A respiratory syncytial virus f protein mutant and uses thereof

Abstract

The application relates to the technical field of viral vaccines, and discloses a respiratory syncytial virus F protein mutant and application, wherein the amino acid sequence of the mutant protein is shown as SEQ ID NO: 3. The F protein mutant designed by the application overcomes the problems of low in-vivo expression and insufficient pre-fusion conformation stability of existing wild-type and mutant proteins; a new mutant with 37.0% higher expression than DS-Cav1 and 98.7% pre-F proportion is provided; the vaccine prepared by using the RN007-3 mutant designed by the application can produce long-acting immunoprotective effects in animals, and has great popularization and application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of viral vaccine, in particular to a respiratory syncytial virus F protein mutant and application thereof. BACKGROUND

[0002] The statements in this section merely provide background information related to the present disclosure and can not constitute the prior art.

[0003] RSV (respiratory syncytial virus) infection causes extensive morbidity and mortality in infants and the elderly, with approximately 64 million cases of infection and 160,000 deaths worldwide each year. RSV infection-induced pneumonia is one of the main causes of clinical death, which highlights the urgent need for RSV vaccines that elicit or provide protective immune responses.

[0004] The full-length genome of RSV is about 15.2 kb, which encodes 11 kinds of proteins, including 2 non-structural proteins and 9 structural proteins; among them, the small hydrophobic (SH), attachment (G) and fusion (F) genes encode three surface proteins of the virus, among which the G and F proteins are two important membrane surface glycoproteins of HRSV, which stimulate the body to produce neutralizing antibodies, the G protein helps the virus to adhere to the host cell, the F protein is responsible for virus fusion and syncytium formation, and the function of the SH protein is not clear, which may be related to viral infection.

[0005] RSV enters the cell by fusion of the viral particle with the membrane of the infected cell mediated by the F protein, and forms a syncytium by fusion of adjacent cells on the surface of the infected cell mediated by the F protein. The full-length sequence of the wild-type F protein contains about 574 amino acids, and the reference sequence is shown as SEQ ID NO: 1. Among them, the first to 513th amino acids are the extracellular region of the F protein, and the 514th to 574th amino acids are the transmembrane region and intracellular region of the F protein (as shown in Figure 1 ). The mature RSV F protein initially exists in a metastable pre-fusion F conformation, and then undergoes a conformational change, resulting in insertion of a hydrophobic fusion peptide into the host cell membrane. Then, the F protein refolds into a stable, elongated post-fusion F conformation, which facilitates fusion of the virus with the host cell membrane. Due to the inherent instability of the pre-fusion F protein, this protein can prematurely transform into the stable post-fusion F form in solution and on the virus surface. Current research has implemented stabilization of the pre-fusion F conformation through protein engineering techniques. Stabilized pre-fusion F induces higher titers of neutralizing antibodies than post-fusion F in animal models. The current main method is to implement stabilization of the pre-fusion F conformation by forming disulfide bonds between different amino acids and adjusting the amino acids in the hydrophobic cavity position. However, the current stabilization mutation scheme has the challenges of low protein expression and insufficient stability of the pre-fusion conformation.

[0006] For example, the prior art CN118078974A discloses an mRNA vaccine of respiratory syncytial virus and its construction method and application, which uses a wild type F protein, although high titer IgG antibodies are produced, but the level of protective antibodies is not high (McLellan et al. 2013). CN118078974A discloses an optimized pre-fusion conformation F protein coding sequence based on the design basis of WO 2022 / 221336 A1, but it is not compared with its reference design WO 2022 / 221336 A1 to confirm its superiority, and the results lack evidence to prove.

[0007] The inventors of the present application are committed to developing mutant F proteins with higher protein expression and higher pre-fusion conformation stability, significantly improving the protein expression of RSV pre-fusion conformation F, and significantly enhancing the stability of the pre-fusion conformation, with strong and stable immunogenicity. SUMMARY

[0008] The present application aims to: in view of the current low protein expression and insufficient pre-fusion conformation stability, a respiratory syncytial virus F protein mutant and application are provided, which has strong immunogenicity, good safety, can produce specific neutralizing antibodies, and can resist RSV virus infection. The total expression of the F protein designed as RN007-3 provided in the present case is 37.0% higher than that of the DS-Cav1 design, and the pre-F proportion is significantly improved to 98.7% compared with the wild type and DS-Cav1.

[0009] The technical solutions of the present application are as follows:

[0010] A respiratory syncytial virus F protein mutant, which is mutated from a wild type respiratory syncytial virus F protein in at least one of the following:

[0011] 1) The p27 polypeptide fragment at positions 106-136 is deleted and replaced by GGGSG;

[0012] 2) The glutamic acid at position 487 is mutated to glutamine (E487Q);

[0013] 3) The valine at position 296 is mutated to phenylalanine (V296F);

[0014] 4) The valine at position 220 is mutated to leucine (V220L);

[0015] 5) The glutamic acid at position 30 is mutated to cysteine (E30C);

[0016] 6) The asparagine at position 388 is mutated to cysteine (N388C);

[0017] 7) The tyrosine at position 44 is mutated to cysteine ​​(Y44C).

[0018] According to a preferred embodiment, the sequence of the RSV virus F protein can be derived from respiratory syncytial virus sequences of type A2, A1, B1, or Along. In addition to the specific sequence used in the examples, it can also be an RSV F protein sequence of the same or different genotypes.

[0019] In one possible implementation, the partial amino acid sequence of the RSV virus F protein receptor-binding region is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the total amino acid sequence of the RSV virus F protein, and the protein encoded by this partial amino acid sequence has the same or substantially the same immunogenicity as the protein encoded by the total amino acid sequence.

[0020] According to a preferred embodiment, the amino acid sequence of the wild-type respiratory syncytial virus F protein is shown in SEQ ID NO:1.

[0021] Another aspect of the present invention provides a respiratory syncytial virus F protein mutant, comprising the aforementioned seven mutations, the amino acid sequence of which is shown in SEQ ID NO:3.

[0022] According to a preferred embodiment, the nucleic acid sequence of the respiratory syncytial virus F protein mutant is a nucleic acid sequence capable of encoding the amino acid sequence shown in SEQ ID NO:3.

[0023] According to a preferred embodiment, the nucleic acid sequence of the respiratory syncytial virus F protein mutant is shown in SEQ ID NO:4.

[0024] Application of an mRNA sequence encoding a mutant of the respiratory syncytial virus F protein as described above in the preparation of a respiratory syncytial virus vaccine.

[0025] According to a preferred embodiment, the mRNA sequence is an mRNA sequence capable of encoding an amino acid sequence as shown in SEQ ID NO:3.

[0026] According to a preferred embodiment, the mRNA sequence is shown in SEQ ID NO:4.

[0027] A respiratory syncytial virus (RSV) mRNA vaccine comprising a mRNA sequence encoding a mutant RSV F protein, as shown in SEQ ID NO:4.

[0028] The respiratory syncytial virus F protein mutant in this application is named RN007-3 mutant.

[0029] According to a preferred embodiment, the mRNA sequence can be a nucleic acid sequence that includes the sequence shown in SEQ ID NO:4 of this application: a nucleic acid sequence encoding a partial or complete amino acid sequence of the receptor-binding region of the RSV virus F protein, a nucleic acid sequence encoding the RSV virus F2 protein, or a nucleic acid sequence encoding the full-length RSV virus F protein.

[0030] The stop codon encoding the RSV viral antigen gene is one or more TGA, TAA, or TAG.

[0031] According to a preferred embodiment, the mRNA sequence in the vaccine can be encapsulated by cationic lipids.

[0032] Preferably, the cationic lipid, in addition to SM-102, can also be Dlin-MC3-DMA or ALC-1059. The encapsulation process used for the cationic lipid is T-mix, microfluidics, or IJPM technology.

[0033] According to a preferred embodiment, the nitrogen-to-phosphorus ratio in the encapsulation process is 3:1-8:1 or 10:1-15:1.

[0034] According to a preferred embodiment, the vaccine further includes PBS or Tris buffer.

[0035] According to a preferred embodiment, the pH range of the vaccine is 6.0-8.0.

[0036] According to a preferred embodiment, the vaccine further includes a protective agent component, which is sucrose.

[0037] According to a preferred embodiment, the RSV virus vaccine is prepared in the form of a nasal spray, an oral dose, or an injection.

[0038] According to a preferred embodiment, an RSV virus vaccine is used for two or more immunizations.

[0039] Compared with existing technologies, the advantages of this invention are:

[0040] 1. A respiratory syncytial virus (RSV) F protein mutant and its application. The total expression level of F protein in the design RN007-3 provided in this case is 37.0% higher than that in the DS-Cav1 design, and the pre-F ratio is significantly increased to 98.7% compared with DS-Cav1. The expression level is significantly better than wild-type F or the internationally recognized DS-Cav1 design, and the pre-F ratio expressed is also significantly better than existing designs.

[0041] 2. A respiratory syncytial virus (RSV) F protein mutant and its application: By detecting IgG antibody titers and neutralizing antibody titers, the RSV F protein pre-fusion conformation mutant RN007-3 mRNA vaccine provided by this invention induces high levels of neutralizing antibodies against the A2 and B1 epidemic strains in mice. The antibody levels and humoral immunity levels are significantly higher than those of vaccines designed by existing technologies.

[0042] 3. A respiratory syncytial virus (RSV) F protein mutant and its application. The mutant RN007-3 mRNA vaccine designed in this application is significantly superior to the wild-type and the internationally recognized DS-Cav1 design. Furthermore, both in vivo and in vitro, the RN007-3 mRNA vaccine designed in this application can produce an immunoprophylactic effect superior to existing technologies. Mouse experiments have confirmed its long-term antiviral effect. It demonstrates complete protection against mixed infection with A2 genotype RSV virus, with protection manifested in high and high neutralizing antibody levels, low viral load in the lungs and nasal turbinates, and low pathological manifestations. All data indicate that the RSV mRNA vaccine designed with RN007-3 has a significantly better protective effect than the wild-type F design and is superior to the existing recognized DS-Cav1 design.

[0043] 4. A respiratory syncytial virus F protein mutant and its application. The process provided by this invention is different from the laboratory process and is a scalable industrial production process. The plasmids and RNA prepared meet the standards for commercial production.

[0044] 5. A respiratory syncytial virus F protein mutant and its application, which has high safety. Its mRNA has no ability to replicate in the cells of animals and humans, and the immunogenicity of the modified mRNA molecule itself is also very limited, making it very safe to use as a vaccine. Attached Figure Description

[0045] Figure 1 This is the transmembrane region and intracellular region of an amino acid profile encoding the F protein of a respiratory syncytial virus (RSV).

[0046] Figure 2 This is a plasmid map of pcDNA3.1;

[0047] Figure 3 The plasmid map of pIVT-D1-Kan-BsaI;

[0048] Figure 4 The plasmid map of pIVT-D1-Kan-BsaI, which contains the F protein mutant;

[0049] Figure 5The image shows the electrophoretic detection pattern after plasmid linearization; lane 1: DNA ladder, lane 2: wild-type F, lane 3: DS-Cav1, lane 4: RN007-3;

[0050] Figure 6 The results of capillary electrophoresis analysis showed that the prepared mRNA had high integrity and purity.

[0051] Figure 7 Dynamic light scattering was used to detect mRNA-LNPs. The LNPs were uniform in size and exhibited a uniform normal distribution (DSL).

[0052] Figure 8 The protein expression of LNP after transfection into HEK293 cells;

[0053] Figure 9 Results of specific IgG antibody induction in mice by RSV mRNA vaccine;

[0054] Figure 10 The mRNA vaccine induced a high level of neutralizing titer against the A2 strain in mice;

[0055] Figure 11 The mRNA vaccine induced a high level of neutralizing titer against the B1 strain in mice;

[0056] Figure 12 The status of neutralizing antibodies in the serum of cotton mice after the second immunization;

[0057] Figure 13 The viral copy number in the lungs of cotton rats after challenge.

[0058] Figure 14 The number of nasal turbinate virus copies after challenge in cotton mice;

[0059] Figure 15 HE staining for lung pathology in cotton rats after viral challenge;

[0060] Figure 16 The pathological scoring of the lungs of cotton rats after viral challenge was performed using a 10-point scale. Detailed Implementation

[0061] The specific embodiments listed in this invention are merely examples, and the invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products. To better illustrate this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of this invention.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0063] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0064] Example 1

[0065] (1) Synthesis of mRNA vaccine coding sequence

[0066] The coding sequence of the fusion protein (F protein) in the mRNA vaccine is based on the F gene sequence SEQ ID NO:1 (Uniport: P03420) of the A2 type RSV epidemic strain.

[0067] The F protein gene coding sequence underwent codon optimization and further design, replacing rare codons in the sequence. While avoiding specific restriction enzyme sites such as XbaI, SalI, and BsaI, and unfavorable motifs, the sequence was modified to improve the translation efficiency of the antigen protein in host cells. The original amino acid sequence was then mutated to design an innovative mutant RN007-3 (SEQ ID NO.3), different from the internationally recognized mutant DS-Cav1 (SEQ ID NO.2), as the coding sequence for the F antigen protein in the RSV mRNA vaccine. This mutant was then synthesized by a gene synthesis company and cloned into the pcDNA3.1 vector. Figure 2 ).

[0068] (2) Construction of mRNA vaccine transcription vector

[0069] The RSV mRNA vaccine in vitro transcription vector pIVT-D1-Kan-BsaI sequence contains the following elements: T7 promoter SEQ ID NO.5, 5' untranslated region (5' UTR) SEQ ID NO.6, S antigen protein encoding gene, 3' untranslated region (3' UTR) SEQ ID NO.7, and polyadenylated nucleotide (Poly A) SEQ ID NO.8. Downstream of the Poly A tail structure is a plasmid-linearized restriction enzyme site (Bsa I). Figure 3 ).

[0070] The synthesized DNA template sequence and pIVT-D1-Kan-BsaI vector were double-digested with XbalI and SalI and ligated overnight with T4 ligase to generate the pIVT-RSV-F recombinant vector. Figure 4 ), and were transformed into stbl3 competent cells to select single clones for preservation.

[0071] (3) Preparation of mRNA in vitro transcription plasmids

[0072] A. The transformant strains of pIVT plasmid were fermented, lysed with 0.2% NaOH and 1% SDS solution, neutralized with 2M acetic acid / potassium acetate solution, and the supernatant was harvested by centrifugation at 8000 rpm for 30 minutes.

[0073] B. 6FF molecular sieve chromatography (purchased from Cytiva): Primarily removes E. coli RNA; elute with 6FF chromatography buffer (100 mmol / L Tris-HCl, 20 mmol / L EDTA, 5 mol / L (NH4)2SO4, pH 7.5±0.1), collect the first elution peak, begin collecting when UV260 rises to 50 mAU or higher, and stop collecting when it falls to 500 mAU or lower. The linear flow rate during chromatography should not exceed 800 cm / h.

[0074] C. PS hydrophobic chromatography (Plasmid Select (PS) purchased from Cytiva): Packing material name: Plasmid Select; mainly binds supercoiled plasmid DNA to remove open circular plasmid DNA; PS chromatography buffer A (100 mmol / L Tris-HCl, 20 mmol / L EDTA, 5 mol / L (NH4)2SO4, pH 7.5±0.1) was used to equilibrate the Plasmid Select (PS) chromatography column;

[0075] Load the 6FF chromatography collection solution onto the packing material at a loading rate not exceeding 1 mg plasmid / mL packing material.

[0076] (4) Linearization

[0077] The plasmid obtained in step (3) was digested with Bsa I (purchased from Nanjing Novizan) restriction endonuclease. The reaction system is shown in Table 1:

[0078] Table 1 Linearized Enzyme Digestion Reaction System

[0079]

[0080] The linearized harvest was purified according to the steps in step (3), which will not be repeated here. The plasmid digestion efficiency was assessed by electrophoresis on a 1% agarose gel at 150 V for 20 min. The results are as follows: Figure 5 As shown, the target band is consistent with the expected size. Figure 5 In the middle, lane 1: DNA ladder, lane 2: wild type F, lane 3: DS-Cav1, lane 4: RN007-3.

[0081] (5) Preparation of mRNA by in vitro transcription

[0082] The raw materials listed in Table 2 below were all purchased from Nanjing Shenji. After the reaction system was prepared, it was reacted at 37 degrees Celsius and 10 rpm for 4 hours. After the reaction was completed, 4 ml of 0.5M EDTA was added to the reaction system to terminate the reaction.

[0083] Table 2 In vitro transcription system

[0084]

[0085] (6) mRNA purification

[0086] dT affinity column chromatography (mRNA chromatography 1, Oligo dT purchased from Thermofisher) is a highly selective method for purifying target mRNAs containing polyA tails, maximizing the removal of template DNA, incompletely transcribed mRNA, proteins, and NTPs in the reaction system. The main chromatographic parameters are:

[0087] RNA chromatography solution A: 20 mmol / L Tris-HCl, pH 7.5, 2 mmol / L EDTA, 500 mmol / L NaCl;

[0088] RNA chromatography solution B: 20 ​​mmol / L Tris-HCl, pH 7.5;

[0089] Add 1 / 9 volume of the transcription product to 4 mol / L NaCl. dT affinity chromatography (packing material: Oligo dT): Equilibrate the Oligo dT column with at least 1 column volume of dT chromatography buffer A. Load the dT chromatography sample solution at a volume not exceeding 0.5 column volumes, with a linear flow rate not exceeding 150 cm / h. After loading, elute with at least 2 column volumes of dT chromatography buffer A, with a linear flow rate not exceeding 150 cm / h. Then elute with at least 2 column volumes of 50% dT chromatography buffer B, with a linear flow rate not exceeding 300 cm / h. Finally, elute with dT chromatography buffer B. Start collecting when UV260 rises to 50 mAU or higher, and stop collecting when it falls to 50 mAU or lower.

[0090] Add 20 KU of DNase I enzyme to the harvest fluid to remove the DNA template.

[0091] The second step is Core400 tomography:

[0092] Equilibrate the Core 400 chromatography column (packing material: Capto Core400; purchased from Cytiva) with at least one column volume of dT chromatography buffer B. Load the DNase I digest solution, with a loading volume not exceeding three column volumes. Begin collecting when the UV260 rises to 50 mAU or higher. After loading, elute with at least two column volumes of dT chromatography buffer B, collecting the target material during the eluent process. Stop collecting when the UV260 drops to 100 mAU or lower; this is the Core chromatography collect solution. The linear flow rate during chromatography should not exceed 160 cm / h.

[0093] The collected chromatographic flow-through samples were first concentrated 5 ± 1 times using a 100 kD tangential flow system, then replaced 10 times with 2.5 mmol / L sodium citrate (pH 6.4). Finally, 1–4 volumes of 2.5 mmol / L sodium citrate (pH 6.4) were added, and after filtration for sterilization, the mRNA concentration was determined using a NanoDrop spectrophotometer. mRNA purity was analyzed by capillary electrophoresis.

[0094] like Figure 6 As shown, this process can achieve an mRNA purity of over 95% and good mRNA integrity.

[0095] (7) Lipid nanoparticle encapsulation

[0096] The raw materials for liposomes are shown in Table 3:

[0097] Table 3 Liposome Components

[0098]

[0099] Take frozen mRNA and thaw it along with its container in crushed ice; remove it when all mRNA has thawed and set aside; dilute with 25 mM sodium citrate buffer to ensure a final mRNA concentration of 167 µg / mL; turn on the microfluidic control instrument; open the settings interface and input the required parameters (Maianna R-SDM chip, flow rate 20 mL / min; aqueous phase: alcohol phase = 3:1; 0.5 mL initial waste liquid, 0 mL final waste liquid; PNI conventional chip, flow rate 18 mL / min; aqueous phase: alcohol phase = 3:1; 0.5 mL initial waste liquid); according to the settings, use a syringe to draw the corresponding lipid alcohol solution / mRNA buffer and install it onto the instrument to start preparing the LNP stock solution (draw 1.5 mL of mRNA solution and 0.5 mL of lipid alcohol solution respectively, and prepare approximately 1.5 mL). LNP stock solution; the collected LNP stock solution was diluted 5 times with PBS buffer (or Tris-HCl buffer) and concentrated back to the original volume using an ultrafiltration tube (centrifugation parameters: 2000g, 10min, 25℃, repeated dilution and ultrafiltration twice); sucrose buffer replacement: the ultrafiltered LNP solution was replaced with sucrose solution until the final sucrose concentration was 5%; the above product was sterile filtered through a 0.22 μm membrane to obtain the finished LNP solution, and the LNP solution was placed in an enzyme-free sterile container; the finished LNP solution can be stored frozen at 2~8℃, -15~-25℃, or -65~-85℃.

[0100] (8) Detection

[0101] Dynamic light scattering technique was used to detect the particle size and distribution of lipid nanoparticles, such as... Figure 7 As shown, the lipid nanoparticles have a uniform diameter distribution between 80-120 nm. The encapsulation efficiency was detected using the Ribogreen kit (purchased from Invitrogen) according to the kit instructions, and the encapsulation efficiency reached over 90%.

[0102] Table 4 Encapsulation rate data

[0103]

[0104] 2 µg of mRNA-LNP was used to infect 80% confluence HEK293 cells in 6-well plates. After 24 hours, samples were collected, lysates were harvested, and protein expression was detected by Western blotting. First, 10% SDS-PAGE was used... PAGE gel was used to perform SDS-PAGE on protein samples. PAGE electrophoresis was performed at a constant voltage of 80 V for 30 min, followed by a constant voltage of 120 V for 60 min. After electrophoresis, the protein was transferred to a PVDF membrane under a constant current of 250 mA for 90 min. The PVDF membrane was then blocked overnight at 4 °C with 5% skim milk powder, followed by washing three times with PBS buffer for 5 min each time. Anti-RSV F protein monoclonal antibody (purchased from Novizan) diluted 1000-fold with PBS buffer was added and incubated at room temperature for 1 h. The membrane was then washed three times with PBST buffer for 10 min each time. Horseradish peroxidase-conjugated goat anti-mouse IgG (purchased from Abcam) diluted 20000-fold was added and incubated at room temperature for 1 h. The membrane was then washed three times with PBST buffer for 10 min each time. Finally, the membrane was washed once with PBS, and ECL chemiluminescence was performed using enhanced ECL solution (purchased from Beyotime Biotechnology) according to the manufacturer's instructions. Results are as follows. Figure 8 As shown, a distinct band of approximately 60 kDa appeared in all experimental groups, consistent with expectations.

[0105] The relative expression level of the F protein in the RN007-3 mRNA invented in this case is significantly higher than that of wild-type F and DS-Cav1 designed mRNA.

[0106] The F protein in different conformations in the cell supernatant was quantitatively analyzed using an F protein antibody (AM14) and an F protein pre-fusion conformation-specific antibody (D25 antibody).

[0107] Take 2 µg / well of AM14 and D25 antibody working solution, add 100 µl / well, and coat overnight at 2-8℃. Discard the coating solution from the overnight coated ELISA plate, add 300 µl of washing buffer to each well and wash 3 times. Invert the plate onto absorbent paper, pat dry any remaining liquid in the wells, add 200 µl / well of blocking buffer, cover with sealing film, and incubate at 37℃ for 1 hour. Dilute the LNP-transfected cell supernatant, add 100 µl / well to the sealed ELISA wells. For the negative control, add 100 µl of control cell supernatant; for the blank control, add 100 µl of diluent; for the complete blank control, add no liquid. Incubate at 37℃ for 2 hours. Remove the ELISA plate, discard the samples, wash 5 times with washing buffer, and pat dry. Add 100 µl of HRP-labeled F protein polyclonal antibody working solution to each well and incubate at 37℃ for 1 hour. The protein was developed using TMB reagent, and the absorbance at 450 nm and 650 nm was measured after development was terminated. The protein content was calculated using a standard curve.

[0108] The test results are shown in Table 5 below:

[0109] Table 5: Protein content detection results

[0110]

[0111] As shown in Table 5, the results demonstrate that the total expression level of F protein in the DS-Cav1 design is approximately 19.3% higher than that of the wild type, and the pre-F expression level is approximately 120.6% higher. Furthermore, the total expression level of F protein in the RN007-3 design provided in this study is 37.0% higher than that of the DS-Cav1 design, and the pre-F proportion is significantly increased to 98.7% compared to both the wild type and DS-Cav1. This proves that the RN007-3 design not only significantly improves the expression level of the RSV F protein sequence compared to the wild type, but also maintains a significant advantage in total expression and pre-F proportion compared to the internationally recognized DS-Cav1 design.

[0112] (9) Safety and efficacy trials of mRNA vaccines in mice

[0113] The encapsulated LNP solution was used as the vaccine stock solution for mouse immunization.

[0114] The experimental groups and immunization doses are shown in Table 6:

[0115] Table 6: Trial Groups and Immunization Doses

[0116]

[0117] To verify the immunogenicity of the RSV mRNA vaccine described in this invention, an animal experiment was conducted in SPF-grade Balb / c mice. Twenty 6-8 week old female Balb / c mice were randomly divided into four groups. Group 1 mice were injected with PBS as a blank control group; Group 2 mice were injected with 5 μg of wild-type F mRNA vaccine; Group 3 mice were injected with 5 μg of DS-Cav1 mRNA vaccine; and Group 4 mice were injected with 5 μg of RN007-3 RNA vaccine. All groups received a booster immunization with the same dose 28 days after the initial immunization. The RSVpre-F specific antibody IgG and the neutralizing titers of A2 and B1 strains of true virus were measured at 14 and 42 days after the initial immunization. Abnormal reactions at the injection site and the animals' mental state were observed during the experimental period.

[0118] a) Detection of Pre-F protein-specific IgG antibodies

[0119] On days 14 and 42 post-primary immunization, blood was collected from the retro-orbital venous plexus of mice to prepare serum. The serum was coated overnight in ELISA plates with 2 µg / ml RSV pre-F protein (purchased from Novizan). Five serum aliquots from each group were serially diluted 200-fold to detect pre-F-specific IgG. Serum was added to ELISA plates and incubated at 37°C for 1 hour, washed five times with 0.05% PBST, and then incubated at 37°C for 30 minutes with 1:2000 dilution of HRP-labeled anti-mouse IgG secondary antibody (purchased from Abcam). The plates were washed five times with 0.05% PBST, and TMB substrate (purchased from Beyotime Biotechnology) was added. After colorimetric termination, the absorbance at 650 nm was read using a microplate reader. The final titer was determined by the highest dilution where the absorbance was higher than the background value of the negative control well by 2.5 times.

[0120] IgG test results as follows Figure 9 As shown, immunization of mice with the mRNA vaccine induced a high level of pre-F specific humoral immune response. Whether after the first immunization (day 14) or the second booster immunization (day 42), the serum pre-F specific antibody levels in mice immunized with the RN007-3 mRNA vaccine were higher than those with the wild-type F vaccine or the DS-Cav1 vaccine, reaching a maximum of 10. 6 The above demonstrates that the RSV vaccine RN007-3 exhibits significantly improved immunogenicity compared to wild-type F or existing recognized pre-F designs.

[0121] b) Detection of neutralizing antibody titer against true virus

[0122] The neutralizing antibody titer of a real virus can be used to verify whether the antibodies produced in the animal body have a neutralizing effect on the virus, thus reflecting the level of protective antibodies. To demonstrate whether the RSV vaccine of this invention is protective, the neutralizing titer of mouse serum against two prevalent RSV strains, genotypes A2 and B1, was tested 14 days after the second immunization (day 42). RSV strains of genotypes A2 and B1 preserved in the laboratory were cultured and virus culture solutions were prepared. The virus solution was then cultured at 200 TCID⁻¹. 50 Mouse serum, serially diluted 4-fold from 100-fold to 100-fold, was neutralized at 37°C for 1 h. 100 µl of the mixture was then added to a monolayer of Vero cells cultured in a 96-well plate. Five replicates were set up for each dilution of each sample. Vero cell pathogenesis was observed daily until stable. Observation was repeated for 3-5 days, and the number of wells showing pathogenesis was recorded. Reed... The neutralizing titer of the test antibody in each group of mice was calculated using the Muench formula, and the results are as follows: Figure 10 , Figure 11 As shown, the mRNA vaccine designed by RN007-3 can induce a high level of activity (greater than 2) against both strains.10 The neutralizing antibody levels of RN007-3 were significantly higher than those of DS-Cav1, demonstrating that RN007-3 not only significantly outperformed existing designs in terms of F protein expression, pre-F expression ratio, and IgG levels at the in vitro cellular level, but also significantly outperformed the DS-Cav1 design in terms of neutralizing antibody induction, and possessed broad-spectrum protection.

[0123] c) Safety of mRNA vaccines

[0124] During the observation period, all mice in each group were normal, and there was no obvious swelling or induration at the injection site, proving that the tested mRNA vaccine was safe.

[0125] (10) In vivo efficacy test of mRNA vaccine in rats

[0126] To verify the efficacy of the vaccine of this invention in animals, an in vivo efficacy verification was conducted using a cotton rat model. Eighteen cotton rats aged 8-10 weeks were enrolled and randomly divided into 3 groups. Different components were immunized on day 0 and day 28 according to Table 7.

[0127] Table 7: Trial Groups and Immunization Doses

[0128]

[0129] Fourteen days after the second immunization, serum samples were collected from rats to detect RSV A2 strain neutralizing antibodies. On day 21 after the second immunization, 10... 7 TCID 50 The A2 strain was administered via nasal droplets to cotton rats. The experimental animals were sacrificed on the 5th day after the challenge, and RNA was extracted from lung and nasal turbinate tissues. The viral genome copy number was detected by RT-qPCR.

[0130] a) Serum neutralizing antibody levels in mice 14 days after booster immunization, as shown in... Figure 12 As shown. The detection method is as described in step 9b). The results show that the RN007-3 and DS-Cav1 designed mRNA vaccines induced high levels of RSV neutralizing antibodies in mice. Overall, the immunogenicity of the RN007-3 designed mRNA vaccine group was significantly better than that of the existing DS-Cav1 vaccine.

[0131] b) After challenging cotton rats with the virus, lung and nasal turbinate tissues were collected to extract total RNA, and the viral genome copy number was detected using RT-qPCR. Results are as follows: Figure 13 , Figure 14 As shown, the data indicates that in each of the 6 experimental animals in the PBS group, the mean value detected was greater than 10. 8The viral load was significantly reduced in the immunized group compared to the PBS control group, with a large number of copies / g of virus in the tissue. The pre-fusion conformation F vaccine of DS-Cav1 and RN007-3 was significantly more effective than the wild-type, undesigned F vaccine. Compared to the DS-Cav1 design, the RN007-3 design of this invention resulted in lower viral loads in immunized animals, reaching the limit of detection. These data demonstrate the significant advantages of the pre-fusion conformation design of the RSV F protein in this invention.

[0132] c) Perform necropsy on the lung tissue of the experimental cotton rats and prepare typical pathological sections, such as... Figure 15 As shown, the PBS group ( Figure 15 A) showed extremely severe septal widening and congestion, with partial alveolar hemorrhage; wild-type F group ( Figure 15 B) Moderate septal widening and congestion were observed; DS-Cav1 design group ( Figure 15 C) and RN007-3 design group ( Figure 15 D) Only a very mild, small amount of inflammatory cell infiltration is observed, or no significant pathological features are observed. Rate the pathological condition on a scale of 1-10. Results are as follows: Figure 16 As shown, the PBS group and wild-type F group exhibited validated or moderate lesions, while the DS-Cav1 group and RN007-3 design group showed no lesions or very mild lesions. These results indicate that both pre-F mRNA vaccines provide strong protection against RSV virus challenge, and the RN007-3 design in this study demonstrates superior efficacy.

[0133] SEQ ID NO:1: Amino acid sequence of wild-type F protein of RSV A2 strain (Uniport: P03420)

[0134] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN*

[0135] SEQ ID NO:2: Amino acid sequence of prefusion F protein DS-Cav1 of RSV A2 strain

[0136] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN*

[0137] SEQ ID NO:3 Amino acid sequence of the pre-fusion F protein mutant RN007-3 of the RSV A2 strain of the invention in this case

[0138] MELLILKANAITTILTAVTFCFASGQNITCEFYQSTCSAVSKGCLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNGGGSGFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETLIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEFLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFCPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDQFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN*

[0139] SEQ ID NO:4 Nucleic acid sequence of prefusion F protein mutant RN007-3 of RSV A2 strain, the invention of this case

[0140]

[0141] SEQ ID NO:5:

[0142] TAATACGACTCACTATAGG

[0143] SEQ ID NO:6:

[0144] ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCC

[0145] SEQ ID NO:7:

[0146] CCCAACGGGCCCTCCTCCCC

[0147] SEQ ID NO:8:

[0148] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0149] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A respiratory syncytial virus (RSV) F protein mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO:

3.

2. The use of an mRNA encoding a respiratory syncytial virus F protein mutant as described in claim 1 in the preparation of a respiratory syncytial virus vaccine.

3. The application according to claim 2, characterized in that, The mRNA encodes the amino acid sequence shown in SEQ ID NO:

3.

4. The application according to claim 3, characterized in that, The sequence of the mRNA is shown in SEQ ID NO:

4.

5. A respiratory syncytial virus mRNA vaccine, characterized in that, It contains mRNA encoding an amino acid sequence as shown in SEQ ID NO:

3.

6. The respiratory syncytial virus mRNA vaccine according to claim 5, characterized in that, The sequence of the mRNA is shown in SEQ ID NO:

4.

7. The respiratory syncytial virus mRNA vaccine according to claim 6, characterized in that, The mRNA in the vaccine is encapsulated by cationic lipids.

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