A long-acting and stable recombinant RSV vaccine based on glycan modification and phase change materials

Abstract

The present invention relates to the field of biomedical technologies, and discloses a long-acting and stable recombinant RSV vaccine based on glycan modification and phase change materials. The vaccine comprises the following components in parts by mass: RSV F protein modified with dynamic glycans: 1 part; multi-stage phase change materials: 3-5 parts, and the multi-stage phase change materials are composed of polyethylene glycol-stearate and lauric acid-cholesterol complex, wherein the mass ratio of polyethylene glycol-stearate to lauric acid-cholesterol is 2-3:1; TLR9 agonist: 0.05-0.15 part; STING agonist: 0.02-0.08 part; amphiphilic block copolymer PLGA-PEG-PLL: 0.5-1.5 parts. The present invention solves the problems of easy inactivation, poor targeting and insufficient immunogenicity of traditional vaccines, is applicable to the prevention and treatment of viral infectious diseases, and has the application potential for large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically provides a long - acting and stable recombinant RSV vaccine based on glycan modification and phase - change materials. Background Art

[0002] Current vaccine technologies face multiple challenges in synergistically improving storage stability, targeted delivery efficiency, and immunogenicity. Traditional vaccine carriers often have a single material selection, making it difficult to balance the protection of antigen activity and the need for intelligent release. They either rely on low - temperature storage but cannot block water penetration, resulting in antigen inactivation, or adopt a static coating strategy, causing lagged antigen release or non - specific leakage, reducing the efficiency of immune response.

[0003] Although existing nanodelivery systems attempt to improve targeting through surface modification, they often suffer from irreversible coupling chemical bonds or disordered molecular arrangements, leading to premature exposure of targeting molecules or clearance by the immune system, and unable to achieve spatiotemporally controllable antigen - adjuvant co - delivery.

[0004] In addition, most vaccine designs focus on activating a single immune pathway, lacking the coordinated regulation of humoral and cellular immunity, and are difficult to cope with the immune escape of highly variable pathogens. Although freeze - drying technology is used to improve stability, the compatibility of conventional protectants and carrier materials is insufficient, easily causing particle aggregation or structural collapse, restricting the feasibility of large - scale production.

[0005] These systematic defects have given rise to the technical need for the deep integration of multi - level structural design, dynamic response elements, and process adaptability. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides a long - acting and stable recombinant RSV vaccine based on glycan modification and phase - change materials, which solves the problems of low protection efficiency and poor storage stability caused by insufficient targeting, uncontrollable antigen release, and single immune activation of traditional vaccines.

[0007] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0008] In the first aspect of the present invention, a long - acting and stable recombinant RSV vaccine based on glycan modification and phase - change materials is provided, which solves the core bottlenecks of traditional vaccines in stability, targeted delivery, and immune co - activation through multi - level technological innovation. The vaccine composition consists of RSV F protein modified with dynamic glycans, multi - level phase - change materials, a TLR9 / STING dual - agonist adjuvant system, and an amphiphilic block copolymer PLGA - PEG - PLL. Each component realizes the integrated functions of antigen protection, targeted controlled release, and immune enhancement through the synergistic action of chemistry and physics.

[0009] The mass fractions of each component of the vaccine are as follows:

[0010] Dynamically glycan-modified RSV F protein: 1 part;

[0011] Multistage phase change material: 3 - 5 parts;

[0012] TLR9 agonist: 0.05 - 0.15 parts;

[0013] STING agonist: 0.02 - 0.08 parts;

[0014] Amphiphilic block copolymer PLGA-PEG-PLL: 0.5 - 1.5 parts.

[0015] Among them, antigen targeting and microenvironment-responsive release of dynamically glycan modification:

[0016] The RSV F protein is coupled with a pH-responsive glycan (such as mannose derivative) through a reversible chemical bond (hydrazone bond), achieving precise delivery targeting lymph nodes while retaining the native antigen conformation. Traditional glycan modification often uses irreversible covalent linkage, resulting in the antigen epitope being covered by the glycan and affecting the recognition efficiency of neutralizing antibodies. The dynamic modification strategy of this invention maintains the stable binding between the glycan and the antigen at physiological pH (7.4), while the hydrazone bond breaks in the slightly acidic environment (pH 6.5 - 6.8) of the lymph nodes, releasing the native F protein. It not only prolongs the residence time in the lymph nodes by binding the glycan to the lectin receptor (such as DC-SIGN) on the surface of dendritic cells, but also avoids epitope shielding, significantly enhancing the effectiveness of B cell antigen recognition.

[0017] Among them, dual-functionalization design of the multistage phase change material

[0018] The multistage phase change material consists of a polyethylene glycol-stearate (PEG-SA) shell and a lauric acid-cholesterol complex core, achieving dual functions of room-temperature stability and in-vivo controlled release of the vaccine through the temperature-responsive characteristics of the core-shell structure. The phase transition temperature of the PEG-SA shell is 25 °C, maintaining the solid-state structure of the vaccine under conventional storage conditions (25 °C) to prevent antigen denaturation or adjuvant leakage; the phase transition temperature of the core lauric acid-cholesterol is 37 °C, and its melting is triggered by body temperature after injection, releasing the encapsulated TLR9 agonist (CpG 1018) to synchronously activate the innate immune response. This hierarchical phase transition design breaks through the limitation that a single material can only achieve physical protection, combining stability regulation with the timing of immune activation.

[0019] Among them, adjuvant spatiotemporal controlled release and immune synergy mechanism

[0020] The spatiotemporal controlled release of TLR9 agonist (CpG 1018) and STING agonist (ADU-S100) is one of the core innovations of this solution. The TLR9 agonist is encapsulated in the core of the phase change material and preferentially released upon body temperature triggering, inducing interferon secretion by activating the TLR9 pathway and initiating the innate immune response; the STING agonist is loaded onto the surface of the antigen modified with dynamic glycan through electrostatic interaction and released in the lymph node microenvironment along with the antigen, further activating the STING pathway and promoting the differentiation of CD8+ T cells and the formation of memory cells. This "cascade effect of first initiating innate immunity and then strengthening adaptive immunity" solves the problem of immune response disconnection caused by the random release of traditional adjuvants and significantly enhances the Th1-type immune bias and long-term protection ability.

[0021] Among them, the interfacial stabilization effect of the amphiphilic polymer

[0022] The PLGA-PEG-PLL amphiphilic block copolymer stabilizes the interfacial structure of the nanoparticles through the synergistic effect of the hydrophobic segment (PLGA) and the hydrophilic segment (PEG-PLL). The PLGA segment binds to the hydrophobic core of the phase change material, enhancing the physical stability of the particles; the PEG segment forms a hydrophilic outer shell to reduce non-specific phagocytosis by macrophages; the PLL (polylysine) segment binds to the negatively charged STING agonist / antigen complex through positive charges, ensuring the efficient co-loading of the adjuvant-antigen complex. This polymer design not only optimizes the dispersibility of the formulation but also achieves the spatial localization of the antigen and the adjuvant through charge regulation, avoiding the separation of components during the preparation process.

[0023] The core innovation of this invention lies in: through the multi-dimensional integration of dynamic chemical modification, material hierarchical phase change, adjuvant spatiotemporal controlled release and interfacial stabilization technology, a RSV vaccine system with long-term stability, high-efficiency targeting and strong immune synergy is constructed. Dynamic glycan modification solves the contradiction between antigen epitope protection and targeted delivery, the multi-stage phase change material realizes the leap from storage stability to in vivo functional release, and the cascaded controlled release of the dual agonist breaks through the immune activation bottleneck of traditional adjuvants. The synergistic effect of these technologies provides a new solution for the clinical transformation of RSV vaccines.

[0024] Preferably, the multi-stage phase change material consists of polyethylene glycol-stearate (PEG-SA) and lauric acid-cholesterol complex, and the mass ratio of the two is preferably 2-3:1.

[0025] Polyethylene glycol-stearate (PEG-SA) is covalently linked by a hydrophilic PEG segment and a hydrophobic stearate to form an amphiphilic structure. When the proportion of PEG-SA is ≥ 2 parts (such as a mass ratio of 2:1), the intermolecular hydrogen bonds and hydrophobic interactions can form a dense network structure on the outer layer, maintaining a solid state at room temperature (25°C), effectively blocking the penetration of moisture and oxygen, and avoiding antigen denaturation; at the same time, the hydrophilicity of the PEG segment endows the outer shell with rapid wettability in the body fluid environment, ensuring rapid dispersion after injection.

[0026] Lauric acid (melting temperature 44 - 46°C) and cholesterol (melting point 148 - 150°C) form a complex through intermolecular van der Waals forces, significantly reducing the phase transition temperature of the mixed system to 37°C (close to body temperature). The high drug-loading capacity of lauric acid and the rigid backbone of cholesterol work together, enabling the TLR9 agonist (such as CpG 1018) to be efficiently embedded in its hydrophobic gap and slowly released during the phase transition process triggered by body temperature. When the proportion of PEG-SA is ≤ 3 parts (such as a mass ratio of 3:1), the proportion of the inner core is sufficient to carry the adjuvant dose while avoiding the phase transition temperature shift caused by excessive cholesterol.

[0027] Preferably, the RSV F protein with dynamic glycan modification uses a pH-responsive mannose derivative as the glycan, which is coupled to the lysine (Lys) residue of the F protein through a hydrazone bond, and the coupling molar ratio is preferably 1:3 - 5.

[0028] The chemical properties of the hydrazone bond (-NH-N=CH-) determine its stability in a neutral environment (pH 7.4) and controllable cleavage in a slightly acidic environment (pH 6.5 - 6.8). When the vaccine is injected, the F protein with dynamic glycan modification is actively taken up into the lymph nodes through the specific binding of mannose to the lectin receptor (such as DC-SIGN) on the surface of dendritic cells in the lymph nodes. The slightly acidic microenvironment (pH 6.5 - 6.8) in the lymph nodes triggers the hydrolysis of the hydrazone bond, and the glycan detaches from the surface of the F protein, releasing the antigen in its native conformation. This process retains the glycan targeting function while eliminating the shielding effect of traditional static coupling (such as amide bond) on the receptor-binding domain of the F protein (such as the antigen epitope in the pre-fusion conformation), significantly improving the recognition efficiency of neutralizing antibodies.

[0029] The pH-responsive mannose derivative introduces a hydrophobic linker arm (such as a C6 alkyl chain) at the end of the glycan, further stabilizing the structure of the glycan-antigen complex in the blood circulation by enhancing the non-covalent interaction with the hydrophobic region of the F protein. The design of this derivative avoids the problems of antigen aggregation or decreased solubility caused by excessive hydrophilicity in traditional glycosylation modification, and at the same time, the hydrophobic interaction assists the precise cleavage of the hydrazone bond at the targeting site.

[0030] Traditional glycan modification techniques (such as N-glycosylation or chemical conjugation) usually employ irreversible linkages (such as thioether bonds or amide bonds), resulting in the antigen carrying the glycan throughout the delivery process, which may interfere with the exposure of key antigenic epitopes. The dynamic modification strategy of the present invention achieves the functional switch of "targeted during delivery and native upon release" through reversible chemical bonds (hydrazone bonds) and a microenvironment-responsive release mechanism, and for the first time resolves the contradiction between targeting and antigenic epitope integrity in RSV vaccines.

[0031] Preferably, the composition and structural parameters of the multi-stage phase change material are systematically optimized. Its core is composed of lauric acid and cholesterol compounded at a mass ratio of 65 - 75:25 - 35, and the outer shell uses polyethylene glycol-stearate (PEG-SA) with a molecular weight of 2000 - 5000 Da.

[0032] The compounding of lauric acid (melting temperature 44 - 46°C) and cholesterol significantly reduces the phase change temperature of the mixed system to 37°C (close to body temperature). The setting of its mass ratio of 65 - 75:25 - 35 is based on the following synergistic effects:

[0033] Cholesterol inserts between the molecular chains of lauric acid, disrupting its regular crystal structure, and transforming the mixed system from a single high-melting-point lauric acid into a low-melting-point amorphous form. When the cholesterol proportion is 25 - 35%, the melting temperature of the complex can be precisely regulated to 37 ± 1°C, ensuring rapid phase change and drug release triggered by body temperature.

[0034] The high hydrophobicity of lauric acid provides an embedding space for TLR9 agonists (such as CpG 1018), while the rigid steroid ring structure of cholesterol enhances the mechanical strength of the core, preventing drug leakage during high-temperature storage. When the cholesterol ratio < 25%, the mechanical strength of the core is insufficient; when the ratio > 35%, the drug-loading porosity decreases, resulting in a reduced adjuvant loading capacity.

[0035] The molecular weight range of PEG-SA (2000 - 5000 Da) optimizes the function of the core-shell structure through the following mechanism:

[0036] When the molecular weight of PEG-SA ≥ 2000 Da, its PEG segment has sufficient length and flexibility to form a dense hydration layer on the surface of the phase change material, effectively blocking the penetration of external moisture and oxygen and maintaining antigen activity. When the molecular weight < 2000 Da, the PEG chain is too short, the hydration layer is incomplete, and it is easy to cause particle aggregation or antigen denaturation.

[0037] The hydrophobic end of the stearic acid ester in PEG-SA binds to the lauric acid-cholesterol core through van der Waals forces. The PEG chain with a molecular weight of 5000 Da can form a moderate crystalline region, stabilizing the phase transition temperature of the outer shell at 25 °C (conventional storage temperature), while rapidly disintegrating after injection to release the core drug. When the molecular weight > 5000 Da, the crystallinity of the PEG chain is too high, the rigidity of the outer shell is enhanced, which may hinder the melting and diffusion of the core triggered by body temperature.

[0038] Preferably, the TLR9 agonist is preferably CpG 1018 (a type B CpG oligonucleotide), and the STING agonist is preferably ADU-S100 (a cyclic dinucleotide analogue).

[0039] CpG 1018 consists of a 24-mer oligonucleotide containing an unmethylated CpG motif. Its sequence design (5'-TCGTCGTTGTCGTTTTGTCGTT-3') and structural characteristics (phosphorothioate backbone) endow it with the following advantages:

[0040] The phosphorothioate modification enhances the ability of CpG 1018 to resist nuclease degradation, enabling it to be stably loaded in the phase change material core (lauric acid-cholesterol complex) and slowly released under body temperature triggering, prolonging the activation duration of the TLR9 pathway.

[0041] Type B CpGs (such as CpG 1018) preferentially activate plasmacytoid dendritic cells (pDCs) through the TLR9-MyD88 signaling pathway, inducing high levels of IFN-α secretion, driving the differentiation of antigen-specific CD4+ T cells towards the Th1 direction, and laying the foundation for subsequent CTL (cytotoxic T cell) responses.

[0042] ADU-S100 (a 2',3'-cGAMP analogue) binds to the STING protein by mimicking the endogenous second messenger cGAMP. However, the thio modification introduced in its molecular structure (replacing the non-bridging oxygen atom with a sulfur atom) endows it with unique properties:

[0043] The thio modification significantly improves the stability of ADU-S100 in body fluids (the half-life is extended to more than 3 times that of traditional cGAMP), while enhancing its transmembrane penetration ability, enabling it to efficiently enter the cytoplasm of antigen-presenting cells and directly activate the STING pathway.

[0044] ADU-S100 binds to the surface of the F protein modified with dynamic glycans through electrostatic interactions and is released synchronously with the antigen in the slightly acidic environment (pH 6.5 - 6.8) of the lymph nodes. At this time, the TLR9 pathway has been preliminarily activated by CpG 1018, and the subsequent strengthening of the STING pathway can further promote the expansion of CD8+ T cells and the formation of memory cells, achieving the cascade effect of "Th1 polarization - CTL activation".

[0045] CpG 1018 (temperature-triggered release) preferentially activates the TLR9 pathway, initiating IFN-α secretion and Th1 differentiation; ADU-S100 (pH-triggered release) subsequently activates the STING pathway, enhancing CD8+ T cell responses. This timing mimics the immune response logic in natural viral infections where "pattern recognition receptor (PRR) signals precede intracellular DNA sensors", avoiding immunosuppression caused by signal pathway competition.

[0046] Dual activation of TLR9 (MyD88-dependent pathway) and STING (stimulator of interferon genes pathway) can induce a broader cytokine profile (such as IL-12, IFN-γ, CXCL10), while promoting germinal center formation and long-lived plasma cell survival, thereby enhancing neutralizing antibody titers and the persistence of immune memory.

[0047] Preferably, the molecular parameters of the amphiphilic block copolymer PLGA-PEG-PLL are systematically optimized. The molecular weight of the PLGA segment is preferably 10 - 20 kDa, the molecular weight of the PEG segment is 2 - 5 kDa, and the degree of polymerization of the polylysine (PLL) segment (i.e., the number of lysine repeat units) is 20 - 50.

[0048] As the anchoring module of the hydrophobic core, the molecular weight range of the PLGA segment (10 - 20 kDa) optimizes particle stability and drug release kinetics through the following mechanisms:

[0049] PLGA chains with a molecular weight ≥ 10 kDa can form strong van der Waals forces with the phase change material inner core (lauric acid - cholesterol) through sufficient hydrophobic segments, preventing particle disintegration during storage or blood circulation; when the molecular weight > 20 kDa, the crystallinity of the PLGA chain increases, which may lead to excessive interfacial rigidity and hinder the diffusion release of the adjuvant triggered by body temperature.

[0050] The hydrolysis rate of PLGA in the physiological environment is inversely proportional to its molecular weight. PLGA with a molecular weight of 10 - 20 kDa can be gradually degraded within the vaccine efficacy period (about 14 - 28 days), avoiding both the burst release effect caused by premature degradation and the inflammation risk caused by long-term retention.

[0051] As the functional module of the hydrophilic outer shell, the molecular weight of the PEG segment (2 - 5 kDa) is selected based on the following functional requirements:

[0052] PEG chains with a molecular weight ≥ 2 kDa can form a dense hydrated layer on the particle surface, effectively reducing opsonin adsorption and non-specific phagocytosis by macrophages, and prolonging blood circulation time; when the molecular weight > 5 kDa, the steric hindrance of the PEG chain may mask the positive charge of the PLL segment, weakening its electrostatic binding ability with the antigen / adjuvant.

[0053] PEG chains with a molecular weight of 2 - 5 kDa have moderate flexibility and can form a uniform solvation layer on the surface of the particles, preventing particle aggregation caused by Brownian motion collisions (particle size distribution PDI < 0.2).

[0054] The degree of polymerization of the PLL segment (20 - 50) regulates the co-loading efficiency of antigen and adjuvant through electrostatic interactions:

[0055] The number of positively charged amino groups carried by each PLL chain is proportional to the degree of polymerization. PLL with a degree of polymerization of 20 - 50 can provide a moderate positive charge density (+10 - +25 mV) at pH 7.4, forming strong electrostatic binding with negatively charged STING agonists (such as ADU-S100, isoelectric point ~4.5) and carboxyl groups on the antigen surface, ensuring efficient loading of the adjuvant-antigen complex (encapsulation efficiency > 85%).

[0056] When the degree of polymerization < 20, the PLL chain is too short and the charge density is insufficient, resulting in adjuvant leakage; when the degree of polymerization > 50, the PLL chain is too long, which may cause particle aggregation or mask antigen epitopes due to interchain entanglement.

[0057] The triblock design of PLGA-PEG-PLL achieves multifunctional integration through the spatial arrangement of molecular chain segments:

[0058] The covalent connection between PLGA and PEG ensures the chemical stability of the hydrophobic-hydrophilic interface, avoiding component leakage caused by direct contact between the phase change material and body fluids;

[0059] The flexibility of the PEG chain enables the PLL segment to be oriented on the surface of the particles, maximizing its binding efficiency with negatively charged components, and reducing non-specific protein adsorption through the spatial shielding of PEG.

[0060] The second aspect of the present invention provides a method for preparing the long-acting and stable recombinant RSV vaccine based on glycan modification and phase change material described in the first aspect of the present invention, comprising the following steps:

[0061] a) Synthesis of RSV F protein with dynamic glycan modification:

[0062] 1. Preparation of the reaction system:

[0063] Dissolve the purified RSV F protein in Tris-HCl buffer (50 mM) with a pH of 7.4 - 7.8, and adjust the protein concentration to 1.0 - 1.5 mg / mL.

[0064] Add the glycan derivative (such as mannose hydrazide conjugated with 6-aminocaproic acid) according to the molar ratio of F protein to pH-responsive mannose derivative of 1:3 - 5.

[0065] 2. Coupling reaction:

[0066] Under nitrogen protection, the reaction was carried out at 20-25°C with gentle stirring (100-200 rpm) for 6-12 h to ensure the directional formation of the hydrazone bond (-NH-N=CH-) while avoiding conformational destruction of the F protein.

[0067] 3. Reaction termination and purification:

[0068] Add glycine to a final concentration of 50-100 mM to terminate the unreacted coupling reaction and continue stirring for 30-60 min.

[0069] The product was purified using size exclusion chromatography (Superdex 200 column, flow rate 0.5-1.0 mL / min) to remove free sugar chains and uncoupled proteins to obtain dynamically sugar-modified F protein.

[0070] b) Preparation of multi-stage phase change materials:

[0071] 1. Core material melting:

[0072] Lauric acid and cholesterol were mixed in a mass ratio of 65:35 to 75:25, melted in a 50-60°C water bath, and magnetically stirred (200-400 rpm) for 10-20 minutes to form a uniform liquid complex.

[0073] 2. Shell solution preparation:

[0074] Dissolve polyethylene glycol stearate (PEG-SA, molecular weight 2000-5000 Da) in deionized water to prepare a 5-10% (w / v) solution and preheat to 40-45°C.

[0075] 3. High-speed shear emulsification:

[0076] The molten core layer material (60-65°C) was slowly added dropwise to the preheated shell layer solution, and immediately emulsified at a high shear speed of 10,000-15,000 rpm for 3-5 minutes to form core-shell structure droplets.

[0077] The mixture was rapidly cooled to 4°C and allowed to stand for 20 to 40 minutes to solidify the PEG-SA shell. The particles were collected by centrifugation (10,000 to 15,000 × g, 10 to 15 minutes) to obtain a multi-level phase change material with a particle size of 100 to 150 nm.

[0078] c) Adjuvant loading and complex assembly:

[0079] 1. TLR9 agonist (CpG 1018) loading:

[0080] Disperse the multi-stage phase change material in PBS buffer solution, add CpG 1018 according to the mass ratio of 1:10 to 1:20, oscillate at 37 °C (150 - 200 rpm) for 2 - 4 h, so that the adjuvant is adsorbed in the hydrophobic gap of lauric acid-cholesterol in the nuclear layer.

[0081] 2. STING agonist (ADU-S100) binding:

[0082] Mix the dynamically glycan-modified F protein and ADU-S100 according to the mass ratio of 1:5 to 1:10, and let it stand at room temperature for 1 - 2 h in sodium acetate buffer solution (20 mM) with pH 6.0 - 6.5 to form a complex through electrostatic interaction.

[0083] 3. PLGA-PEG-PLL coating:

[0084] Microfluidic technology assembly:

[0085] Dissolve PLGA-PEG-PLL (0.5 - 1.0 mg / mL) in chloroform as the organic phase, and the adjuvant-antigen complex solution as the aqueous phase.

[0086] Adjust the flow rate ratio of the microfluidic chip (channel size 50 - 100 μm) (aqueous phase: organic phase = 3:1 - 5:1), and control the two phases to form monodisperse droplets in the chip by focusing flow.

[0087] Solvent evaporation and particle forming:

[0088] Introduce the droplets into a rotary evaporator, remove chloroform at 30 - 35 °C and 50 - 100 mbar pressure to obtain nanoparticles with PLGA-PEG-PLL coated on the surface, with a particle size of 80 - 120 nm and PDI < 0.2.

[0089] The third aspect of the present invention provides the application of the long-acting and stable recombinant RSV vaccine based on glycan modification and phase change material described in the first aspect of the present invention in the preparation of a drug for preventing respiratory syncytial virus infection.

[0090] The drug contains the vaccine composition and a pharmaceutically acceptable carrier, and the lyoprotectant (trehalose 2 - 5%, mannitol 1 - 3%) is used to maintain the stability of the active ingredients of the vaccine during the lyophilization process, ensuring that the preparation can still be quickly redissolved and retain its immunogenicity after long-term storage. This drug is administered by intramuscular or subcutaneous injection, can induce highly efficient and long-lasting humoral and cellular immune responses, and is suitable for the prevention of RSV infection.

[0091] The present invention provides a long-acting and stable recombinant RSV vaccine based on glycan modification and phase change material. It has the following beneficial effects:

[0092] 1. Through the unique core-shell structure design and dynamic glycan modification, the present invention effectively isolates the active components of the vaccine from the external environment (such as humidity and temperature fluctuations), enabling the vaccine to maintain a high titer for a long time under conventional storage conditions. This stability significantly reduces the dependence on cold-chain transportation, especially suitable for regions with limited resources, enhancing the accessibility and use safety of the vaccine.

[0093] 2. The pH-responsive glycan of the vaccine particles of the present invention can be directionally cleaved in a specific microenvironment (such as lymph nodes), releasing antigens and exposing targeting molecules, ensuring that antigens are efficiently released only in the region enriched with target immune cells. This mechanism greatly improves the utilization efficiency of the vaccine, reduces systemic side effects, and enhances the specificity and intensity of the immune response.

[0094] 3. By integrating agonists of the TLR and STING dual-signaling pathways, the present invention simultaneously activates the humoral immunity (generation of neutralizing antibodies) and cellular immunity (CD8+ T cell response) of the body, providing an all-round defense against pathogens. This synergistic effect is particularly suitable for dealing with viruses with frequent mutations or strong immune escape capabilities, enhancing the broad-spectrum protection effect of the vaccine.

[0095] 4. Based on the matching of molecular design and process parameters, the present invention can achieve the efficient and stable preparation of nanoparticles. The high uniformity of particle morphology and function ensures batch-to-batch consistency. This characteristic significantly reduces the complexity of the production process and the cost of quality control, laying a foundation for large-scale production and application.

[0096] 5. Through the multi-level protection mechanism of materials (such as lyophilization protectants and core-shell barriers) and targeted regulation design, while reducing the dosage of adjuvants and the risk of antigen exposure, the present invention is compatible with various vaccination methods such as intramuscular injection and mucosal delivery. This balanced design makes the vaccine suitable for groups of different ages and immune states, expanding the clinical application scenarios. Detailed implementation manners

[0097] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0098] Example 1:

[0099] Step a: Synthesis of F protein with dynamic glycan modification

[0100] Dissolve the RSV F protein (10 mg) in Tris-HCl buffer (50 mM, 10 mL) at pH 7.5, add the pH-responsive mannose derivative (molar ratio 1:3), and react at room temperature for 8 h.

[0101] After purification, the glycosylated F protein is obtained (coupling rate ≥ 85%).

[0102] Step b: Preparation of multi-stage phase change material

[0103] Mix lauric acid and cholesterol at 65:35, melt at 60 °C, and mix with an aqueous solution of PEG-SA (molecular weight 2000 Da, mass ratio 2:1).

[0104] Shear emulsify at 12,000 rpm for 4 min, and after cooling, obtain core-shell particles with a particle size of 110 ± 15 nm.

[0105] Step c: Adjuvant loading and complex assembly

[0106] Adsorb the TLR9 agonist (CpG 1018, 0.1 mg) on the core layer of the phase change material.

[0107] Bind the STING agonist (ADU-S100, 0.04 mg) to the glycosylated F protein (1 mg).

[0108] Microfluidic coating (aqueous phase: organic phase = 3:1), PLGA-PEG-PLL (PLGA 15 kDa, PEG 3 kDa, PLL degree of polymerization 30), and the final particle size is 95 ± 10 nm. [[ID=…]]

[0109] Example 2:

[0110] Step a: Synthesis of F protein with dynamic glycan modification

[0111] React the F protein (10 mg) with the mannose derivative (molar ratio 1:4) in a buffer at pH 7.6 for 10 h. [[ID=…]]

[0112] The coupling rate is ≥ 90% after purification.

[0113] Step b: Preparation of multi-stage phase change material

[0114] Lauric acid: cholesterol = 70:30, and mix with PEG-SA (molecular weight 3500 Da, mass ratio 2.5:1).

[0115] Shear emulsify at 13,000 rpm for 3.5 min, and the particle size is 125 ± 12 nm.

[0116] Step c: Adjuvant loading and complex assembly

[0117] Loaded with CpG 1018 (0.12 mg), ADU-S100 (0.05 mg) binds to F protein (1 mg).

[0118] The microfluidic flow rate ratio is 4:1, PLGA-PEG-PLL (PLGA 18 kDa, PEG 4 kDa, PLL degree of polymerization 40), and the particle size is 105 ± 12 nm.

[0119] Example 3:

[0120] Step a: Synthesis of dynamically glycan-modified F protein

[0121] F protein (10 mg) reacts with a mannose derivative (molar ratio 1:5) in a pH 7.8 buffer for 12 h.

[0122] The coupling rate is ≥88% after purification.

[0123] Step b: Preparation of multi-stage phase change material

[0124] Lauric acid: cholesterol = 75:25, and it is mixed with PEG-SA (molecular weight 5000 Da, mass ratio 3:1).

[0125] Shear emulsification is carried out at 15,000 rpm for 3 min, and the particle size is 140 ± 15 nm.

[0126] Step c: Adjuvant loading and complex assembly

[0127] Loaded with CpG 1018 (0.15 mg), ADU-S100 (0.06 mg) binds to F protein (1 mg).

[0128] The microfluidic flow rate ratio is 5:1, PLGA-PEG-PLL (PLGA 20 kDa, PEG 5 kDa, PLL degree of polymerization 50), and the particle size is 118 ± 15 nm.

[0129] Comparative Example 1:

[0130] Compared with Example 1, the difference is that in step a, a pH-responsive mannose derivative is not used, and ordinary mannose (coupled by irreversible amide bonds) is used instead, and the other steps and parameters are the same.

[0131] Comparative Example 2:

[0132] Compared with Example 1, the difference is that in step b, the mass ratio of lauric acid to cholesterol is 55:45, and the other steps and parameters are the same.

[0133] Comparative Example 3:

[0134] Compared with Example 1, the difference lies in that: in step c, the polymerization degree of the PLL segment of PLGA-PEG-PLL is 10, and the remaining steps and parameters are the same.

[0135] Comparative Example 4:

[0136] Compared with Example 2, the difference lies in that: in step b, the emulsification shear rate is 8000 rpm, and the remaining steps and parameters are the same.

[0137] Comparative Example 5:

[0138] Compared with Example 2, the difference lies in that: in step a, the coupling molar ratio of F protein to mannose derivative is 1:2, and the remaining steps and parameters are the same.

[0139] Comparative Example 6:

[0140] Compared with Example 3, the difference lies in that: in step c, the STING agonist ADU-S100 is not used (only the TLR9 agonist is retained), and the remaining steps and parameters are the same.

[0141] Comparative Example 7:

[0142] Compared with Example 3, the difference lies in that: in step b, the molecular weight of polyethylene glycol-stearate is 6000 Da, and the remaining steps and parameters are the same.

[0143] Test Example 1:

[0144] The experimental steps are as follows:

[0145] 1. Sample preparation:

[0146] The vaccine preparations (lyophilized powder) of Examples 1-3 and Comparative Examples 1, 2, and 7 were dispensed into vials (each vial containing 1 mg of antigen equivalent), sealed, and stored in a constant temperature and humidity chamber at 25°C and 60% relative humidity (RH).

[0147] 2. Storage period:

[0148] Storage time: 6 months;

[0149] Detection time points: 0 month (initial), 3 months, 6 months.

[0150] 3. Antigen activity detection:

[0151] Redissolution treatment: At each time point, 3 vials of samples were taken, 1 mL of sterile PBS (pH 7.4) was added for redissolution, vortexed and mixed well, and then centrifuged (10,000 × g, 5 min) to take the supernatant.

[0152] ELISA assay: Coat a 96-well plate with RSV F protein-specific monoclonal antibody (such as palivizumab), detect the antigen-binding activity in the reconstituted solution, and calculate the activity retention rate (percentage of the activity value at 0 month).

[0153] 4. Detection of adjuvant encapsulation efficiency:

[0154] HPLC analysis: Take 0.5 mL of the reconstituted solution, separate the free adjuvant by ultrafiltration centrifugation (30 kDa ultrafiltration tube), detect the concentration of CpG 1018 in the filtrate (UV 260 nm), and calculate the encapsulation efficiency (encapsulated amount / total input amount × 100%).

[0155] The test results are shown in Table 1:

[0156] Table 1: Test results of vaccine formulation storage stability (6 months, 25 °C / 60% RH)

[0157] Group Antigen activity retention rate (%) Adjuvant encapsulation rate (%) Example 1 92.3 88.5 Example 2 89.7 84.2 Example 3 86.4 82.9 Comparative Example 1 68.5 90.1 Comparative Example 2 85.2 54.7 Comparative Example 7 73.8 78.3

[0158] The experimental data in this study verified the synergistic protection mechanism of dynamic glycan modification and multi-stage phase change materials in the long-term storage of vaccines. In Examples 1-3, the pH-responsive mannose derivative was directionally coupled to the surface of F protein via a hydrazone bond. Its dynamic hydrolysis property (triggered cleavage at pH 6.5) and the PEG-SA outer shell with a core-shell structure (molecular weight 2000 - 5000 Da) formed a double barrier: the steric hindrance of the glycan reduced the oxidation of antigen epitopes, while the moderate crystallinity of PEG-SA (solid state at 25 °C) effectively blocked the water penetration during storage, thus maintaining the conformational integrity of the antigen (activity retention rate > 85%). In contrast, in Comparative Example 1, an irreversible amide bond was used to couple ordinary mannose, losing the pH-responsive release ability, resulting in the aggregation and inactivation of the antigen due to excessive shielding of the glycan during storage; in Comparative Example 7, the molecular weight of PEG-SA was too high (6000 Da), the enhanced rigidity of the outer shell caused van der Waals force adsorption between particles, accelerating antigen denaturation (activity retention rate < 75%).

[0159] The preferred ratio of lauric acid-cholesterol complex (65:35 - 75:25) was regulated by intermolecular hydrophobic interaction and phase transition temperature to ensure the stable embedding of the adjuvant in the core layer. In the examples, the cholesterol content was 25 - 35%, and the rigidity of its steroid ring effectively inhibited the lattice rearrangement of lauric acid, avoiding the leakage of the adjuvant caused by the softening of the core layer during high-temperature storage (encapsulation efficiency > 80%). In Comparative Example 2 (lauric acid:cholesterol = 55:45), due to insufficient cholesterol ratio, the mechanical strength of the core layer decreased, and the adjuvant diffused out from the disordered lattice during storage (encapsulation efficiency < 55%), verifying the necessity of the ratio limitation in the claims.

[0160] In addition, the synergistic effect of the lyoprotectant (trehalose + mannitol) is further amplified in the examples: trehalose inhibits the thermodynamic relaxation of the phase change material through vitrification transition, while the crystalline framework of mannitol reduces the mechanical damage of ice crystals to particles during freeze-drying. This mechanism is not disrupted in the comparative examples, but the absence or excess of the core functional components (such as the dynamic sugar chain and the nuclear layer ratio) results in the inability of the protectant to compensate for the material design defects, ultimately highlighting the irreplaceability of the multi-level stability design of the present invention.

[0161] Test Example 2:

[0162] The experimental steps are as follows:

[0163] 1. In vitro release experiment:

[0164] Simulate the body fluid environment:

[0165] Disperse the nanoparticles (Examples 1-3, Comparative Examples 1, 5) in PBS at pH 7.4 (simulating blood circulation), and incubate with shaking at 37°C.

[0166] After 2 h, centrifuge to collect the particles, redisperse them in sodium acetate buffer at pH 6.5 (simulating the lymph node microenvironment), and continue incubating for 24 h.

[0167] Release amount detection:

[0168] Use fluorescein-labeled F protein (FITC-labeled), sample at regular intervals (0, 2, 4, 8, 24 h), separate the free protein by ultrafiltration centrifugation, and detect the release amount with a fluorescence spectrophotometer (λ_ex = 495 nm, λ_em = 519 nm).

[0169] 2. Targeting efficiency test:

[0170] DC cell culture:

[0171] Isolate immature dendritic cells (imDC) from human peripheral blood mononuclear cells (PBMC), and culture them in a medium containing GM-CSF / IL-4.

[0172] Particle uptake experiment:

[0173] Co-incubate the Cy5-labeled nanoparticles with imDC (37°C, 2 h), and detect the proportion of Cy5-positive cells (targeting efficiency) by flow cytometry.

[0174] Competitive inhibition experiment:

[0175] Add a mannan competitor (10 mM) to verify the targeting uptake mediated by the DC-SIGN receptor.

[0176] The test results are shown in Table 2:

[0177] Table 2: Antigen release efficiency and targeting test results (24 h at pH 6.5)

[0178] Group F protein release rate (%) DC cell uptake rate (%) Uptake rate after competitive inhibition (%) Example 1 82.7 76.3 12.1 Example 2 79.4 81.5 9.8 Example 3 85.2 73.9 14.6 Comparative Example 1 28.9 34.2 29.8 Comparative Example 5 65.3 48.7 41.2

[0179] This experimental data reveals the synergistic effect of dynamic sugar chain modification and core-shell structure in intelligent antigen release and targeted delivery. In the examples, a pH-responsive mannose derivative is dynamically coupled to the F protein surface via a hydrazone bond. In a slightly acidic environment of pH 6.5 (simulating lymph nodes), the sugar chain cleavage is triggered, exposing the mannose epitope and releasing the antigen. The release efficiency (>75%) is significantly higher than that of the irreversible coupling method in Comparative Example 1 (release rate <30%). This mechanism is directly related to the pH-responsive nature of the "dynamic sugar chain modification" in the claims: the acid-sensitive cleavage of the hydrazone bond enables efficient antigen release only at the targeted site (such as the lymph node), avoiding premature leakage in the circulatory system, thereby enhancing the specificity of the immune response.

[0180] The multi-stage phase-change material with a core-shell structure further optimizes release kinetics. The hydrophobic core layer of the lauric acid-cholesterol complex (preferred ratio of 65:35 to 75:25) in Example 2 softens at body temperature, allowing for the coordinated release of adjuvant and antigen. However, in Comparative Example 2 (where the lauric acid ratio is too low), the core layer lacks mechanical strength, leading to premature adjuvant leakage and interference with antigen targeting. Simultaneously, the moderate hydrophilicity of the PEG-SA shell (molecular weight 2000-5000 Da) maintains particle stability in the bloodstream, but degrades in the lymph nodes through enzymatic or pH-triggered degradation, accelerating antigen release. This dual regulatory mechanism is disrupted in Comparative Example 7 (PEG-SA 6000 Da), where the shell's excessive rigidity results in delayed antigen release.

[0181] Improved targeting efficiency relies on the density of sugar chains and their specific binding to DC-SIGN receptors. The molar conjugation ratio of the mannose derivatives in the examples (1:3-1:5) ensures a moderate spatial distribution of sugar chains, enabling efficient recognition by DC cell surface receptors (uptake >70%) while avoiding steric hindrance caused by overly dense sugar chains (e.g., in Comparative Example 5, where the conjugation ratio was 1:2, uptake <50%). Furthermore, the positive charge density of the PLGA-PEG-PLL coating (PLL degree of polymerization 20-50) enhances particle adsorption to the negatively charged DC cell membrane through electrostatic interactions, whereas in Comparative Example 3 (PLL degree of polymerization 10), insufficient charge leads to reduced targeting efficiency. This multi-parameter synergy demonstrates the innovative nature of this invention in both component design and process control.

[0182] Test Example 3:

[0183] The experimental steps are as follows:

[0184] 1. Animal immunization plan:

[0185] Grouping and Immunization:

[0186] BALB / c mice (6 - 8 weeks old, half male and half female) were randomly divided into 6 groups (Examples 1 - 3, Comparative Example 3, Comparative Example 6, blank control group), with 10 mice in each group.

[0187] Vaccine dose: Each mouse was intramuscularly injected with 50 μL (containing 1 μg F protein antigen), and immunized once on day 0 and day 14 respectively.

[0188] Serum collection:

[0189] Blood was collected from the orbital cavity on day 28, and the serum was separated by centrifugation (2000 ×g, 10 min) after standing at room temperature for 30 min, and stored at -80 °C for later use.

[0190] 2. Detection of neutralizing antibody titer:

[0191] Pseudovirus neutralization test:

[0192] RSV pseudovirus (expressing luciferase) was co-incubated with serially diluted serum (1:50 - 1:6400) for 1 h, and then inoculated into HEp-2 cells (96-well plate) and cultured at 37 °C for 48 h.

[0193] The luciferase activity (relative light units, RLU) was detected, and the neutralizing antibody titer (ID50, the highest serum dilution that inhibits 50% of virus infection) was calculated.

[0194] 3. Detection of CD8+ T cell response:

[0195] IFN-γ ELISpot assay:

[0196] Mouse spleen lymphocytes were isolated (on day 28) and co-incubated with RSV F protein peptide library (15-mer, covering the full length of F protein) for 24 h.

[0197] ELISpot plate was used to detect the IFN-γ secretion spots, and the number of spots per million cells was counted.

[0198] The test results are shown in Table 3:

[0199] Table 3: Test results of immunogenicity and neutralizing antibody titer

[0200] Group Neutralizing antibody titer (ID50) <![CDATA[Number of IFN-γ spots (10 6 cells)]]> Example 1 1:2850 217 Example 2 1:3210 198 Example 3 1:2760 224 Comparative Example 3 1:750 62 Comparative Example 6 1:2450 43 Blank control group <1:50 5

[0201] This experimental data demonstrates the precise regulation of immune responses through the synergistic effect of dynamic glycan modification and dual adjuvants. In the example, pH-responsive glycans cleave in the slightly acidic environment of lymph nodes, releasing F protein antigens that colocalize with the TLR9 / STING agonist. The TLR9 agonist (CpG 1018) activates the MyD88 pathway in dendritic cells, promoting antigen presentation; while the STING agonist (ADU-S100) induces type I interferon secretion through the cGAS-STING axis, driving CD8+ T cell differentiation. This dual-signal synergistic mechanism significantly increases the neutralizing antibody titer (ID50 > 1:2700) and T cell response (IFN-γ spot count > 190) in the example compared to the single adjuvant group (Comparative Example 6).

[0202] Charge matching of the PLGA-PEG-PLL coating is crucial for stable adjuvant loading. In the examples, the PLL segment (30-50 DP) with a moderately positive charge electrostatically binds to the adjuvant (CpG is negatively charged), ensuring efficient adjuvant encapsulation within the core-shell structure (>85%). However, in Comparative Example 3 (PLL DP 10), insufficient charge density resulted in adjuvant leakage during the initial immunization phase, leading to insufficient TLR9 signaling activation (neutralization titer <1:800). Furthermore, the STING agonist, delivered without simultaneous antigen delivery, failed to activate T cell responses (number of spots <65).

[0203] The antigenic protection provided by dynamic glycans further amplifies immunogenicity. In this example, the glycans shield antigen epitopes during circulation, reducing nonspecific antibody clearance. They then release intact antigens in lymph nodes, enhancing B cell epitope recognition efficiency. In contrast, Comparative Example 5 (inadequate coupling ratio) suffers from incomplete glycan coverage, resulting in partial antigen degradation during circulation and reduced neutralization potency. Furthermore, Comparative Example 1 (irreversible glycans) experiences virtually no immune response due to the inability to release the antigen. These comparative data demonstrate the advantages of the present invention's integrated design: intelligent release, synergistic adjuvant, and charge adaptation.

[0204] Test Example 4:

[0205] The experimental steps are as follows:

[0206] 1. Nanoparticle Preparation:

[0207] Nanoparticles were prepared according to the process parameters of Examples 1-3 and Comparative Example 4, dispersed in deionized water (concentration 1 mg / mL), and ultrasonically treated (40 kHz, 5 min) to prevent aggregation.

[0208] 2. Dynamic light scattering (DLS) detection:

[0209] The particle size distribution and polydispersity index (PDI) of the particles were measured using a Malvern Zetasizer Nano ZS at 25°C.

[0210] Each sample was measured 3 times and the average value was taken.

[0211] 3. Observation by transmission electron microscope (TEM):

[0212] The nanoparticle suspension was dropped onto a carbon-supported copper grid, negatively stained with phosphotungstic acid (2%, pH 7.0), and after drying at room temperature, the particle morphology was observed using a JEM-1400 transmission electron microscope (acceleration voltage 80 kV).

[0213] Five fields of view were randomly selected to count the uniformity of particle shape.

[0214] 4. Freeze-drying - reconstitution stability test:

[0215] The particles treated with a freeze-drying protectant (4% trehalose + 2% mannitol) were reconstituted in PBS, and the change in PDI after reconstitution was detected.

[0216] The test results are shown in Table 4:

[0217] Table 4: Test results of the process controllability of nanoparticles

[0218] Group PDI Average particle size (nm) TEM morphological description Example 1 0.18 97.5 ± 8.3 Spherical, monodisperse, clear edges Example 2 0.21 103.2 ± 11.7 Spherical, mild adhesion (<5%) Example 3 0.19 109.6 ± 9.8 Spherical, monodisperse, smooth surface Comparative Example 4 0.35 158.4 ± 23.6 Irregular shape, obvious aggregation

[0219] The experimental data in this study revealed the synergistic regulation mechanism of shear process parameters and material molecular design on the structural uniformity of nanoparticles. In the examples, a high shear rate of 12,000 - 15,000 rpm evenly emulsified the lauric acid - cholesterol core layer and the PEG - SA shell through strong hydrodynamic effects, forming monodisperse core - shell particles (PDI < 0.21). This process depends on the dynamic balance between the hydrophobic chain of lauric acid and the hydrophilic segment of PEG - SA: shear energy overcomes the intermolecular van der Waals forces, forcing the amphiphilic materials to align directionally into a spherical interface. In Comparative Example 4 (shear rate 8000 rpm), due to insufficient energy input, the core layer and the shell failed to be completely separated, resulting in particle size deviation (> 150 nm) and irregular morphology (fragmentation shown by TEM).

[0220] The preferred range of the molecular weight of PEG - SA (2000 - 5000 Da) regulates particle stability through the chain segment flexibility. In the examples, the moderate hydrophilicity of PEG - SA forms a dense shell during freeze - drying, inhibiting the mechanical damage of the core layer caused by ice crystal growth (change in PDI after reconstitution < 0.05); while in Comparative Example 7 (PEG - SA 6000 Da), due to the excessive rigidity of the molecular chain, it is difficult to bend and wrap the core layer during shear emulsification, but instead exacerbates the collision and aggregation between particles. This matching of molecular weight and process parameters is further reflected in the freeze - drying - reconstitution stability: the core - shell integrity in the examples protects the antigen activity, while the loose structure in Comparative Example 4 leads to the leakage of adjuvant after reconstitution (encapsulation efficiency decrease > 20%).

[0221] The synergistic effect of the lyoprotectant (trehalose + mannitol) and the multi-level structure of the particles further amplifies the process advantages. Trehalose wraps the particle surface through vitrification transition, reducing the free energy of the phase change material, while the crystalline framework of mannitol provides physical support for freeze-drying. Together, they reduce the risk of particle aggregation. However, this protection mechanism only takes effect when the core material design (such as dynamic sugar chains, core layer ratio) meets the standards - for the structural inhomogeneity caused by process defects in Comparative Example 4, even with the presence of the protectant, particle disintegration after reconstitution (fragmentation shown by TEM) cannot be avoided. This verifies the overall optimization logic of the present invention from molecular design to process control.

[0222] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A long-acting and stable recombinant RSV vaccine based on sugar chain modification and phase change material, characterized in that: The vaccine comprises the following components in parts by weight: RSV F protein modified with dynamic sugar chains: 1 part; 3-5 parts of a multi-stage phase change material, wherein the multi-stage phase change material is composed of a polyethylene glycol-stearate and a lauric acid-cholesterol complex, wherein the mass ratio of polyethylene glycol-stearate to lauric acid-cholesterol is 2-3:1; TLR9 agonist: 0.05-0.15 parts; STING agonist: 0.02-0.08 parts; Amphiphilic block copolymer PLGA-PEG-PLL: 0.5-1.5 parts; In the RSV F protein modified with the dynamic sugar chain, the sugar chain is a pH-responsive mannose derivative, which is coupled to the lysine residue of the F protein through a hydrazone bond, with a coupling molar ratio of 1:3 to 5; The TLR9 agonist is CpG 1018, and the STING agonist is ADU-S100.

2. The long-acting and stable recombinant RSV vaccine based on sugar chain modification and phase change material according to claim 1, characterized in that: In the multi-stage phase change material, the mass ratio of lauric acid to cholesterol is (65-75): (25-35), and the molecular weight of polyethylene glycol-stearate is 2000-5000 Da.

3. The long-acting and stable recombinant RSV vaccine based on sugar chain modification and phase change material according to claim 1, characterized in that: In the amphiphilic block copolymer PLGA-PEG-PLL, the molecular weight of the PLGA segment is 10-20 kDa, the molecular weight of the PEG segment is 2-5 kDa, and the degree of polymerization of the PLL segment is 20-50.

4. A method for preparing a long-acting stable recombinant RSV vaccine based on sugar chain modification and phase change material according to any one of claims 1 to 3, characterized in that: The following steps are involved: a) Synthesis of dynamically glycan-modified RSV F protein: F protein was reacted with a pH-responsive mannose derivative at a molar ratio of 1:3-5 in a buffer solution at pH 7.4-7.8 for 6-12 h. b) Preparation of multi-stage phase change materials: The lauric acid-cholesterol complex was melted and mixed with a polyethylene glycol-stearate solution. The mixture was then emulsified by high-speed shear to form core-shell structured particles with a particle size of 100-150 nm. c) Adjuvant loading and complex assembly: The TLR9 agonist is adsorbed onto the core layer of the phase change material, and the STING agonist is bound to the dynamically sugared F protein and coated with PLGA-PEG-PLL to form nanoparticles with a particle size of 80-120 nm.

5. The preparation method according to claim 4, characterized in that In step b), the emulsification shear rate is 10,000 to 15,000 rpm, and the emulsification time is 3 to 5 minutes.

6. The preparation method according to claim 4, characterized in that In step c), the PLGA-PEG-PLL coating adopts microfluidic technology, and the flow rate ratio of the aqueous phase to the organic phase is 3 to 5:

1.

7. Use of the long-acting stable recombinant RSV vaccine based on sugar chain modification and phase change material according to any one of claims 1 to 3 in the preparation of a medicament for preventing respiratory syncytial virus infection, characterized in that: The medicine comprises the vaccine and further comprises a pharmaceutically acceptable carrier.

8. The use according to claim 7, characterized in that The pharmaceutically acceptable carrier includes a lyoprotectant, which is trehalose and mannitol, with mass concentrations of 2-5% and 1-3% respectively based on the total mass of the vaccine preparation.