.5±5.1 surface

[0148] From Table 2, we can obtain:

[0149] Data shows that, under simulated physiological conditions, both 3D-MLA and QS-21 exhibited sustained release from the in-situ gel of Example 1, with no burst release occurring within 96 hours. This sustained-release behavior directly demonstrates the effectiveness of the adjuvant formulation technology.

[0150] The mechanism lies in the fact that the in-situ formed polyethylene glycol-modified sodium alginate hydrogel has a three-dimensional network structure that physically encapsulates adjuvant molecules within it. The release of these molecules is primarily controlled by their diffusion rate within the gel network. This diffusion process is constrained by various factors, including the pore size of the gel network and the interactions between the polymer chains and the adjuvant molecules, thus slowing the rate at which the adjuvant enters the surrounding environment.

[0151] This sustained-release effect generated by the gel reservoir, known as the reservoir effect, is the core mechanism of this invention. It allows the adjuvant and antigen to coexist at the injection site for a longer period, prolonging the contact window between the immune system and both, and providing continuous stimulating signals for subsequent immune cell recruitment, antigen presentation, and lymphocyte activation, among other immune response processes. The in vitro sustained-release data in this test case provide direct experimental evidence that this adjuvant system can achieve controlled release of the adjuvant in vivo, thereby enhancing the immune response.

[0152] Test Example 3:

[0153] Experimental materials and equipment: Samples to be tested: formulations of Example 1, Comparative Example 4, and Comparative Example 5.

[0154] Biological material: fresh rabbit anticoagulated whole blood.

[0155] Reagent: Phosphate-buffered saline (PBS, pH 7.4).

[0156] Equipment: Low-temperature centrifuge, constant temperature water bath, ultraviolet-visible spectrophotometer (or microplate reader), vortex mixer.

[0157] Preparation of red blood cell suspension: Fresh anticoagulated whole blood from rabbits was washed three times with PBS. After each centrifugation, the supernatant and the brownish-yellow layer of erythrocyte sedimentation rate were discarded. After the final centrifugation, the precipitated red blood cells were prepared into a 2% (v / v) red blood cell suspension with PBS for later use.

[0158] Sample preparation: The formulations of Example 1, Comparative Example 4, and Comparative Example 5 were diluted with PBS to ensure that the final concentration of saponin QS-21 in each dilution was uniformly 50 μg / mL. PBS was set up as a negative control group, and deionized water as a positive control group.

[0159] Incubation reaction: In a centrifuge tube, mix 1.0 mL of 2% erythrocyte suspension with 1.0 mL of each test sample (or control). After gently vortexing to mix, incubate all centrifuge tubes in a 37°C water bath for 2 hours.

[0160] Measurement: After incubation, centrifuge all centrifuge tubes at 2000 rpm for 5 minutes. Carefully aspirate the supernatant and use a spectrophotometer to measure the absorbance (OD value) of free hemoglobin in the supernatant at a wavelength of 540 nm.

[0161] Hemolysis rate calculation: The hemolysis rate of each group is calculated according to the following formula: Hemolysis rate (%) The test results are shown in Table 3.

[0162] Table 3: In vitro hemolysis rate of each test group

[0163] Test group absorbance value (OD) 540 Hemolysis rate (%) Positive control (deionized water) 1.853±0.071100 Negative control (PBS) 0.042±0.0080 Example 1 0.065±0.013 1.3±0.7 Comparative example 4 1.237±0.088 66.0±4.8 Comparative example 5 0.081±0.019 2.2±1.1 surface

[0164] From Table 3, we can obtain:

[0165] Comparative Example 4 (free QS-21) induced hemolysis of up to 66.0% upon contact with erythrocytes, verifying that the saponin adjuvant in its free state has significant cell membrane disrupting activity. In contrast, the hemolysis rate of the formulation in Example 1 was only 1.3%, with no significant difference from the negative control group.

[0166] The underlying mechanism of this result lies in the adjuvant delivery of the present invention. The saponin QS-21 molecule is physically encapsulated in an in-situ formed three-dimensional network structure of sodium alginate hydrogel. This structure restricts the direct interaction between the saponin molecule and the erythrocyte membrane, thereby shielding its molecular-level membrane-breaking activity.

[0167] Comparative Example 5 (using unmodified sodium alginate) also showed an extremely low hemolysis rate (2.2%), which further confirms that the main mechanism for reducing hemolytic toxicity is the physical barrier effect of the sodium alginate hydrogel matrix, rather than polyethylene glycol modification.

[0168] The results of this test case confirm that the technical solution can safely integrate bioactive saponin adjuvants into the delivery system, reduce their in vitro hemolytic toxicity, and thus significantly improve the biocompatibility and application safety of the compound adjuvant formulation.

[0169] Test Example 4:

[0170] Experimental animals and grouping: SPF-grade female BALB / c mice, 6-8 weeks old, were randomly divided into 13 groups, with 10 mice in each group. The grouping was as follows: Examples 1-6, Comparative Examples 1-6, and PBS blank control group.

[0171] Immunization schedule: On day 0 (primary immunization) and day 14 (booster immunization), mice in each group were subcutaneously injected with 100 μL of the corresponding formulation. All antigen-containing formulations were pre-adjusted to ensure that the dose of antigen (gE or Pre-F) in a single injection was consistent across all relevant groups.

[0172] Sample collection: On day 28 of the experiment, whole blood was collected from the retro-orbital venous plexus of mice. After standing at room temperature, the blood was centrifuged to prepare serum, which was then frozen at -80°C for later use. After blood collection, mice were euthanized by cervical dislocation, and their spleens were harvested under aseptic conditions for subsequent cellular immunological analysis.

[0173] Serum antibody titer detection (ELISA method):

[0174] Dilute the recombinant antigen (gE or Pre-F) to 2 μg / mL, coat a 96-well microplate, and incubate overnight at 4°C.

[0175] The next day, the plate was washed with washing buffer (PBS containing 0.05% Tween-20), and then blocking buffer (PBS containing 5% skim milk powder) was added and blocked at 37°C for 2 hours.

[0176] After washing the plate, add serially diluted mouse serum samples and incubate at 37°C for 1.5 hours.

[0177] Wash the plate and add HRP-labeled goat anti-mouse IgG, IgG1 or IgG2a secondary antibody working solution respectively, and incubate at 37°C for 1 hour.

[0178] After washing the plate again, add TMB substrate solution and incubate at room temperature in the dark for 15 minutes to develop color. Then add stop solution to terminate the reaction.

[0179] The absorbance was measured at 450 nm using an ELISA reader. The antibody titer was defined as the highest serum dilution that produced an absorbance 2.1 times higher than the mean of the negative control.

[0180] Antigen-specific T-cell response assay (ELISpot method):

[0181] The spleen was aseptically ground, and a single-cell suspension was prepared by passing it through a cell sieve. After removing red blood cells with red blood cell lysis buffer, the cells were counted.

[0182] Follow the instructions for the mouse IFN-γ and IL-4 ELISpot kit. Prepare spleen cell suspension (2 × 10⁻⁶). 5 Add (each well) to a PVDF plate pre-coated with IFN-γ or IL-4 capture antibody.

[0183] Each well was stimulated with the corresponding antigen (10 μg / mL), with ConA as a positive control and the culture medium as a negative control.

[0184] Place the plate in a 37℃, 5% CO2 incubator and incubate for 20-24 hours.

[0185] After terminating the culture, add the biotinylated detection antibody, enzyme-labeled streptavidin, and substrate sequentially according to the kit steps until clear spots appear.

[0186] Spot forming units (SFUs) were counted using an ELISpot analyzer. The test results are shown in Tables 4 and 5.

[0187] Table 4: Antigen-specific antibody titers in serum of mice in each group

[0188] Test Group Total IgG Titer IgG1 Titer IgG2a Titer Example 1 5.38±0.29 4.95±0.31 5.21±0.28 Example 2 5.81±0.33 5.23±0.35 5.67±0.30 Example 3 4.69±0.41 4.28±0.38 4.55±0.40 Example 4 5.45±0.27 4.88±0.30 5.33±0.26 Example 5 5.29±0.32 4.81±0.34 5.15±0.31 Example 6 5.31±0.30 4.90±0.32 5.18±0 .29 Comparative Example 1 3.98±0.45 3.81±0.43 3.65±0.48 Comparative Example 2 4.52±0.38 4.39±0.37 3.51±0.42 Comparative Example 3 4.71±0.35 4.45±0.39 4.18±0.41 Comparative Example 4 3.65±0.42 3.42±0.40 3.19±0.46 Comparative Example 5 4.89±0.36 4.55±0.35 4.68±0.39 Comparative Example 6 5.11±0.31 5.28±0.29 3.48±0.44 PBS Control <2.0 <2.0 <2.0 surface

[0189] From Table 4, we can obtain:

[0190] The antigen-specific total IgG, IgG1, and IgG2a antibody levels induced in Examples 1-6 were all higher than those in all comparative groups and the PBS control group. Compared with Comparative Example 2 (lacking QS-21), Comparative Example 3 (lacking 3D-MLA), and Comparative Example 4 (free component), Example 1 showed a significant advantage in total IgG titer, especially in IgG2a subtype titer. This indicates that the complete formulation of the present invention is crucial for inducing high levels of humoral immunity, and that the adjuvant components 3D-MLA and QS-21 play a key synergistic role in this process. Compared with Comparative Example 6 (aluminum adjuvant), the Example 1 group had a higher IgG2a antibody titer, and the IgG2a / IgG1 ratio was significantly higher than that of Comparative Example 6. This indicates that the adjuvant formulation of the present invention can induce a Th1-biased humoral immune response, different from that of conventional aluminum adjuvants.

[0191] Table 5: Levels of Antigen-Specific Cytokines Secreted by Spleen Cells in Each Group of Mice

[0192] Test Group IFN-γIL-4 Example 1 588±6531±9 Example 2 751±8245±12 Example 3 326±5125±8 Example 4 605±7135±11 Example 5 572±6833±10 Example 6 581±6632±9 Comparative Example 1 151±4219±7 Comparative Example 2 203±4828±8 Comparative Example 3 345±5524±7 Comparative Example 4 112±3815±6 Comparative Example 5 421±6030±9 Comparative Example 6 98±35115±33 PBS Control <10 <10 surface

[0193] From Table 5, we can obtain:

[0194] High levels of antigen-specific IFN-γ secreting cells were detected in all six groups of Examples 1-6, with numbers far exceeding those of all comparative groups and the PBS control group. The number of IFN-γ secreting cells in each group of Examples 1 was significantly higher than the number of IL-4 secreting cells, indicating that the formulation of this invention can effectively induce a strong Th1-type cellular immune response. Conversely, the number of IL-4 secreting cells in Comparative Example 6 (aluminum adjuvant) was significantly higher than its number of IFN-γ secreting cells, exhibiting a typical Th2-type immune bias. The IFN-γ levels in Comparative Example 2 (lacking QS-21) and Comparative Example 4 (free component) were significantly lower than in Example 1, demonstrating the necessity of the adjuvant component and the in-situ gel delivery system for effectively activating cellular immunity.

[0195] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A 3D-MLA and QS-21 composite adjuvant, characterized in that, The compound adjuvant comprises the following components in parts by weight: synthetic monophospholipid A: 0.1-1.0 parts; saponin QS-21: 0.2-10.0 parts; polyethylene glycol-modified sodium alginate: 15.0-220.0 parts; pH-sensitive calcium chloride encapsulated particles: 0.3-22.0 parts; vaccine antigen: 0.1-5.5 parts; wherein the synthetic monophospholipid A is 3D-MLA.

2. The 3D-MLA and QS-21 composite adjuvant according to claim 1, characterized in that, The PEGylated sodium alginate is obtained by grafting sodium alginate with a molecular weight of 100-300 kDa onto polyethylene glycol with a molecular weight of 5,000-20,000 Da, and the grafting rate of polyethylene glycol is 5%-20% w / w.

3. The 3D-MLA and QS-21 composite adjuvant according to claim 1, characterized in that, The pH-sensitive calcium chloride encapsulated particles are formed by encapsulating calcium chloride as a multivalent ion source in a pH-sensitive polymer carboxymethyl cellulose-acrylic acid copolymer. The average particle size distribution of the particles is 100-500 nanometers, and the calcium chloride loading rate is 10%-30%w / w.

4. The 3D-MLA and QS-21 composite adjuvant according to claim 3, characterized in that, The pH-sensitive polymer carboxymethyl cellulose-acrylic acid copolymer is obtained by copolymerizing sodium carboxymethyl cellulose and acrylic acid in a molar ratio of 1:0.1-0.

5. The sodium carboxymethyl cellulose has a molecular weight of 90-250 kDa and a degree of substitution of 0.6-0.

9.

5. A method for preparing a composite adjuvant of 3D-MLA and QS-21, characterized in that, The preparation of a 3D-MLA and QS-21 composite adjuvant according to any one of claims 1-4 comprises the following steps: S1. Preparation of polyethylene glycol-modified sodium alginate: Activating sodium alginate and reacting it with methoxy polyethylene glycol-N-hydroxysuccinimide ester, purifying and freeze-drying to obtain polyethylene glycol-modified sodium alginate; S2. Preparation of pH-sensitive calcium chloride encapsulated particles: Synthesizing a pH-sensitive polymer carboxymethyl cellulose-acrylic acid copolymer, mixing it with calcium chloride, and preparing pH-sensitive calcium chloride encapsulated particles by spray drying; S3. Preparation of adjuvant active ingredient solution: Dissolving synthetic monophospholipid A and saponin QS-21 in phosphate buffered saline, and then sonicating until fully soluble. After dissolution, an adjuvant active ingredient solution is obtained. The amount of phosphate buffered saline is used to control the total concentration of the adjuvant active ingredient within the range of 0.1-6.0 mg / mL. S4. Preparation of adjuvant-modified polymeric composite precursor formulation: Polyethylene glycolated sodium alginate is dissolved in phosphate buffered saline to prepare a polyethylene glycolated sodium alginate solution. Then, the adjuvant active ingredient solution is added to the polyethylene glycolated sodium alginate solution and stirred to obtain the adjuvant-modified polymeric composite precursor formulation. S5. Formulation of final adjuvant formulation: The vaccine antigen is added to the adjuvant-modified polymeric composite precursor formulation, and then pH-sensitive calcium chloride encapsulated particles are added and mixed evenly to obtain the 3D-MLA and QS-21 composite adjuvant.

6. The method for preparing a 3D-MLA and QS-21 composite adjuvant according to claim 5, characterized in that, In step S1, the step of preparing polyethylene glycol-modified sodium alginate includes: dissolving sodium alginate, adding an activator and stirring at pH 5.0-6.0 for 2-4 hours; subsequently, adding methoxy polyethylene glycol-N-hydroxysuccinimide ester and reacting at pH 6.0-7.0 for 12-24 hours; finally, purifying the reaction product by dialysis and freeze-drying to obtain the polyethylene glycol-modified sodium alginate.

7. The method for preparing a 3D-MLA and QS-21 composite adjuvant according to claim 6, characterized in that, In step S2, the step of preparing pH-sensitive calcium chloride encapsulated particles includes: reacting sodium carboxymethyl cellulose, acrylic acid, and an initiator at 60-80°C for 3-6 hours to obtain the pH-sensitive polymer carboxymethyl cellulose-acrylic acid copolymer; subsequently, mixing the pH-sensitive polymer carboxymethyl cellulose-acrylic acid copolymer with calcium chloride, and preparing the pH-sensitive calcium chloride encapsulated particles by spray drying, wherein the inlet temperature of the spray drying is 120-150°C and the outlet temperature is 70-90°C.

8. The method for preparing a 3D-MLA and QS-21 composite adjuvant according to claim 7, characterized in that, In step S4, the step of preparing the adjuvant-modified polymeric composite precursor formulation includes: dissolving the polyethylene glycolated sodium alginate in phosphate buffered saline to prepare a polyethylene glycolated sodium alginate solution with a concentration of 0.5%-2.0% w / v; subsequently, under a stirring rate of 100-300 rpm, slowly adding the adjuvant active ingredient solution to the pre-prepared polyethylene glycolated sodium alginate solution at a rate of 50-200 μL / min, and continuing to stir for 30-60 minutes to obtain the adjuvant-modified polymeric composite precursor formulation.

9. The method for preparing a 3D-MLA and QS-21 composite adjuvant according to claim 8, characterized in that, In step S5, the step of preparing the final adjuvant formulation includes: adding the vaccine antigen to the adjuvant-modified polymeric composite precursor formulation and mixing for 15-30 minutes at a temperature of 4-25°C and a stirring rate of 50-250 rpm; subsequently, adding the pH-sensitive calcium chloride encapsulated particles and continuing to mix for 5-15 minutes to obtain the 3D-MLA and QS-21 composite adjuvant.

10. The use of a 3D-MLA and QS-21 composite adjuvant as described in any one of claims 1-4 in the preparation of a vaccine adjuvant or vaccine composition for the prevention or treatment of viral infectious diseases.