Recombinant RSV vaccine composition based on fusion protein multimer and TLR / STING dual agonist

Abstract

The invention relates to the technical field of biological pharmacy, and discloses a recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist, and the recombinant RSV vaccine composition comprises the following component materials: the fusion protein polymer, a TLR7 / 8 agonist, STING, a liposome and a heat stabilizer; the fusion protein polymer comprises an RSV F protein (PreF conformation) and an adenovirus Pentamer protein; the liposome comprises phospholipid and cholesterol; the heat stabilizer comprises trehalose, glycerol and cane sugar. According to the present invention, the fusion antigen is constructed through the Pre-F and the Pentamer according to the optimization ratio, and the heat stabilizer is supplemented, such that the completeness of the antigen conformation can be maintained while the humoral immunity and cellular immunity induction ability of the vaccine on the target antigen can be effectively enhanced without introducing the exogenous adjuvant, and the consistency and the immune protection level of the vaccine can be easily improved.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and specifically to a recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist. Background Art

[0002] At present, in the research of subunit vaccines for respiratory syncytial virus (RSV), the construction of fusion proteins is widely used to simulate the natural conformation of the virus to enhance immunogenicity. Pre-F protein has become an important antigen design object because of its enrichment in central epitopes. In existing technologies, Pre-F is often fused with a polymer auxiliary structure (such as Pentamer protein) to form a virus-like structure to enhance the immune system's ability to recognize key conformational epitopes. In addition, in order to further enhance the cellular and humoral immune responses of the vaccine, researchers introduced immune agonists into the formula, especially TLR7 / 8 small molecules and STING pathway agonists. At the same time, in order to achieve room temperature transportation and long-term storage of vaccines, the freeze-drying process combined with certain stabilizers has become a mature technical means in the field of vaccine preparation.

[0003] Although relevant technologies have made beneficial progress in many aspects, there is still room for further optimization in key links such as the ratio of fusion protein construction, the synergistic strategy of immune agonists, and the guarantee of freeze-drying stability. The existing literature on the ratio design of Pre-F and auxiliary structural proteins is mostly based on preliminary screening, and there is a lack of systematic correlation studies on the ratio of antigen conformation stability and immune activation effect. In terms of the combined use of immune agonists, although there have been attempts to adopt a multi-pathway synergistic strategy, the release rhythm of the agonists in the body and the delivery stability have not been fully matched, which may affect their synergistic activation effect. In addition, during the freeze-dried storage of vaccines, the response characteristics of different components to drying stress vary greatly, and the existing stabilizer combinations are often based on a single protection mechanism, which makes it difficult to simultaneously maintain the integrity of protein conformation and liposome structure. It can be seen that in terms of antigen construction, immune enhancement and storage stability, more synergistic and structurally reasonable technical solutions are still needed. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a recombinant RSV vaccine composition based on fusion protein polymers and TLR / STING dual agonists, which solves the problems of unreasonable fusion protein ratio, unstable synergistic effect of immune agonists, and easy loss of antigen conformation and delivery system during freeze-drying.

[0005] To achieve the above objectives, the present invention is implemented by the following technical scheme: a recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist comprises the following component materials in parts by weight: Fusion protein polymer 100-250 copies; TLR7 / 8 agonist 10-20 copies; STING agonist 5-10 copies; Liposomes 75-250 parts; Heat stabilizer 70-100 parts.

[0006] Furthermore, the fusion protein polymer is composed of RSV F protein Pre-F conformation and adenovirus Pentamer protein, wherein the mass ratio of the two is 5-7:3-5, which is conducive to the formation of a structurally complete and stably presented polymer; RSV F protein Pre-F conformation, as the core antigen of the present invention, has a high neutralizing epitope stability and is a key conformation for inducing broad-spectrum neutralizing antibodies, but its in vitro stability is poor and it is easily converted into a Post-F form, resulting in loss of antigenicity; by recombination with adenovirus Pentamer protein, the Pre-F conformation can be displayed in a multivalent manner, simulating the virus-like particle structure (VLPs), improving its lymphatic tissue retention ability in the body, and enhancing the cross-linking efficiency with B cell receptors, thereby significantly improving the antibody affinity and neutralization efficiency, achieving the dual effects of antigen stability and immune enhancement; The TLR7 / 8 agonist is Resiquimod, which is added in an amount of 10-20 parts. It is a typical small molecule immunomodulator that can activate the TLR7 and TLR8 signaling pathways in myeloid cells (such as dendritic cells and macrophages), causing the activation of NF-κB and IRF pathways, promoting the release of cytokines such as IL-12 and TNF-α, and enhancing Th1 responses and antiviral immunity. As a vaccine adjuvant, it can guide the immune response to shift in the direction of cellular immunity, prevent RSV vaccines from inducing adverse Th2 bias, and thus reduce the risk of immune enhancement disease (VAERD). The STING agonist is ADU-S100, which is added in an amount of 5-10 copies. It acts on the cGAS-STING signaling axis in host cells and is an innate immune agonist that has been studied more in recent years. After being recognized by STING in the cytoplasm, ADU-S100 can induce TBK1 and IRF3 phosphorylation, further upregulating type I interferons and chemokines such as IFN-β and CXCL10, thereby enhancing the antiviral CD8+T cell response. Combining it with a TLR7 / 8 agonist can synergistically activate multiple immune pathways, improve the broad spectrum and durability of the vaccine in clearing the virus, and both are small molecules, which facilitates uniform distribution and delivery control in the vaccine system. Liposomes are vaccine delivery carriers and adjuvant structures. They are composed of phospholipids and cholesterol in a mass ratio of 6-8:2-4, and have good membrane structure stability and hydrophilic and hydrophobic dual encapsulation capacity. The role of liposomes in the present invention is not limited to physical carriers. More importantly, they can encapsulate or adsorb immune agonists to form a sustained-release system, regulate the release rate of agonists, and reduce local immunotoxicity. At the same time, their virus-like particle size (100-200nm) can enhance the binding and endocytosis efficiency with APCs. In addition, liposomes themselves also have adjuvant functions, which can enhance antigen presentation and lymph node targeting distribution, and are one of the important means to improve the effectiveness of vaccines. The thermal stabilizer is composed of trehalose, glycerol and sucrose in a mass ratio of 5-7:1-2:1-2. This combination has a good protective effect during the freeze-drying and reconstitution of the vaccine; trehalose can form an amorphous glassy coated protein antigen during the freeze-drying process to avoid its structural collapse and aggregation during the sublimation drying process; sucrose can stabilize the liposome structure and prevent the phospholipid bilayer from rupturing during low-temperature dehydration; glycerol plays a role in humidity regulation after freeze-drying, reducing particle instability caused by excessive drying; this ternary system can effectively improve the storage stability of the vaccine composition at room temperature and cold chain conditions, extend its shelf life and improve the retention rate of biological activity after reconstitution of the preparation.

[0007] Preferably, the fusion protein polymer includes RSV F protein Pre-F conformation and adenovirus Pentamer protein, and the mass ratio of RSV F protein Pre-F conformation to adenovirus Pentamer protein is 5-7:3-5.

[0008] Preferably, the liposome comprises phospholipids and cholesterol, and the mass ratio of the phospholipids to cholesterol is 6-8:2-4.

[0009] Preferably, the specific model of the TLR7 / 8 agonist is Resiquimod, and the specific model of the STING agonist is ADU-S100.

[0010] Preferably, the thermal stabilizer comprises trehalose, glycerol and sucrose, and the mass ratio of the trehalose, glycerol and sucrose is 5-7:1-2:1-2.

[0011] A method for preparing a recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist comprises the following steps: S1, recombinantly expressing RSV F protein Pre-F conformation and adenovirus Pentamer protein to obtain fusion protein polymers; S2, dissolving the TLR7 / 8 agonist Resiquimod and the STING agonist ADU-S100 in a sterile phosphate buffer solution with the aid of an ultrasonic water bath for 5-10 minutes, and filtering and sterilizing to obtain an agonist solution; S3, mixing phospholipids and cholesterol according to the proportion and preparing liposomes using a thin film method; S4, mixing trehalose, glycerol and sucrose in proportion to prepare a heat stabilizer solution; S5. Precool the fusion protein polymer, TLR7 / 8 agonist, STING agonist, liposome and thermal stabilizer to 4° C., and stir them at a temperature of 2-8° C. according to a mass ratio, and stir for 20-60 minutes to obtain a recombinant RSV vaccine composition; S6. Freeze-drying the obtained composition to obtain the final vaccine preparation.

[0012] Specifically, in step S1, the RSV F protein Pre-F conformation and the adenovirus Pentamer protein are respectively used to construct a recombinant expression system in CHO cells, and after culture, the supernatant containing the target protein is obtained by cell lysis, and the expression product with the His tag is captured by Ni-NTA affinity chromatography, and then the host impurities and oligomers are removed by anion exchange chromatography and gel filtration chromatography, and finally a high-purity, high-aggregation fusion protein polymer is obtained; this process can ensure that the antigen conformation is stable, the structure is controllable, and the size of the polymer particles is uniform, providing a structural basis for the subsequent uniform mixing of components and immune efficacy; In step S2, the solubility and uniformity of the agonist are improved by physical means, while avoiding the degradation of the small molecule structure caused by solvent residue or high temperature heating, providing a dispersed state with good biocompatibility for subsequent liposome adsorption and mixing, while ensuring the sterility and safety of the system; In step S3, the formed liposome membrane has a stable structure and is suitable for subsequent encapsulation of immune agonists or mixing with protein antigens to form a composite vaccine system; In step S4, the prepared solution has reasonable composition and moderate osmotic pressure, which can protect the protein, liposome and small molecule structures from being destroyed during the freeze-drying process, and effectively inhibit the denaturation of biomacromolecules caused by heat and freezing stress; In step S5, controlling temperature and shear force during the stirring process is the key to preventing protein inactivation, liposome rupture, and agonist aggregation. Low temperature and low-speed stirring modes can achieve full fusion of the components without causing structural damage, ensuring the stability and immune consistency of the finished product. In step S6, the freeze-drying process can maintain the structural integrity and immune function of various active ingredients in the vaccine to the greatest extent, and is easy to transport and store, meeting the dual requirements of industrial preparations for long-term stability and rapid reconstitution.

[0013] Preferably, in the step S1, the recombinant expression comprises the following steps: RSV F protein Pre-F conformation and adenovirus Pentamer protein were recombinantly expressed in CHO cells; Cultivate in an expression system and obtain a recombinant protein solution by cell lysis; Purify the target protein by affinity chromatography using Ni-NTA resin; Impurities are removed by ion exchange chromatography and gel permeation chromatography to obtain high-purity fusion protein polymers.

[0014] Preferably, in the step S3, the liposome preparation specifically comprises the following steps: The phospholipids and cholesterol are mixed in a mass ratio of 6-8:2-4 and dissolved in a chloroform-methanol mixed solvent; The dissolved phospholipid and cholesterol solution is treated using a thin film method to remove the solvent and form a thin film; The film is redissolved in water to form a liposome precursor solution; Ultrasonic treatment is used to make the liposome particles uniform and reach a particle size of 100-200 nanometers; The unencapsulated components were removed by filtration to obtain the final liposome solution.

[0015] Preferably, in the step S4, the preparation of the heat stabilizer specifically comprises the following steps: Mix trehalose, glycerol and sucrose in a mass ratio of 5-7:1-2:1-2; Dissolve the mixture in an appropriate amount of water, ensuring that all components are completely dissolved; Use high pressure homogenization to improve solution homogeneity; The resulting solution was sterilized by filtration to ensure that the solution was sterile and free of particulate matter.

[0016] Preferably, in step S6, the moisture content of the vaccine powder obtained after freeze-drying is controlled at 1.0%-3.0%, and the powder is packaged in a glass freeze-drying bottle and stored at -20°C.

[0017] The present invention provides a recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist. It has the following beneficial effects: 1. The present invention uses a fusion antigen constructed by optimizing the ratio of Pre-F and Pentamer, supplemented with a heat stabilizer, to maintain the integrity of the antigen conformation while effectively enhancing the humoral immunity and cellular immunity induction ability of the vaccine to the target antigen without introducing exogenous adjuvants. Compared with the case where no heat stabilizer is added or the antigen ratio is adjusted, the scheme of the present invention shows a synergistic optimization effect in terms of antigen presentation efficiency, immune response intensity and conformational stability, which helps to improve the consistency of the vaccine and the level of immune protection.

[0018] 2. The present invention uses a combination of TLR7 / 8 agonists and STING agonists, and cooperates with a structurally stable liposome system to achieve sequential synergistic activation of immune signaling pathways in vivo, significantly enhancing antigen-specific cellular immune responses. Compared with the comparative examples that use only a single agonist or lack structural assistance, this design not only improves the retention stability of the agonist in the local tissue, but also enhances the efficiency of antigen delivery, effectively stimulating the synergistic linkage of innate immunity and adaptive immunity.

[0019] 3. The present invention optimizes freeze-drying process parameters and introduces a multi-component thermal stabilization system, which significantly improves the long-term stability of the antigen conformation and the integrity of the liposome structure while achieving vaccine dry storage. Compared with the comparative examples that do not use stabilizers or have insufficient sublimation conditions, this solution can effectively prevent the structural collapse and loss of biological activity of vaccine components during storage and transportation, ensure the rapid recovery and uniformity of vaccine performance after reconstitution, and is suitable for storage requirements under various temperature environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a flow chart of the preparation steps of the present invention. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Please see attached Figure 1 , Example 1: In this embodiment, the vaccine composition is composed of the following components: 250 parts of fusion protein polymer, in which the mass ratio of Pre-F protein to Pentamer protein is 7:3; 20 parts of Resiquimod agonist; 10 parts of ADU-S100 agonist; 250 parts of liposomes, in which the mass ratio of phospholipids to cholesterol is 8:2; 100 parts of thermal stabilizer, in which the mass ratio of trehalose, glycerol and sucrose is 7:2:2.

[0023] Preparation steps: During the preparation of fusion protein polymers, expression vectors were constructed for RSV F protein Pre-F conformation and adenovirus Pentamer protein, respectively, and transfected into CHO cells. The culture medium was harvested after 96 hours of culture, and the cells were frozen and lysed. The protein solution was first enriched by Ni-NTA affinity chromatography for the target protein, and then DEAE weak anion exchange chromatography was used to remove impurity proteins. Finally, the volume was screened by Superdex-200 gel chromatography to obtain a high-purity polymer protein solution. After concentration and quantification, the products were mixed in a mass ratio of 7:3 to form 250 fusion protein polymers.

[0024] Resiquimod and ADU-S100 agonist were weighed 20 and 10 parts respectively and dissolved in sterile PBS with a target concentration of 1 mg / mL. The dissolution process used an ultrasonic water bath for 8 minutes to accelerate the dissolution rate. After treatment, they were filtered through a 0.22 μm polyethersulfone membrane to obtain clear sterile agonist solutions.

[0025] In the preparation of liposomes, 8 g of phosphatidylcholine and 2 g of cholesterol were added to 80 mL of chloroform-methanol (volume ratio 2:1) and fully dissolved, and evaporated to dryness in a rotary evaporator at 40°C to form a lipid film. 20 mL of PBS buffer was added to re-dissolve, and placed in an ultrasonic water bath for 12 minutes to control the particle size within 160 nm to form a liposome with stable structure, and 250 parts were quantitatively obtained.

[0026] When preparing the heat stabilizer, 7 g of trehalose, 2 g of glycerol, and 2 g of sucrose were weighed and added into 50 mL of water for injection. After magnetic stirring for 25 minutes, the mixture was treated three times with a high-pressure homogenizer (pressure was 1000 bar), and sterilized by filtration with a 0.22 μm membrane to obtain 100 portions of clear and uniform heat stabilizer.

[0027] After all components are cooled to 4°C, they are added to the mixing bottle in sequence under clean conditions. The mixing order is fusion protein polymer, liposome, agonist, and thermal stabilizer. The mixing temperature is controlled at 5°C, the stirring rate is 400rpm, and the time is 60 minutes to obtain a stable vaccine combination liquid. The liquid is put into a freeze-drying bottle, pre-frozen at -40°C for 3 hours, and then sublimated and dried in the temperature range of -15°C to 0°C for 36 hours after entering the freeze dryer to obtain a freeze-dried vaccine powder with a moisture content of 1.2%. It is packaged in a neutral glass bottle and stored for a long time at -20°C.

[0028] Embodiment 2: In this embodiment, the component ratios of the vaccine composition are as follows: 180 parts of fusion protein polymer, the mass ratio of Pre-F protein to Pentamer protein is 6:3; 15 parts of Resiquimod agonist; 8 parts of ADU-S100 agonist; 160 parts of liposomes, the mass ratio of phospholipids to cholesterol is 7:3; 85 parts of thermal stabilizer, the mass ratio of trehalose, glycerol and sucrose is 6:1.5:1.5.

[0029] Preparation steps: The production of fusion protein multimers uses HEK293 cell transient transfection expression system to express Pre-F and Pentamer proteins respectively, and after purification by Ni-NTA affinity chromatography, charged impurities are removed by DEAE chromatography, and then the high molecular weight polymer part is collected by Superdex200 gel chromatography. The two proteins are mixed at a mass ratio of 6:3 and the concentration is adjusted to obtain 180 copies of fusion protein multimers.

[0030] Resiquimod and ADU-S100 were weighed in 15 and 8 portions respectively, added into PBS buffer to prepare a 0.8 mg / mL solution, and treated in an ultrasonic water bath at 25°C for 7 minutes to ensure complete dissolution, then filtered and sterilized at 0.22 μm. The filtrate was clear and free of impurities and used as a vaccine immune activating factor.

[0031] In the preparation of liposomes, 7 g of phosphatidylcholine and 3 g of cholesterol were added to 60 mL of a chloroform-methanol mixed solvent, and a dry film was formed by rotary evaporation for 30 minutes. After redissolving in PBS, the film was treated with a probe type ultrasound for 10 minutes. The particle size was adjusted to 140 ± 10 nm, and 160 liposomes were obtained after filtration.

[0032] Weigh 6 g of trehalose, 1.5 g of glycerol and 1.5 g of sucrose in the heat stabilizer, add them into 40 mL of water for injection and stir for 20 minutes, homogenize twice under high pressure (800 bar), and then sterilize and filter to obtain 85 parts of heat stabilizer, which is clear and has no precipitation and is suitable for freeze-dried vaccine system.

[0033] The five components were pre-cooled at 4°C in turn, and each component was prepared separately before mixing, and then slowly added at a ratio of 180:15:8:160:85 at 6°C, with the stirring rate set at 450rpm, and continued to mix for 45 minutes to obtain a uniform vaccine suspension. It was divided into freeze-dried bottles, frozen at -40°C for 2 hours, and then transferred to a freeze dryer for sublimation drying for 30 hours. The freeze-dried powder had a moisture content of 1.6%, and was stored in a refrigerator with stable performance.

[0034] Embodiment 3: The vaccine composition contains 150 parts of fusion protein polymers, of which the mass ratio of Pre-F protein to Pentamer protein is 5:3; 10 parts of Resiquimod agonist; 5 parts of ADU-S100 agonist; 120 parts of liposomes, of which the mass ratio of phospholipids to cholesterol is 6:4; 70 parts of thermal stabilizer, and the mass ratio of trehalose, glycerol and sucrose is 5:1:1.

[0035] Preparation steps: The CHO cell expression system was used in the preparation of the fusion protein. After expression and culture, Ni-NTA purification was used, followed by DEAE column chromatography to remove negatively charged impurities, and finally Superdex-200 separation of polymers. 100 parts of Pre-F protein and 50 parts of Pentamer protein were taken and mixed in a ratio of 5:3 to obtain 150 parts of fusion protein stock solution.

[0036] Weigh Resiquimod (10 parts) and ADU-S100 (5 parts) and add them to PBS to prepare a 0.5 mg / mL working solution. Treat with an ultrasonic water bath for 5 minutes, sterilize and filter, and set aside for use as an immune activation factor.

[0037] In the preparation of liposomes, 6 g of phosphatidylcholine and 4 g of cholesterol were added to 50 mL of chloroform-methanol and mixed, evaporated at 40°C for 25 minutes to form a dry film, redissolved in PBS and subjected to water bath ultrasound for 8 minutes to form liposomes with a particle size of about 120 nm. After filtering through a microporous membrane, 120 portions of liposomes were obtained.

[0038] The heat stabilizer was prepared as 5 g of trehalose, 1 g of glycerol and 1 g of sucrose, added into 30 mL of water for injection and stirred for 15 minutes. After high pressure homogenization once, it was filtered through 0.22 μm to obtain 70 portions of clear liquid for vaccine freeze-drying.

[0039] All materials were precooled to 4°C and mixed, and added to the mixing bottle one by one in a ratio of 150:10:5:120:70. Stirred at 4°C for 30 minutes at a speed of 350rpm. After mixing evenly, the mixture was immediately packaged. The freeze-drying operation included freezing at -40°C for 2 hours and sublimation drying for 25 hours. The moisture content of the finished freeze-dried powder was about 2.8%. It was packaged and refrigerated to meet the requirements of storage and transportation and on-site reconstitution.

[0040] Comparative Example 1: This comparative example is based on Example 1, and the other parts remain unchanged, only the STING agonist ADU-S100 in the immune agonist part is replaced with Poly (I: C), the dosage is still 10 parts, and after being dissolved in PBS, ultrasonically treated for 5 minutes, sterilized and filtered, it is added to the vaccine combination liquid. The formula, preparation method and mixing process of the fusion protein polymer, liposome, and thermal stabilizer are the same as those in Example 1.

[0041] Comparative Example 2: This comparative example is based on Example 1, and other conditions remain unchanged, only the mass ratio of RSVPre-F to Pentamer in the fusion protein polymer is changed from 7:3 to 3:7, and the total is still 250 parts. That is, the Pre-F protein is 75 parts and the Pentamer is 175 parts. After mixing, the subsequent mixing and lyophilization steps are entered, and the agonist, liposome, and thermal stabilizer are kept unchanged in the amount and preparation method of Example 1.

[0042] Comparative Example 3: This comparative example is based on Example 2, except that cholesterol is removed from the liposome preparation, and only 7 g of phosphatidylcholine is used to prepare the liposome alone, and other process conditions remain unchanged, and the final liposome particle size is maintained at about 140 nm, and 160 liposomes are obtained. The remaining fusion protein, agonist, and thermal stabilizer composition and operation are the same as in Example 2.

[0043] Comparative Example 4: This comparative example is based on Example 2, only 15 parts of a single agonist Resiquimod is used, and no STING agonist ADU-S100 is added. The liposome still encapsulates the agonist solution, and the ratio, structure and mixing process are consistent with Example 2. The remaining fusion protein, liposome and thermal stabilizer ratio and preparation method are exactly the same.

[0044] Comparative Example 5: This comparative example is based on Example 3, except that the heat stabilizer is not added at all and the component is omitted in the mixing step. The rest of the components including the fusion protein, agonist, liposome component ratio, preparation steps and freeze-drying conditions are the same as those in Example 3.

[0045] Comparative Example 6: This comparative example is based on Example 3, and the freeze-drying stage is adjusted to shorten the sublimation time to 10 hours, and the drying is not sufficient. All component proportions, preparation processes and mixing parameters are kept consistent with Example 3, and only the freeze-drying parameters are changed, and the -40°C pre-freezing treatment is still used, and the sublimation temperature is set to 0°C.

[0046] Comparative experiment 1: Experimental description: This experiment aims to evaluate the effects of the ratio of fusion antigen Pre-F to Pentamer and the addition of thermal stabilizer on the immunogenicity of the vaccine. Using Example 1 as the standard group, two groups of comparative examples were set up: one group only changed the antigen ratio (Comparative Example 1-B), and the other group only removed the thermal stabilizer (Comparative Example 1-A) to achieve single variable control.

[0047] Experimental steps: Experimental animal groups: BALB / c mice aged 6-8 weeks were selected and randomly divided into 3 groups, with 6 mice in each group.

[0048] Vaccine formulation settings: Example 1: Pre-F: Pentamer = 7:3, containing a heat stabilizer (trehalose: sucrose: glycerol = 7:2:2), combined with liposomes and dual agonists to form a complete vaccine combination.

[0049] Comparative Example 1: Pre-F: Pentamer = 7:3, the heat stabilizer is removed, and the other components remain unchanged.

[0050] Comparative Example 2: Pre-F: Pentamer = 3:7, the heat stabilizer is consistent with other components.

[0051] Inoculation and sampling: Each mouse was subcutaneously injected with 50 µL of the vaccine in the groin for a single vaccination. On the 14th day, serum was collected for ELISA to detect the anti-Pre-F antibody titer, and on the 21st day, spleen cells were isolated and the IFN-γ production level was analyzed using ELISPOT; at the same time, the retention of the Pre-F protein conformation was evaluated by Western blot.

[0052] Data processing: All data are presented as mean values ​​and subjected to one-way analysis of variance (ANOVA). p<0.05 was considered significant. Example 1, Comparative Example 1 and Comparative Example 2 were grouped together, and each group was tested 3 times. The last two repeated experiments were distinguished by the suffixes "(Repeat 1)" and "(Repeat 2)" (see Table 1 for specific data).

[0053] Table 1: Group Pre-F Antibody Titer (OD value) Number of IFN-γ Spots Pre-F Conformational Integrity Example 1 1.25 72 Remain Intact Comparative Example 1 1.01 53 Significantly Damaged Comparative Example 2 0.89 50 Partially Inactivated Example 1 (Repeated 1) 1.32 77 Remain Intact Comparative Example 1 (Repeated 1) 0.95 49 Severely Lost Comparative Example 2 (Repeated 1) 0.91 47 Partially Inactivated Example 1 (Repeated 2) 1.28 75 Remain Intact Comparative Example 1 (Repeated 2) 0.99 55 Conformation Unstable Comparative Example 2 (Repeated 2) 0.87 52 Partially Inactivated Summarize: The experimental results showed that when Pre-F and Pentamer were co-expressed at a ratio of 7:3, they could effectively enhance the humoral and cellular immune responses induced by the vaccine. At this ratio, the Pre-F conformational stability was the strongest and highly matched with the antibody binding ability, suggesting that the correct antigen ratio helps to construct the virus-like particle structure, thereby enhancing immune recognition.

[0054] After removing the heat stabilizer, the antibody titer and IFN-γ level decreased significantly, and the Pre-F conformation showed a strong denaturation trend in the test. This shows that the heat stabilizer plays a key conformational protection role during the freeze-drying process, and its absence will weaken the antigen activity and affect the final immune effect.

[0055] After reducing the Pre-F ratio to 3:7, the overall immune response decreased, suggesting that the relative abundance of Pre-F plays a decisive role in antigen presentation and B cell recognition. In summary, the fusion ratio and stabilizer play a synergistic role in maintaining protein structure and inducing immunity, and neither factor can be dispensable.

[0056] Comparative experiment 2: Experimental description: This experiment evaluates the effects of agonist combinations and liposome structures on immune activation ability. C57BL / 6 mice, aged 8 weeks, were used and randomly divided into groups of 6 mice each. The three groups were vaccinated with the vaccines in Example 2, Comparative Example 2-A and Comparative Example 2-B, respectively. The injection method was a single lateral thigh muscle injection, with an injection dose of 50µL per mouse. The vaccines were all freeze-dried and reconstituted before use, and no exogenous adjuvant was added.

[0057] The experimental setup is as follows: The vaccine of Example 2 contains the dual agonists of Resiquimod (15 parts) and ADU-S100 (8 parts), and the liposomes contain phospholipids and cholesterol (7:3); The vaccine used in Comparative Example 3 removed the cholesterol in the liposomes, and the agonist combination remained unchanged; The vaccine of Comparative Example 4 uses only Resiquimod single agonist, and the liposomes are normal.

[0058] On the 7th day, the draining lymph nodes were taken from the foot pad on the injection side, and the total RNA was extracted. The expression levels of Ifnb and Cxcl10 were analyzed by qPCR to reflect the activation efficiency of the STING pathway. On the 14th day, splenocytes were taken and re-stimulated with recombinant Pre-F protein, and the positive rate of IFN-γ expression in CD8+T cells was detected by flow cytometry to evaluate the cytotoxic response. Within 24 hours after inoculation, serum and mouse lymph were collected, and the drug distribution of ADU-S100 and Resiquimod was detected by LC-MS / MS, and the delivery efficiency and tissue enrichment were analyzed. Example 2, Comparative Example 3 and Comparative Example 4 were a group, and each group was repeated twice. The last two experiments were distinguished by the suffix "(Repeat 1)" (see Table 2 for specific data).

[0059] Table 2: Group Ifnb Relative Expression CXCL10 Expression <![CDATA[CD8 + IFN-γ(%)]]> Agonist Serum Concentration (ng / mL) Example 2 9.2 6.8 4.7 12.4 Comparative Example 3 4.5 3.2 2.6 19.1 Comparative Example 4 5.1 2.9 1.8 8.7 Example 2 (Repeated 1) 8.4 7.0 4.2 11.8 Comparative Example 3 (Repeated 1) 3.9 2.5 2.2 17.4 Comparative Example 4 (Repeated 1) 5.4 2.8 2.0 9.2 Summarize: The data showed that the combination of TLR7 / 8 agonist Resiquimod and STING agonist ADU-S100 can achieve synergistic immune enhancement effects at the molecular and cellular levels. In the implementation group using dual agonists, the expression levels of immune signaling molecules such as IFNB and CXCL10 were significantly increased, and the IFN-γ positivity rate in CD8⁺T cells was also significantly improved. This shows that the combination not only enhances the conduction of innate immune signals, but also promotes effective adaptive immune activation.

[0060] In the comparative example, after removing the STING agonist or adjusting the liposome composition, the immune indicators all decreased to varying degrees. Among them, the liposome after removing cholesterol caused the agonist to release faster in serum, reduce the delivery stability, and ultimately weaken the immune activation efficiency. This shows that the structure of the delivery system plays a decisive role in controlling the distribution of the agonist and cellular uptake.

[0061] Overall, this experiment supports the effectiveness of the combined activation strategy of TLR and STING signaling axes, and emphasizes the role of delivery system design in achieving effective immune rhythms and targeted release. The synergistic application of immune agonists requires matching a stable carrier system to form an immune activation mechanism with biological sustainability and timeliness.

[0062] Comparative experiment 3: Experimental description: This experiment focuses on whether the freeze-drying process is sufficient and whether the heat stabilizer is added to evaluate the physical stability and antigen retention capacity of the vaccine at different storage temperatures and times. Example 3 was used as the control group, and Comparative Example 3-A (no heating stabilizer) and Comparative Example 3-B (inadequate freeze-drying sublimation time) were used as experimental comparison groups.

[0063] All three groups of vaccine samples were freeze-dried. After preparation, they were packaged and sealed and stored at -20°C, 4°C, and 25°C respectively. Each group was stored for 3 months, and samples were taken monthly for reconstitution and analysis. Three bottles of samples were tested in each group each time, and the average value was taken.

[0064] Before reconstitution, observe whether the appearance of the freeze-dried cake collapses and absorbs moisture. After reconstitution, record the clarification time and whether it is turbid. Then detect the content retention rate of the antigen Pre-F (ELISA method) and measure the change in liposome particle size (dynamic light scattering DLS). In addition, the degree of protein structure change is evaluated by the change in the ultraviolet absorption peak position. Example 3, Comparative Example 5 and Comparative Example 6 are a group, each group is tested 3 times, and the last two repeated experiments are distinguished by adding the suffixes "(Repeat 1)" and "(Repeat 2)" (see Table 3 for specific data).

[0065] Table 3: Group Storage Condition Reconstitution Time (s) Particle Size Change (nm) Pre-F Retention Rate (%) UV Peak Position Drift Example 3 -20℃ 18 +12 94.6 None Comparative Example 5 -20℃ 35 +45 72.8 Slight Offset Comparative Example 6 -20℃ 28 +33 80.1 Minor Offset Example 3 (Repeated 1) 4℃ 22 +18 91.3 None Comparative Example 5 (Repeated 1) 4℃ 43 +68 66.4 Significant Drift Comparative Example 6 (Repeated 1) 4℃ 35 +41 75.5 Offset Example 3 (Repeated 2) 25℃ 26 +25 88.7 Slight Offset Comparative Example 5 (Repeated 2) 25℃ 50 +80 58.2 Strong Drift Comparative Example 6 (Repeated 2) 25℃ 42 +56 67.5 Significant Drift Summarize: The results showed that the optimization of the freeze-drying process and the rational addition of thermal stabilizers can significantly improve the antigen stability and physical integrity of the vaccine under different storage conditions. In the implementation group, the conformation of the antigen Pre-F was well maintained, the particle size changed slightly, the reconstitution time was short, and there was no obvious drift in the protein UV absorption peak, indicating that its structural stability was high.

[0066] In the control group without adding thermal stabilizer, the protein retention rate decreased, the particle size increased significantly, and the absorption peak shifted, indicating that the protein conformation changed. At the same time, under the condition of insufficient freeze-drying sublimation, residual water may destroy the liposome membrane structure, thereby affecting the antigen encapsulation efficiency and resolubility performance.

[0067] The experimental results verify the protective effect of thermal stabilizers (such as trehalose, glycerol, and sucrose) on protein and liposome structures during vaccine drying and storage. The coordinated design of freeze-drying process parameters and stabilizers helps maintain the activity of vaccines during long-term storage, providing technical support for room temperature transportation and terminal applications.

[0068] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist, characterized in that: The following components are included in the following parts by weight: Fusion protein polymer 100-250 copies; TLR7 / 8 agonist 10-20 copies; STING agonist 5-10 copies; Liposomes 75-250 parts; Heat stabilizer 70-100 parts.

2. The recombinant RSV vaccine composition based on fusion protein polymer and TLR / STING dual agonist according to claim 1, characterized in that: The fusion protein polymer includes RSV F protein Pre-F conformation and adenovirus Pentamer protein, and the mass ratio of RSV F protein Pre-F conformation to adenovirus Pentamer protein is 5-7:3-5.

3. The recombinant RSV vaccine composition based on fusion protein polymer and TLR / STING dual agonist according to claim 1, characterized in that: The liposome comprises phospholipid and cholesterol, and the mass ratio of the phospholipid to cholesterol is 6-8:2-4.

4. The recombinant RSV vaccine composition based on fusion protein polymer and TLR / STING dual agonist according to claim 1, characterized in that: The specific model of the TLR7 / 8 agonist is Resiquimod, and the specific model of the STING agonist is ADU-S100.

5. The recombinant RSV vaccine composition based on fusion protein polymer and TLR / STING dual agonist according to claim 1, characterized in that: The thermal stabilizer includes trehalose, glycerol and sucrose, and the mass ratio of the trehalose, glycerol and sucrose is 5-7:1-2:1-2.

6. A method for preparing a recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist, according to the recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, recombinantly expressing RSV F protein Pre-F conformation and adenovirus Pentamer protein to obtain fusion protein polymers; S2, dissolving the TLR7 / 8 agonist Resiquimod and the STING agonist ADU-S100 in a sterile phosphate buffer solution with the aid of an ultrasonic water bath for 5-10 minutes, and filtering and sterilizing to obtain an agonist solution; S3, mixing phospholipids and cholesterol according to the proportion and preparing liposomes using a thin film method; S4, mixing trehalose, glycerol and sucrose in proportion to prepare a heat stabilizer solution; S5. Precool the fusion protein polymer, TLR7 / 8 agonist, STING agonist, liposome and thermal stabilizer to 4° C., and stir them at a temperature of 2-8° C. according to a mass ratio, and stir for 20-60 minutes to obtain a recombinant RSV vaccine composition; S6. Freeze-drying the obtained composition to obtain the final vaccine preparation.

7. The method for preparing a recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist according to claim 6, characterized in that: In the S1 step, the recombinant expression comprises the following steps: RSV F protein Pre-F conformation and adenovirus Pentamer protein were recombinantly expressed in CHO cells; Cultivate in an expression system and obtain a recombinant protein solution by cell lysis; Purify the target protein by affinity chromatography using Ni-NTA resin; Impurities are removed by ion exchange chromatography and gel permeation chromatography to obtain high-purity fusion protein polymers.

8. The method for preparing a recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist according to claim 6, characterized in that: In the step S3, the liposome preparation specifically comprises the following steps: The phospholipids and cholesterol are mixed in a mass ratio of 6-8:2-4 and dissolved in a chloroform-methanol mixed solvent; The dissolved phospholipid and cholesterol solution is treated using a thin film method to remove the solvent and form a thin film; The film is redissolved in water to form a liposome precursor solution; Ultrasonic treatment is used to make the liposome particles uniform and reach a particle size of 100-200 nanometers; The unencapsulated components were removed by filtration to obtain the final liposome solution.

9. The method for preparing a recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist according to claim 6, characterized in that: In the step S4, the preparation of the thermal stabilizer specifically comprises the following steps: Mix trehalose, glycerol and sucrose in a mass ratio of 5-7:1-2:1-2; Dissolve the mixture in an appropriate amount of water, ensuring that all components are completely dissolved; Use high pressure homogenization to improve solution homogeneity; The resulting solution was sterilized by filtration to ensure that the solution was sterile and free of particulate matter.

10. The method for preparing a recombinant RSV vaccine composition based on a fusion protein polymer and a TLR / STING dual agonist according to claim 6, characterized in that: In the step S6, the moisture content of the vaccine powder obtained after freeze-drying is controlled at 1.0%-3.0%, and the powder is packaged in a glass freeze-drying bottle and stored at -20°C.

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