A nanoparticle for preparing a syncytial virus vaccine

Abstract

The application relates to the technical field of syncytial viruses, in particular to a nanoparticle for preparing a syncytial virus vaccine. The application designs an RSV F protein antigen which can be well stabilized in a pre-fusion conformation and is favorable for inducing stronger neutralizing antibodies; the RSV F protein antigen is self-assembled with a multimeric complex HPF protein nanoparticle by using a GvTagOpti / SdCatcher system to form a spherical 24-mer SP-VE-NP protein nanoparticle, the RSV F protein antigen is displayed on the surface of the nanoparticle, stronger immune responses can be effectively caused, and antibodies preventing respiratory syncytial viruses from invading target cells are generated. The 24-mer SP-VE-NP protein nanoparticle of the application significantly improves the titer of neutralizing antibodies through a multi-antigen display mode.

Description

Technical Field

[0001] This application relates to the field of respiratory syncytial virus (RSV) technology, and in particular to nanoparticles for preparing RSV vaccines. Background Technology

[0002] Respiratory syncytial virus (RSV) is an enveloped, single-stranded, negative-sense RNA virus belonging to the family Paramyxoviridae and the genus Pneumovirus. RSV respiratory infections pose a high risk to infants and the elderly. The RSV envelope consists of a lipid bilayer of the host cell membrane, within which two embedded surface glycoproteins, F and G, are crucial for viral infection.

[0003] The G protein is responsible for helping the virus attach to host cells, primarily through its interaction with host cell heparan sulfate. Based on differences in the G protein, RSV is divided into two main subtypes: type A and type B. The F protein is responsible for viral fusion with the host cell membrane, facilitating the entry of viral RNA into the host cell. The F protein exhibits 89% homology in both subtypes, significantly higher than the 53% homology of the G protein. This makes the F protein a primary candidate antigen for vaccine development.

[0004] The F protein is a trimeric glycoprotein that mediates viral entry into host cells. The monomeric precursor peptide chain, designated F0, contains an N-terminal signal peptide sequence (amino acids 1–25) that is subdivided into two main segments, F2 (amino acids 26–109) and F1 (amino acids 136–524). These two segments are separated by an intermediate peptide named Pep27, flanked by two Furin sites. At the C-terminus, there is also a transmembrane domain (amino acids 525–550) and an intracellular domain (amino acids 551–574). During maturation, F0 is activated by proteolytic cleavage at the two Furin sites, leading to the removal of Pep27. F1 and F2 remain bound together by two disulfide bonds. Figure 1 ).

[0005] The F1 protein contains a highly hydrophobic fusion peptide FP at its N-terminus, located within a hydrophobic cavity and protected from external hydrophilic environments. When the F protein appears on the surface of a virus or cell, its structure is unstable, existing in a high-energy metastable pre-fusion conformation. Subsequently, the N-terminus of the F1 protein undergoes dramatic structural changes, with its secondary structural components α2, α3, α4, and α5 forming a larger α-helix structure named the hepta-repetitive sequence (HRA). This series of rearrangements ultimately leads to the insertion of the N-terminal FP of the trimerized F1 protein into adjacent cells. The three HRAs in the trimer pair with the three hepta-repetitive sequences (HRBs) at the other end, forming a six-helix bundle (6HB) through the folding of the F protein. This process induces membrane fusion, thereby achieving viral infection of cells. Simultaneously, this process also causes the F protein to change from its pre-fusion conformation (pre-F). Figure 2 ) transforms into a postfusion conformation (postfusion F, post-F, Figure 3 The pre-fusion conformation contains several important neutralizing antibody-binding epitopes and is a crucial antigenic form for inducing a highly effective protective immune response. However, the pre-fusion conformation of the native RSV F protein is relatively unstable and prone to conversion to the post-fusion conformation, leading to the loss of antigenic epitopes and reduced immunogenicity.

[0006] Research on RSV F protein vaccines has seen significant progress in structure-based vaccine design in recent years. In 2013, McLellan et al. first resolved the pre-fusion conformation (Pre-F) of the RSV F protein, a breakthrough that laid the foundation for vaccine development. Using X-ray crystallography, they successfully determined the three-dimensional structure of the RSV F protein and designed the stable pre-fusion protein DS-Cav1 based on it. The development of DS-Cav1 has led the trend of Pre-F protein-based vaccine design and has become the core basis for many vaccine candidates. Although DS-Cav1 has made significant progress in enhancing the stability of the Pre-F protein, its stability is insufficient under certain conditions, affecting the long-term storage and transport of the protein. Subsequently, researchers have developed a variety of F protein-based candidate vaccines, including Pfizer's bivalent subunit vaccine and Moderna's mRNA vaccine, which are currently in clinical development. However, RSV vaccines are not yet available in China.

[0007] The A2 strain is a classic A subtype of respiratory syncytial virus (RSV) and is widely used in scientific research and vaccine / drug development.

[0008] D25 monoclonal antibody is a neutralizing antibody that targets the site Ø of the pre-F protein of respiratory syncytial virus (RSV) fusion.

[0009] Palivizumab monoclonal antibody is a neutralizing antibody that targets the pre-fusion F protein (Pre-F) and post-fusion site II (Site II) of respiratory syncytial virus (RSV).

[0010] DS-Cav1 is a stabilized pre-fusion F protein (Pre-F) antigen of respiratory syncytial virus (RSV). By introducing specific disulfide bond mutations, the stability of the pre-fusion conformation of the F protein is improved, and its transformation to the post-fusion conformation is reduced, thereby maintaining the specific antigenic epitope of the pre-fusion F protein.

[0011] DS-Cav1 can be used as a positive reference molecule for RSV Pre-F antigen stabilization design to evaluate the conformational retention and stability of engineered RSVF proteins. Summary of the Invention

[0012] The purpose of this invention is to overcome the shortcomings of the prior art and provide nanoparticles for preparing syncytial virus vaccines.

[0013] The first objective of this invention is to provide a fusion protein.

[0014] A second objective of this invention is to provide a nucleic acid molecule.

[0015] A third objective of this invention is to provide a biomaterial.

[0016] A fourth object of the present invention is to provide a fusion protein composition.

[0017] The fifth objective of this invention is to provide a protein nanoparticle.

[0018] The sixth objective of this invention is to provide a method for preparing protein nanoparticles.

[0019] A seventh objective of this invention is to provide the application of the protein nanoparticles in the preparation of a novel coronavirus vaccine.

[0020] The eighth object of the present invention is to provide a composition.

[0021] A ninth object of the present invention is to provide another composition.

[0022] The tenth object of the present invention is to provide the use of the composition in the preparation of a syncytial virus vaccine.

[0023] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention claims protection for a fusion protein having an amino acid sequence as shown in any one of SEQ ID NO:4 to 12.

[0024] Among them, the recombinant proteins with amino acid sequences such as SEQ ID NO:7 to 9 are based on amino acid sequences such as SEQ ID NO:4 to 6 with a Gv-Tag added to the C-terminus for constructing protein nanoparticles; the recombinant proteins with amino acid sequences such as SEQ ID NO:10 to 12 are based on amino acid sequences such as SEQ ID NO:4 to 6 with a T4 trimerization domain (T4 Foldon) added to the C-terminus for constructing trimers.

[0025] Preferably, the amino acid sequence is as shown in SEQ ID NO:5 or 8, which improves the stability of the fusion protein.

[0026] The present invention also claims protection for a nucleic acid molecule that encodes the gene for the fusion protein, or the reverse complementary sequence of the gene for the fusion protein.

[0027] This invention claims protection for a biological material, which is any one of the following: (1) An expression cassette containing the nucleic acid molecule; (2) A recombinant vector containing the nucleic acid molecule or the expression cassette described in (1); (3) A recombinant microorganism containing the nucleic acid molecule, the expression cassette described in (1) or the recombinant vector described in (2); (4) A cell line containing the nucleic acid molecule, the expression cassette in (1), the recombinant vector in (2), or the recombinant microorganism in (3).

[0028] This invention claims protection for a fusion protein composition comprising an HPF fusion protein with an amino acid sequence as shown in SEQ ID NO. 17 and a fusion protein with an amino acid sequence as shown in any one of SEQ ID NO. 7 to 9.

[0029] This invention claims protection for a protein nanoparticle obtained by self-assembly of an HPF fusion protein with an amino acid sequence as shown in SEQ ID NO.17 and a fusion protein with an amino acid sequence as shown in any one of SEQ ID NO.7 to 9.

[0030] This invention claims protection for a method for preparing protein nanoparticles, wherein the HPF fusion protein with an amino acid sequence as shown in SEQ ID NO.17 and any of the fusion proteins shown in SEQ ID NO.7 to 9 are incubated in a buffer solution to obtain the nanoparticles.

[0031] This invention claims protection for the use of the protein nanoparticles in the preparation of a novel coronavirus vaccine.

[0032] This invention claims protection for a composition comprising the fusion protein composition and an adjuvant.

[0033] This invention claims protection for a composition comprising the protein nanoparticles and an adjuvant.

[0034] This invention claims protection for the use of the composition in the preparation of syncytial virus vaccines.

[0035] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes Pymol and RF diffusion to design an RSV F protein antigen that remains well-stabilized in its pre-fusion conformation, facilitating the induction of stronger neutralizing antibodies. Using the GvTagOpti / SdCatcher system (Gv / Sd system, see Chinese Patent CN113621031A), this RSV F protein antigen self-assembles with multimeric HPF protein nanoparticles, forming spherical 24-dimer SP-VE-NP protein nanoparticles. Displaying the RSV F protein antigen on the surface of these nanoparticles effectively induces a stronger immune response, generating antibodies that prevent respiratory syncytial virus (RSV) from invading target cells. The 24-dimer SP-VE-NP protein nanoparticles of this invention significantly improve neutralizing antibody titers through multi-antigen display. Immunization of mice and rats via intramuscular injection and intranasal administration revealed that the prepared RSV nanoparticle vaccine induced high titers of neutralizing antibodies, and intramuscular injection and intranasal administration protected mice and rats from RSV A2 subtype infection. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the structure of the syncytial virus F0 protein.

[0037] Figure 2 This is the pre-fusion conformation of the respiratory syncytial virus F protein trimer.

[0038] Figure 3 This is the conformation of the syncytial virus F protein trimer after fusion.

[0039] Figure 4This paper presents a method for engineering RSV pre-fusion F protein based on conformational hotspot remodeling and a multi-step stabilization strategy. A) By comparing the 3D structural differences between the pre-fusion conformation (PDB: 7UJ3) and post-fusion conformation (PDB: 3RKI) of RSV F protein, the structural hotspot regions during the pre-fusion transition are identified, providing targets for subsequent stabilization design. B) The spatial location of Region 1 (amino acids 73–170) in the pre-fusion trimer structure of RSV F protein is shown, clarifying its location in a key region affecting the stability of the pre-fusion conformation. C) The specific location and amino acid range of Region 1 in the RSV F protein monomer structure are further shown, identifying the target fragment for engineering. D) Region 1 is redesigned using the RFdiffusion protein structure generation algorithm, generating new structural fragments while maintaining the overall pre-fusion conformation of RSV F protein. E) The structural quality of the first round of Region 1 redesign results is evaluated using pLDDT and RMSD indicators, screening candidate mutants with high structural confidence and good conformational matching. F) The first round of engineered RSV is detected by ELISA. The binding ability of the F protein mutant to the pre-fusion specific antibodies D25 and Palivizumab was used to screen for candidates that maintain the pre-fusion antigenic epitope; G was to further restrict the protein structure flexibility and improve the pre-fusion conformation stability of RSV F protein by introducing a new disulfide bond linkage design based on the first round of optimization; H was to evaluate the binding activity of D25 and Palivizumab of the RSV F protein mutant after the second round of disulfide bond optimization by ELISA and screen for further stabilized engineered proteins; I was to introduce a Gv tag at amino acid 513 at the C-terminus of the optimized RSV F protein; J was to evaluate the stability and stress tolerance of the final engineered RSV F protein through freeze-thaw cycles, long-term low-temperature storage and high-temperature stress treatment experiments.

[0040] Figure 5 The specific sequence description for the modification of the F protein.

[0041] Figure 6 A schematic diagram of the structure of the respiratory syncytial virus F protein multipolymer complex SP-VE-NP, constructed by linking M-SC7-CC14-Gv (SP-VE) with multipolymer complex HPF protein nanoparticles.

[0042] Figure 7 Results of expression and purification of WT-F, DS-Cav1, SP-VE, SP-VE-T4, 24-tetrameric complex FPF protein nanoparticles, and 24-tetrameric complex SP-VE-NP protein nanoparticles after non-reducing SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

[0043] Figure 8Characterization of the prepared respiratory syncytial virus SP-VE-NP protein nanoparticles: A: Transmission electron microscopy (TEM) image of SP-VE-NP; B: Dynamic light scattering (DLS) detection and analysis results of SP-VE-NP and HPF; C: Size exclusion chromatography (SEC) elution curves of SP-VE, SP-VE-T4, HPF and SP-VE-NP, with the detection wavelength at 280 nm UV absorption and the elution volume corresponding to the characteristic peak position of the nanoparticles.

[0044] Figure 9 BALB / c mice were immunized by intramuscular injection; A: immunization strategy; B: detection of serum neutralizing antibody (nAb) titers using RSV A2 strain, defined as the highest serum dilution that reduces the number of infected cells by 50%.

[0045] Figure 10 Mice (BALB / c) and cotton rats (Cotton Rat) were immunized via nasal immunization (in) and intramuscular immunization (im) for respiratory system inoculation. A represents the immunization and challenge strategies. B represents the induction of anti-RSV A2 neutralizing antibodies against the virus by SP-VE-NP in mice, with the neutralizing antibody titer defined as the highest serum dilution that reduces the number of infected cells by 50%. C represents the induction of anti-RSV A2 neutralizing antibodies against the virus by SP-VE-NP in cotton rats, with the neutralizing antibody titer defined as the highest serum dilution that reduces the number of infected cells by 50%. D represents the viral load (RSVN gene copy number) in the lung tissue of BALB / c mice. E represents the viral load (RSVN gene copy number) in the lung tissue of cotton rats.

[0046] In the following embodiments, the data is... Mean ± Standard Error (mean ± SEM) The corrected p-value was calculated using one-way ANOVA combined with Tukey's multiple comparison test.

[0047] p < 0.05 p < 0.01, p < 0.001, p < 0.0001, ns = not significant. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0049] Example 1: Engineering Method for Pre-Fusion F Protein of RSV Based on Conformational Hotspot Region Remodeling and Multi-Step Stabilization Strategy I. Analysis of Conformational Differences in RSV F Protein and Determination of Target Regions To screen key structural regions affecting the stability of the pre-fusion F protein (Pre-F), this embodiment performed structural alignment analysis on the pre-fusion F protein monomer structure (PDB: 7UJ3) and the post-fusion F protein monomer structure (PDB: 3RKI). Using PyMOL software, spatial superposition of the two conformations revealed regions of significant structural difference during the transition from the pre-fusion to the post-fusion conformation of the RSV F protein, defined as Difference 1 and Difference 2 regions, respectively. Figure 4 (A in the middle).

[0050] Further analysis revealed that the Difference 1 and Difference 2 regions correspond to the previously reported refolding regions RR1 and RR2, respectively, suggesting that these regions may be involved in the conformational transition of the RSV F protein. Since the Difference 2 region contains multiple natural disulfide bonds, its strong structural constraints hinder structural redesign. Therefore, the more flexible and engineerable Difference 1 region was chosen as the primary target for stabilization.

[0051] By comparing the spatial differences between different conformations, regions exhibiting significant structural changes are screened. Regions with marked structural changes are defined as candidate hotspot regions and then subjected to subsequent stabilization design. In this diagram, SP represents the signal peptide region, TM represents the transmembrane domain, and CD represents the cytoplasmic domain. The numbers in the diagram represent the corresponding amino acid residue numbers.

[0052] Based on the structural analysis results, the region of amino acid residues from position 73 to position 170 was selected from the pre-RSV fusion F protein trimer structure and defined as Region 1. Figure 4 (B and C in the original text). Region 1 is located at a key spatial position in the prefusion trimer structure of the RSV F protein, and this region is involved in maintaining the stability of the prefusion conformation. Among them, amino acid positions 136 to 170 cover the Difference 1 region, and amino acid positions 104 to 147 are further identified as the region for subsequent structural remodeling.

[0053] II. Restructuring and Filtering of Region 1 Based on RFdiffusion 1. Experimental Methods (1) To improve the conformational stability of RSV F protein before fusion, this embodiment uses a protein structure generation algorithm based on RFdiffusion to redesign the flexible region in Region 1. While keeping the overall conformational framework of RSV F protein unchanged before fusion, only the local part of Region 1 is regenerated to generate new candidate structural fragments.

[0054] like Figure 4 As shown in D, Region 1 is inpainted using the RFdiffusion protein redesign algorithm to obtain multiple candidate design fragments, where the purple area represents the regenerated structural fragment.

[0055] (2) The AlphaFold structure prediction method is used to evaluate the generated candidate structures. The reliability of the local structure is assessed by the predicted local distance difference test (pLDDT), and the root mean square deviation (RMSD) is used to evaluate the consistency between the designed structure and the reference structure before fusion. A higher pLDDT score indicates that the predicted structure has higher reliability. Generally, pLDDT > 80 represents a higher structure prediction reliability. A lower RMSD value indicates that the designed structure has higher similarity to the reference structure. Generally, RMSD < 2 Å indicates that the two have a good structural match.

[0056] like Figure 4 As shown in E, after calculation and screening, nine candidate mutants, namely SC1 to SC9, were obtained from 2045 design schemes. The only difference between the nine candidate fragments is the linker between F1 and F2.

[0057] Commonly used flexible and rigid linkers were used as linkers between F1 and F2 (NC1-NC6 as controls), while WT-F (i.e. F protein without engineered stability mutations) was used as a control. Specific information is shown in Table 1.

[0058] Table 1

[0059] (3) The coding sequence of the RSV F protein mutant in Table 1, along with WT-F, was cloned into the pcDNA3.1 plasmid, and a 6× His tag was added to the C-terminus. Small-scale expression was then carried out by transient transfection of HEK293T cells. The specific method is as follows: Vector construction: The RSV F protein mutant, along with the coding DNA sequence of WT-F, was cloned into the eukaryotic expression vector pcDNA3.1 to construct a recombinant expression vector. During protein expression, six his tags were attached to its C-terminus.

[0060] Expression of syncytial virus antigen: The recombinant expression vector was transfected into HEK293T cells; then, the cells were cultured at 37°C and 5% CO2 for 2 days, and the supernatant of HEK293T cells was harvested for subsequent experiments.

[0061] (4) The binding ability of each RSV F protein mutant to D25 was detected in the harvested HEK293T cell supernatant using the double antibody sandwich method (F protein conformation specificity detection). The specific method is as follows: D25 mAb (amino acid sequence as shown in SEQ ID NO:15) was diluted to 5 μg / ml and coated onto 96-well plates, 50 μl per well, and incubated overnight at 4°C. The plates were then washed three times with phosphate-buffered saline (PBST) containing Tween 20, blocked with 100 μl of 5% skim milk powder / PBS solution at room temperature for 1 hour, and washed again with PBST.

[0062] 100 μl of supernatant from HEK293T cells transiently overexpressing each RSV F protein mutant was added to each well, and the cells were incubated at 37°C for 2 hours. After washing three times, 100 μl of HRP-labeled Palivizumab (amino acid sequence shown in SEQ ID NO:16) diluted 1:4000 was added to each well, and the cells were incubated at 37°C for 1 hour. After washing again, tetramethylbenzidine (TMB) and hydrogen peroxide solution were added, and the reaction was carried out at room temperature for 5 minutes, then stopped with sulfuric acid. Finally, the absorbance was measured at 450 nm.

[0063] (4) The binding ability of each RSV F protein mutant to Palivizumab was detected in the supernatant of the harvested HEK293T cells using the double-antibody sandwich method. The specific method is as follows: PalivizumabmAb was diluted to 5 μg / ml and coated onto 96-well plates, 50 μl per well. The plates were incubated overnight at 4°C. Afterwards, the plates were washed three times with phosphate-buffered saline (PBST) containing Tween 20, blocked with 100 μl of 5% skim milk powder / PBS solution at room temperature for 1 hour, and washed again with PBST.

[0064] 100 μl of HEK293T cell supernatant transiently overexpressing each RSV F protein mutant was added to each well, and the cells were incubated at 37°C for 2 hours. After washing three times, 100 μl of HRP-labeled His tag antibody diluted 1:4000 was added to each well, and the cells were incubated at 37°C for 1 hour. After washing again, tetramethylbenzidine (TMB) and hydrogen peroxide solution were added, and the reaction was carried out at room temperature for 5 minutes, then stopped with sulfuric acid. Finally, the absorbance was measured at 450 nm.

[0065] 2. Experimental Results The binding affinity of each RSV F protein mutant to D25 and to Pavilionb is shown in the table below. Figure 4 F in the middle.

[0066] The results showed that SC1–SC9 had a much higher binding capacity to D25 than ordinary flexible linkers and rigid linkers (NC1–NC6) and unmodified WT-F. However, in terms of the binding capacity to Pavilizumab, apart from NC1 and NC5, there was no significant difference between the RF Diffusion design, ordinary flexible linkers and rigid linkers (NC1–NC6), and unmodified WT-F. This indicates that the RF Diffusion design stabilized most of the protein in its pre-fusion conformation, and SC3 to SC9 were selected as the seven designs for subsequent disulfide bond mutation site design.

[0067] III. Second round of modification of RSV F protein mutant (introduction of disulfide bond mutation) and antigenicity evaluation 1. Experimental Methods To further limit the local structural flexibility of Region 1, new cysteine ​​pairing sites are introduced on top of SC3 to SC9, causing additional disulfide bonds to form in the local protein structure, thereby limiting the protein's structural flexibility and improving the stability of the prefusion conformation. Figure 4 In G, the red area represents the newly designed disulfide bond structure.

[0068] The specific information on the RSV F protein mutant is shown in Table 2: Table 2:

[0069] Following the method described above, the 18 RSV F protein mutants in Table 2 were expressed on a small scale by transiently transfecting HEK293T cells. The supernatant of HEK293T cells was harvested to detect the binding ability of the 18 RSV F protein mutants to D25 and Palivizumab, respectively.

[0070] 2. Experimental Results The binding affinity of each RSV F protein mutant to D25 and to Pavilionb is shown in the table below. Figure 4 H in the middle.

[0071] The results showed that all 18 RSV F protein mutants could bind to D25 and Pavilizumab. Regarding binding to Pavilizumab, most RSV F protein mutants showed little difference in stability. However, regarding binding to D25, only SC7-CC11, SC7-CC12, SC7-CC13, SC7-CC14, SC8-CC15, SC8-CC16, SC8-CC17, and SC9-CC18 exhibited superior stability. The SC7-CC11, SC7-CC14, and SC8-CC17 mutants were selected for subsequent experiments.

[0072] IV. Oligomeric State Optimization and Stability Evaluation 1. Experimental Methods (1) Design of RSV F protein mutants SC7-CC11, SC7-CC14, and SC8-CC17 can form trimers due to the presence of the T4-foldon trimerization domain at residue F1 513. Based on SC7-CC11, SC7-CC14 and SC8-CC17, the C-terminal trimerization motif was removed to obtain M-SC7-CC11, M-SC7-CC14 and M-SC7-CC17; Furthermore, Gv-Tag tags were added to the C-terminus of M-SC7-CC11, M-SC7-CC14, and M-SC7-CC17 (resulting in M-SC7-CC11-Gv, M-SC7-CC14-Gv, and M-SC7-CC17-Gv). Figure 4 As shown in I), so as to couple with HPF (connected to SD) and assemble into nanoparticles.

[0073] See Table 3 for details.

[0074] Table 3

[0075] (2) Expression of RSV F protein mutant Following the method described above, the coding sequences of various RSV F protein mutants in Table 3 were cloned into the pcDNA3.1 plasmid, and a 6× His tag was added to the C-terminus. Small-scale expression was then achieved by transiently transfecting HEK293T cells, and the HEK293T cell supernatant was collected. (3) Storage stability assessment of RSV F protein mutant Following the method described above, the various RSV F protein mutants listed in Table 3, along with WT-F (amino acid sequence as shown in SEQ ID NO: 14) and DS-Cav1 (amino acid sequence as shown in SEQ ID NO: 13), were subjected to three freeze-thaw cycles or stored at 4°C for two weeks. Afterward, the binding affinity of the RSV F protein mutants to D25 was measured using HEK293T cell supernatant as a sample, as described above. The ratio of antibody reactivity between the treated and untreated samples was defined as the storage stability of the RSV F protein mutants.

[0076] (4) Heat stress assessment of the stability of RSV F protein mutants.

[0077] Following the method described above, the various RSV F protein mutants listed in Table 3, along with WT-F (amino acid sequence as shown in SEQ ID NO: 14) and DS-Cav1 (amino acid sequence as shown in SEQ ID NO: 13), were incubated at 50°C or 60°C for 1 hour. Afterward, the binding affinity of the RSV F protein mutants to D25 was measured using HEK293T cell supernatant as a sample, following the method described above. The ratio of antibody reactivity between the treated and untreated samples was defined as the storage stability of the RSV F protein mutants.

[0078] 2. Experimental Results Experimental results are as follows Figure 4 As shown in J and Table 4, all RSV F mutants exhibited stronger binding affinity to D25 compared to WT-F. Regarding heat stress, monomers showed stronger binding affinity than trimers, with M-SC7-CC14 and M-SC7-CC14-Gv showing the strongest binding. All of the above demonstrates that the design of the M-SC7-CC14 and M-SC7-CC14-Gv mutants allows the RSVF protein to be well stabilized in its pre-fusion conformation. The M-SC7-CC14-Gv mutant is named "SP-VE", and the SC-CC14 mutant is named "SP-VE-T4".

[0079] Table 4

[0080] RSV F protein is expressed in its precursor form F0, and during maturation, it is cleaved at two protease cleavage sites. Figure 5 (As shown by the middle arrow), F1 and F2 subunits are formed, which are connected by disulfide bonds to maintain the fused active structure.

[0081] Figure 5 The upper sequence alignment area shows the structural remodeling results obtained using the RFdiffusion protein redesign method: using the RSV A2 strain F0 protein fragment as a reference sequence, the target flexible region was reconstructed using computation-assisted protein redesign. The orange-marked areas represent newly added or replaced amino acid sequences relative to the natural RSV A2 F0 sequence. By introducing artificially designed sequences, the local structural regions of the pre-fusion F protein were optimized to improve pre-fusion conformational stability.

[0082] Figure 5 In the lower sequence alignment region, an engineered design scheme in the form of soluble truncated monomers is shown: while retaining the key pre-fusion F protein structural region, the transmembrane domain and related membrane anchoring regions are removed, and a C-terminal Gv tag and a trimerized structural motif derived from fibritin (marked in red) are further introduced to achieve stable protein expression, oligomeric state regulation, and subsequent nanoparticle assembly.

[0083] Among them, SP-VE is the engineered pre-fusion form of F protein without the foldon domain; SP-VE-T4 is the engineered pre-fusion form of F protein containing the T4 foldon trimerization domain; WT-F is the unmodified T4 foldon trimerization F protein, which is obtained by removing the transmembrane domain from F0 and introducing T4 Folden at the C-terminus.

[0084] The amino acid sequence of F0 is shown in SEQ ID NO:1; the amino acid sequence of Gv-Tag is shown in SEQ ID NO:2; the amino acid sequence of T4-foldon is shown in SEQ ID NO:3; the amino acid sequence of M-SC7-CC11 is shown in SEQ ID NO:4; the amino acid sequence of M-SC7-CC14 is shown in SEQ ID NO:5; the amino acid sequence of M-SC8-CC17 is shown in SEQ ID NO:6; the amino acid sequence of M-SC7-CC11-Gv is shown in SEQ ID NO:7; the amino acid sequence of M-SC7-CC14-Gv is shown in SEQ ID NO:8; the amino acid sequence of M-SC8-CC17-Gv is shown in SEQ ID NO:9; the amino acid sequence of SC7-CC11 is shown in SEQ ID NO:10; the amino acid sequence of SC7-CC14 is shown in SEQ ID NO:11; the amino acid sequence of SC8-CC17 is shown in SEQ ID NO:12; and the amino acid sequence of DS-Cav1 is shown in SEQ ID NO:1. As shown in NO:13, the amino acid sequence of WT-F is shown in SEQ ID NO:14, the amino acid sequence of D25 is shown in SEQ ID NO:15, and the amino acid sequence of Palivizumab is shown in SEQ ID NO:16.

[0085] SEQ ID NO:1 (F0) MELLILKANAITTILTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN (three mutations P102A, I379V and M447V are introduced on the basis of native A2); SEQ ID NO:2 (Gv-Tag) SKVGNTIVMVDKLKEVPTPGSG; SEQ ID NO:3 (T4-foldon) GYIPEAPRDGQAYVRKDGEWVLLSTFL; SEQ ID NO:4 (M-SC7-CC11) MELGLSWIFLLAILKGVQCGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATSPCVELTGDKSCEEAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL; SEQ ID NO:5(M-SC7-CC14) MELGLSWIFLLAILKGVQCGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATSPLCELTGDKSLCEAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL; SEQ ID NO:6(M-SC8-CC17) MELGLSWIFLLAILKGVQCGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATEPLCALSPNKEECAAAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL; SEQ ID NO:7(M-SC7-CC11-Gv) MELGLSWIFLLAILKGVQCGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATSPCVELTGDKSCEEAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLGGGGGGSGGGGSKVGNTIVMVDKLKEVPTPGSG; SEQ ID NO:8(M-SC7-CC14-Gv) MELGLSWIFLLAILKGVQCGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATSPLCELTGDKSLCEAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLGGGGGGSGGGGSKVGNTIVMVDKLKEVPTPGSG; SEQ ID NO:9(M-SC8-CC17-Gv) MELGLSWIFLLAILKGVQCGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATEPLCALSPNKEECAAAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRRLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLGGGGGSGGGGSKVGNTIVMVDKLKEVPTPGSG; SEQ ID NO:10(SC7-CC11) MELGLSWIFLLAILKGVQCGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATSPCVELTGDKSCEEAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGG; SEQ ID NO:11(SC7-CC14) MELGLSWIFLLAILKGVQCGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATSPLCELTGDKSLCEAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGG; SEQ ID NO:12(SC8-CC17) MELGLSWIFLLAILKGVQCGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATEPLCALSPNKEECAAAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGG; SEQ ID NO:13(DS-Cav1) MGILPSPGMPALLSLVSLLSVLLMGCVAGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGS; SEQ ID NO:14(WT-F) MGILPSPGMPALLSLVSLLSVLLMGCVAGSGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGS; SEQ ID NO:15(D25) MEFGLSWVFLVALFRGVQCDIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSGATNFSLLKQAGDVEENPGPMGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; SEQ ID NO:16(Palivizumab) .

[0086] Example 2 Preparation of protein nanoparticles The respiratory syncytial virus antigen SP-VE fusion protein was assembled onto the surface of HPF-based nanoparticles to form respiratory syncytial virus antigen-twenty-tetrameric complex SP-VE-NP protein nanoparticles.

[0087] I. Experimental Methods 1. The expression of respiratory syncytial virus antigen SP-VE fusion protein is shown in Example 1.

[0088] 2. Preparation of HPF fusion protein HPF protein was expressed and purified in Escherichia coli (E. coli) using the following method: (1) Construction of recombinant vector The encoding DNA sequence of the HPF fusion protein (linked with SD) shown in SEQ ID NO.17 was cloned into the prokaryotic expression vector PET28a to construct the expression vector PET28a-HPF.

[0089] SEQ ID NO.17 (HPF) SGETGQSGNTTIEEDSTTHVKFSKRDINGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDSTLIVGDDPI GGGGSGGGGSDIIKLLNEQVNKEMQSSNLYMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSLER.

[0090] (2) Expression of HPF fusion protein The expression vector PET28a-HPF was transformed into BL21 (Takara) cells; then, single clones were amplified by shaking culture in LB medium containing kanamycin at 37°C; isopropyl-β-D-thiogalactoside (IPTG) was added to the bacterial culture to induce protein expression; after 18 hours of induction, the bacteria expressing the protein were harvested and subjected to high-pressure disruption.

[0091] (3) Purification of HPF fusion protein The supernatant was initially purified using a 10K ultrafiltration tube (Millipore), followed by incubation on a Ni-NTA agarose column (GE) to enrich the His-labeled HPF fusion protein. The obtained HPF fusion protein was washed with Tris buffer containing 500 mM Imidazole (20 mM Tris-HCl and 50 mM NaCl, pH 7.5) to remove the protein. The purified protein was concentrated, and the buffer was replaced with standard Imidazole-free Tris buffer (20 mM Tris-HCl and 50 mM NaCl, pH 7.5). Finally, the concentration of the HPF fusion protein was determined using the BCA assay.

[0092] 3. Self-assembly of respiratory syncytial virus nanoparticle antigens The prepared SP-VE fusion protein (SEQ ID NO.8) and HPF fusion protein (SEQ ID NO.17) were placed in a buffer solution (20 mM Tris-HCl, 50 mM NaCl, pH 7.5) without any enzymes and incubated at 25°C for 15 hours. The SP-VE fusion protein and HPF fusion protein were covalently bound through the GvTagOpti / Sdcatcher system (Gv / Sd, see Chinese Patent CN113621031A) to prepare 24-tetrameric complex SP-VE-NP protein nanoparticles.

[0093] HPF fusion protein (SEQ ID NO.17) was placed in a buffer solution (20 mM Tris-HCl, 50 mM NaCl, pH 7.5) without any enzymes and incubated at 25°C for 15 hours to prepare 24-tetrameric HPF protein nanoparticles.

[0094] Then, the SP-VE-NP protein nanoparticles were purified by gel chromatography using a Siperose6 Increase10 / 300 GL column (GE) to obtain high-purity tetratrimethylenetetrameric complex (SP-VE-NP). The storage solvent for the purified SP-VE-NP protein nanoparticles after gel chromatography was a buffer solution containing 20 mM Tris-HCl and 50 mM NaCl at pH 7.5.

[0095] Subsequently, the prepared WT-F, DS-Cav1, SP-VE-T4 (SC7-CC14), HPF protein nanoparticles (tetrameric complex), SP-VE fusion protein, and SP-VE-NP protein nanoparticles (tetrameric complex) were subjected to SDS-PAGE electrophoresis.

[0096] 4. Transmission electron microscopy (TEM) detection Negative staining transmission electron microscopy (TEM) was performed using a nanoparticle-supported grid of the 24-tetrameric complex SP-VE-NP protein. 5 μL of each sample (0.1 μg / μL) was added to a TEM grid coated with a continuous carbon film after glow discharge treatment, and negatively stained with 2% uranium acetate. Images were then acquired using a Thermo Fisher Scientific Talos TEM at an accelerating voltage of 120 kV; the imaging magnification was 73,000x, and the defocus was set to 1.5 μm.

[0097] 5. Dynamic Light Scattering (DLS) Detection The hydrodynamic particle size of SP-VE-NP and HPF was determined using a Bettersize nanoparticle size and Zeta potential analyzer (Bettersize Instrument Co., Ltd.) based on the principle of dynamic light scattering (DLS). The specific methods are as follows: The refractive index (RI) and absorption coefficient of the material were set to 1.45 and 0.001, respectively; the refractive index and viscosity of the dispersion medium were set to 1.33 and 0.8936 mPa·s, respectively. A 671 nm laser was used for analysis, with a scattering angle of 173°. Data acquisition was performed in general mode, with an equilibration time of 120 s. The final reported particle size data is the average hydrodynamic diameter obtained based on number distribution analysis.

[0098] The measurements were taken at a constant temperature of 25 ℃.

[0099] 6. Size exclusion chromatography (SEC) detection The test samples were incubated in phosphate-buffered saline (PBS) at room temperature for 70 hours. After incubation, size exclusion chromatography (SEC) was performed for separation, with an elution flow rate of 0.5 mL / min. The target protein elutes in the range of 10–12 mL elution volume.

[0100] II. Experimental Results See the schematic diagram of the SP-VE-NP structure. Figure 6 Using SDS-PAGE results Figure 7 As shown, WT-F, DS-Cav1, SC7-CC14 (SP-VE-T4), HPF protein nanoparticles (a tetratrimester complex), SP-VE fusion protein, and SP-VE-NP protein nanoparticles (a tetratrimester complex) were successfully prepared, and their sizes were consistent with expectations.

[0101] Transmission electron microscopy (TEM) Figure 8 (A) Dynamic light scattering (DLS), Figure 8 (B) and size exclusion chromatography (SEC) Figure 8 The results from both C and D studies confirm that SP-VE-NP exhibits high purity, good particle uniformity, and forms a uniformly shaped nanoparticle population. Furthermore, it possesses excellent protein purity.

[0102] Example 3: Immunization of mice with muscle tissue using 24-tetrameric complex SP-VE-NP protein nanoparticles. I. Immunity in mice BALb / C mice were immunized by intramuscular injection to test the immunoprotective effect of the 24-tetrameric complex SP-VE-NP protein nanoparticle vaccine prepared in Example 2.

[0103] (1) Various immunogens and aluminum adjuvants are mixed in a mass ratio of 1:1 to obtain the vaccine for immunization.

[0104] The immunogens were: the 24-tetrameric complex SP-VE-NP protein nanoparticles prepared in Example 2, HPF prepared in Example 2, and WT-F and DS-Cav1 prepared in Example 1.

[0105] (2) Immune strategy Figure 9 In section A): Each mouse was inoculated with 10 μg of antigen and immunized twice (on day 0 and day 28). Serum was collected on day 42.

[0106] II. Neutralization Experiment (FRNT) 50 ) 1. Experimental Methods Human respiratory syncytial virus (RSV) 300 PFU (purchased from ATCC, VR-1540) was mixed with collected serum (diluted) at a 1:1 ratio and incubated at 37°C for 1 hour. The mixture was then added to confluent Hep-2 cells and incubated for 1 hour. After three days, cells were fixed with 4% paraformaldehyde (PFA). RSV infection was detected by fluorescence focusing assay using palilizumab as the capture antibody and Alexa Fluor® 488-labeled goat anti-human IgG as the detection antibody. Fluorescently labeled virus spots were counted using a CTL Immunospot analyzer. FRNT50 titers were determined by nonlinear regression analysis of the inhibition rate in the dilution series using GraphPad Prismv.9.0.

[0107] 2. Experimental Results See results Figure 9 The antibodies induced by B, SP-VE-NP in FRNT exhibit excellent neutralizing ability against wild-type RSV A2 strains. 50 The potency reached 447.7, which was higher than that of the DS-Cav1 group (218.0).

[0108] The above data demonstrate that the 24-tetrameric complex SP-VE-NP protein nanoparticles prepared in Example 2 have excellent neutralizing and protective efficacy.

[0109] Example 4: Muscle immunization of mice and cotton rats with 24-tetrameric complex SP-VE-NP protein nanoparticles. I. Immunity in mice and cotton rats BALb / C mice and cotton mice were immunized via nasal and intramuscular immunization (im) through the respiratory system to test the immunoprotective effect of the 24-tetrameric complex SP-VE-NP protein nanoparticle vaccine prepared in Example 2.

[0110] (1) Prepare the immunogen and aluminum adjuvant at a mass ratio of 1:1 to obtain the vaccine for intramuscular injection.

[0111] The immunogen was the 24-tetrameric complex SP-VE-NP protein nanoparticles prepared in Example 2.

[0112] (2) Prepare a vaccine for nasal immunization by mixing the immunogen with C-di-GMP adjuvant at a dose of 5 μL per mouse.

[0113] The immunogen was the 24-tetrameric complex SP-VE-NP protein nanoparticles prepared in Example 2.

[0114] Immune strategy ( Figure 10 In section A): Each mouse and cotton swab was immunized with a dose of 10 μg of antigen using a two-dose immunization strategy (day 0 and day 28). Serum was collected on day 42, and the experimental animals were challenged with respiratory syncytial virus A2 strain (RSV A2) (BALB / c 1.2 × 10⁻⁶ per animal). 6 PFU, 1.0 × 10⁻⁶ per cotton mouse 5 PFU); Animals were euthanized 3 days after challenge with BALB / c or 5 days after challenge with cotton rats, and lung tissue was separated and harvested for final testing.

[0115] PBS served as the control group, with the vehicle as the reference.

[0116] II. Neutralization Experiment (FRNT) 50 ) 1. Experimental Methods Human respiratory syncytial virus (RSV) 300 PFU (purchased from ATCC, VR-1540) was mixed with collected serum (diluted) at a 1:1 ratio and incubated at 37°C for 1 hour. The mixture was then added to confluent Hep-2 cells and incubated for 1 hour. After three days, cells were fixed with 4% paraformaldehyde (PFA). RSV infection was detected by fluorescence focusing assay using palilizumab as the capture antibody and Alexa Fluor® 488-labeled goat anti-human IgG as the detection antibody. Fluorescently labeled virus spots were counted using a CTL Immunospot analyzer. FRNT50 titers were determined by nonlinear regression analysis of the inhibition rate in the dilution series using GraphPad Prismv.9.0.

[0117] 2. Experimental Results The results show that ( Figure 10 Consistent with B and C in the study, mice and cotton rats inoculated with SP-VE-NP via intranasal (in) or intramuscular (im) administration showed significantly elevated serum neutralizing antibody titers compared to the control group. In BALB / c mice, there was no significant difference in the neutralizing capacity of antibodies induced by intramuscular (im) and respiratory (in) administration against the RSV A2 strain, but in cotton rats, intramuscular (im) administration induced significantly higher neutralizing antibodies than respiratory (in) administration.

[0118] III. Viral load detection 1. Experimental Methods Lung tissue samples were collected from BALB / c mice and cotton rats after infection and challenge, and the tissues were homogenized. Total RNA was then extracted using the RNeasy Mini Kit (QIAGEN, catalog number: 4104) according to the kit instructions.

[0119] The extracted RNA was further detected by quantitative reverse transcription polymerase chain reaction, and the viral RNA copy number in lung tissue was analyzed using the AceQ qPCRProbe Master Mix Kit (PCR-Fluorescence Probing) (Vazyme, Q112-02).

[0120] For quantitative calibration, a respiratory syncytial virus (RSV) nucleoprotein (N) gene fragment (SEQ ID NO:18) was cloned and inserted into the pcDNA3.1 vector to construct a standard template and establish a standard curve. The N gene standard with known copy numbers was serially diluted 10-fold and then detected using the same qRT-PCR kit to obtain the standard curve.

[0121] The number of RSV N gene RNA copies in the sample was calculated based on the standard curve to assess the viral load in the lung tissue after infection.

[0122] SEQ ID NO:18 (Nucleoprotein gene standard) AACCTAGACATCGCATATAACATACCTATTAACCCAGTGAATTTATGATTAGCATCTTCTGTGATTAATAACATGCCACATAACTTATTGATGTGTTTCTGCACATCATAATTAGGAGTATCAATACTATCTCCTGTGCT CCGTTGGATGGTGTATTTGCTGGATGACAGAAGTTGATCTTTGTTGAGTGTATCATTCAACTTGACTTTGCTAAGAGCCATCTTTGTATTTGCCCCATCTTCTATCTTATATCTCTCCTTAATTTTAAATTACTATAATT.

[0123] 2. Experimental Results The results show that ( Figure 10 (D and E in the data), viral load assays revealed that SP-VE-NP immunization effectively protected BALB / c mice and cotton rats from RSV infection. Compared with the control group, the RSV viral replication level in the lung tissue of the SP-VE-NP immunized group was significantly reduced, indicating that the vaccine can induce an effective protective immune response against RSV.

[0124] In summary, the SP-VE-NP vaccine can induce potent protection against RSV infection, with nasal administration showing particularly outstanding inhibitory effects on viral replication in the respiratory tract.

Claims

1. A fusion protein, characterized in that, Its amino acid sequence is shown as any one of SEQ ID NO:4 to 12.

2. A nucleic acid molecule, characterized in that, The encoding gene of the fusion protein of claim 1, or the reverse complementary sequence of the encoding gene.

3. A biomaterial, characterized in that, It is any one of the following: (1) An expression cassette containing the nucleic acid molecule of claim 2; (2) A recombinant vector containing the nucleic acid molecule of claim 2 or the expression cassette of (1); (3) A recombinant microorganism containing the nucleic acid molecule of claim 2, the expression cassette of (1), or the recombinant vector of (2); (4) A cell line containing the nucleic acid molecule of claim 2, the expression cassette of (1), the recombinant vector of (2), or the recombinant microorganism of (3).

4. A fusion protein composition, characterized in that, This includes HPF fusion proteins with amino acid sequences as shown in SEQ ID NO.17 and fusion proteins with amino acid sequences as shown in any one of SEQ ID NO.7 to 9.

5. A protein nanoparticle, characterized in that, It was obtained by self-assembly using the HPF fusion protein with the amino acid sequence shown in SEQ ID NO.17 and the fusion protein with the amino acid sequence shown in any one of SEQ ID NO.7 to 9.

6. A method for preparing protein nanoparticles, characterized in that, The HPF fusion protein with the amino acid sequence shown in SEQ ID NO.17 and any of the fusion proteins shown in SEQ ID NO.7 to 9 are obtained by incubating in a buffer solution.

7. The use of the protein nanoparticles according to claim 6 in the preparation of a novel coronavirus vaccine.

8. A composition, characterized in that, It contains the fusion protein composition of claim 4 and an adjuvant.

9. A composition, characterized in that, It contains the protein nanoparticles of claim 5 and an adjuvant.

10. Use of the composition of claim 8 or 9 in the preparation of a syncytial virus vaccine.

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