An HMPV vaccine composition

Abstract

This application relates to the field of biomedicine and discloses an HMPV vaccine composition comprising the following components: a pre-fusion HMPV F protein, lipid nanoparticles, an immune adjuvant, a stabilizer, and a buffer; the lipid nanoparticles comprising cholesterol, phospholipids, and PEGylated lipids; the immune adjuvant comprising a CpG oligonucleotide and monophosphoryl lipid A; the stabilizer comprising sucrose, mannitol, and polysorbate 80; and the buffer comprising sodium dihydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and water for injection. By optimizing the conformation of the F protein and selecting a suitable delivery vehicle, the antigen is able to better expose its immune epitopes, thereby enhancing the immunogenicity of the vaccine. In particular, the use of the pre-fusion F protein demonstrated significant antibody titers and T cell activation abilities, significantly superior to those of the post-fusion F protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and 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 become more 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 research and development due to their excellent biocompatibility and strong antigen delivery ability. In addition, the design of antigens 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 antigens are fully exposed and thus enhance immunogenicity has become one of the key issues in vaccine research and 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 triggering unnecessary immune responses. In addition, conventional antigen design often ignores the degree of exposure of antigen epitopes, resulting in limited recognition ability 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 comprehensive immune needs against specific pathogens. Summary of the Invention

[0004] In response to the deficiencies of the existing technology, the present invention provides an HMPV vaccine composition, which solves the problems of poor stability of lipid nanoparticles, non-optimized antigen conformation design, and poor adjuvant combination effect in existing vaccine delivery systems.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: an HMPV vaccine composition, comprising the following component materials in parts by weight:

[0006] 1.0-5.0 copies of HMPV F protein in the prefusion conformation;

[0007] 1.0-10.0 parts of lipid nanoparticles;

[0008] 0.1-1.0 parts of immune adjuvant;

[0009] 2.0-10.0 parts of stabilizer;

[0010] 50-100.0 parts of buffer.

[0011] Furthermore, the HMPV F protein in its pre-fusion conformation, as the main antigenic component of this vaccine, is in a pre-fusion state before the virus infects cells and possesses antigenic epitopes in its native conformation, which can induce the production of neutralizing antibodies. The pre-fusion conformation of the F protein exposes more neutralizing epitopes than the post-fusion conformation, effectively mimicking the surface structure of the natural virus, thereby enhancing the vaccine-induced immune response. By stabilizing its trimer structure, its conformation can be maintained stable, preventing it from transitioning to a post-fusion conformation with lower immunological activity.

[0012] Lipid nanoparticles, as a delivery system, mimic the cell membrane environment and complex with F protein through electrostatic or hydrophobic interactions, effectively encapsulating antigen proteins and improving their stability and cellular uptake efficiency. PEG modification can increase the particle's circulation time in the body and reduce immune clearance. The lipid structure can also promote transmembrane delivery of antigens to antigen-presenting cells, enhancing T cell activation.

[0013] 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 combines safety with strong immune-enhancing ability. The two synergistically stimulate dendritic cell maturation and antigen presentation, significantly improving the quality and breadth of the immune response and effectively stimulating cellular and humoral immunity.

[0014] Stabilizers include sucrose, mannitol, and polysorbate 80. Sucrose and mannitol can act as osmotic pressure regulators and protein protectants to maintain the structural stability of the vaccine during low-temperature storage and freeze-drying. Polysorbate 80 can reduce protein adsorption and interfacial denaturation, enhancing vaccine uniformity and injectability.

[0015] The buffer solution is composed of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection; this PBS-like 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] A method for preparing an HMPV vaccine composition comprises the following steps:

[0021] S1. Expression and purification of the prefusion conformation of HMPV F protein;

[0022] S2, preparation of lipid nanoparticles;

[0023] S3, forming a protein-lipid complex with the HMPV F protein in the prefusion conformation and lipid nanoparticles in a buffer solution by magnetic stirring for 30-60 minutes;

[0024] S4. Add the immune adjuvant and stabilizer in sequence under the condition that the complex is at a temperature of 4-8°C, stir for 30-40 minutes, mix to obtain the final mixture, and adjust the volume to the target concentration;

[0025] S5. The final mixture is sterilized by filtration at 0.22-0.25 μm, then divided into sterile bottles and stored at 2-8°C.

[0026] Furthermore, in the S1 step, the mammalian system ensures correct protein glycosylation and conformation, and cross-linking and stabilization treatment prevents the protein from loosening or aggregating during preparation and storage, thereby improving immunogenicity and long-term stability.

[0027] The S2 step ensures uniform lipid film formation and assembly into stable nanostructures; high-pressure homogenization effectively controls particle size and uniformity, ensuring consistent vaccine delivery and immune response after injection.

[0028] In step S3, the low temperature environment helps 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;

[0029] In step S4, slow dropwise addition is performed to prevent precipitation caused by excessive local concentration. Adding the adjuvant first helps it adsorb evenly on the surface of the complex, enhancing the immune stimulation effect. Adding the stabilizer at the end protects the structure of the final mixture, making it suitable for subsequent filling and storage.

[0030] 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.

[0031] Preferably, in step S1, the HMPV F protein in the pre-fusion conformation is obtained by transfecting a mammalian cell expression system, and the purification process adopts a method of affinity chromatography combined with molecular sieve chromatography. The obtained F protein is stabilized in its trimeric conformation by a cross-linking agent, and the protein concentration is adjusted to 1.0-5.0 mg / mL.

[0032] Preferably, in step S2, lipid nanoparticles are prepared from cholesterol, phospholipids and PEGylated lipids using a thin film hydration method, specifically comprising:

[0033] Each lipid was dissolved in an organic solvent and rotary evaporated at 40-50°C to form a lipid film;

[0034] Add PBS buffer to hydrate for 30-60 minutes, then sonicate for 10-15 minutes and homogenize under high pressure at 800-1200 bar for 3-5 times;

[0035] Finally, uniform lipid nanoparticles with a particle size of 50-150 nm and a PDI of less than 0.2 were obtained.

[0036] Preferably, in step S4, the immune adjuvant is a combination of CpG oligonucleotide and monophosphoryl lipid A, which are dissolved in a buffer solution in proportion and then added dropwise and stirred for 15-30 minutes.

[0037] 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.

[0038] The present invention provides an HMPV vaccine composition. It has the following beneficial effects:

[0039] 1. By optimizing the conformation of the F protein and selecting an appropriate delivery vehicle, the present invention allows the antigen to better expose its immune epitopes, thereby enhancing the immunogenicity of the vaccine. Compared with traditional immunization strategies, the vaccine, which combines the F protein with lipid nanoparticles after low-temperature compounding, can effectively induce higher levels of neutralizing antibodies and F protein-specific IgG. In particular, the vaccine using the prefusion conformation of the F protein demonstrated significant antibody titers and T cell activation, significantly superior to the postfusion conformation of the F protein.

[0040] 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 vivo. The PEGylated lipid particles not only improve the dispersibility of the vaccine in vivo 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 vaccine's immune response, particularly in terms of activation of memory T cells and establishment of immune bias.

[0041] 3. Through a low-temperature, slow compounding process, the vaccine formulation of the present invention effectively reduces protein aggregation and adverse reactions, ensuring better biocompatibility. In immunotherapy experiments, the use of a surfactant-based stabilizer system effectively reduced redness, swelling, and induration at the injection site, reducing local reactions associated with traditional vaccine formulations. These improvements enhance vaccine safety and provide assurance for clinical application.

[0042] 4. By rationally combining two adjuvants, the present invention not only enhances the immune response but also optimizes the bias of the immune response. The CpG adjuvant effectively promotes the establishment of a Th1 immune response, strengthens the cell-mediated immune response, and increases the proportion of memory T cells. Experimental data show that the vaccine using this combination not only improves the IgG1 / IgG2a ratio but also significantly increases the activation level of specific T cells, enhances immune memory, and provides long-term immune protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a flow chart of the preparation steps of the present invention. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the present specification. 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 making creative efforts are within the scope of protection of the present invention.

[0045] Please see the attached Figure 1 , Example 1:

[0046] Vaccine group allocation ratio:

[0047] HMPV F protein in prefusion conformation: 5.0 copies;

[0048] Lipid nanoparticles: 10.0 parts;

[0049] Immune adjuvant: 1.0 part;

[0050] Stabilizer: 10.0 parts;

[0051] Buffer: 100.0 parts;

[0052] Lipid nanoparticle composition:

[0053] Cholesterol:phospholipid:PEGylated lipid is 3.5:5:1.5;

[0054] Immune adjuvant composition:

[0055] CpG oligonucleotide:monophosphoryl lipid A 1:1;

[0056] Stabilizer composition:

[0057] Sucrose: mannitol: polysorbate is 6:3:1;

[0058] Buffer composition:

[0059] Sodium dihydrogen phosphate: disodium hydrogen phosphate: sodium chloride: water for injection = 1:1.5:9:88.5;

[0060] Preparation steps:

[0061] HEK293F cells were cultured in suspension and transfected with the F protein gene. The culture supernatant was harvested 72 hours later. The trimer was purified using Ni-NTA affinity chromatography coupled with Superdex200 molecular sieves, collected, and cross-linked with glutaraldehyde to a final concentration of 5.0 mg / mL.

[0062] 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. The film was then hydrated with PBS (pH 7.4) for 50 minutes and sonicated on ice for 10 minutes. The film was homogenized five times using a 1200 bar high-pressure homogenizer to obtain an average particle size of approximately 90 nm and a PDI of 0.18.

[0063] The F protein solution was slowly added dropwise to the lipid nanoparticles and magnetic stirring was maintained at 4°C for 50 minutes to form a uniform complex.

[0064] Separately, dissolve CpG and MPLA, mix them, and add them dropwise to the complex. Stir for 20 minutes. Then, add sucrose, mannitol, and polysorbate 80 in that order, stirring for another 30 minutes. Add buffer to the final volume of 100 parts.

[0065] 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.

[0066] Example 2:

[0067] Vaccine group allocation ratio:

[0068] HMPV F protein in prefusion conformation: 3.0 copies;

[0069] Lipid nanoparticles: 5.0 parts;

[0070] Immune adjuvant: 0.5 parts;

[0071] Stabilizer: 6.0 parts;

[0072] Buffer: 75.0 parts;

[0073] Lipid nanoparticle composition:

[0074] Cholesterol:phospholipid:PEGylated lipid is 2.5:5:1;

[0075] Immune adjuvant composition:

[0076] CpG oligonucleotide:monophosphoryl lipid A 3:2;

[0077] Stabilizer composition:

[0078] Sucrose: mannitol: polysorbate is 5:1.5:0.5;

[0079] Buffer composition:

[0080] Sodium dihydrogen phosphate: disodium hydrogen phosphate: sodium chloride: water for injection is 1:1.2:8.5:89.3;

[0081] Preparation steps:

[0082] A stable expression line was constructed using CHO-K1 cells and cultured in glutamine-containing medium. After 72 hours of expression, the supernatant was collected by centrifugation. The protein was purified using a Strep-tag affinity column and then chromatographed on a Superdex200 column, concentrating to 3.0 mg / mL. BS3 crosslinker was used to stabilize its conformation.

[0083] Lipids were mixed in a 2.5:5:1 ratio and dissolved in ethanol. Rotary evaporation was performed at 45°C for 20 minutes to form a lipid film, which was then hydrated with PBS buffer for 40 minutes. After 12 minutes of ice-water bath sonication, high-pressure homogenization was performed four times at 1000 bar to obtain lipid particles with an average particle size of 110 nm and a PDI of 0.16.

[0084] 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.

[0085] 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. Finally, PBS was added to make the volume 75 parts.

[0086] Sterilize using a 0.25 μm filter, place in a 5 mL sterile injection vial, and store at 2-8°C without freezing.

[0087] Example 3:

[0088] Vaccine group allocation ratio:

[0089] HMPV F protein in prefusion conformation: 1.0 part

[0090] Lipid nanoparticles: 1.0 part

[0091] Immune adjuvant: 0.1 part

[0092] Stabilizer: 2.0 parts

[0093] Buffer: 50.0 parts

[0094] Lipid nanoparticle composition:

[0095] Cholesterol:phospholipid:PEGylated lipid is 2:4:0.5;

[0096] Immune adjuvant composition:

[0097] CpG oligonucleotide:monophosphoryl lipid A 7:3;

[0098] Stabilizer composition:

[0099] Sucrose: mannitol: polysorbate is 6:2.5:1.5;

[0100] Buffer composition:

[0101] Sodium dihydrogen phosphate: disodium hydrogen phosphate: sodium chloride: water for injection is 1:1:7.5:90.5;

[0102] Preparation steps:

[0103] F protein was expressed in a small-scale expression system using FreeStyle 293 cells in suspension and cultured for 48 hours. Initial purification was performed using Protein A affinity chromatography, followed by treatment with Superose 6 molecular sieves and concentration to 1.0 mg / mL. No crosslinking agent was used for this conformation.

[0104] The lipid preparation process was simplified, utilizing a thin-film hydration and short homogenization protocol. The lipid mixture was dissolved in ethanol, dried at 40°C for 10 minutes, and then rehydrated with PBS for 30 minutes. Ultrasonic treatment was performed for 5 minutes, followed by homogenization at 800 bar pressure three times. The particle size was controlled at 120 nm, with a PDI of 0.19.

[0105] 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.

[0106] CpG and MPLA were pre-dissolved in the appropriate proportions and added to the mixture, gently stirred for 10 minutes, and then the stabilizer components were added to make up the total volume of the buffer to 50 parts.

[0107] The composite liquid was quickly sterilized by 0.22 μm filter membrane, dispensed into 1 mL sterile brown vials, and stored under conventional refrigeration.

[0108] Comparative Example 1:

[0109] This comparative example is based on Example 1, with only the antigen source modified. The F protein was replaced by a monomeric expression product in the post-fusion conformation. The expression system and purification procedures remained the same, and no trimer stabilization or cross-linking treatment was performed. After adjusting the protein concentration to 5.0 mg / mL, it was complexed with lipid nanoparticles. Subsequent steps were consistent with Example 1, including the lipid component ratios, adjuvant addition, stabilizer, and buffer ratios.

[0110] Comparative Example 2:

[0111] This comparative example, based on Example 1, replaced the lipid nanoparticles with a traditional cationic oil-in-water adjuvant emulsion (e.g., a DDA / aluminum adjuvant system). DDA lipid (a cationic lipid) was emulsified with MCT oil to form an oil-in-water system, into which the antigen was directly dispersed. CpG / MPLA were also added, and the stabilizer ratio was consistent with that in the previous example. No lipid complexation strategy was employed, and the F protein was not entrapped within the particle structure; it was simply physically suspended.

[0112] Comparative Example 3:

[0113] In this comparative example, the lipid nanoparticles consisted of cholesterol and phospholipids, without DSPE-PEG2000 or other PEG-modified lipids. The lipid mixture ratio was adjusted to 3:6 cholesterol:phospholipid, and the preparation method (thin film hydration followed by high-pressure homogenization) remained the same. Adjuvants and stabilizers were added after protein complexation, but the absence of PEG predisposed the particles to aggregation and reduced circulation stability. All other process parameters were consistent with those in Example 2.

[0114] Comparative Example 4:

[0115] In this comparative example, all the same processes and formulations as in Example 2 were used, except that the adjuvant component was simplified from a combination of CpG and MPLA to a single MPLA (0.5 parts). MPLA was dissolved in buffer and added to the protein-lipid complex. Subsequent stabilizer and buffer addition procedures remained unchanged. CpG was omitted to simulate the effects of a traditional adjuvant.

[0116] Comparative Example 5:

[0117] This comparative example uses Example 3 as a reference, with only the stabilizer system adjusted: sucrose and mannitol are retained, while polysorbate 80 is omitted. The stabilizer ratio is 1.5 parts sucrose to 0.5 parts mannitol. The remaining procedures are consistent with Example 3, including the F protein expression method, lipid ratio, adjuvant combination, and mixing method.

[0118] Comparative Example 6:

[0119] In this comparative example, the F protein and lipid particles were not subjected to slow, low-temperature complexation. Instead, they were simply physically mixed: the protein and lipid nanoparticles were directly suspended without magnetic stirring, dropwise addition, or temperature control. The adjuvant and stabilizer were added in the same manner. This method ignores the interfacial affinity between the protein and the particles and represents a conventional "blending mode."

[0120] Comparative experiment:

[0121] Experiment 1:

[0122] Purpose:

[0123] 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.

[0124] Animal models and groups:

[0125] 6-8 week old BALB / c mice were selected, half male and half female, and divided into 3 groups, with 10 mice in each group.

[0126] Group G1: vaccine of Example 1 (prefusion F protein + lipid nanoparticles + CpG / MPLA);

[0127] Group G2: Comparative Example 1 vaccine (post-fusion F protein + lipid nanoparticles + CpG / MPLA);

[0128] Group G3: Comparative Example 2 vaccine (prefusion F protein + oil-in-water adjuvant + CpG / MPLA).

[0129] Immunization schedule and vaccination method:

[0130] The inoculation dose is 100 μL / vial, administered intraperitoneally. The primary immunization is administered on day 0, and the booster immunization is administered on day 21.

[0131] Sampling and testing time points:

[0132] Blood was collected from the tail vein for serum collection on days 14, 28, and 42. Some mice were sacrificed on day 28, and splenocytes were isolated for ELISPOT and flow cytometric analysis.

[0133] Detection method:

[0134] Neutralizing antibody titer: Pseudovirus neutralization method, determination of IC50 value;

[0135] F protein-specific IgG titer: ELISA method, OD450 quantification;

[0136] ELISPOT assay: IFN-γ spot-forming unit (SFU) counting after stimulation;

[0137] Flow cytometry: the proportions of CD4+CD44hiCD62Llo and CD8+CD44hi memory T cells in splenocytes (see Table 1 for specific values).

[0138] Table 1

[0139] 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

[0140] Summarize:

[0141] In this experiment, the vaccine after the pre-fusion conformation of F protein was combined with lipid nanoparticles showed a significant immune effect. This result shows that the F protein can more effectively expose its antigenic epitopes without undergoing conformational changes, enhancing the production of antibodies and the activation of T cells. 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 efficiency of antigen delivery, thereby enhancing the immune response, especially in the activation of memory T cells. By combining with the CpG / MPLA adjuvant, the immune response was effectively promoted, verifying the role of the dual adjuvant system in enhancing the immune response.

[0142] Experiment 2:

[0143] Purpose of the experiment:

[0144] This experiment aims 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, revealing the actual effect of the synergistic effect of particle structure and adjuvant in immunization strategies.

[0145] Animal models and groups:

[0146] C57BL / 6 mice were selected, 8 mice per group, and divided into 3 groups:

[0147] Group G4: formulation of Example 2 (PEG-modified lipid particles + CpG / MPLA);

[0148] Group G5: Comparative Example 3 (no PEG-modified particles + CpG / MPLA);

[0149] Group G6: formulation of comparative example 4 (PEG-modified particles + MPLA single adjuvant).

[0150] Dosage regimen:

[0151] Subcutaneous injection, 50 μL / side, once on each side of the back, primary immunization on day 0, booster immunization on day 21. The dosage should remain the same.

[0152] Sampling and analysis plan:

[0153] On the 28th day, all animals were sacrificed, and spleen, lymph nodes, and tissues at the injection site were collected and tested separately.

[0154] Testing items and methods:

[0155] Particle size and zeta potential: DLS analysis was performed before and after inoculation to observe the effect of PEG modification on particle stability;

[0156] IgG1 / IgG2a subtype ratio: Th1 / Th2 immune bias was assessed using typing ELISA;

[0157] In vivo distribution tracking: using DiR fluorescently labeled particles and real-time monitoring with a small animal imaging system;

[0158] Cytokine expression: RNA was extracted from splenocytes, and the transcript levels of IL-12p40 and TNF-α were detected by qPCR (see Table 2 for specific values).

[0159] Table 2

[0160] 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

[0161] Summarize:

[0162] PEG-modified lipid nanoparticles significantly improved the stability and delivery effect of vaccines. Through PEG modification, the particles maintained better dispersibility in the body, avoiding the precipitation phenomenon caused by the aggregation of unmodified particles in the blood. Experimental results showed that PEG modification not only improved the stability of the particles, but also enhanced the biocompatibility of the vaccine and reduced 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.

[0163] Experiment 3:

[0164] Purpose of the experiment:

[0165] 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 the actual impact on the physical stability, storage performance and immunogenicity of the finished vaccine product.

[0166] Animal models and groups:

[0167] Adult New Zealand white rabbits (weighing 2.0-2.5 kg) were used, with 6 rabbits in each group, divided into 3 groups:

[0168] Group G7: Example 3 (complete stabilizer + low temperature compound);

[0169] Group G8: Comparative Example 5 (removal of polysorbate 80);

[0170] Group G9: Comparative Example 6 (no compounding, direct blending).

[0171] Inoculation and sampling setup:

[0172] A single injection of 100 μL was given intramuscularly in each anterior thigh on day 0. The observation period was 4 weeks.

[0173] Observe injection site reactions weekly to assess changes in redness, swelling, and induration;

[0174] On day 28, blood was collected for IgG testing;

[0175] 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.

[0176] Evaluation items and methods:

[0177] Particle size change: DLS dynamic detection;

[0178] Solution appearance: Visually record clarity, flocculation, and precipitation;

[0179] Redness and swelling reaction: Induration area (mm 2 ) Record + duration days;

[0180] IgG titer: ELISA method, detecting the level of F protein-specific antibodies (specific values are shown in Table 3).

[0181] Table 3

[0182] 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 Relatively clear + partial precipitation 4 19 1.07

[0183] Summarize:

[0184] The compounding process and the choice of stabilizer have a significant impact on vaccine quality and immune efficacy. In this experiment, low-temperature compounding and the use of a surfactant (polysorbate 80) significantly improved the vaccine's physical stability and immunogenicity. Low-temperature, slow-drop compounding not only ensured a tight bond between the lipid particles and the F protein but also effectively maintained particle stability, preventing macromolecular aggregation. Polysorbate 80, as a stabilizer, provided surface activity, reduced particle aggregation, and maintained system homogeneity, significantly improving the vaccine's storage stability. In contrast, removing polysorbate 80 or using other compounding methods significantly reduced vaccine stability, manifested by particle aggregation and precipitation. Immune response results also demonstrated that a stable vaccine formulation facilitated enhanced activation of specific antibodies and memory T cells, suggesting the role of stabilizers and compounding processes in promoting immune efficacy.

[0185] While 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 these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An HMPV vaccine composition, characterized in that: It is composed of the following components in parts by mass: 1.0-5.0 copies of HMPV F protein in the prefusion conformation; 1.0-10.0 parts of lipid nanoparticles, composed of cholesterol, phospholipids and PEGylated lipids, wherein the weight ratio of cholesterol, phospholipids and PEGylated lipids is 2-4:4-6:0.5-1.5; 0.1-1.0 parts of an immune adjuvant, consisting of a CpG oligonucleotide and monophosphoryl lipid A, wherein the mass ratio of the CpG oligonucleotide to the monophosphoryl lipid A is 1-5:1-5; 2.0-10.0 parts of a stabilizer, consisting of sucrose, mannitol, and polysorbate 80, wherein the mass ratio of sucrose, mannitol, and polysorbate 80 is 4-7:2-4:0.5-1.5; 50-100.0 parts of a buffer solution, consisting of 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; The preparation method of the HMPV vaccine composition comprises the following steps: S1. Expression and purification of the prefusion conformation of HMPV F protein; S2, preparation of lipid nanoparticles; S3, forming a protein-lipid complex with the HMPV F protein in the prefusion conformation and lipid nanoparticles in a buffer solution by magnetic stirring for 30-60 minutes; S4. Add the immune adjuvant and stabilizer in sequence under the condition that the complex is at a temperature of 4-8°C, stir for 30-40 minutes, mix 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, aliquoted, and stored in sterile bottles at 2-8°C. In step S3, the F protein solution was slowly added dropwise to the lipid nanoparticles, and magnetic stirring was maintained at 4° C. for 50 minutes to form a uniform complex.

2. The HMPV vaccine composition according to claim 1, characterized in that: In step S1, the HMPV F protein in the pre-fusion conformation is obtained by transfecting a mammalian cell expression system. During the purification process, affinity chromatography combined with molecular sieve chromatography is used. The obtained F protein is stabilized in its trimeric conformation by a cross-linking agent, and the protein concentration is adjusted to 1.0-5.0 mg / mL.

3. The HMPV vaccine composition according to claim 1, characterized in that: In step S2, lipid nanoparticles are prepared from cholesterol, phospholipids and PEGylated lipids using a thin film hydration method, specifically comprising: Each lipid was 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 sonicate for 10-15 minutes and homogenize under high pressure at 800-1200 bar for 3-5 times; Finally, uniform lipid nanoparticles with a particle size of 50-150 nm and a PDI of less than 0.2 were obtained.

4. The HMPV vaccine composition according to claim 1, 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 solution in a certain proportion and then added dropwise and stirred for 15-30 minutes.

5. An HMPV vaccine composition according to claim 4, characterized in that: 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.

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