Construction and application of RSV F-eVLP that spontaneously forms enveloped virus-like particles and its mRNA vaccine, adenovirus vector vaccine and recombinant protein vaccine

Abstract

The present invention provides a kind of RSV F-eVLP that spontaneously forms enveloped virus-like particles and its mRNA vaccine, adenocarcinoma vector vaccine and the construction and application of recombinant protein vaccine, which belongs to the field of innovative vaccine technology. The coding sequence of the RSV F protein spontaneously forming enveloped virus-like particles provided by the present invention is composed of the coding sequence of the RSV F protein or its mutant and the coding motif fusion related to the formation of enveloped virus-like particles. The vaccine with the coding RSV F protein that spontaneously forms enveloped virus-like particles, compared with the vaccine of the RSV F protein or its mutant without the motif related to the formation of enveloped virus-like particles, has a stronger and more lasting RSV neutralizing antibody level and virus-specific memory B and memory T cell immune response induced in the mouse model, thereby achieving efficient and long-term protection against RSV infection and morbidity.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and in particular to a RSV F mutant that spontaneously forms enveloped virus-like particles and the construction and application of its mRNA vaccine, vector vaccine and recombinant protein vaccine. Background Art

[0002] Respiratory syncytial virus (RSV) is one of the most common pathogens causing acute respiratory infections in infants, the elderly, and immunocompromised individuals. In 2019, nearly 32 million children under the age of five worldwide were infected with RSV and developed lower respiratory tract infections, 10% of whom required hospitalization and were prone to developing obstructive airway disease and asthma. RSV also imposes a significant burden on immunocompromised and elderly individuals. Furthermore, RSV infection fails to elicit a sustained antibody response, leading to reinfection and a significant global medical and economic burden. RSV belongs to the family Paramyxoviridae, the subfamily Pneumovirinae, and the genus Pneumovirus. It is a nonsegmented, single-stranded, negative-sense RNA enveloped virus. The virion consists of a lipid envelope encapsidated by the viral nucleocapsid. Virions can be spherical with a diameter of 100-350 nm or fibrillar with a diameter of 60-200 nm. The virus particle consists of an envelope, a nucleocapsid, and a core. The envelope is inlaid with three transmembrane proteins: G, F, and SH; the nucleocapsid is composed of capsomeres; and the viral core is a complex composed of viral genomic RNA and nonstructural proteins such as N, P, and L. The F and G proteins mediate contact and fusion between the virus and cells during RSV infection and are currently the primary targets of vaccine design. The F protein is highly conserved among different subtypes, while the G protein is highly variable. RSV is divided into subtypes A and B based on differences in the sequence of the second hypervariable region of the G protein. Existing research indicates that the F protein exists in at least two states in the body: the metastable prefusion F protein (PreF) and the stable postfusion F protein (PostF).

[0003] Vaccines are an effective means of preventing RSV infection. mRNA plays a key role in the translation of protein-encoding deoxyribonucleic acid (DNA) into protein. mRNA vaccines utilize the host cell's protein synthesis machinery to translate mRNA into the corresponding antigen within the cell, thereby mimicking the viral infection process and inducing effective humoral and cellular immune responses. Their advantages, including safety, strong immunogenicity, rapid production, and wide application, have rapidly become a hot topic in the biopharmaceutical industry. With the outbreak of SARS-CoV-2, mRNA vaccines were first approved for human use, and their safety and efficacy have been widely demonstrated. Existing studies have demonstrated that mRNA vaccines based on the RSV F protein elicit strong humoral immune responses in vivo. Furthermore, compared to traditional protein vaccines, mRNA vaccines also induce robust CD4+ and CD8+ T cell responses, suggesting potential advantages for mRNA vaccines in activating cellular immune responses.

[0004] eVLP (enveloped virus-like particle) vaccines present viral antigens on the cell surface or as virus-assembled nanoparticles, which is more conducive to B cell activation and induces strong B cell immune responses, potentially leading to long-term protection. eVLP vaccines enable precise antigen presentation. Through genetic engineering, specific antigens can be displayed on the VLP surface in an optimal conformation. eVLPs can also carry multiple antigens or epitopes simultaneously, achieving multi-antigen synergistic stimulation of the immune system. These diverse antigen combinations mimic the complex antigenic composition of viruses, stimulating a comprehensive and long-lasting immune response and increasing the vaccine's protective efficacy against different viral strains or variants. eVLP vaccines not only activate adaptive immunity but also innate immunity. Their virus-like structure is recognized by pattern recognition receptors (PRRs) of the innate immune system, such as Toll-like receptors (TLRs). When eVLPs are taken up by antigen-presenting cells, they trigger a series of innate immune signaling pathways, releasing cytokines and chemokines, creating a favorable environment for the activation of adaptive immunity. This dual activation of innate and adaptive immunity allows for more effective viral clearance and enhances the vaccine's immune efficacy.

[0005] Human clinical studies have shown that existing PreF-based mRNA vaccines do not provide strong sustained protection, possibly because they do not induce long-lasting memory T cell and B cell responses. They overstimulate short-term antibody responses without fully activating the generation of memory cells. Memory B cells and memory T cells are essential for long-term immunity. If the vaccine cannot effectively induce the formation and maintenance of these cells, the immune system may not be able to respond quickly and effectively when exposed to RSV again. Therefore, combining mRNA technology with eVLP technology can induce stronger neutralizing antibody levels and memory B and memory T cell immune responses in mouse models than mRNA vaccines.

[0006] ESCRT can recruit related pathway proteins to target proteins, causing them to self-assemble into eVLPs. This pathway is driven by the EGE motif. The EGE motif consists of three parts: EPM, GS linker, and EABR. The EPM is used to retain the target protein on the cell surface to prevent endocytosis. The GS linker connects the EPM and EABR. The EABR contains one or both of the ALIX and TSG101 binding domains. However, different EGE motifs induce different immune effects. Therefore, the selection of the EGE motif is of paramount importance in improving the immune efficacy of RSV mRNA vaccines. Summary of the Invention

[0007] The purpose of the present invention is to provide a new, efficient and highly safe RSV mRNA vaccine to address the problems of insufficient RSV mRNA vaccines and insufficient long-term protection in the existing technology. The antigens produced by this technology can also be widely used in adenovirus vectors.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a RSV PreF-eVLP that spontaneously forms enveloped virus-like particles, wherein the target gene in the PreF-eVLP mainly consists of a nucleotide sequence of RSV F protein and an EGE motif.

[0010] Preferably, the RSV F protein is the original F protein, and the amino acid sequence of the original F protein is shown in SEQ ID NO: 1.

[0011] Preferably, the RSV F protein is a modified F protein mutant.

[0012] Preferably, the modified F protein mutant includes an optimized PreF protein or a truncated PreFΔCT protein.

[0013] Preferably, the optimization method of the optimized PreF protein comprises one or more of signal peptide replacement, P27 fragment deletion and amino acid mutation.

[0014] Preferably, the replacement signal peptide includes one of the signal peptide sequences shown in SEQ ID NOs: 10 to 14; the amino acid mutation site includes one or more of N67I, R106Q, F137S, S215P and E487Q.

[0015] Preferably, the amino acid sequence of the optimized PreF protein is shown in SEQ ID NOs: 2, 4, 6, and 8, and the corresponding nucleotide sequences are shown in SEQ ID NOs: 3, 5, 7, and 9, respectively.

[0016] Preferably, the truncated PreFΔCT protein is truncated with any 1 to 23 amino acids at the C-terminus.

[0017] Preferably, the amino acid sequence of the truncated PreFΔCT protein is shown in SEQ ID NO: 15, and the nucleotide sequence of the truncated PreFΔCT protein is shown in SEQ ID NO: 16.

[0018] Preferably, the EGE motif includes EPM, linker and EABR; the EPM is the nucleotide sequence shown in any one of SEQ ID NOs: 18, 20, and 22; the linker is GS, (GS)n or (GGGGS)n, wherein n represents the number of repetitions, n=1~4; the EABR is the nucleotide sequence shown in any one of SEQ ID NOs: 24, 26, 28, 30, 32, 34, and 36.

[0019] Preferably, the EPM is the nucleotide sequence shown in SEQ ID NO: 18, the linker is GS, and the EABR is the nucleotide sequence shown in SEQ ID NO: 36.

[0020] The present invention also provides a method for constructing RSV PreF-eVLP that spontaneously forms enveloped virus-like particles, wherein the target gene is constructed into an expression plasmid, cell transfection is performed, the cells are cultured and the supernatant is collected to obtain RSV PreF-eVLP that spontaneously forms enveloped virus-like particles.

[0021] The present invention also provides a use of the truncated PreFΔCT protein in the RSV PreF-eVLP that spontaneously forms enveloped virus-like particles, wherein the use of the truncated PreFΔCT protein is to increase the expression level of the F protein on the membrane.

[0022] The present invention also provides an RSV mRNA vaccine platform constructed by the spontaneously formed enveloped virus-like particles RSV PreF-eVLP, characterized in that the RSV mRNA vaccine contains the target gene.

[0023] Preferably, the nucleotide sequence of the RSV mRNA is shown as SEQ ID NO: 37 or SEQ ID NO: 38; the nucleotide sequence of the optimized RSV mRNA is shown as SEQ ID NO: 39.

[0024] The present invention also provides a method for constructing the RSV mRNA vaccine platform, including but not limited to the following steps:

[0025] (1) constructing the nucleotide sequence of the target gene or RSV mRNA into an expression vector to obtain a recombinant plasmid;

[0026] (2) linearizing the recombinant plasmid and using it as a transcription template to obtain in vitro transcribed RSV mRNA;

[0027] (3) The in vitro transcribed RSV mRNA is digested, capped, purified, and then encapsulated in liposomes to obtain an RSV mRNA vaccine.

[0028] Preferably, the expression vector is pCDNA3.1(+) expression plasmid, and the liposome is liposome SM102.

[0029] The present invention also provides a liposome RSV mRNA vaccine obtained by the construction method, wherein the liposome RSV mRNA vaccine is in the form of liquid or freeze-dried powder.

[0030] The present invention also provides an RSV adenovirus vector vaccine comprising the spontaneously formed enveloped virus-like particles RSV PreF-eVLP.

[0031] Preferably, the adenovirus vectors in the adenovirus vector vaccine include human adenovirus vectors and chimpanzee adenovirus vectors; the human adenovirus vectors include but are not limited to human adenovirus type 5 and human adenovirus type 26; the chimpanzee adenovirus vectors include but are not limited to AdC9 and AdC68.

[0032] Preferably, the nucleotide sequence of the target gene in the RSV adenovirus vector vaccine is shown as SEQ ID NO: 43 or SEQ ID NO: 45, and the amino acid sequence is shown as SEQ ID NO: 44 or SEQ ID NO: 46.

[0033] The present invention also provides the use of the RSV PreF-eVLP that spontaneously forms enveloped virus-like particles, the RSV mRNA vaccine platform, the liposomal RSV mRNA vaccine, or the RSV adenovirus vector vaccine in the preparation of a respiratory syncytial virus vaccine.

[0034] This invention provides a novel VLP self-assembly technology that forms eVLPs by adding an EGE motif to the cytoplasmic terminus of the PreF protein. This technology, rather than introducing other backbone sequences for co-transfection, addresses the scientific issue of weak cellular immunity induction with single nanoparticle vaccines. It also leverages the advantages of VLP vaccines in antibody responses and enhances the B cell immune response of mRNA vaccines, addressing the issue of short-lasting protection with existing mRNA vaccines. This technology is the first to be applied to RSV and marks the first application of an RSV mRNA vaccine.

[0035] The present invention has the following beneficial effects compared to the prior art:

[0036] (1) The present invention successfully developed an eVLP technology based on RSV PreF protein, which can be self-assembled and classified as a type of extracellular vesicle.

[0037] (2) The present invention successfully established the eVLP technology, which can achieve the production of high-quality antibodies, and the neutralizing antibody titer is increased by 2.5 to 5 times compared with a single PreF.

[0038] (3) The present invention successfully combines eVLP technology with mRNA technology and adenovirus technology to construct RSV mRNA vaccine platforms and adenovirus platforms based on eVLP technology, which induce strong immune responses in mice and protect them from RSV attack. The protective dose is far lower than that of PreF. At the same time, the present invention can also combine eVLP technology with recombinant protein technology to obtain recombinant protein vaccines.

[0039] (4) Compared with the original PreF, the eVLP technology established in the present invention can significantly stimulate B cell immune responses, including germinal center B cells and memory B cells, providing ideas for further exploration and development of long-acting respiratory syncytial virus vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the plasmid construction in Example 1;

[0041] Figure 2The expression of PreF protein on the membrane surface and in the supernatant of the plasmid containing PreF, PreFΔCT, PreFΔCT-GE (SEQ ID NO: 8), PreFΔCT-EGE-5 or PreFΔCT-EGE-12 in Example 1;

[0042] Figure 3 The immunoelectron microscopy results of eVLPs expressed in the supernatant in Example 1 are shown in Figures 2 and 3. Both figures are immunoelectron microscopy images of eVLPs, showing different sizes.

[0043] Figure 4 The results of Western blotting for the identification of CD81, CD9, CD63, and Calnexin markers in supernatant eVLPs in Example 1 are shown;

[0044] Figure 5 This is the situation in Example 3 where mRNA-PreF and mRNA-eVLP stimulate mice to produce binding antibodies against PreF;

[0045] Figure 6 The results of Example 3 show the production of neutralizing antibodies against RSV Long (upper panel) and RSV B1 (lower panel) stimulated by mRNA-PreF and mRNA-eVLP in mice.

[0046] Figure 7 is the viral copy number in lung tissue and nasal tissue after challenge in Example 3;

[0047] Figure 8 HE and PAS staining results of lung tissue after challenge with poison in Example 3;

[0048] Figure 9 The pathological scoring results after the challenge in Example 3;

[0049] Figure 10 is the germinal center B cell response and follicular helper T cell response in Example 3, wherein A is the frequency of GC B cells in the draining lymph nodes, B is the frequency of F-specific GC B cells in the draining lymph nodes, and C is the frequency of follicular helper T lymphocytes in the spleen;

[0050] Figure 11 is the memory B cell response in Example 3;

[0051] Figure 12 This is the situation in Example 4 where AdC68-PreF and AdC68-eVLP stimulate mice to produce binding antibodies against PreF;

[0052] Figure 13 This is the situation in Example 4 where AdC68-PreF and AdC68-eVLP stimulated mice to produce neutralizing antibodies against PreF. DETAILED DESCRIPTION

[0053] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] In this example, the membrane-bound F protein (SEQ ID NO: 1) was used as the PreF protein. Its signal peptide was replaced with the tPA signal peptide. The P27 segment was deleted (RRELPRFMNYTLNNTKNTNVTLSKKRKRR) and replaced with a GSGSGR linker. Point mutations N67I, R106Q, F137S, S215P, and E487Q (underlined amino acids) were also added to maintain the prefusion conformation. The amino acid sequence of the optimized PreF protein is shown in SEQ ID NO: 2. Other optimized sequences are shown in SEQ ID NOs: 4, 6, and 8, but are not limited to these and are presented here for illustrative purposes only. eVLPs are formed by adding an EGE motif to the C-terminus of the PreF protein. The C-terminus was modified to increase the PreF protein's surface presentation and facilitate eVLP formation by arbitrarily truncating the C-terminal 1-23 amino acids (ARSTPVTLSKDQLSGINNIAFSN). The sequences involved in eVLP construction are shown below:

[0056] F protein amino acid sequence (NCBI: APW77972.1, SEQ ID NO: 1)

[0057] MELLILKTNAITTILAAVTLYFASS QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKE N KCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANS R ARRELPRFMNYTLNNTKNTNVTLSKKRKRR F LGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI SNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSD E FDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN*。

[0058] Optimized amino acid sequence of PreF protein

[0059] Amino acid sequence of optimized Sequence 1, as shown in SEQ ID NO: 2:

[0060] MDAMKRGLCCVLLLCGAVFVSP QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANS Q ARGSGSGR S LGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI P NIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSD QFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN*.

[0061] The nucleotide sequence of the optimized sequence 1 is shown in SEQ ID NO: 3;

[0062] The amino acid sequence of the optimized sequence 2 is shown in SEQ ID NO: 4;

[0063] The optimized nucleotide sequence of sequence 2 is shown in SEQ ID NO: 5;

[0064] The amino acid sequence of the optimized sequence three is shown in SEQ ID NO: 6;

[0065] The nucleotide sequence of the optimized sequence three is shown in SEQ ID NO: 7;

[0066] The amino acid sequence of the optimized sequence four is shown in SEQ ID NO: 8;

[0067] The nucleotide sequence of the optimized sequence four is shown in SEQ ID NO:9.

[0068] Signal peptide sequence:

[0069] tPA signal peptide sequence: as shown in SEQ ID NO: 10. Alternative signal peptides also include:

[0070] Human insulin signal peptide sequence, as shown in SEQ ID NO: 11; Igκ light chain signal peptide, as shown in SEQ ID NO: 12; Human CD33 signal peptide sequence, as shown in SEQ ID NO: 13; Human IL-2 signal peptide sequence, as shown in SEQ ID NO: 14.

[0071] The amino acid sequence of the truncated PreFΔCT protein is shown in SEQ ID NO: 15, and the nucleotide sequence of the truncated PreFΔCT protein is shown in SEQ ID NO: 16.

[0072] The EPM sequence is as follows:

[0073] (1) Human low-affinity immunoglobulin gamma Fc region receptor II-b1:

[0074] ALPGYPECREMGETLPEKP (SEQ ID NO: 17); the corresponding nucleotide sequence is shown in SEQ ID NO: 18.

[0075] (2) Mouse low affinity immunoglobulin gamma Fc region receptor II-b1:

[0076] DLPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY (SEQ ID NO: 19); the corresponding nucleotide sequence is shown in SEQ ID NO: 20;

[0077] ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY (SEQ ID NO: 21); the corresponding nucleotide sequence is shown in SEQ ID NO: 22.

[0078] GS linker (n represents the number of repeats, n = 1-4): (GGGGS)n or (GS)n, the corresponding nucleotide sequences are (gggggaggaggaagt) n and (ggaagt) n

[0079] The EABR sequence is derived from one of the following sequences:

[0080] Table 1 EABR sequences and sources

[0081]

[0082] The nucleotide sequence of the above sequence was Figure 1 The fragments were constructed as shown and inserted into the pCDNA3.1(+) expression plasmid between the Not I and Kpn I restriction sites (synthesis commissioned by GenScript). PreF is the nucleotide sequence set forth in SEQ ID NO: 3; PreFΔCT is the nucleotide sequence set forth in SEQ ID NO: 16; the EABR sequence in PreFΔCT-GE is the nucleotide sequence set forth in SEQ ID NO: 24; the EPM and EABR combinations in PreFΔCT-EGE are shown in Table 2; all linkers used were GSLinker. The above sequence fragments were directly ligated and constructed into the pCDNA3.1(+) expression plasmid.

[0083] Table 2 Combination of EPM and EABR

[0084]

[0085] The above-constructed expression vector was expressed in vitro, identified, and eVLPs were detected by electron microscopy as follows:

[0086] a. Cell transfection

[0087] One day before transfection, 293T cells were cultured with DMEM+10% FBS complete medium at a rate of 4×10 5 Plate cells / well in a six-well plate and incubate overnight at 37°C, 5% CO2. The next day, cells were grown to 80% confluence for plasmid transfection. 2.5 μg / well of the constructed plasmid containing PreF, PreF△CT, PreF△CT GE, or PreF△CT EGE was added using the transfection reagent X-tremeGENE™ HP DNA:plasmid (μl:μg) ratio of 2:1. After gentle shaking, the cells were incubated at 37°C, 5% CO2 for 48 hours.

[0088] b. Enrichment of supernatant eVLPs

[0089] 48 hours after transfection, cells were centrifuged at 400 × g for 10 minutes. The cell pellet was lysed with lysis buffer and stored at -20°C until use. The supernatant was passed through a 0.45 mm syringe filter to further remove cellular debris and then ultracentrifuged at 135,000 × g for 2 hours over a 20% (w / v) sucrose cushion. The supernatant was removed, and the pellet was resuspended in PBS overnight. After overnight, the sample was centrifuged at 10,000 × g for 10 minutes, and the supernatant was collected again for analysis.

[0090] c. Western Blot detection of antigen expression

[0091] Electrophoresis sample preparation

[0092] 5x SDS loading buffer was added to the supernatant and the cell pellet samples to a final concentration of 1x. Before loading, the samples were heated at 100°C for 10 min to denature the proteins.

[0093] SDS-PAGE

[0094] Electrophoresis was performed using 5% stacking gel and 10% separating gel at 80 V for 30 min and 150 V for 30 min.

[0095] Transfer

[0096] Use PVDF membrane, 0.4mA, transfer for 1h, and control the heat with ice packs.

[0097] Blockade and immune response

[0098] The sections were blocked with 5% skim milk powder for 2 hours at room temperature, washed three times with TBST buffer, and hybridized overnight at 4°C with appropriately diluted primary antibody (human anti-RSV F). The sections were then washed three times with TBST on a rocking platform for 10 minutes each. The secondary antibody, goat anti-human HRP, was incubated on a rocking platform for 1 hour at room temperature. The sections were then washed three times with TBST for 10 minutes each.

[0099] Development and exposure

[0100] According to the instructions of the ECL kit, the color developing solution was evenly added to the membrane surface, and then exposed and detected in a chemiluminescence image analyzer.

[0101] The results are as follows Figure 2 As shown, the membrane-expressed protein PreF△CT remained expressed on the cell surface even after truncation, with increased cell surface expression. Addition of EABR allowed expression in the supernatant. Addition of the EPM motif increased the expression of PreF△CT-EGE-5 on the membrane surface and in the supernatant, while PreF△CT-EGE-12 expression on the membrane surface did not increase but decreased. This indicates that different EGE sequence combinations have unpredictable effects on PreF△CT protein expression. For the EPM and EABR combination, the EGE-5 sequence (Group 5 in Table 2) showed the highest expression among all combinations.

[0102] d. eVLP morphology identification

[0103] 1. Add an equal volume of 4% paraformaldehyde to the extracted supernatant sample (eVLP) and fix for at least 4 hours.

[0104] 2. Pipette 30 μl of liquid onto the wax film and negatively stain with a carbon-coated nickel grid for 30 minutes;

[0105] 3. Absorb excess liquid with filter paper and wash with 0.05M PB three times, 1 minute each time;

[0106] 4. Block with 0.05M glycine for 10 minutes and 1% BSA for 1 hour.

[0107] 5. Incubate with primary antibody (antibody titration method, negative control replaced with 1% BSA) for 1 hour, then overnight at 4°C;

[0108] 6. Wash the nickel mesh with PB 6 times, 1 min each time;

[0109] 7. Block with 1% BSA for 10 minutes and incubate with secondary antibody (1:100, 10 nm colloidal gold) for 3 hours;

[0110] 8. Wash with PB 6 times, 1 min each time;

[0111] 9. Fix with 2.5% glutaraldehyde for 15 minutes and wash four times with ddH2O;

[0112] 10. Stain with 2% uranyl acetate for 5 min, rinse four times with ddH2O, air dry, and observe and photograph using JEOL-1200.

[0113] The results are as follows Figure 3As shown, F protein particles (black particles in the figure) appear on the surface of eVLP, and the size of eVLP is between 100-150nm.

[0114] e. eVLP classification and identification

[0115] eVLPs originate from cells recruiting ESCRT-related pathway proteins to self-assemble into eVLPs, so WB was used to further identify eVLPs by staining CD81, CD9, CD63, and Calnexin markers. Figure 4 As shown, eVLP presents CD9+CD81+CD63+Calnexin-, which is a type of extracellular vesicle.

[0116] Example 2

[0117] The mRNA vaccine sequences are shown in SEQ ID NOs: 37 to 39. SEQ ID NO: 37 is the sequence of the RSV PreF mRNA vaccine, SEQ ID NO: 38 is the sequence of the RSV PreFΔCT-EGE mRNA vaccine (EGE-5 group), and SEQ ID NO: 39 is the sequence of the further optimized RSV PreFΔCT-EGE mRNA, the characteristics of which are shown in Table 3 below:

[0118] Table 3 Comparison of RSV PreF△CT-EGE mRNA sequence parameters before and after optimization

[0119]

[0120] The above sequence was sent to GenScript for sequence synthesis and synthesized into the pCDNA.1-BsaI plasmid.

[0121] 1. Plasmid Linearization

[0122] Prepare the plasmid linearization system according to Table 4 and digest at 37°C for 16 h. Then run on a 1% agarose gel at 150 V for 20 min.

[0123] Table 4 Plasmid linearization system

[0124]

[0125] 2. Recovery of linearized plasmid: Purify and recover the plasmid using the EZNAGel Extraction Kit, and determine the concentration using Nanodrop.

[0126] 3. mRNA in vitro transcription

[0127] Prepare the mRNA transcription system according to Table 5 and incubate at 37°C for 4 h.

[0128] Table 5 mRNA transcription system

[0129]

[0130] 4. Template Digestion

[0131] Add 30 μl of DNase I (2 U / μl) to the reaction system in step 3 and react at 37°C for 30 min.

[0132] 5. Agarose Gel Electrophoresis

[0133] Dilute the transcript after treatment in step 4 23-fold. Add 1 μl of the diluted product to 4 μl of DEPC and 5 μl of 2xLoading. Incubate at 90°C for 2 minutes. After denaturation, perform agarose gel electrophoresis at 180V for 25 minutes. The results show a single band with no visible tail, indicating that the mRNA sequence has not been degraded.

[0134] 6. mRNA capping

[0135] 6.1 Denaturation: Dilute 430 μl of DNase-treated RNA (from step 4) with 570 μl of DEPC water. Heat the diluted RNA at 65°C for 5 min and place on ice for 5 min.

[0136] 6.2 Configuring the Capping System

[0137] The capping system was prepared according to Table 6 and reacted at 42°C for 60 min.

[0138] Table 6 Capping system

[0139]

[0140] 7. mRNA purification:

[0141] 7.1 Mix 4 mL of capped product, 4 mL of DEPC water, and 4 mL of LiCl, aliquot 1 mL into each tube, and incubate at -20°C for 30 min.

[0142] 7.2 15000 rpm, 4℃, 15min;

[0143] 7.3 Wash three times with 70% pre-cooled ethanol;

[0144] 7.4 Place in a clean bench and air dry (the precipitate turns from white to transparent);

[0145] 7.5 Add a total of 2 mL of DEPC water to dissolve and obtain the mRNA vaccine.

[0146] 8. Package

[0147] mRNA lipid nanoparticles were prepared using a microfluidic mixing method, using the SM102 liposome formulation. Liposomes (SM102: DSPC: Cholesterol: PEG2000-DMG = 50:10:38.5:1.5, molar ratio) were dissolved in ethanol (final concentration: 90% ethanol) with an N:P ratio of 6. This liposome solution served as the organic phase. 2 mg of mRNA was dissolved in citric acid buffer to a final concentration of 1 μg / μl, serving as the aqueous phase. Encapsulation was performed using a NanoAssemblr microfluidic system in a 3:1 aqueous:organic ratio at a flow rate of 9 L / min. After encapsulation, the product was concentrated and exchanged with PBS buffer for storage at 4°C.

[0148] The mRNA liposomes were successfully encapsulated, and their physicochemical properties are shown in Table 7 below:

[0149] Table 7 Physicochemical properties of mRNA liposomes

[0150]

[0151] Example 3

[0152] immunity:

[0153] Animals: The animals used were Balb / C mice, 6-8 weeks old, SPF grade, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0154] Immunization and sampling:

[0155] According to the experimental requirements, 0.5-2.5 μg / mouse of mRNA vaccine (Table 8) was injected intramuscularly for a total of two immunizations, with an interval of 14 days. Blood was collected from the cheek before the first immunization and every two weeks thereafter. The mouse blood was placed at 37°C for 1 hour, and then centrifuged at 10,000 rpm for 10 minutes at 4°C to separate the serum, which was then packaged and stored in a -80°C freezer.

[0156] Table 8 Experimental groups

[0157]

[0158] 1. Antibody testing:

[0159] (1) Serum binding antibody ELISA assay

[0160] a. Antigen coating: Dilute the antigen (PreF protein) to 100 ng / 50 μl / well using Na2CO3-NaHCO3 buffer (pH 9.6). Mix thoroughly and add to a 96-well ELISA plate using a dispenser. Cover with plastic wrap and place in a refrigerator at 4°C overnight for coating.

[0161] b. Wash: Discard the solution and wash the plate three times with 0.05% PBST buffer for 5 minutes each time, 200 μl / well;

[0162] c. Blocking: Discard the solution and add 5% skim milk powder (prepared in 1× DPBS) at 200 μl / well and block at 37°C for 1 hour.

[0163] d. Wash: Discard the solution and wash the plate three times with 0.05% PBST buffer for 5 minutes each time, 200 μl / well;

[0164] e. Primary Antibody Binding: Serum was serially diluted in 2% skim milk (prepared in 1× DPBS) in a 96-well serum dilution plate, starting at a 1:100 dilution, followed by 3-fold dilutions, 50 μl / well, and incubated at 37°C for 2 hours.

[0165] f. Wash: Discard the solution and wash the plate three times with 0.05% PBST buffer for 5 minutes each time, 200 μl / well;

[0166] g. Secondary antibody binding: Dilute Goat anti-mouse IgG H&L (HRP) in 0.05% PBST, 1:30,000 dilution, 50 μl / well, incubate at 37°C for 1 hour;

[0167] h. Wash: Discard the solution and wash the plate three times with 0.05% PBST buffer for 5 minutes each, 200 μl / well;

[0168] i. Color development: Discard the solution, pat dry the ELISA plate, add 50 μl of Solebro TMB substrate to each well, and place in a drawer in the dark for 10 minutes.

[0169] j. Stop: After color development is complete, add 50 μl of 2 M H2SO4 to each well to stop the color development.

[0170] k. Reading on the microplate reader: Use a microplate reader to read the OD value at a wavelength of 450nm (OD450) and the OD value at a reference wavelength of 630nm (OD630);

[0171] Result determination: First, subtract the reference wavelength OD630 from the OD450 value to obtain the corrected OD value. Use the logarithmic value of the serum dilution factor as the horizontal axis and the OD values ​​measured at different dilution factors as the vertical axis. Fit the curve: For the antigen-antibody reaction gradient S-shaped curve, take the OD upper limit of approximately 2.0 and the lower limit of approximately 0.2. Take the logarithmic linear portion to calculate the horizontal axis value when the vertical axis is 0, which is the endpoint titer of the mouse serum specific antibody. For low OD values ​​at the starting dilution gradient, if the antigen-antibody reaction gradient S-shaped curve cannot be drawn, all titers with OD < 0.1 are determined to be 10, and all titers with 0.1 ≤ OD ≤ 0.2 are determined to be the starting dilution gradient.

[0172] The results show Figure 5 Both mRNA-PreF and mRNA-PreF△CT-EGE-eVLP (referred to as mRNA-eVLP in the figure) can stimulate mice to produce strong binding antibodies against PreF (the LNP group did not produce antibodies), but mRNA-eVLP takes effect faster than mRNA-PreF.

[0173] (2) Neutralizing antibody detection:

[0174] a. Serum dilution: Serum was serially diluted in DMEN serum-free medium in a 96-well cell culture plate, starting at a 1:20 dilution ratio, with 100 μl / well for 2-fold dilution.

[0175] b. Neutralization: Dilute RSV Long and RSV B1 viruses to a final concentration of 1×10 4 PFU / mL, 100 μl / well. Incubate at 37°C for 2 h.

[0176] c. Cell preparation: When the confluence of Hep-2 cells in T75 culture flasks reaches 85%~90%, they can be plated. After cell digestion, the density is adjusted to 1×10 6 Add 50 μl of cell suspension into a 96-well cell culture plate, shake gently, and culture in a 37°C, 5% CO2 cell culture incubator for 72 h.

[0177] d. Fixation: After the incubation period, discard the culture medium, wash twice with DPBS, and then add pre-cooled methanol (200 μl / well) and fix at -20°C for 20 min.

[0178] e. Washing: Discard the fixative and wash the plate three times with DPBS buffer, 5 minutes each time, 200 μl / well;

[0179] f. Blocking: Discard the solution and add 5% skim milk powder (prepared in 1× DPBS) at 200 μl / well and block at 37°C for 1 hour.

[0180] g. Wash: Discard the solution and wash the plate three times with 0.05% PBST buffer, 5 minutes each time, 200 μl / well;

[0181] h. Primary antibody binding: Dilute palizumab antibody with 2% skim milk (prepared in 1× DPBS) at a dilution of 1:5000, 100 μl / well, and incubate at 37°C for 1 hour.

[0182] i. Wash the plate: Discard the solution and wash the plate three times with 0.05% PBST buffer for 5 minutes each time, 200 μl / well;

[0183] j. Secondary antibody binding: Dilute Goat anti-human IgG H&L (HRP) secondary antibody in 0.05% PBST at a dilution of 1:5000, 100 μl / well, and incubate at 37°C for 1 hour.

[0184] k. Wash the plate: Discard the solution and wash the plate three times with 0.05% PBST buffer for 5 minutes each time, 200 μl / well;

[0185] 1. Color development: Discard the solution, pat the ELISA plate dry, add 50 μl of TrueBlue Peroxidase Substrate to each well, and place in a drawer in the dark for 10 minutes.

[0186] m. Observe the neutralizing activity under a microscope and determine the neutralizing titer as follows: The neutralizing antibody titer (NT50) is the reciprocal of the highest dilution gradient at which the viral plaques are reduced by 50% or more compared to the negative control wells after mixed infection with serum and virus.

[0187] The results are as follows Figure 6 As shown, eVLP technology stimulated stronger neutralizing antibodies against RSV Long (top) and RSV B1 (bottom) in mice compared to PreF, with an increase of approximately 2.5-5 times. Furthermore, only the eVLP vaccine produced strong neutralizing antibody levels two weeks after the initial immunization, while PreF did not produce any at low doses.

[0188] 2. Anti-virus protection

[0189] Three weeks after the booster immunization, the challenge protection test was carried out, and each mouse was intranasally dripped with 2×10 6 After challenge with PFU of RSV Long strain, body weight was measured. Five days after challenge, lung and nasal turbinates were isolated and analyzed for lung histopathology and viral copy number in lung and nasal tissues. A naïve group served as an unchallenged control, and a FI-RSV group received formaldehyde-inactivated vaccine as a positive control for the challenge experiment.

[0190] (1) Viral load detection:

[0191] a. Prepare lung and nasal turbinate homogenates:

[0192] Place lung tissue and nasal concha tissue in homogenization tubes, add 1 mL of pre-cooled PBS, and use a high-throughput tissue grinder to homogenize the lung tissue or nasal concha tissue until there are no obvious particles. Centrifuge at 8000 rpm for 5 min at 4°C, and aspirate the supernatant into a 1.5 mL EP tube.

[0193] b. Nucleic Acid Extraction

[0194] Nucleic acids were extracted using the FastPure® Cell / Tissue Total RNA Isolation Kit V2, and RNA concentration was measured using Nanodrop.

[0195] c. q-PCR detection of viral copy number

[0196] Real-time fluorescence quantitative PCR was performed using the HiScript II One Step qRT-PCR Probe Kit, and a plasmid carrying the L gene was used as a standard curve.

[0197] Table 9 Primer probe design

[0198]

[0199] Table 10 PCR reaction system

[0200]

[0201] The detection was performed using a Tianlong Real-Time PCR instrument, and the reaction program was set as follows:

[0202] Table 11 Reaction procedure

[0203]

[0204] Result analysis:

[0205] The viral load per gram of tissue was calculated based on the standard curve. The log10 of the copy number was used to express the viral load. When the viral copy number was below the lower limit of detection (LLOD), the viral copy number of the sample was considered to be 1 / 2 LLOD. The LLOD is the copy number detected in the lung tissue of a blank mouse.

[0206] The results are as follows Figure 7As shown, at doses of 1 μg and 2.5 μg, both mRNA-PreF and mRNA-eVLP could significantly reduce the number of viral copies in lung tissue compared with the control group (mRNA-LNP) and there was no significant difference with the non-immunized and non-challenged group (Naïve group). However, at the 0.5 μg dose group, mRNA-eVLP had a greater advantage than mRNA-PreF in reducing the number of viral copies in lung tissue.

[0207] (2) Pathological evaluation of mouse lung tissue after challenge:

[0208] Lung tissue was fixed with 4% paraformaldehyde and sliced. HE staining and PAS staining were performed to evaluate bronchiolitis, alveolitis, and interstitial alveolitis in lung tissue. The scoring criteria are shown in Table 12 below.

[0209] Table 12 Scoring criteria

[0210]

[0211] The results are as follows Figure 8 and Figure 9 As shown, the positive control group (FI-RSV) caused interstitial pneumonia, bronchiolitis and perivascular inflammatory cell infiltration in mice after infection, while the vaccine group at doses of 1 μg and 2.5 μg could significantly reduce lung tissue inflammation in mice, and the mRNA-eVLP was lower than the mRNA-PreF group in the inflammatory response.

[0212] (3) B cell immune response detection:

[0213] Inguinal lymph nodes and spleen tissues were collected from mice 7 days after immunization to detect germinal center B cell responses and follicular helper T cell responses, and spleens were collected 18 weeks after immunization to detect memory B cell responses.

[0214] a. Remove mouse spleen tissue and inguinal lymph nodes, place in PBS + 2% FBS, store on ice, and process within 8 hours.

[0215] b. Place mouse spleen tissue and lymph nodes on a 70 μm nylon mesh sieve and grind with a syringe to form a single-cell suspension.

[0216] c. Immediately transfer the lymphocyte suspension to a 15 mL centrifuge tube and centrifuge at 500 g for 10 minutes to collect the cells (lymph node lymphocytes).

[0217] d. Wash: Add 10 ml of wash buffer to the centrifuge tube, mix the cells, and centrifuge at 250g for 10 minutes.

[0218] e. Lyse red blood cells: Discard the supernatant, add 2 ml of red blood cell lysis buffer, and lyse for 5 minutes; add an equal volume of RPMI1640 complete medium to mix the cells, terminate lysis, and centrifuge at 500g for 5 minutes;

[0219] f. Wash: Discard the supernatant, add 5 ml of RPMI 1640 complete medium to the centrifuge tube, mix the cells, and centrifuge at 500g for 5 minutes;

[0220] g. Resuspend and count: Discard the supernatant and resuspend the cells in 200 μl of complete culture medium. Count the cells using a cell counting plate before proceeding with flow cytometry staining.

[0221] h. Wash: Add 200 μl of DPBS, mix gently by pipetting, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant.

[0222] i. Blocking and cell death and viability staining: Dilute Anti-CD16 / CD32 antibody (1:300) and cell death and viability stain (1:1000) in DPBS, add 100 μl to each well, mix by pipetting, and incubate on ice for 30 minutes; centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant;

[0223] j. Wash: Add 200 μl Stain Buffer, mix gently by pipetting, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant.

[0224] k. Staining: Use Stain Buffer to dilute the antibodies PerCP-Cy5.5 CXCR5 (1:100), BV421-CD4 (1:250), BV650-CD62L (1:250), BV786-B220 (1:250), FITC-RSV F (1:100), PerCP-Cy5.5-CD185 (1:250), PE-PD-1 (1:250), PE-Dazzle 594-CD19 (1:250), PE-Cy7-Fas (1:250), APC-GL7 (1:250), and APC-Cy7-CD44 (1:250). Add 100 μl to each well, mix well by pipetting, and incubate on ice for 30 minutes.

[0225] 1. Wash: Add 250 μl of Stain Buffer, mix gently by pipetting, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant.

[0226] m. Fixation: Add 200 μl of 2% paraformaldehyde fixative, gently pipette to mix, and incubate on ice for 15 minutes. Centrifuge at 500g, 4°C for 8 minutes and discard the supernatant.

[0227] Wash: Add 250 μl of Stain Buffer, mix gently by pipetting, centrifuge at 500 g, 4°C for 8 minutes, and discard the supernatant.

[0228] Resuspend: Add 200 μl of 1× PBS to resuspend the cells and transfer them to a 5 ml flow cytometer. Perform cell analysis and collect data using an LSR Fortessa instrument.

[0229] p. Analysis: FlowJo 10.0.7 software was used to analyze the cells and calculate the percentage of positive cells.

[0230] The results are as follows Figure 10 As shown in the results, mRNA-eVLP can stimulate a strong GC B cell response in the inguinal lymph nodes compared with mRNA-PreF, especially stimulate F-specific GC B cell response, while there is no significant difference between the two in Tfh cell response.

[0231] Memory B cell responses such as Figure 11 As shown in Figure 3, at week 18 after immunization, eVLPs generated stronger F-specific MBC responses, which may mean that they can stimulate more lasting immune protection.

[0232] As can be seen from the above examples, the present invention provides an RSV mRNA vaccine with spontaneously formed enveloped virus-like particles based on mRNA technology and eVLP technology, which can induce stronger neutralizing antibody levels and memory B and memory T cell immune responses than PreF in a mouse model, achieving the purpose of efficient and long-term protection.

[0233] Example 4

[0234] The above-mentioned eVLP formation technology can also be applied to adenovirus vector vaccines. This embodiment uses chimpanzee adenovirus AdC68 vector as an example.

[0235] AdC68 vector was used. SEQ ID NOs: 43-44 are the target sequences of the RSV AdC68-PreF vaccine, and SEQ ID NOs: 45-46 are the target sequences of the RSV AdC68-PreF-eVLP vaccine (EGE-5 group).

[0236] Adenoviral vector construction

[0237] The pAdC6-empty vector was linearized using I-CeuI and PI-SceI restriction enzymes. The reaction was incubated in a 37°C water bath for 2 hours, followed by inactivation at 65°C for 20 minutes. The linearized vector was obtained for later use. Using the pCDNA3.1-PreF and pCDNA3.1-PreF-eVLP plasmids constructed in Example 1 as templates, primers specific for the linearized pAdC6 vector were designed to include the homology arms. The target fragment, including the CMV promoter, PreF / PreF-eVLP genes, and BGH polyA, was amplified. A homologous recombination kit was used to homologously recombine the target fragment with the linearized vector. The recombinant product was transformed into Stbl2 competent cells, activated, plated on LB plates containing ampicillin, and incubated inverted at 30°C overnight. Single colonies were selected and the correct insertion sequence was verified by Sanger sequencing. One colony that was positive for PCR and Sanger sequencing was selected for expansion and plasmid extraction using an endotoxin removal kit. The extracted plasmid was digested with three restriction endonucleases, AleⅠ, Xho, and XcmⅠ, respectively. The size of the digested product was identified by 0.8% agarose gel electrophoresis and compared with the standard map to verify the integrity and correctness of the entire adenovirus vector (pAdC68-PreF or pAdC68-PreF-eVLP).

[0238] Adenovirus packaging, amplification, purification, and genome characterization

[0239] ① Adenovirus packaging

[0240] a. HEK293A cell culture and plating:

[0241] When the confluence of HEK293A cells in the T75 culture flask reached about 90%, the cells were digested and resuspended, and the single cell suspension was counted using a cell counting plate. 1×10 6 Add 100 cells dropwise into the prepared 6-well plate containing 2 ml of fresh DMEM complete medium, shake gently, and culture in a 37°C, 5% CO2 cell culture incubator overnight.

[0242] b. Linearization of pAdC68-PreF or pAdC68-PreF-eVLP adenoviral vector:

[0243] The recombinant adenovirus vectors pAdC68-PreF and pAdC6-PreF-eVLP were linearized with pacⅠ restriction endonuclease, digested at 37°C for 2 hours, and inactivated at 65°C for 20 minutes to obtain linearized large fragments with exposed long terminal repeat sequences at the 5' and 3' ends. They can be directly used for the next transfection or stored in a -20°C refrigerator overnight.

[0244] c. Transfection:

[0245] In a biosafety cabinet, transfer the linearized pAdC68-PreF and pAdC6-PreF-eVLP vectors to sterile EP tubes, add 180 μl of Opti-MEM medium, and mix thoroughly. Then, add 5 μl of X-tremeGENE HP transfection reagent (DNA:transfection reagent = 1:2.5) to 200 μl of the transfection system. Gently flick the tube to mix thoroughly. Let it stand at room temperature for 20 minutes before adding the tube dropwise to a 6-well plate containing HEK293A cells. Observe the cells daily for 5 days after transfection. Harvest the cells and supernatant when plaques appear in over 60% of the wells.

[0246] ②Adenovirus amplification

[0247] Viruses collected from 6-well plates were amplified three times from P0 to P3 by repeated freeze-thaw cycles between room temperature (25°C) and -80°C. The adenovirus amplification ratio was controlled between 1:6 and 1:10. After obtaining 30 150mm culture dishes of cells and virus, the cells were centrifuged at 500g for 15 minutes, the supernatant discarded, and the cells were resuspended in 8mL of antibiotic- and serum-free DMEM medium. The cells were repeatedly frozen and thawed three times between room temperature and -80°C, and centrifuged at 3000g for 20 minutes. The final viral supernatant was collected.

[0248] ③Adenovirus purification

[0249] a. Ultracentrifugation:

[0250] Purify adenovirus using CsCl density gradient centrifugation using a discontinuous gradient (1.4 g / mL and 1.2 g / mL) at 25,000 rpm and 4°C for 2.5 hours. After centrifugation, two distinct adenovirus bands will be visible, with the upper band representing incomplete adenovirus. Use a 1 ml syringe to pierce the tube slightly below the lower band, aspirate the lower band, cap the syringe, and place on ice.

[0251] b. Desalination:

[0252] The purified virus solution was desalted using desalting gel and replaced with PBS, and glycerol was added to a final concentration of 10%.

[0253] c. Determination of adenovirus particle number:

[0254] The A260 absorbance value was measured using Nanodrop. The instrument was first calibrated using PBS containing 10% glycerol as a blank control. The final virus concentration was calculated as A260 value × 1.1 × 10 12 After recording, the virus was stored in a -80℃ refrigerator.

[0255] (4) Adenovirus infection titer determination

[0256] Virus titer was titrated according to the QuickTiter™ Adenovirus Titer Immunoassay Kit instructions.

[0257] Table 13 Adenovirus infection titer determination results

[0258]

[0259] immunity:

[0260] Animals: The animals used were Balb / C mice, 6-8 weeks old, SPF grade, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0261] Immunization and sampling:

[0262] According to the experimental requirements, AdC68-PreF and AdC68-PreF-eVLP vaccines were injected intramuscularly, and empty AdC68-empty was used as a control for immunization (Table 14). Blood was collected before immunization and cheek blood was collected every three weeks thereafter. After the mouse blood was placed at 37°C for 1 hour, it was centrifuged at 10,000 rpm for 10 minutes at 4°C to separate the serum, which was then packaged and stored in a -80°C freezer.

[0263] Table 14 Experimental groups

[0264]

[0265] The subsequent binding antibody and neutralizing antibody detection methods are the same as those in Example 3.

[0266] Binding antibody results such as Figure 11 As shown, AdC68-PreF-eVLP stimulated higher levels of binding antibodies and required a lower dose to stimulate antibody production compared to AdC68-PreF.

[0267] Neutralizing antibody results Figure 12 As shown in Figure 2, eVLP technology stimulated the production of stronger neutralizing antibodies against RSV Long in mice compared with PreF. 6 IFU administration can induce neutralizing antibody levels, while at the same dose, AdC68-PreF does not produce neutralizing antibodies, and AdC68-PreF-eVLP 10 6 The neutralizing antibody of IFU was higher than that of AdC68-PreF at 10 7 For IFU administration, the dose difference was more than tenfold.

[0268] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A RSV PreF-eVLP that spontaneously forms enveloped virus-like particles, characterized in that The target gene in the PreF-eVLP mainly consists of the nucleotide sequence of RSV F protein and EGE motif; The RSV F protein is a truncated PreFΔCT protein, the amino acid sequence of the truncated PreFΔCT protein is shown in SEQ ID NO: 15, and the nucleotide sequence is shown in SEQ ID NO: 16; The EGE motif includes EPM, linker and EABR, the EPM is the nucleotide sequence shown in SEQ ID NO: 18, the linker is GS, and the EABR is the nucleotide sequence shown in SEQ ID NO:

36.

2. A method for constructing a RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 1, characterized in that: The target gene is constructed into an expression plasmid, and cell transfection is performed. The cells are cultured and the supernatant is collected to obtain spontaneously formed enveloped virus-like particles RSV PreF-eVLP.

3. A use of the truncated PreFΔCT protein in the RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 1, characterized in that: The purpose of the truncated PreFΔCT protein is to increase the expression level of the F protein on the membrane.

4. Use of the RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 1 in the preparation of a respiratory syncytial virus vaccine.

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