HMPV vaccine composition

Abstract

The invention relates to the technical field of biological medicines, and discloses an HMPV vaccine composition which comprises the following component materials: HMPV F protein of prefusion conformation, lipid nanoparticles, an immunologic adjuvant, a stabilizer and a buffer solution, the lipid nanoparticles comprise cholesterol, phospholipid and PEG (Polyethylene Glycol) lipid, the immunologic adjuvant is prepared from CpG oligonucleotide and monophosphoryl lipid A, the stabilizer comprises cane sugar, mannitol and polysorbate 80; the buffer solution comprises sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection. By optimizing the conformation of the F protein and selecting a proper delivery carrier, the antigen can better expose the immune epitope, so that the immunogenicity of the vaccine is enhanced, and particularly, under the use of the conformation F protein before fusion, the vaccine shows remarkable antibody titer and T cell activation capability which are obviously superior to those of the conformation F protein after fusion.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to an HMPV vaccine composition. Background Art

[0002] With the continuous advancement of vaccine research and development technology, vaccine preparation methods have gradually tended to be refined and diversified. Modern vaccine delivery systems generally use nanoparticle carriers to improve antigen delivery efficiency and immunogenicity. Lipid nanoparticles, as a more commonly used delivery carrier, have been widely used in vaccine development due to their excellent biocompatibility and strong antigen delivery ability. In addition, antigen design and the use of adjuvants in vaccines also play a vital role. The selection of antigens, conformational design, and the combination of adjuvants can greatly affect the immune response and immune effect of the vaccine. Especially for protein vaccines, how to ensure that the immune epitopes of the antigen are fully exposed and thus enhance immunogenicity has become one of the key issues in vaccine development.

[0003] However, although the existing technology has achieved certain results in vaccine research and development, there are still obvious deficiencies in some aspects. Although lipid nanoparticles have played a role in improving the effect of antigen delivery as a delivery system, their stability problem has not been effectively solved. Particles that have not been surface-modified are prone to aggregation in the body, resulting in a decrease in delivery efficiency and inducing unnecessary immune responses. In addition, conventional antigen designs often ignore the degree of exposure of antigen epitopes, resulting in limited recognition of the immune system and reduced immunogenicity. In terms of adjuvants, although the existing CpG and MPLA adjuvants can enhance the immune response, it is often difficult to accurately guide the bias of the immune response when used alone, and cannot meet the full range of immunization needs against specific pathogens. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an HMPV vaccine composition, which solves the problems of poor stability of lipid nanoparticles, unoptimized antigen conformation design and poor adjuvant matching effect in existing vaccine delivery systems.

[0005] To achieve the above objectives, the present invention is implemented by the following technical solutions: an HMPV vaccine composition, comprising the following component materials in parts by weight: 1.0-5.0 copies of HMPV F protein in the prefusion conformation; Lipid nanoparticles 1.0-10.0 parts; Immunoadjuvant 0.1-1.0 part; Stabilizer 2.0-10.0 parts; Buffer 50-100.0 parts.

[0006] Furthermore, the HMPV F protein in the pre-fusion conformation is the main antigen component of the vaccine. It is in the pre-fusion state before the virus infects cells, has antigenic epitopes in the natural conformation, and can induce the production of neutralizing antibodies; the pre-fusion conformation of the F protein exposes more neutralizing epitopes than the post-fusion conformation, which can effectively simulate the surface structure of the natural virus, thereby enhancing the immune response induced by the vaccine; by stabilizing its trimer structure, its conformation can be kept stable to prevent it from transforming into a post-fusion conformation with lower immunological activity; As a delivery system, lipid nanoparticles simulate the cell membrane environment and complex with F protein through electrostatic or hydrophobic interactions to effectively encapsulate antigen proteins and improve their stability and cellular uptake efficiency. PEG modification can increase the circulation time of particles in the body and reduce immune clearance. The lipid structure can also promote transmembrane delivery of antigens to antigen-presenting cells and enhance T cell activation. The immune adjuvant is a combination of CpG oligonucleotide and MPLA. CpG is a TLR9 agonist that can induce Th1 immune bias; MPLA is a TLR4 agonist that has both safety and strong immune enhancement capabilities. The two synergistically stimulate dendritic cell maturation and antigen presentation, significantly improving the quality and breadth of immune responses, and effectively stimulating cellular and humoral immunity. Stabilizers include sucrose, mannitol and polysorbate 80. Sucrose and mannitol can be used as osmotic pressure regulators and protein protectors to maintain the structural stability of the vaccine during low-temperature storage and freeze-drying. Polysorbate 80 can reduce protein adsorption and interface denaturation, and enhance vaccine uniformity and injectability. The buffer solution is composed of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection; this PBS-type buffer system has good physiological compatibility, can maintain protein conformation and nanoparticle stability, and prevent component aggregation or degradation; a reasonable pH value is also conducive to maintaining adjuvant activity and the overall stability of the vaccine.

[0007] Preferably, the lipid nanoparticles include cholesterol, phospholipids and PEGylated lipids, and the mass ratio of cholesterol, phospholipids and PEGylated lipids is 2-4:4-6:0.5-1.5.

[0008] Preferably, the immune adjuvant comprises CpG oligonucleotide and monophosphoryl lipid A, and the mass ratio of the CpG oligonucleotide to the monophosphoryl lipid A is 1-5:1-5.

[0009] Preferably, the stabilizer includes sucrose, mannitol and polysorbate 80, and the mass ratio of sucrose, mannitol and polysorbate 80 is 4-7:2-4:0.5-1.5.

[0010] Preferably, the buffer comprises sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection, and the mass ratio of the sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection is 0.5-1.5:1-2:7-10:85-95.

[0011] A method for preparing an HMPV vaccine composition comprises the following steps: S1. Expression and purification of HMPV F protein in the prefusion conformation; S2, preparing lipid nanoparticles; S3, forming a protein-lipid complex by magnetically stirring the HMPV F protein in the pre-fusion conformation with the lipid nanoparticles in a buffer for 30-60 minutes; S4. When the temperature of the complex is 4-8°C, add the immune adjuvant and the stabilizer in sequence, stir for 30-40 minutes, mix well to obtain the final mixture and adjust the volume to the target concentration; S5. The final mixture is sterilized by filtration at 0.22-0.25 μm, then divided and stored in sterile bottles at 2-8°C.

[0012] Furthermore, in the S1 step, the mammalian system ensures that the protein glycosylation and conformation are correct, and the cross-linking stabilization treatment prevents the protein from loosening its structure or aggregating during preparation and storage, thereby improving immunogenicity and long-term stability; The S2 step ensures that lipids are uniformly film-formed and assembled into stable nanostructures; high-pressure homogenization can effectively control particle size and uniformity, ensuring that the vaccine has consistent delivery performance and immune response after injection; In step S3, the low temperature environment helps to maintain the stability of the protein conformation; slow stirring can increase the interaction time between lipids and proteins, which is conducive to the formation of efficient complexes and enhances the delivery effect; In step S4, slow dripping is performed to prevent precipitation caused by excessive local concentration; adding the adjuvant first helps it to be evenly adsorbed on the surface of the complex and enhance the immunostimulatory effect; adding the stabilizer at the end can protect the structure of the final mixture and make it suitable for subsequent filling and storage; In the S5 step, microporous filtration ensures the sterility of the vaccine and meets clinical injection requirements; low-temperature storage can effectively extend the shelf life of the vaccine and maintain the stability of the adjuvant and antigen structure.

[0013] Preferably, in step S1, the HMPV F protein in the pre-fusion conformation is obtained by transfecting a mammalian cell expression system, and the method of affinity chromatography combined with molecular sieve chromatography is used in the purification process. The obtained F protein is stabilized in its trimer conformation by a cross-linking agent, and the protein concentration is adjusted to 1.0-5.0 mg / mL.

[0014] Preferably, in step S2, lipid nanoparticles are prepared from cholesterol, phospholipids and PEGylated lipids by a thin film hydration method, which specifically includes: Each lipid is dissolved in an organic solvent and rotary evaporated at 40-50°C to form a lipid film; Add PBS buffer to hydrate for 30-60 minutes, then ultrasonicate for 10-15 minutes and high-pressure homogenize at a pressure of 800-1200 bar for 3-5 times; Finally, uniform lipid nanoparticles with a particle size of 50-150nm and a PDI of less than 0.2 were obtained.

[0015] Preferably, in step S4, the immune adjuvant is a combination of CpG oligonucleotide and monophosphoryl lipid A, which is dissolved in a buffer solution in proportion and then added dropwise and stirred for 15-30 minutes.

[0016] Preferably, in step S4, the buffer solution is prepared by mixing sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection in a certain proportion, and the pH value is adjusted to 7.2-7.6.

[0017] The present invention provides an HMPV vaccine composition having the following beneficial effects: 1. The present invention optimizes the conformation of the F protein and selects a suitable delivery carrier, so that the antigen can better expose its immune epitope, thereby enhancing the immunogenicity of the vaccine. Compared with the traditional immunization strategy, the vaccine after the low-temperature composite treatment of the F protein combined with lipid nanoparticles can effectively induce higher levels of neutralizing antibodies and F protein-specific IgG. In particular, under the use of the pre-fusion conformation F protein, the vaccine showed significant antibody titers and T cell activation ability, which was significantly better than the post-fusion conformation F protein.

[0018] 2. The present invention significantly improves the physical stability of the vaccine by using PEG-modified lipid nanoparticles, avoiding the aggregation and precipitation of traditional nanoparticles in the body. PEGylated lipid particles not only improve the dispersibility of the vaccine in the body, but also prolong the duration of its immune effect. Experiments have shown that the combination of PEG-modified particles and dual adjuvants not only ensures stability, but also effectively enhances the immune response of the vaccine, especially in terms of activation of memory T cells and establishment of immune bias.

[0019] 3. The vaccine preparation of the present invention effectively reduces protein aggregation and adverse reactions through the low-temperature slow compounding process, ensuring better biocompatibility. In the immune experiment, the stabilizer system using surfactants effectively reduces the redness, swelling and induration reactions of the vaccine preparation at the injection site, reducing the local reactions caused by traditional vaccine formulations. These improvements enhance the safety of the vaccine and provide guarantees for clinical application.

[0020] 4. The present invention optimizes the bias of immune response while enhancing immune response by rationally matching dual adjuvants. CpG adjuvant effectively promotes the establishment of Th1 immune response, enhances cell-mediated immune response, and increases the proportion of memory T cells. Experimental data show that the use of this combination of vaccines can not only improve the IgG1 / IgG2a ratio, but also significantly increase the activation level of specific T cells, enhance immune memory, and provide long-term immune protection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a flow chart of the preparation steps of the present invention. DETAILED DESCRIPTION

[0022] 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.

[0023] Please see attached Figure 1 , Example 1: Vaccine group allocation ratio: HMPV F protein in prefusion conformation: 5.0 copies; Lipid nanoparticles: 10.0 parts; Immunoadjuvant: 1.0 part; Stabilizer: 10.0 parts; Buffer: 100.0 parts; Lipid nanoparticle composition: Cholesterol:phospholipid:PEGylated lipid is 3.5:5:1.5; Immunoadjuvant composition: CpG oligonucleotide: monophosphoryl lipid A 1:1; Stabilizer composition: Sucrose: mannitol: polysorbate is 6:3:1; Buffer composition: Sodium dihydrogen phosphate: disodium hydrogen phosphate: sodium chloride: water for injection = 1:1.5:9:88.5; Preparation steps: HEK293F cells were cultured in suspension and transfected with the F protein gene, and the culture supernatant was harvested after 72 hours. Ni-NTA affinity chromatography combined with Superdex200 molecular sieve purification was used to collect the trimer structure, which was cross-linked with glutaraldehyde and the final concentration was adjusted to 5.0 mg / mL.

[0024] Cholesterol, phospholipids and DSPE-PEG2000 were dissolved in chloroform at a mass ratio of 3.5:5.0:1.5, and rotary evaporated at 45°C to form a uniform lipid film. PBS (pH 7.4) was added for hydration for 50 minutes, and ultrasonicated in an ice bath for 10 minutes. The mixture was treated with a 1200 bar high-pressure homogenizer for 5 times to obtain an average particle size of about 90 nm and a PDI of 0.18.

[0025] The F protein solution was slowly added dropwise to the lipid nanoparticles and magnetic stirring was maintained at 4°C for 50 min to form a uniform complex.

[0026] CpG and MPLA were dissolved separately and mixed, and then added dropwise to the composite system and stirred for 20 minutes. Sucrose, mannitol, and polysorbate 80 were then added in sequence and stirred for another 30 minutes. The buffer was added to make up to 100 parts of volume.

[0027] The composite mixture was sterilized by filtration through a 0.22 μm PES membrane, dispensed into sterile glass bottles, and stored at 2-8°C in the dark.

[0028] Embodiment 2: Vaccine group allocation ratio: HMPV F protein in prefusion conformation: 3.0 copies; Lipid nanoparticles: 5.0 parts; Immune adjuvant: 0.5 parts; Stabilizer: 6.0 parts; Buffer: 75.0 parts; Lipid nanoparticle composition: Cholesterol:phospholipid:PEGylated lipid is 2.5:5:1; Immunoadjuvant composition: CpG oligonucleotide:monophosphoryl lipid A:3:2; Stabilizer composition: Sucrose: mannitol: polysorbate is 5:1.5:0.5; Buffer composition: Sodium dihydrogen phosphate: disodium hydrogen phosphate: sodium chloride: water for injection is 1:1.2:8.5:89.3; Preparation steps: A stable expression system was constructed using CHO-K1 cells, and the cells were amplified and cultured in a glutamine-containing medium. After 72 hours of expression, the supernatant was collected by centrifugation. The protein was purified by a Strep-tag affinity column and then chromatographed on Superdex200 and concentrated to 3.0 mg / mL. The conformation was stabilized using a BS3 crosslinker.

[0029] The lipids were mixed in a ratio of 2.5:5:1 and dissolved in ethanol. The lipid film was formed by rotary evaporation at 45°C for 20 minutes and hydrated with PBS buffer for 40 minutes. After ice-water bath ultrasound for 12 minutes, high-pressure homogenization was performed at 1000 bar for 4 times to obtain lipid particles with an average particle size of 110 nm and a PDI of 0.16.

[0030] The protein solution and lipid particles were mixed in a pH 7.3 buffer environment at a volume ratio of 1:1 and stirred at 4°C for 45 minutes.

[0031] The CpG / MPLA mixture was slowly added dropwise to the system and stirred for 15 minutes; the stabilizer combination was added and stirred for another 20 minutes; and finally PBS was added to make the volume 75 parts.

[0032] Sterilize using a 0.25 μm filter, fill into a 5 mL sterile injection bottle, and store at 2-8°C without freezing.

[0033] Embodiment 3: Vaccine group allocation ratio: HMPV F protein in prefusion conformation: 1.0 Lipid nanoparticles: 1.0 part Immune adjuvant: 0.1 Stabilizer: 2.0 parts Buffer: 50.0 parts Lipid nanoparticle composition: Cholesterol:phospholipid:PEGylated lipid is 2:4:0.5; Immunoadjuvant composition: CpG oligonucleotide:monophosphoryl lipid A:7:3; Stabilizer composition: Sucrose: mannitol: polysorbate is 6:2.5:1.5; Buffer composition: Sodium dihydrogen phosphate: disodium hydrogen phosphate: sodium chloride: water for injection is 1:1:7.5:90.5; Preparation steps: In a small-scale expression system, the F protein was expressed in suspension using FreeStyle 293 cells, cultured for 48 hours, initially purified using Protein A affinity chromatography, and concentrated to 1.0 mg / mL after treatment with Superose 6 molecular sieves. The conformation was not treated with a crosslinker.

[0034] The lipid preparation steps were simplified, using a thin film hydration + short-time homogenization scheme. The lipid mixture was dissolved in ethanol, dried at 40°C for 10 minutes, then hydrated with PBS for 30 minutes, ultrasonically treated for 5 minutes, and homogenized only 3 times with 800 bar pressure. The particle size was controlled at 120 nm, and the PDI was 0.19.

[0035] Rapid mixing method: Inject F protein directly into the lipid suspension and rotate and mix at room temperature for 30 minutes to form a crude complex.

[0036] CpG and MPLA were pre-dissolved in proportion and added to the mixture, and gently stirred for 10 minutes. Then, the three components of the stabilizer were added to make up the buffer to a total volume of 50 parts.

[0037] The composite liquid was quickly sterilized through a 0.22 μm filter membrane, dispensed into 1 mL sterile brown vials, and stored in conventional refrigeration.

[0038] Comparative Example 1: This comparative example is based on Example 1, with only the source of the antigen being adjusted. The F protein is replaced by a monomeric expression product of the post-fusion conformation, the expression system and purification process remain the same, and no trimer stabilization or cross-linking treatment is performed. After the protein concentration is adjusted to 5.0 mg / mL, it is compounded with lipid nanoparticles, and the subsequent steps are consistent with Example 1, including the lipid component ratio, adjuvant addition, stabilizer and buffer ratio.

[0039] Comparative Example 2: In this comparative example, based on Example 1, the lipid nanoparticles are replaced with a traditional cationic water-in-oil adjuvant emulsion (such as a DDA / aluminum adjuvant system). DDA lipid (cationic lipid) and MCT oil are emulsified to form an water-in-oil system, and the antigen is directly dispersed therein. CpG / MPLA are also added, and the stabilizer ratio is consistent with the example. The overall lipid complex strategy is not adopted, and the F protein is not encapsulated with the particle structure, but only physically suspended.

[0040] Comparative Example 3: In this comparative example, the composition of lipid nanoparticles is composed of cholesterol and phospholipids, and does not contain DSPE-PEG2000 or other PEG-modified lipids. The lipid mixing ratio is adjusted to cholesterol: phospholipid = 3:6, and the preparation method (thin film hydration + high pressure homogenization) remains consistent. Adjuvants and stabilizers continue to be added after protein compounding, but the lack of PEG makes the particles easy to aggregate and the circulation stability decreases. The remaining process parameters are consistent with Example 2.

[0041] Comparative Example 4: In this comparative example, all the same processes and formulations as in Example 2 were used, except that the immune adjuvant part was simplified from a combination of CpG and MPLA to a single MPLA (0.5 parts). MPLA was dissolved in a buffer and added to the protein-lipid complex, and the subsequent stabilizer and buffer addition steps remained unchanged. CpG was not used to simulate the effect comparison under the traditional adjuvant mode.

[0042] Comparative Example 5: This comparative example uses Example 3 as a reference, and only adjusts the stabilizer system: sucrose and mannitol are retained, and polysorbate 80 is not added. The stabilizer ratio is 1.5 parts of sucrose and 0.5 parts of mannitol. The rest of the process is consistent with Example 3, including the F protein expression method, lipid ratio, adjuvant combination and mixing method, etc.

[0043] Comparative Example 6: In this comparative example, the F protein and lipid particles are not slowly compounded at low temperature, but simply physically mixed, that is, the protein and lipid nanoparticles are directly suspended, without magnetic stirring or dropwise addition, and without temperature control. The addition method of adjuvant and stabilizer remains consistent. This method ignores the interfacial affinity between protein and particles and belongs to the conventional "blending mode".

[0044] Comparative experiment: Experiment 1: Purpose: The differences in the effects of the pre-fusion conformation trimeric F protein (Example 1) and the post-fusion conformation F protein (Comparative Example 1) in inducing neutralizing antibodies and T cell immunity were evaluated, and the effects of the particle delivery system and the traditional water-in-oil emulsion system (Comparative Example 2) in vaccine immune enhancement were compared.

[0045] Animal models and groups: BALB / c mice aged 6-8 weeks were selected, half male and half female, and divided into 3 groups, with 10 mice in each group.

[0046] Group G1: vaccine of Example 1 (prefusion F protein + lipid nanoparticles + CpG / MPLA); Group G2: Comparative Example 1 vaccine (post-fusion F protein + lipid nanoparticles + CpG / MPLA); Group G3: Comparative Example 2 vaccine (pre-fusion F protein + oil-in-water adjuvant + CpG / MPLA).

[0047] Immunization schedule and vaccination method: The inoculation dose is 100 μL / vial, injected intraperitoneally. The primary immunization is set on day 0, and the booster immunization is set on day 21.

[0048] Sampling and testing time points: Blood was collected from the tail vein on days 14, 28, and 42 to collect serum. Some mice were sacrificed on day 28 to isolate spleen cells for ELISPOT and flow cytometry analysis.

[0049] Detection method: Neutralizing antibody titer: Pseudovirus neutralization method, IC50 value determination; F protein-specific IgG titer: ELISA method, OD450 quantification; ELISPOT assay: IFN-γ spot-forming unit (SFU) counts after stimulation; Flow cytometry: the percentage of CD4+CD44hiCD62Llo and CD8+CD44hi memory T cells in spleen cells (see Table 1 for specific values).

[0050] Table 1 Group Neutralizing antibody titer (IC50) IgG antibody OD450 <![CDATA[IFN-γSFU / 10 6 Splenocytes]]> <![CDATA[CD4 + Memory T cells (%)]]> <![CDATA[CD8 + Memory T cells (%)]]> G1 1426 1.78 392 18.3 12.5 G2 317 0.86 123 9.1 5.7 G3 688 1.10 189 11.6 7.8 Summarize: In this experiment, the vaccine after the pre-fusion conformation of F protein combined with lipid nanoparticles showed significant immune effects. This result shows that the F protein can more effectively expose its antigenic epitopes, enhance antibody production and T cell activation without undergoing conformational changes. In contrast, the F protein in the post-fusion conformation showed lower immunogenicity, which may be due to the shielding of its antigenic epitopes or conformational changes, resulting in a decrease in the immune system's recognition ability. In addition, the use of lipid nanoparticles significantly improved the delivery efficiency of antigens and enhanced the immune response, especially in the activation of memory T cells. By combining with CpG / MPLA adjuvants, the immune response was effectively promoted, verifying the role of the dual adjuvant system in improving the immune response.

[0051] Experiment 2: Purpose: The purpose of this experiment is to evaluate the effects of PEG-modified lipid nanoparticles on vaccine delivery efficiency, immune bias and in vivo stability, and to compare the types of immune responses stimulated by dual adjuvants and single adjuvants to reveal the actual effect of the synergistic effect of particle structure and adjuvant in immunization strategies.

[0052] Animal models and groups: C57BL / 6 mice were selected, 8 mice in each group, and divided into 3 groups: Group G4: formulation of Example 2 (PEG-modified lipid particles + CpG / MPLA); Group G5: Comparative Example 3 formulation (no PEG-modified particles + CpG / MPLA); Group G6: Comparative Example 4 formulation (PEG-modified particles + MPLA single adjuvant).

[0053] Dosage regimen: Subcutaneous injection, 50 μL / side, once on both sides of the back, primary immunization on day 0, booster immunization on day 21. The dosage should be consistent.

[0054] Sampling and analysis plan: On the 28th day, all animals were sacrificed, and spleen, lymph nodes, and tissues at the injection site were obtained and tested item by item.

[0055] Testing items and methods: Particle size and Zeta potential: DLS analysis was used before and after inoculation to observe the effect of PEG modification on particle stability; IgG1 / IgG2a subtype ratio: Th1 / Th2 immune bias was assessed using typing ELISA; In vivo distribution tracking: using DiR fluorescently labeled particles and real-time monitoring by small animal imaging system; Cytokine expression: RNA was extracted from spleen cells, and the transcription levels of IL-12p40 and TNF-α were detected by qPCR (see Table 2 for specific values).

[0056] Table 2 Group Particle size change (nm) Zeta potential (mV) IgG1 / IgG2a ratio IL-12p40 expression (fold) DiR fluorescence intensity (ROI) G4 106→114 -13.2 0.78 4.6 21,385 G5 104→153 -6.7 1.61 2.2 11,904 G6 110→118 -12.8 1.04 1.7 20,127 Summarize: PEG-modified lipid nanoparticles significantly improve the stability and delivery effect of vaccines. Through PEG modification, the particles maintain better dispersibility in the body and avoid precipitation caused by aggregation of unmodified particles in the blood. Experimental results show that PEG modification not only improves the stability of the particles, but also enhances the biocompatibility of the vaccine and reduces adverse reactions in the immune response. The choice of adjuvant has a decisive influence on the establishment of immune bias. The use of CpG adjuvant promotes the formation of Th1-type immune response, while MPLA fails to significantly favor a certain type of immune response when used alone. Therefore, the combination of PEG-modified lipid particles and CpG / MPLA exhibits a more excellent immune effect, providing an effective reference for vaccine development.

[0057] Experiment 3: Purpose: This experiment is used to explore whether the F protein and lipid nanoparticles are subjected to slow low-temperature complexing treatment and whether the stabilizer system contains a surfactant (polysorbate 80), and their actual effects on the physical stability, storage performance and immunogenicity of the finished vaccine product.

[0058] Animal models and groups: Adult New Zealand white rabbits (weight 2.0-2.5 kg) were used, 6 rabbits per group, divided into 3 groups: Group G7: Example 3 (complete stabilizer + low temperature compound); Group G8: Comparative Example 5 (removal of polysorbate 80); Group G9: Comparative Example 6 (direct blending without compounding).

[0059] Inoculation and sampling setup: 100 μL of each drug was injected intramuscularly into the anterior thigh on each side, as a single injection, on day 0. The observation period was 4 weeks.

[0060] Observe injection site reactions weekly and assess changes in redness, swelling, and nodules; On the 28th day, blood was drawn for IgG testing; After preparation, the vaccine was stored at room temperature for 1 week, 2 weeks, and 4 weeks, and the particle size, clarity, and precipitation state were tested.

[0061] Evaluation items and methods: Particle size change: DLS dynamic detection; Solution appearance: Visually record clarity, flocculation, and precipitation; Redness and swelling reaction: Nodule area (mm 2 ) Record + duration days; IgG titer: ELISA method, detecting the level of F protein-specific antibodies (see Table 3 for specific values).

[0062] Table 3 Group Particle size change on day 14 (nm) Appearance Status (Day 28) Duration of induration (days) <![CDATA[Maximum induration area (mm 2 )]]> IgG OD450 (D28) G7 112→119 Clear microemulsion 2.5 12 1.61 G8 110→147 Weak turbidity + visible flocculation 6 28 0.94 G9 109→138 Clear + partial precipitation 4 19 1.07 Summarize: The choice of compounding process and stabilizer has an important influence on the quality and immune effect of the vaccine. In this experiment, the use of low-temperature compounding and surfactant (polysorbate 80) significantly improved the physical stability and immunogenicity of the vaccine. Low-temperature slow-drop compounding not only ensures the close binding between lipid particles and F protein, but also effectively maintains the stability of the particles and avoids the aggregation of macromolecules. As a stabilizer, polysorbate 80 provides surface activity, reduces particle aggregation and maintains the uniformity of the system, significantly improving the storage stability of the vaccine. In contrast, the stability of the vaccine is significantly reduced by removing polysorbate 80 or using other compounding methods, which is manifested as particle aggregation and precipitation. The results of the immune response also show that stable vaccine preparations help to better activate specific antibodies and memory T cells, suggesting the promoting effect of stabilizers and compounding processes on immune effects.

[0063] 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 HMPV vaccine composition, characterized in that: The following components are included in the following parts by weight: 1.0-5.0 copies of HMPV F protein in the prefusion conformation; Lipid nanoparticles 1.0-10.0 parts; Immunoadjuvant 0.1-1.0 part; Stabilizer 2.0-10.0 parts; Buffer 50-100.0 parts.

2. A HMPV vaccine composition according to claim 1, characterized in that: The lipid nanoparticles include cholesterol, phospholipids and PEGylated lipids, and the mass ratio of the cholesterol, phospholipids and PEGylated lipids is 2-4:4-6:0.5-1.

5.

3. The HMPV vaccine composition according to claim 1, characterized in that: The immune adjuvant comprises CpG oligonucleotide and monophosphoryl lipid A, and the mass ratio of the CpG oligonucleotide to the monophosphoryl lipid A is 1-5:1-5.

4. The HMPV vaccine composition according to claim 1, characterized in that: The stabilizer includes sucrose, mannitol and polysorbate 80, and the mass ratio of sucrose, mannitol and polysorbate 80 is 4-7:2-4:0.5-1.

5.

5. The HMPV vaccine composition according to claim 1, characterized in that: The buffer comprises sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection, wherein the mass ratio of the sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection is 0.5-1.5:1-2:7-10:85-95.

6. A method for preparing a HMPV vaccine composition, according to the HMPV vaccine composition of claims 1-5, characterized in that: The following steps are involved: S1. Expression and purification of HMPV F protein in the prefusion conformation; S2, preparing lipid nanoparticles; S3, forming a protein-lipid complex by magnetically stirring the HMPV F protein in the pre-fusion conformation with the lipid nanoparticles in a buffer for 30-60 minutes; S4. When the temperature of the complex is 4-8°C, add the immune adjuvant and the stabilizer in sequence, stir for 30-40 minutes, mix well to obtain the final mixture and adjust the volume to the target concentration; S5. The final mixture is sterilized by filtration at 0.22-0.25 μm, then divided and stored in sterile bottles at 2-8°C.

7. The method for preparing a HMPV vaccine composition according to claim 6, characterized in that: In step S1, the HMPV F protein in the pre-fusion conformation is obtained by transfecting a mammalian cell expression system, and the method of affinity chromatography combined with molecular sieve chromatography is used in the purification process. The obtained F protein is stabilized in its trimer conformation by a cross-linking agent, and the protein concentration is adjusted to 1.0-5.0 mg / mL.

8. The method for preparing a HMPV vaccine composition according to claim 6, characterized in that: In step S2, lipid nanoparticles are prepared from cholesterol, phospholipids and PEGylated lipids by thin film hydration method, which specifically includes: Each lipid is dissolved in an organic solvent and rotary evaporated at 40-50°C to form a lipid film; Add PBS buffer to hydrate for 30-60 minutes, then ultrasonicate for 10-15 minutes and high-pressure homogenize at a pressure of 800-1200 bar for 3-5 times; Finally, uniform lipid nanoparticles with a particle size of 50-150nm and a PDI of less than 0.2 were obtained.

9. The method for preparing a HMPV vaccine composition according to claim 6, characterized in that: In step S4, the immune adjuvant is a combination of CpG oligonucleotide and monophosphoryl lipid A, which is dissolved in a buffer according to a certain proportion and then added dropwise and stirred for 15-30 minutes.

10. The method for preparing a HMPV vaccine composition according to claim 6, characterized in that: In the step S4, the buffer solution is prepared by mixing sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection in a certain proportion, and the pH value is adjusted to 7.2-7.6.

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