RSV F-eVLP capable of spontaneously forming enveloped virus-like particles and construction and application of mRNA vaccine, gland vector vaccine and recombinant protein vaccine of RSV F-eVLP

Abstract

The invention provides RSV F-eVLP capable of spontaneously forming enveloped virus-like particles and construction and application of an mRNA vaccine, a gland vector vaccine and a recombinant protein vaccine of the RSV F-eVLP, and belongs to the technical field of innovative vaccines. The coding sequence of the enveloped virus-like particle spontaneously formed by the RSV F protein is formed by fusing a coding sequence of the RSV F protein or a mutant thereof and a coding motif related to the formation of the enveloped virus-like particle. Compared with vaccines of RSV F proteins or mutants of RSV F proteins which do not have enveloped virus-like particles and form related motifs, the vaccines which encode the RSV F proteins and spontaneously form enveloped virus-like particles have the advantages that RSV neutralizing antibody level induced by a mouse model and immune response of memory B and memory T cells of virus specificity are stronger and more durable; the purpose of efficient and long-term protection of RSV infection and morbidity is achieved.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and in particular to an 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 5 years old globally were infected with RSV and developed lower respiratory tract infections, 10% of whom required hospitalization and were prone to secondary obstructive airway diseases and asthma. RSV also imposes a great disease burden on immunodeficient and elderly populations. In addition, RSV infection cannot trigger a lasting antibody response, resulting in reinfection, bringing a huge medical and economic burden to the world. RSV belongs to the family Paramyxoviridae, subfamily Pneumovirinae, and genus Pneumovirus, a non-segmented single-stranded negative-strand RNA enveloped virus. The outer layer of the virus particle is a lipid envelope, and the inside is the viral nucleocapsid. The virus particles have two different morphologies, spherical shapes with a diameter of 100-350 nm, or filamentous shapes with a diameter of 60-200 nm. The virus particles are composed of an envelope, a nucleocapsid, and a core. The envelope is embedded with three transmembrane proteins, G, F, and SH; the nucleocapsid is composed of capsomeres; the virus core is a complex composed of viral genomic RNA and non-structural proteins such as N, P, and L. The F protein and the G protein mediate the contact and fusion of the virus with cells during RSV infection and are also the main targets for current vaccine design. The F protein is highly conserved among different subtypes, while the G protein is highly variable. RSV can be divided into two subtypes, A and B, according to the difference in the sequence of the second highly variable region of the G protein. Existing studies have shown that the F protein exists in at least two states in vivo: the metastable prefusion F protein (PreF) and the stable postfusion F protein (PostF).

[0003] Vaccines are one of the effective measures for preventing RSV infection. mRNA plays a key role in the process of translating deoxyribonucleic acid (DNA) encoding proteins into proteins. The principle of mRNA vaccines is to utilize the protein synthesis mechanism of host cells to translate mRNA into corresponding antigens intracellularly, thus mimicking the process of virus infection and inducing effective humoral and cellular immune responses. With advantages such as good safety, strong immunogenicity, fast production speed, and wide application range, it has rapidly become a hot development direction in the biomedical industry. With the outbreak of SARS-Cov2, mRNA vaccines were first approved for human use, and their safety and effectiveness have been widely confirmed. Existing research shows that mRNA vaccines based on the RSV F protein induce strong humoral immune responses in vivo. In addition, compared with traditional protein vaccines, mRNA vaccines also induce strong CD4+ and CD8+ T cell responses, suggesting potential advantages of mRNA vaccines in activating cellular immune responses.

[0004] eVLPs (enveloped virus-like particles) vaccines can present viral antigens on the cell surface or assemble viral nanoparticles, which is more conducive to the activation of B cells and thus induces strong B cell immune responses to obtain long-term protection. eVLP vaccines can precisely display antigens. Through genetic engineering techniques, specific antigens can be displayed on the surface of VLPs in the optimal conformation. eVLP can also carry multiple antigens or antigenic epitopes simultaneously to achieve multi-antigen co-stimulation of the immune system. These different antigen combinations can mimic the complex antigen composition of the virus, stimulate the immune system to produce comprehensive and lasting immune responses, and increase the protective efficacy of the vaccine against different virus strains or virus variants. eVLP vaccines can not only activate adaptive immunity but also stimulate innate immunity. Their virus-like structures can be recognized by pattern recognition receptors (PRRs) of the innate immune system, such as Toll-like receptors (TLRs). When eVLP is taken up by antigen-presenting cells, it triggers a series of innate immune signaling pathways, releasing cytokines and chemokines, thus creating a favorable environment for the initiation of adaptive immunity. This dual activation of innate and adaptive immunity can more effectively clear the virus and enhance the immune effect of the vaccine.

[0005] Existing mRNA vaccines based on PreF have shown weak persistent protection in human clinical studies. The possible reason is that they fail to effectively induce long-lasting memory T and B cell responses, overstimulating short-term antibody responses while insufficiently activating the generation of memory cells. Memory B cells and memory T cells are crucial for long-term immunity. If a vaccine cannot effectively induce the formation and maintenance of these cells, the immune system may not be able to respond quickly and effectively upon re-exposure to RSV. 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 compared to mRNA vaccines.

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

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

[0008] To achieve the above object of the invention, the present invention provides the following technical solutions: The present invention provides an RSV PreF-eVLP that spontaneously forms enveloped virus-like particles. The target gene in the PreF-eVLP mainly consists of the nucleotide sequences of the RSV F protein and the EGE motif.

[0009] Preferably, the RSV F protein is the native F protein, and the amino acid sequence of the native F protein is as shown in SEQ ID NO: 1.

[0010] Preferably, the RSV F protein is a modified F protein mutant.

[0011] Preferably, the modified F protein mutant includes an optimized PreF protein or a truncated PreF△CT protein.

[0012] Preferably, the optimization method of the optimized PreF protein includes one or more of signal peptide replacement, deletion of the P27 fragment, and amino acid mutation.

[0013] Preferably, the replacement signal peptide includes one of the signal peptide sequences shown in SEQ ID NO: 10 to 14; the amino acid mutation sites include one or more of N67I, R106Q, F137S, S215P, and E487Q.

[0014] Preferably, the amino acid sequence of the optimized PreF protein is as shown in SEQ ID NO: 2, 4, 6, 8, and the corresponding nucleotide sequences are as shown in SEQ ID NO: 3, 5, 7, 9 in sequence.

[0015] Preferably, the truncated PreF△CT protein is truncated at 1 to 23 random amino acids at the C-terminus.

[0016] Preferably, the amino acid sequence of the truncated PreF△CT protein is as shown in SEQ ID NO: 15, and the nucleotide sequence of the truncated PreF△CT protein is as shown in SEQ ID NO: 16.

[0017] Preferably, the EGE motif includes EPM, linker, and EABR; the EPM is the nucleotide sequence shown in any one of SEQ ID NO: 18, 20, 22; the linker is GS, (GS)n, or (GGGGS)n, where n represents the number of repetitions, and n = 1 to 4; the EABR is the nucleotide sequence shown in any one of SEQ ID NO: 24, 26, 28, 30, 32, 34, 36.

[0018] 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.

[0019] The present invention also provides a method for constructing RSV PreF-eVLP that spontaneously forms envelope virus-like particles. The target gene is constructed into an expression plasmid, the cells are transfected, and the cells are cultured and the supernatant is collected to obtain RSV PreF-eVLP that spontaneously forms envelope virus-like particles.

[0020] The present invention also provides a use of the truncated PreF△CT protein in the RSV PreF-eVLP that spontaneously forms envelope virus-like particles. The use of the truncated PreF△CT protein is to increase the expression level of the F protein on the membrane.

[0021] The present invention also provides an RSV mRNA vaccine platform constructed with the self - assembled enveloped virus - like particle RSV PreF - eVLP as described above, characterized in that the RSV mRNA vaccine contains the target gene as described above.

[0022] Preferably, the nucleotide sequence of the RSV mRNA is as shown in SEQ ID NO: 37 or SEQ ID NO: 38; the optimized nucleotide sequence of the RSV mRNA is as shown in SEQ ID NO: 39.

[0023] The present invention also provides a method for constructing the RSV mRNA vaccine platform as described above, including but not limited to the following steps: (1) Construct the target gene or the nucleotide sequence of RSV mRNA into an expression vector to obtain a recombinant plasmid; (2) Linearize the recombinant plasmid as a transcription template to obtain in vitro transcribed RSV mRNA; (3) After digestion, capping, and purification of the in vitro transcribed RSV mRNA, it is encapsulated with liposomes to obtain the RSV mRNA vaccine.

[0024] Preferably, the expression vector is the pCDNA3.1(+) expression plasmid, and the liposome is liposome SM102.

[0025] The present invention also provides the liposomal RSV mRNA vaccine obtained by the construction method as described above, and the liposomal RSV mRNA vaccine is in the form of a liquid or lyophilized powder.

[0026] The present invention also provides an RSV adenovirus vector vaccine constructed with the self - assembled enveloped virus - like particle RSV PreF - eVLP as described above.

[0027] Preferably, the adenovirus vector in the adenovirus vector vaccine includes 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.

[0028] Preferably, the nucleotide sequence of the target gene in the RSV adenovirus vector vaccine is as shown in SEQ ID NO: 43 or SEQ ID NO: 45, and the amino acid is as shown in SEQ ID NO: 44 or SEQ ID NO: 46.

[0029] The present invention also provides the use of RSV PreF-eVLP that spontaneously forms an enveloped virus-like particle, or the RSV mRNA vaccine platform described above, or the liposomal RSV mRNA vaccine, or the RSV adenovirus vector vaccine in the preparation of a respiratory syncytial virus vaccine.

[0030] The present invention provides a novel VLP self-assembly technology. By adding an EGE motif to the cytoplasmic terminus of the PreF protein, self-assembly is induced to form eVLP, rather than introducing other backbone sequences for co-transfection to form eVLP. The present invention also combines the nanoparticle vaccine technology with the existing mRNA vaccine platform, solves the scientific problem that a single nanoparticle vaccine has weak induction of cellular immunity, and at the same time gives full play to the advantages of the VLP vaccine in antibody response, enhancing the B cell immune response of the mRNA vaccine to solve the problem of the short-lived protection of existing mRNA vaccines. Moreover, this technology is applied to RSV for the first time and is also the first application of the first RSV mRNA vaccine.

[0031] The present invention has the following beneficial effects compared with the prior art: (1) The present invention has successfully developed an eVLP technology based on the RSV PreF protein. The eVLP can self-assemble and can be classified as a type of extracellular vesicle.

[0032] (2) The present invention has successfully established an eVLP technology, which can achieve the production of high-quality antibodies. Compared with single PreF, the neutralizing antibody titer has increased by 2.5 to 5 times.

[0033] (3) The present invention has successfully combined the eVLP technology with the mRNA technology and the adenovirus technology to construct an RSV mRNA vaccine platform and an adenovirus platform relying on the eVLP technology, which can cause a strong immune response in mice to protect the mice from RSV attack. In terms of the protective dose, it is much lower than that of PreF. At the same time, the present invention can also combine the eVLP technology with the recombinant protein technology to obtain a recombinant protein vaccine.

[0034] (4) The eVLP technology established by the present invention can significantly stimulate the B cell immune response compared with the original PreF, 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

[0035] Figure 1 It is a schematic diagram of plasmid construction in Example 1; Figure 2Expression of PreF protein in the membrane surface and supernatant of plasmids containing PreF, PreFΔCT, PreFΔCT-GE (SEQ ID NO: 8), PreFΔCT-EGE-5 or PreFΔCT-EGE-12 in Example 1; Figure 3 Immuno-electron microscopy identification results of eVLP expressed in the supernatant in Example 1. Both figures are immuno-electron micrographs of eVLP, showing different sizes; Figure 4 Identification results of CD81, CD9, CD63 and Calnexin markers in supernatant eVLP in Example 1 by WB; Figure 5 Situation of mRNA-PreF and mRNA-eVLP stimulating mice to produce binding antibodies against PreF in Example 3; Figure 6 Situation of mRNA-PreF and mRNA-eVLP stimulating the production of neutralizing antibodies against RSV Long (upper figure) and RSV B1 (lower figure) in mice in Example 3; Figure 7 Virus copy numbers in lung tissue and nasal tissue after virus challenge in Example 3; Figure 8 HE and PAS staining results of lung tissue after virus challenge in Example 3; Figure 9 Pathological scoring results after virus challenge in Example 3; Figure 10 Germinal center B cell response and follicular helper T cell response in Example 3. Among them, A is the frequency of GC B cells in the draining lymph node, B is the frequency of F-specific GC B cells in the draining lymph node, and C is the frequency of follicular helper T lymphocytes in the spleen; Figure 11 Memory B cell response in Example 3; Figure 12 Situation of AdC68-PreF and AdC68-eVLP stimulating mice to produce binding antibodies against PreF in Example 4; Figure 13 Situation of AdC68-PreF and AdC68-eVLP stimulating mice to produce neutralizing antibodies against PreF in Example 4. Detailed implementation mode

[0036] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0037] Example 1 In this example, the PreF protein is selected as the membrane-bound F protein (SEQ ID NO: 1). Its signal peptide is replaced with the tPA signal peptide. Meanwhile, the P27 fragment (RRELPRFMNYTLNNTKNTNVTLSKKRKRR) is deleted and replaced with the GSGSGR linker, and point mutations N67I, R106Q, F137S, S215P, E487Q (underlined single amino acids) are made to maintain its pre-fusion 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 NO: 4, 6, 8, etc., but not limited to these. Here is just an exemplary display. The formation of eVLP is achieved by adding the EGE motif to the C-terminus of the PreF protein. Corresponding modifications are made to the C-terminus to make the PreF protein more present on the cell surface for the formation of eVLP. Such modifications are formed by arbitrarily truncating the 1-23 amino acids (ARSTPVTLSKDQLSGINNIAFSN) at the C-terminus. The sequences involved in the eVLP construction are as follows: Amino acid sequence of F protein (NCBI: APW77972.1, SEQ ID NO: 1) MELLILKTNAITTILAAVTLYFASS QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKE N KCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANS R ARRELPRFMNYTLNNTKNTNVTLSKKRKRR F LGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI S NIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSD EFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN*。

[0038] Optimized amino acid sequence of PreF protein Amino acid sequence of optimized Sequence 1, as shown in SEQ ID NO: 2: MDAMKRGLCCVLLLCGAVFVSP QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANS Q ARGSGSGR S LGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSI P NIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSD Q FDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN*。

[0039] Nucleotide sequence of optimized Sequence 1, as shown in SEQ ID NO: 3; Amino acid sequence of optimized Sequence 2, as shown in SEQ ID NO: 4; Nucleotide sequence of optimized Sequence 2, as shown in SEQ ID NO: 5; Amino acid sequence of optimized Sequence 3, as shown in SEQ ID NO: 6; Nucleotide sequence of optimized Sequence 3, as shown in SEQ ID NO: 7; The amino acid sequence of the optimized Sequence 4, as shown in SEQ ID NO: 8; The nucleotide sequence of the optimized Sequence 4, as shown in SEQ ID NO: 9.

[0040] Signal peptide sequence: tPA signal peptide sequence: as shown in SEQ ID NO: 10. Alternative signal peptides also include: 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.

[0041] The amino acid sequence of the truncated PreFΔCT protein, as shown in SEQ ID NO: 15, and the nucleotide sequence of the truncated PreFΔCT protein, as shown in SEQ ID NO: 16.

[0042] The EPM sequence is as follows: (1) Human low-affinity immunoglobulin γ Fc region receptor II - b1: ALPGYPECREMGETLPEKP (SEQ ID NO: 17); the corresponding nucleotide sequence is as shown in SEQ ID NO: 18.

[0043] (2) Mouse low-affinity immunoglobulin γ Fc region receptor II - b1: DLPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY (SEQ ID NO: 19); the corresponding nucleotide sequence is as shown in SEQ ID NO: 20; ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY (SEQ ID NO: 21); the corresponding nucleotide sequence is as shown in SEQ ID NO: 22.

[0044] GS linker (n represents the number of repetitions, n = 1 - 4): (GGGGS)n or (GS)n, and the corresponding nucleotide sequences are (gggggaggaggaagt) n and (ggaagt) n The EABR sequence is derived from one of the following sequences: Table 1 EABR sequences and their sources

[0045] The nucleotide sequences of the above sequences were constructed into the pCDNA3.1(+) expression plasmid as shown Figure 1 between the two restriction enzyme sites of Not I and Kpn I (commissioned by GenScript). Among them, PreF is the nucleotide sequence shown in SEQ ID NO: 3; PreF△CT is the nucleotide sequence shown in SEQ ID NO: 16; the EABR sequence in PreF△CT-GE is the nucleotide sequence shown in SEQ ID NO: 24; the combination of EPM and EABR in PreF△CT-EGE is shown in Table 2; all the Linkers used are GSLinker. After the above sequence fragments are directly connected, they are constructed into the pCDNA3.1(+) expression plasmid.

[0046] Table 2 Combinations of EPM and EABR

[0047] The above-constructed expression vectors were expressed, identified, and subjected to electron microscopy detection of eVLP in vitro, as follows: a. Cell transfection One day before transfection, 293T cells were seeded in a six-well plate at a density of 4×10 5 / well with DMEM + 10% FBS complete medium and cultured overnight at 37°C, 5% CO 2 . The next day, when the cells reached 80% confluence, plasmid transfection was performed. The constructed plasmids containing PreF, PreF△CT, PreF△CT GE, or PreF△CT EGE were added at 2.5 μg / well, and the transfection reagent X-tremeGENE™ HP DNA: plasmid (μl:μg) = 2:1. After gently shaking, the cells were incubated at 37°C, 5% CO 2 for 48 h.

[0048] b. Enrichment of supernatant eVLP 48 h after transfection, the cells were centrifuged at 400×g for 10 minutes. The centrifuged cell pellet was lysed with lysis buffer and stored at -20°C for later use. The supernatant after centrifugation was passed through a 0.45 mm syringe filter to further remove cell debris, and then ultracentrifuged at 135,000×g for 2 h on a 20% (w / v) sucrose cushion. The supernatant was removed, and the pellet after ultracentrifugation was resuspended in PBS overnight. After overnight incubation, the sample was centrifuged at 10,000×g for 10 min, and then the supernatant was collected again for detection.

[0049] c. Detection of antigen expression by Western Blot Electrophoresis sample preparation Add 5x SDS loading buffer to the supernatant sample and the sample after cell pellet lysis to a final concentration of 1x. Heat the samples at 100 °C for 10 min before loading to denature the proteins.

[0050] SDS-PAGE For electrophoresis, use a 5% stacking gel and a 10% separating gel. Set the voltage at 80 V for 30 min and 150 V for 30 min during electrophoresis.

[0051] Transfer membrane Select a PVDF membrane, transfer at 0.4 mA for 1 h, and control the heat with an ice pack.

[0052] Blocking and immunoreaction Block with 5% non-fat milk powder at room temperature for 2 h. After washing three times with TBST buffer, add an appropriate amount of diluted primary antibody (human anti-RSVF) and hybridize overnight at 4 °C. Then wash three times with a shaker using TBST solution, 10 min each time. The secondary antibody is goat anti-human-HRP, incubate at room temperature on a shaker for 1 h. Subsequently, wash three times with TBST, 10 min each time.

[0053] Developing and exposure According to the instructions of the ECL kit, evenly drip the developing solution onto the membrane surface, and then detect by exposure in a chemiluminescence image analyzer.

[0054] The results are as Figure 2 shown. After truncation, the membrane-expressed protein PreF△CT is still expressed on the cell surface and its cell surface expression level increases. After adding EABR, it can be expressed in the supernatant. The addition of the EPM motif increases the expression of PreF△CT-EGE-5 on the cell surface and in the supernatant, while the expression level of PreF△CT-EGE-12 on the cell surface not only does not increase but decreases instead. It can be seen that different EGE sequence combinations have unpredictable results on the expression of the PreF△CT protein. For the combination of EPM and EABR, the EGE-5 sequence (the 5th group in Table 2) has the highest expression among all combinations.

[0055] d. Identification of eVLP morphology 1. Add an equal volume of 4% paraformaldehyde to the above-mentioned extracted supernatant sample (eVLP) and fix for at least 4 h; 2. Pipette 30 μl of the liquid onto a wax film and perform negative staining with a carbon-coated nickel mesh for 30 min; 3. Absorb the excess liquid with filter paper and wash three times with 0.05 M PB, 1 min each time; 4. Block with 0.05 M glycine for 10 min and block with 1% BSA for 1 h, 5. Incubate with the primary antibody (antibody titration method, use 1% BSA instead for the negative control) for 1 h and overnight at 4 °C; 6. Wash the nickel mesh with PB 6 times, 1 min each time; 7. Block with 1% BSA for 10 min, incubate with secondary antibody (1:100, 10 nm colloidal gold) for 3 h; 8. Wash with PB 6 times, 1 min each time; 9. Fix with 2.5% glutaraldehyde for 15 min, wash 4 times with ddH 2 O; 10. Stain with 2% uranyl acetate for 5 min, wash 4 times with ddH 2 O, air dry naturally, observe and photograph with JEOL-1200.

[0056] The results are as Figure 3 shown. F protein particles (black particles in the figure) are presented on the surface of eVLP, and the size of eVLP is between 100 - 150 nm.

[0057] e. Classification and identification of eVLP eVLP is formed by the self-assembly of cell-recruited ESCRT-related pathway proteins. Therefore, WB is used to further identify eVLP with CD81, CD9, CD63, and Calnexin markers. The results are as Figure 4 shown. eVLP shows CD9+CD81+CD63+Calnexin-, and belongs to a type of extracellular vesicle.

[0058] Example 2

[0059] The mRNA vaccine sequences are as shown in SEQ ID NO: 37 - 39. Among them, SEQ ID NO: 37 is the sequence of the RSV PreF mRNA vaccine, SEQ ID NO: 38 is the sequence of the RSV PreF△CT-EGE mRNA vaccine (EGE-5 group), and SEQ ID NO: 39 is the further optimized sequence of RSV PreF△CT-EGE mRNA, and its characteristics are shown in Table 3 below: Table 3 Comparison of parameters before and after optimization of the RSV PreF△CT-EGE mRNA sequence

[0060] Send the above sequences to GenScript for sequence synthesis and synthesize them into the pCDNA.1-BsaI plasmid.

[0061] 1. Plasmid linearization Configure the plasmid linearization system according to Table 4, digest with enzymes at 37 °C for 16 h. Then 1% agarose gel: 150 V, 20 min.

[0062] Table 4 Plasmid linearization system

[0063] 2. Linearized plasmid recovery: Purify and recover using the E.Z.N.A. Gel Extraction Kit, and measure the concentration with Nanodrop after recovery.

[0064] 3. In vitro transcription of mRNA Prepare the mRNA transcription system according to Table 5 and react at 37 °C for 4 h.

[0065] Table 5 mRNA transcription system

[0066] 4. Template digestion Add 30 μl of DNase I (2 U / μl) to the reaction system in Step 3 and react at 37 °C for 30 min.

[0067] 5. Agarose gel electrophoresis Dilute the transcription product after the treatment in Step 4 by 23 times. Take 1 μl of the diluted product + 4 μl of DEPC + 5 μl of 2×Loading, react at 90 °C for 2 min, and perform agarose gel electrophoresis after denaturation at 180 V for 25 min. The result shows a single band without trailing, indicating that the mRNA sequence is not degraded.

[0068] 6. Capping of mRNA 6.1 Denaturation: Add 570 μl of DEPC water to 430 μl of DNase-treated RNA (the product of Step 4) for dilution. After dilution, heat at 65 °C for 5 min for denaturation, and then place on ice for 5 min after completion.

[0069] 6.2 Prepare the capping system Prepare the capping system according to Table 6 and react at 42 °C for 60 min.

[0070] Table 6 Capping system

[0071] 7. Purification of mRNA: 7.1 Mix 4 mL of the capped product + 4 mL of DEPC water + 4 mL of LiCL, aliquot 1 mL / tube, and place at -20 °C for 30 min; 7.2 Centrifuge at 15000 rpm at 4 °C for 15 min; 7.3 Wash three times with 70% pre-cooled ethanol; 7.4 Air dry in the laminar flow hood (the precipitate turns from white to transparent); 7.5 Dissolve with a total of 2 mL of DEPC water to obtain the mRNA vaccine.

[0072] 8. Encapsulation mRNA lipid nanoparticles were prepared using microfluidic mixing, and the liposome was selected with the SM102 formulation. The liposome (SM102:DSPC:Cholesterol:PEG2000-DMG = 50:10:38.5:1.5, molar ratio) was dissolved in an ethanol solution (final concentration of 90% ethanol). The N:P of the LNP was 6 to obtain the required liposome solution as the organic phase; and 2 mg of mRNA was dissolved in citrate buffer to a final concentration of 1 μg / μl as the aqueous phase. Microfluidic encapsulation was carried out in NanoAssemblr according to the aqueous phase:organic phase (3:1, mass ratio), with the flow rate set at 9 L / min. After encapsulation, the product was concentrated and the buffer was exchanged, and it was placed in PBS buffer and stored at 4°C.

[0073] The mRNA liposomes were successfully encapsulated, and their physicochemical properties are shown in Table 7 below: Table 7 Physicochemical properties of mRNA liposomes

[0074] Example 3

[0075] Immunization: Animals: The animals used were Balb / C mice, 6 - 8 weeks old, SPF grade, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0076] Immunization and sampling: According to the experimental requirements, the mRNA vaccine (Table 8) was intramuscularly injected at 0.5 - 2.5 μg / mouse, and immunization was carried out twice with a 14-day interval; blood was collected before the first immunization and cheek blood was collected every two weeks thereafter. After the mouse blood was placed at 37°C for 1 h, it was centrifuged at 10000 rpm for 10 min at 4°C to separate the serum, which was then aliquoted and stored in a -80°C freezer.

[0077] Table 8 Experimental grouping

[0078] 1. Antibody detection: (1) Serum binding antibody ELISA assay a. Coating antigen: The antigen (PreF protein) was diluted to 100 ng / 50 μl / well with Na 2 CO 3 -NaHCO 3 buffer with PH = 9.6. After mixing, it was added to a 96-well ELISA plate using a multi-channel pipette. After sealing with plastic wrap, it was placed in a 4°C refrigerator overnight for coating; b. Plate washing: Discard the solution, wash the plate 3 times with 0.05% PBST buffer for 5 minutes each time, 200 μl / well; c. Blocking: Discard the solution, add 5% non-fat milk (prepared with 1×DPBS), 200 μl / well, block at 37°C for 1 hour; d. Plate washing: Discard the solution, wash the plate 3 times with 0.05% PBST buffer for 5 minutes each time, 200 μl / well; e. Primary antibody binding: Dilute the serum serially in a 96-well serum dilution plate using 2% non-fat milk (prepared with 1×DPBS), starting dilution ratio is 1:100, 3-fold serial dilution, 50 μl / well, incubate at 37°C for 2 hours; f. Plate washing: Discard the solution, wash the plate 3 times with 0.05% PBST buffer for 5 minutes each time, 200 μl / well; g. Secondary antibody binding: Dilute Goat anti-mouse IgG H&L (HRP) with 0.05% PBST, 1:30000 dilution, 50 μl / well, incubate at 37°C for 1 hour; h. Plate washing: Discard the solution, wash the plate 3 times with 0.05% PBST buffer for 5 minutes each time, 200 μl / well; i. Color development: Discard the solution, pat dry the ELISA plate, add 50 μl of TMB substrate from Solarbio to each well, place in a drawer to develop color in the dark for 10 minutes; j. Termination: After color development is completed, add 50 μl of 2 M H 2 SO 4 to terminate; k. Reading on the machine: Use an enzyme-linked immunosorbent assay reader to read the OD value at 450 nm wavelength (OD450), and at the same time read the OD value at the reference wavelength of 630 nm (OD630); Result determination: First subtract the OD630 of the reference wavelength from the OD450 value to obtain the corrected OD value. Use the logarithm of the serum dilution ratio as the abscissa, and the OD values measured at different dilution ratios as the ordinate to fit a curve: For the antigen-antibody reaction gradient S-shaped curve, take the upper limit of OD to be about 2.0 and the lower limit to be about 0.2, and take the logarithmic linear part to calculate the abscissa value when the ordinate is 0, that is, the endpoint titer of mouse serum specific antibody; For the starting dilution gradient with a low OD value at which an antigen-antibody reaction gradient S-shaped curve cannot be made, all OD < 0.1 are judged to have a titer of 10, and all 0.1 ≤ OD ≤ 0.2 are judged to have a titer of the starting dilution gradient.

[0079] Result display 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 (no antibodies were produced in the LNP group), but mRNA-eVLP takes effect faster than mRNA-PreF.

[0080] (2) Neutralizing antibody detection: a. Serum dilution: Use DMEN serum-free medium to serially dilute the serum in a 96-well cell culture plate. The starting dilution factor is 1:20, with a 2-fold serial dilution, 100 μl / well.

[0081] b. Neutralization: Dilute the viruses RSV Long and RSV B1 to a final concentration of 1×10 4 PFU / mL, 100 μl / well. Incubate in a 37°C incubator for 2 h.

[0082] c. Cell preparation: When the confluence of Hep-2 cells in a T75 culture flask reaches 85% - 90%, plating can be carried out. After cell digestion, adjust the density to 1×10 6 cells / ml, add 50 μl of the cell suspension to a 96-well cell culture plate, gently shake well, and culture in a 37°C, 5% CO 2 cell incubator for 72 h.

[0083] d. Fixation: After the culture is completed, discard the medium, add DPBS and wash twice, then add pre-cooled methanol, 200 μl / well, and fix at -20°C for 20 min.

[0084] e. Plate washing: Discard the fixing solution, wash the plate 3 times with DPBS buffer, 5 min each time, 200 μl / well; f. Blocking: Discard the solution, add 5% non-fat milk (prepared with 1×DPBS), 200 μl / well, and block at 37°C for 1 h; g. Plate washing: Discard the solution, wash the plate 3 times with 0.05% PBST buffer, 5 min each time, 200 μl / well; h. Primary antibody binding: Dilute the palizumab antibody with 2% non-fat milk (prepared with 1×DPBS), 1:5000 dilution, 100 μl / well, and incubate at 37°C for 1 h; i. Plate washing: Discard the solution, wash the plate 3 times with 0.05% PBST buffer, 5 min each time, 200 μl / well; j. Secondary antibody binding: Dilute the Goat anti-human IgG H&L (HRP) secondary antibody with 0.05% PBST, 1:5000 dilution, 100 μl / well, and incubate at 37°C for 1 h; k. Plate washing: Discard the solution, wash the plate 3 times with 0.05% PBST buffer for 5 minutes each time, 200 μl per well; l. Color development: Discard the solution, pat the ELISA plate dry, add 50 μl of TrueBlue Peroxidase Substrate to each well, and place it in a drawer for 10 minutes for color development in the dark; m. Observe the neutralization activity under a microscope and determine the neutralization titer. The determination method is as follows: The neutralizing antibody titer (NT50) is the reciprocal of the highest dilution gradient at which the viral plaque is reduced by 50% or more after the serum-virus mixed infection compared to the negative control well.

[0085] The results are as Figure 6 shown. The eVLP technology stimulates the production of stronger neutralizing antibodies against RSV Long (upper figure) and RSV B1 (lower figure) in mice compared to PreF, with a growth multiple of approximately 2.5 to 5 times. And only the eVLP vaccine produces a strong neutralizing antibody level two weeks after the primary immunization, while PreF does not produce at low doses.

[0086] 2. Virus challenge protection The virus challenge protection test was carried out three weeks after the booster immunization. Each mouse was instilled with 2×10 6 PFU of RSV Long strain by nasal drip. After the virus challenge, the body weight was measured. On the fifth day after the virus challenge, the mouse lung tissue and nasal turbinate tissue were isolated for detecting lung tissue pathology, lung tissue virus copy number, and nasal tissue virus copy number. At the same time, the Naïve group was set as the non-virus-challenged control group, and the FI-RSV group was the formalin-inactivated vaccine, used as the positive control for the virus challenge experiment.

[0087] (1) Virus load detection: a. Prepare lung tissue and nasal turbinate tissue homogenates: Place the lung tissue and nasal turbinate tissue in homogenization tubes respectively, add 1 mL of pre-cooled PBS, and use a high-throughput tissue grinder to homogenize the lung tissue or nasal turbinate tissue until there are no obvious particles. Centrifuge at 8000 rpm for 5 minutes at 4°C, and aspirate the supernatant into a 1.5 mL EPP tube.

[0088] b. Nucleic acid extraction Use the FastPure® Cell / Tissue Total RNA Isolation Kit V2 extraction kit to extract nucleic acids, and use Nanodrop to detect the RNA concentration.

[0089] c. q-PCR detection of virus copy number Use the HiScript II One Step qRT-PCR Probe Kit kit for real-time fluorescence quantitative PCR, and use the plasmid carrying the L gene as the standard curve.

[0090] Table 9 Primer and Probe Design

[0091] Table 10 PCR Reaction System

[0092] Detection was performed using the Tianlong Real-Time PCR instrument, and the reaction program was set as follows: Table 11 Reaction Program

[0093] Result Analysis: According to the standard curve, the viral load per gram of tissue was calculated, and the viral load was expressed using the log10 of the copy number. When the viral copy number was below the lower limit of detection (LLOD), the viral copy number of this sample was considered to be 1 / 2 LLOD. LLOD was the copy number of the blank mouse lung tissue for detection.

[0094] The results were as Figure 7 shown. At the doses of 1 μg and 2.5 μg, compared with the control group (mRNA-LNP), both mRNA-PreF and mRNA-eVLP could significantly reduce the viral copy number in the lung tissue and there was no significant difference from the non-immunized and non-challenged group (Naïve group). However, in the 0.5 μg dose group, mRNA-eVLP had a greater advantage in reducing the viral copy number in the lung tissue compared with mRNA-PreF.

[0095] (2) Histopathological Evaluation of Mouse Lung Tissue after Viral Challenge: The lung tissue was fixed with 4% paraformaldehyde. After sectioning, HE staining and PAS staining were performed, and the bronchiolitis, alveolitis, and interstitial alveolitis in the lung tissue were evaluated. The scoring criteria are shown in Table 12 below.

[0096] Table 12 Scoring Criteria

[0097] The results were as Figure 8 and Figure 9 shown. After viral challenge, the positive control group (FI-RSV) caused interstitial pneumonia, bronchiolitis, and perivascular inflammatory cell infiltration in mice, while the vaccine groups at the doses of 1 μg and 2.5 μg could significantly reduce the inflammation in the mouse lung tissue, and the mRNA-eVLP was lower than the mRNA-PreF group in the inflammatory response.

[0098] (3) Detection of B Cell Immune Response: On the 7th day after immunization of the mice, inguinal lymph nodes and spleen tissues were collected to detect germinal center B cell response and follicular helper T cell response. On the 18th week after immunization, the spleen was collected to detect memory B cell response.

[0099] a. Mouse spleen tissues and inguinal lymph nodes were placed in PBS + 2% FBS, stored on ice, and processed within 8 h.

[0100] b. Mouse spleen tissues and lymph nodes were placed in a 70-μm nylon mesh sieve and ground with a syringe through the sieve to form a single-cell suspension.

[0101] c. The lymphocyte-containing separation solution was immediately transferred to a 15-mL centrifuge tube and centrifuged at 500 g for 10 min to collect cells (lymph node lymphocytes).

[0102] d. Washing: Add 10 ml of washing solution to the centrifuge tube, mix the cells well, and centrifuge at 250 g for 10 min. e. Lysing red blood cells: Discard the supernatant, add 2 ml of red blood cell lysate, and lyse for 5 min; add an equal volume of complete RPMI 1640 medium to mix the cells well, terminate lysis, and centrifuge at 500 g for 5 min. f. Washing: Discard the supernatant, add 5 ml of complete RPMI 1640 medium to the centrifuge tube, mix the cells well, and centrifuge at 500 g for 5 min. g. Resuspending and counting: Discard the supernatant, resuspend the cells with 200 μl of complete medium, count the cells using a cell counting chamber, and then flow cytometry staining can be performed.

[0103] h. Washing: Add 200 μl of DPBS, gently pipette to mix well, and centrifuge at 500 g at 4°C for 5 min, then discard the supernatant. i. Blocking and cell viability staining: Dilute Anti-CD16 / CD32 antibody (1:300) and viability dye (1:1000) with DPBS, add 100 μl to each well, pipette to mix well, and incubate on ice for 30 min; centrifuge at 500 g at 4°C for 5 min, then discard the supernatant. j. Washing: Add 200 μl of Stain Buffer Buffer, gently pipette to mix well, and centrifuge at 500 g at 4°C for 5 min, then discard the supernatant. k. Surface staining: 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), APC-Cy7-CD44 (1:250) with Stain Buffer. Add 100 μl to each well, pipette gently to mix, and incubate on ice for 30 minutes.

[0104] l. Washing: Add 250 μl of Stain Buffer, pipette gently to mix, centrifuge at 500 g for 5 minutes at 4°C, and discard the supernatant. m. Fixation: Add 200 μl of 2% paraformaldehyde fixative, pipette gently to mix, and incubate on ice for 15 minutes. Centrifuge at 500 g for 8 minutes at 4°C and discard the supernatant. n. Washing: Add 250 μl of Stain Buffer, pipette gently to mix, centrifuge at 500 g for 8 minutes at 4°C, and discard the supernatant. o. Resuspension: Resuspend the cells in 200 μl of 1×PBS and transfer to a 5-ml flow tube. Analyze the cells using an LSR Fortessa instrument and collect data. p. Analysis: Analyze using FlowJo 10.0.7 software and count the percentage of positive cells.

[0105] The results are as Figure 10 shown. mRNA-eVLP can stimulate a stronger GC B cell response in the inguinal lymph nodes compared to mRNA-PreF, especially stimulating the F-specific GC B cell response, while there is no significant difference between the two in the Tfh cell response.

[0106] The memory B cell response is as Figure 11 shown. At 18 weeks post-immunization, eVLP generates a stronger F-specific MBC response, which may indicate that it can stimulate more persistent immune protection.

[0107] As can be seen from the above examples, the present invention provides an RSV mRNA vaccine with self-assembled enveloped virus-like particles based on mRNA technology and eVLP technology, which can induce a stronger neutralizing antibody level and memory B and memory T cell immune responses compared to PreF in a mouse model, achieving the purpose of efficient and long-term protection.

[0108] Example 4 The above-mentioned eVLP formation technology can also be applied to adenovirus vector vaccines. In this example, the chimpanzee adenovirus AdC68 vector is taken as an example.

[0109] The AdC68 vector is adopted. SEQ ID NO: 43-44 are the target sequences of the RSV AdC68-PreF vaccine, and SEQ ID NO: 45-46 are the target sequences of the RSV AdC68-PreF-eVLP vaccine (EGE-5 group).

[0110] Construction of adenovirus vector The pAdC6-empty vector was linearized using I-CeuⅠ and PI-SceⅠ restriction endonucleases, reacted in a 37°C water bath for 2 hours, and then inactivated in a 65°C water bath for 20 minutes to obtain the linearized vector for standby. Using the pCDNA3.1-PreF and pCDNA3.1-PreF-eVLP plasmids constructed in Example 1 as templates, primers specific to and including the homologous arms of the linearized pAdC6 vector were designed to amplify the target fragments including the CMV promoter, PreF / PreF-eVLP gene, and BGH polyA. The target fragments were homologously recombined with the linearized vector using a homologous recombination kit, and the recombinant products were transformed into Stbl2 competent cells. After activation, they were spread on an LB plate containing ampicillin and cultured overnight at 30°C in an inverted position. Single colonies were picked, and Sanger sequencing was used to verify whether the inserted sequences were correct. One colony with positive identification by colony PCR and correct Sanger sequencing was selected for large-scale culture, and the plasmid was extracted using an endotoxin-free large-scale extraction kit. The extracted plasmid was digested and identified using three restriction endonucleases, AleⅠ, Xho, and XcmⅠ, respectively. The size of the digested products was identified using 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).

[0111] Adenovirus packaging, amplification, purification, and genome identification ① Adenovirus packaging a. HEK293A cell culture and plating: When the confluence of HEK293A cells in a T75 culture flask reaches about 90%, the cells are digested and resuspended. The single-cell suspension is counted using a cell counting plate, and 1×10 6 cells are added drop by drop to a 6-well plate containing 2 ml of fresh DMEM complete medium that has been prepared. Gently shake well and place it in a 37°C, 5% CO 2 cell incubator for overnight culture.

[0112] b. Linearization of pAdC68-PreF or pAdC68-PreF-eVLP adenovirus vector: The recombinant adenovirus vectors pAdC68-PreF and pAdC6-PreF-eVLP were linearized with PacⅠ restriction endonuclease respectively, digested at 37°C for 2 hours, inactivated at 65°C for 20 minutes, and then large linear fragments with 5' and 3' long terminal repeats exposed were obtained. They could be directly used for the next transfection or stored in a -20°C refrigerator overnight.

[0113] c. Transfection: The linearized pAdC68-PreF and pAdC6-PreF-eVLP vectors were transferred into sterile EP tubes respectively in a biosafety cabinet, mixed with 180 μl of Opti-MEM medium. Then, 5 μl of transfection reagent X-tremeGENE HP (DNA: transfection reagent = 1:2.5) was added to the 200 μl transfection system, gently flicked to mix evenly, and left to stand at room temperature for 20 minutes. Then, it was added drop by drop to a 6-well plate seeded with HEK293A cells. The cells were observed daily after transfection for 5 days. When more than 60% of plaques appeared, the cells and supernatant were collected.

[0114] ② Adenovirus amplification The virus collected from the 6-well plate was repeatedly frozen and thawed 3 times between room temperature (25°C) and -80°C for three consecutive amplifications, from P0 to P3 generations. The amplification ratio of adenovirus was controlled between 1:6 and 1:10. Finally, after obtaining the cells and virus in 30 150-mm culture dishes, they were centrifuged at 500 g for 15 minutes, the supernatant was discarded, and finally the cells were resuspended in 8 mL of DMEM medium without antibiotics and serum, repeatedly frozen and thawed 3 times between room temperature and -80°C, and centrifuged at 3000 g for 20 minutes to collect the final virus supernatant.

[0115] ③ Adenovirus purification a. Ultracentrifugation: CsCl density gradient centrifugation was used to purify adenovirus, and the gradient was a discontinuous gradient (1.4 g / mL and 1.2 g / mL). The centrifugation parameters were set as: 25000 rpm, centrifuged at 4°C for 2.5 hours. After centrifugation, two obvious adenovirus bands could be seen with the naked eye. Among them, the upper band was incomplete adenovirus. A 1-ml syringe was used to pierce the wall of the centrifuge tube slightly below the lower band, suck out the lower virus band, cover the syringe needle cap, and place it on ice.

[0116] b. Desalting: The purified virus solution was desalted and exchanged with a desalting gel, and it was replaced with PBS and glycerol was added to a final concentration of 10%.

[0117] c. Determination of adenovirus particle number: The A260 absorbance value was measured using a Nanodrop. First, PBS containing 10% glycerol was used as a blank control to calibrate the instrument. Calculate the final virus concentration as A260 value × 1.1 × 10 12 vp / ml. After recording, the virus was stored in a -80 °C refrigerator.

[0118] (4)Determination of adenovirus infection titer Virus titer titration was performed according to the instructions of the QuickTiter™ Adenovirus Titer Immunoassay Kit Table 13 Results of adenovirus infection titer determination

[0119] Immunization: Animals: The animals used were Balb / C mice, 6 - 8 weeks old, SPF grade, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0120] Immunization and sampling: According to the experimental requirements, AdC68 - PreF and AdC68 - PreF - eVLP vaccines were intramuscularly injected, and at the same time, immunization was carried out with empty - vector AdC68 - empty as a control (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 h, it was centrifuged at 10000 rpm for 10 min at 4 °C to separate the serum, which was then aliquoted and stored in a -80 °C low - temperature refrigerator.

[0121] Table 14 Experimental groups

[0122] Subsequently, the detection methods for binding antibodies and neutralizing antibodies were the same as in Example 3.

[0123] The results of binding antibodies are as Figure 11 shown. Compared with AdC68 - PreF, AdC68 - PreF - eVLP stimulates higher levels of binding antibodies and requires a lower dose to stimulate antibody production.

[0124] The results of neutralizing antibodies are as Figure 12 shown. The eVLP technology stimulates stronger neutralizing antibodies against RSV Long in mice compared to PreF. It can induce neutralizing antibody levels at a dose of 10 6 IFU, while at the same dose, AdC68 - PreF does not produce neutralizing antibodies, and the neutralizing antibodies of AdC68 - PreF - eVLP at a dose of 10 6 IFU are higher than those of AdC68 - PreF at 10 7IFU administration, with a dose difference of more than tenfold.

[0125] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope 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 is mainly composed of the nucleotide sequence of RSV F protein and EGE motif.

2. The RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 1, characterized in that: 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.

3. The RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 1, characterized in that: The RSV F protein is a modified F protein mutant.

4. The RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 3, characterized in that: The modified F protein mutant includes an optimized PreF protein or a truncated PreFΔCT protein.

5. The RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 4, characterized in that: The optimization method of the optimized PreF protein includes one or more of signal peptide replacement, P27 fragment deletion and amino acid mutation.

6. The RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 5, characterized in that: 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.

7. The RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 6, characterized in that: The amino acid sequences of the optimized PreF proteins are 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.

8. The RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 4 or 7, characterized in that: The truncated PreF△CT protein is a truncation of any 1 to 23 amino acids at the C-terminus.

9. The RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 8, characterized in that: 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.

10. The RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 2, characterized in that: The EGE motif includes EPM, linker and EABR; the EPM is a 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 a nucleotide sequence shown in any one of SEQ ID NOs: 24, 26, 28, 30, 32, 34, and 36.

11. The RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to claim 10, characterized in that: 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.

12. A method for constructing a RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to any one of claims 1 to 11, characterized in that: The target gene is constructed into an expression plasmid, cell transfection is performed, the cells are cultured and the supernatant is collected to obtain spontaneously formed enveloped virus-like particles RSV PreF-eVLP.

13. Use of the truncated PreFΔCT protein in the RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to any one of claims 1 to 11, characterized in that: The purpose of the truncated PreFΔCT protein is to increase the expression level of the F protein on the membrane.

14. A RSV mRNA vaccine platform constructed by spontaneously forming enveloped virus-like particles RSV PreF-eVLP according to any one of claims 1 to 11, characterized in that: The RSV mRNA vaccine contains the target gene described in any one of claims 1 to 11.

15. The RSV mRNA vaccine platform of claim 14, wherein: The nucleotide sequence of the RSV mRNA is shown in SEQ ID NO: 37 or SEQ ID NO: 38; the nucleotide sequence of the optimized RSV mRNA is shown in SEQ ID NO:

39.

16. The method for constructing the RSV mRNA vaccine platform according to claim 14 or 15, characterized in that: Including but not limited to the following steps: (1) constructing the nucleotide sequence of the target gene or RSV mRNA into an expression vector to obtain a recombinant plasmid; (2) linearizing the recombinant plasmid and using it as a transcription template to obtain in vitro transcribed RSV mRNA; (3) The in vitro transcribed RSV mRNA is digested, capped, purified, and then encapsulated in liposomes to obtain an RSV mRNA vaccine.

17. The construction method according to claim 16, characterized in that: The expression vector is pCDNA3.1(+) expression plasmid, and the liposome is liposome SM102.

18. The liposomal RSV mRNA vaccine obtained by the construction method of claim 16 or 17, characterized in that: The liposomal RSV mRNA vaccine is in the form of liquid or lyophilized powder.

19. An RSV adenovirus vector vaccine constructed by containing the RSV PreF-eVLP, a spontaneously formed enveloped virus-like particle according to any one of claims 1 to 11.

20. The RSV adenovirus vector vaccine according to claim 19, characterized in that 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.

21. The RSV adenovirus vector vaccine according to claim 20, characterized in that The nucleotide sequence of the target gene in the RSV adenovirus vector vaccine is shown in SEQ ID NO: 43 or SEQ ID NO: 45, and the amino acid sequence is shown in SEQ ID NO: 44 or SEQ ID NO:

46.

22. Use of the RSV PreF-eVLP that spontaneously forms enveloped virus-like particles according to any one of claims 1 to 11, or the RSV mRNA vaccine platform according to claim 14, or the liposomal RSV mRNA vaccine according to claim 18, or the RSV adenovirus vector vaccine according to any one of claims 19 to 21 in the preparation of a respiratory syncytial virus vaccine.

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