Mutant of RSV pre-fusion F protein, nucleic acid and mRNA vaccine

Abstract

The invention discloses an RSV pre-fusion F protein mutant, nucleic acid and an mRNA vaccine. The mutant is obtained by mutating the full length or partial region of a wild type RSV pre-fusion F protein. The RSV pre-fusion F protein mutant disclosed by the invention can ensure stable conformation before fusion, and is beneficial to inducing generation of a higher-level neutralizing antibody; the respiratory syncytial virus mRNA vaccine can effectively induce generation of a neutralizing antibody aiming at RSV pre-fusion F protein, and after being immunized by the respiratory syncytial virus mRNA vaccine, a host can generate extremely high respiratory syncytial virus F protein specific T cell immune response; the antigen compatibility of the combined mRNA vaccine is good, and high titer aiming at the respiratory syncytial virus and the new crown mutant strain EG5.1 can be realized at the same time.

Description

[0001] The present invention claims priority to Chinese patent application No. 202410307151.5 filed with the Patent Office of China on March 18, 2024, with application number 202410307151.5 and invention name “Mutants, nucleic acids and mRNA vaccines of RSV pre-fusion F protein”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present application belongs to the field of mRNA vaccine technology, and specifically relates to a mutant of RSV pre-fusion F protein, a nucleic acid and an mRNA vaccine. Background Art

[0003] Human respiratory syncytial virus (RSV) belongs to the genus Pneumovirus of the family Paramyxoviridae. It mainly causes lower respiratory tract infections such as bronchiolitis and pneumonia in infants under 6 months old, and upper respiratory tract infections such as rhinitis and colds in older children and adults. RSV is spherical in shape, has an envelope, and has a diameter of about 120nm to 300nm. The genome of RSV is a single-stranded negative-strand RNA, which mainly encodes 10 proteins, namely fusion glycoprotein F, adhesion glycoprotein G, small hydrophobic protein SH, M1 matrix protein, M2 matrix protein, N protein, P protein, L protein, NS1 non-structural protein and NS2 non-structural protein. Fusion glycoprotein F (F) is one of the main viral antigens that stimulate the body to produce protective antibodies.

[0004] RSV is an important viral pathogen that causes lower respiratory tract infections in infants and young children. The vast majority of infants and young children have been infected with RSV at least once before the age of 2. For infants and young children under 1 year old, the number of deaths caused by RSV infection accounts for 6.7% of the total number of deaths, second only to malaria. For infants and young children under 5 years old, the number of deaths caused by RSV infection accounts for 6.29% of the total number of deaths, second only to pneumococcus. In my country, 26.5% of acute respiratory tract infections in infants and young children are caused by RSV infection. The elderly and immunocompromised patients are also susceptible to severe infections caused by RSV, and people with a history of RSV infection are prone to lung diseases such as abnormal lung function and bronchial asthma.

[0005] RSV vaccine is one of the effective measures to prevent RSV infection. With the rapid development of mRNA vaccines, its advantages such as good safety, strong immune effect, fast production speed and wide application range have quickly become a hot method for the development of the biopharmaceutical industry. However, at present, mRNA vaccines for RSV are generally prepared based on the pre-fusion F protein of wild-type RSV, which makes it difficult to ensure that the antigen has a stable pre-fusion conformation to induce a higher level of neutralizing antibodies. At the same time, there is no mRNA combination vaccine for multiple respiratory viruses on the market. Summary of the invention

[0006] The purpose of the present application is to provide a mutant, nucleic acid and mRNA vaccine of RSV prefusion F protein, so as to solve the technical problem that the existing mRNA vaccines against RSV in the prior art are generally prepared based on the prefusion F protein of wild-type RSV, and it is difficult to ensure that the antigen has a stable prefusion conformation to induce the production of higher levels of neutralizing antibodies.

[0007] In order to achieve the above purpose, a technical solution adopted in this application is:

[0008] Provided is a mutant of RSV prefusion F protein, wherein the mutant is obtained by mutation of the full length or a partial region of the wild-type RSV prefusion F protein, wherein the mutation comprises one or more of the following amino acid substitutions: S155C, S190F, V207L, S290C, L373R, A149C, Y458C, S46G, N67I, E92D, S215P, K465Q.

[0009] In one or more embodiments, the mutation comprises the following amino acid substitutions: S155C, S190F, V207L, S290C, L373R, A149C, Y458C, S46G, N67I, E92D, S215P, K465Q.

[0010] In one or more embodiments, the mutant is obtained by the mutation occurring in the full length of the wild-type RSV pre-fusion F protein, and the mutant includes the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence shown in SEQ ID NO.1.

[0011] In one or more embodiments, the mutant is obtained by the mutation in the extracellular domain of the wild-type RSV pre-fusion F protein, and the mutant also includes a trimeric peptide, which includes the amino acid sequence as shown in SEQ ID NO.2, or an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence as shown in SEQ ID NO.2; the mutant includes the amino acid sequence as shown in SEQ ID NO.3, or an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence as shown in SEQ ID NO.3.

[0012] In order to achieve the above purpose, a technical solution adopted in this application is:

[0013] A nucleic acid is provided, comprising a nucleotide sequence for encoding the mutant described in one embodiment above, wherein the nucleic acid comprises the nucleotide sequence as shown in SEQ ID NO.4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.4.

[0014] In order to achieve the above purpose, a technical solution adopted in this application is:

[0015] A nucleic acid is provided, comprising a nucleotide sequence for encoding the mutant described in one embodiment above, wherein the nucleic acid comprises the nucleotide sequence as shown in SEQ ID NO.5, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.5.

[0016] In order to achieve the above purpose, a technical solution adopted in this application is:

[0017] Provided is a ribonucleic acid molecule comprising the nucleic acid described in any one of the above embodiments, further comprising a structural element, wherein the structural element comprises one or more of a 5'UTR sequence, a 3'UTR sequence and a polyA tail sequence.

[0018] In one or more embodiments, the 5'UTR sequence comprises a nucleotide sequence as shown in SEQ ID NO.6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.6.

[0019] In one or more embodiments, the 3'UTR sequence comprises a nucleotide sequence as shown in SEQ ID NO.7, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.7.

[0020] In one or more embodiments, the polyA tail sequence includes at least 100 A nucleotides; preferably, the polyA tail sequence includes a nucleotide sequence as shown in SEQ ID NO.8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.8.

[0021] In order to achieve the above purpose, a technical solution adopted in this application is:

[0022] A DNA molecule is provided, comprising a nucleotide sequence for transcribing the ribonucleic acid molecule described in any of the above embodiments.

[0023] In order to achieve the above purpose, a technical solution adopted in this application is:

[0024] A recombinant plasmid is provided, comprising the DNA molecule described in any one of the above embodiments.

[0025] In order to achieve the above purpose, a technical solution adopted in this application is:

[0026] Provided is a use of the ribonucleic acid molecule described in any of the above embodiments, the DNA molecule described in any of the above embodiments, or the recombinant plasmid described in any of the above embodiments in the preparation of a respiratory syncytial virus mRNA vaccine.

[0027] In order to achieve the above purpose, a technical solution adopted in this application is:

[0028] Provided is a respiratory syncytial virus mRNA vaccine, characterized in that it comprises the ribonucleic acid molecules, vaccine carriers and excipients described in any one of the above embodiments, wherein the vaccine carrier is a lipid nanoparticle.

[0029] In order to achieve the above purpose, a technical solution adopted in this application is:

[0030] Provided is a method for preparing a respiratory syncytial virus mRNA vaccine, comprising:

[0031] Synthesizing a DNA fragment encoding the mutant described in any of the above embodiments and a DNA fragment encoding the structural element described in any of the above embodiments, and cloning them into a first plasmid respectively, so as to obtain a target gene fragment by connecting them on the first plasmid;

[0032] Recovering the target gene fragment from the first plasmid, connecting it to the second plasmid, transforming the first competent cell for cultivation;

[0033] Recovering the bacterial cells, extracting the second plasmid, transforming the second competent cells after sequencing, obtaining the expression plasmid based on the stability of the passage, extracting and linearizing the expression plasmid in sequence after culturing, and recovering the DNA fragments after enzyme digestion to obtain an in vitro transcription template;

[0034] Performing in vitro transcription, and then removing the in vitro transcription template to obtain an mRNA stock solution;

[0035] Dissolving the mRNA stock solution in citrate buffer to obtain an aqueous working solution;

[0036] The aqueous phase working solution is mixed with the lipid phase working solution, and then the solvent is replaced by TFF tangential flow filtration to obtain the respiratory syncytial virus mRNA vaccine.

[0037] In order to achieve the above purpose, a technical solution adopted in this application is:

[0038] Provided is a combined mRNA vaccine for a novel coronavirus and respiratory syncytial virus, characterized in that it comprises:

[0039] The RNA molecule according to any one of the above embodiments;

[0040] A ribonucleic acid molecule comprising a nucleotide sequence for encoding a recombinant spike protein, wherein the recombinant spike protein is derived from a novel coronavirus spike protein and has an S-2P site mutation relative to a wild-type novel coronavirus spike protein;

[0041] Vaccine carrier and excipients, wherein the vaccine carrier is lipid nanoparticles.

[0042] In one or more embodiments, the recombinant spike protein has an S-2P site mutation relative to the spike protein of the novel coronavirus Omicron EG.5.1 strain, and the recombinant spike protein includes an amino acid sequence as shown in SEQ ID NO.11, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence as shown in SEQ ID NO.11.

[0043] In one or more embodiments, the ribonucleic acid molecule of the nucleotide sequence encoding the recombinant spike protein includes a nucleotide sequence as shown in SEQ ID NO.12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.12.

[0044] Different from the prior art, the beneficial effects of this application are:

[0045] The mutant of the RSV pre-fusion F protein of the present application is obtained based on mutations in the full length or partial region of the wild-type RSV pre-fusion F protein, which can ensure the stability of the pre-fusion conformation and help induce the production of higher levels of neutralizing antibodies;

[0046] The respiratory syncytial virus mRNA vaccine of the present application can effectively induce the production of neutralizing antibodies against RSV pre-fusion F protein. After being immunized with the respiratory syncytial virus mRNA vaccine of the present application, the host can produce a very high respiratory syncytial virus F protein-specific T cell immune response;

[0047] The combined mRNA vaccine of the novel coronavirus and respiratory syncytial virus of the present application has good antigenic compatibility and can simultaneously achieve high titers against respiratory syncytial virus and the novel coronavirus mutant strain EG5.1;

[0048] The preparation method of the respiratory syncytial virus mRNA vaccine of the present application can effectively ensure high delivery efficiency, help achieve better immune effects, and help induce the production of higher levels of neutralizing antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0050] Figure 1 This is an electron micrograph of the mRNA vaccine prepared in Example 1 of the present application;

[0051] Figure 2 This is a data graph of IgG antibody titer of mice immunized with mRNA vaccine in Example 5 of the present application;

[0052] Figure 3 This is a data graph of the neutralizing antibody titer of mice immunized with mRNA vaccine in Example 5 of the present application;

[0053] Figure 4This is a data chart of immune cytokine indicators of mice after immunization with mRNA vaccine in Example 5 of the present application. DETAILED DESCRIPTION

[0054] In order to solve the problem that the current mRNA vaccines against respiratory syncytial virus (RSV) are all developed based on wild-type RSV pre-fusion F protein, which cannot ensure the stability of the antigen conformation and affects the level of neutralizing antibody production, the applicant has developed a mutant, nucleic acid and mRNA vaccine of RSV pre-fusion F protein.

[0055] Among them, the mutant of RSV prefusion F protein has a mutation in at least one site relative to the wild-type RSV prefusion F protein to ensure that the mutant is stabilized in the prefusion F protein to effectively induce the production of higher levels of neutralizing antibodies.

[0056] Furthermore, based on the mutant, a nucleic acid and ribonucleic acid for encoding the mutant are provided, and an mRNA vaccine comprising the ribonucleic acid for encoding the mutant is provided.

[0057] In the following embodiments of the present application, "RSV pre-fusion F protein" means preF glycoprotein, which is a metastable glycoprotein of fusion glycoprotein F, that is, natural fusion glycoprotein F mainly includes metastable preF glycoprotein and stable post-fusion F glycoprotein. PreF glycoprotein exists on the surface of RSV particles and in the cytoplasm of RSV-infected cells. In different maturation stages of RSV, preF glycoprotein exists in different glycosylation forms.

[0058] "Similarity" refers to the correlation between two amino acid sequences or two nucleotide sequences, wherein the numerical value of similarity corresponding to at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity can be an integer, and can also be further understood as the corresponding numerical value of similarity being a decimal less than 100%, for example 80.1%, 81.2%, 82.3%, 83.4%, 84.5%, 85.6%, 86.7%, 87.8%, 88.9%, 89.8%, 90.3%, 91.7%, 92.2%, 93.5%, 94.8%, 95.9%, 96.6%, 97.5%, 98.4% or 99.9%.

[0059] Specifically, the mutant of the RSV prefusion F protein of the present application is obtained by mutation of the full-length or partial region of the wild-type RSV prefusion F protein, and the mutation includes one or more of the following amino acid substitutions: S155C, S190F, V207L, S290C, L373R, A149C, Y458C, S46G, N67I, E92D, S215P, K465Q.

[0060] Here, "the full length or partial region of RSV prefusion F protein" refers to the entire domain or a partial fragment of the preF glycoprotein. For example, the partial region can be the extracellular domain, intracellular domain, etc. of the preF glycoprotein.

[0061] In one embodiment, in order to further ensure the conformational stability of the mutant, the mutant of the RSV prefusion F protein of the present application can be obtained by subjecting the whole length or part of the wild-type RSV prefusion F protein to amino acid substitutions of S155C, S190F, V207L, S290C, L373R, A149C, Y458C, S46G, N67I, E92D, S215P, and K465Q.

[0062] Furthermore, in one embodiment, the mutant can be obtained by the above-mentioned mutation of the full length of the wild-type RSV pre-fusion F protein, and the mutant can include an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence as shown in SEQ ID NO.1.

[0063] In another embodiment, the mutant can be obtained by subjecting the extracellular domain of the wild-type RSV pre-fusion F protein to the above mutations.

[0064] In order to ensure the stability of the mutant obtained by the above-mentioned mutation of the extracellular domain of the wild-type RSV pre-fusion F protein, in this embodiment, the mutant may also include a trimer peptide, which includes the amino acid sequence as shown in SEQ ID NO.2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence as shown in SEQ ID NO.2.

[0065] In this embodiment, the mutant may include an amino acid sequence as shown in SEQ ID NO.3, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence as shown in SEQ ID NO.3.

[0066] The present application also provides a nucleic acid capable of encoding the mutants of each of the above embodiments. Specifically, the nucleic acid may include a nucleotide sequence encoding the mutants of each of the above embodiments.

[0067] In one embodiment, when the mutant is obtained by the above-mentioned mutation of the full length of the wild-type RSV pre-fusion F protein, the nucleic acid may include the nucleotide sequence shown in SEQ ID NO.4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence shown in SEQ ID NO.4.

[0068] In another embodiment, when the mutant is obtained by the above-mentioned mutation in the extracellular domain of the wild-type RSV pre-fusion F protein, the nucleic acid may include a nucleotide sequence as shown in SEQ ID NO.5, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.5.

[0069] The present application also provides a ribonucleic acid molecule comprising the nucleic acids of the above-mentioned embodiments. In order to ensure the stability and translation efficiency of the ribonucleic acid molecule, the ribonucleic acid molecule may also include structural elements, and the structural elements may include one or more of a 5'UTR sequence, a 3'UTR sequence and a polyA tail sequence.

[0070] In one embodiment, preferably, the RNA molecule may include a 5'UTR sequence, a 3'UTR sequence and a polyA tail sequence at the same time.

[0071] The 5'UTR sequence may include a nucleotide sequence as shown in SEQ ID NO.6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.6.

[0072] The 3'UTR sequence may include the nucleotide sequence shown in SEQ ID NO.7, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence shown in SEQ ID NO.7.

[0073] The polyA tail sequence may include at least 100 A nucleotides; preferably, the polyA tail sequence includes the nucleotide sequence as shown in SEQ ID NO.8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.8.

[0074] Specifically, in one embodiment, when the mutant is obtained by mutation of the full length of the wild-type RSV prefusion F protein, the ribonucleic acid molecule may include a nucleotide sequence as shown in SEQ ID NO.9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.9;

[0075] In another embodiment, when the mutant is obtained by mutation of the extracellular domain of the wild-type RSV pre-fusion F protein, the ribonucleic acid molecule may include a nucleotide sequence as shown in SEQ ID NO.10, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.10.

[0076] The present application also provides a DNA molecule, which includes a nucleotide sequence for transcribing the ribonucleic acid molecule of any of the above embodiments.

[0077] The present application also provides a recombinant plasmid, which includes the DNA molecules of the above embodiments.

[0078] The present application also provides a respiratory syncytial virus mRNA vaccine, which includes the RNA molecule, vaccine vector and excipients of any of the above embodiments.

[0079] In one embodiment, the vaccine carrier may be a lipid nanoparticle.

[0080] Among them, excipients can be excipients and additives used in the production of pharmaceutical compositions and the preparation of prescriptions, which have important functions such as excipients, protection of active ingredients, improvement of stability, solubilization, solubilization, and sustained and controlled release, so that the pharmaceutical composition can achieve a certain shelf life and bioavailability, thereby improving the safety and effectiveness of the pharmaceutical composition. Excipients that can be co-administered with the mRNA vaccines of each embodiment of the present application include, but are not limited to, sugars, proteins, amino acids, and high molecular polymers.

[0081] Furthermore, the present application also provides a combined mRNA vaccine for the new coronavirus and respiratory syncytial virus, which comprises an RNA molecule of any of the above embodiments, an RNA molecule of a nucleotide sequence encoding a recombinant spike protein, a vaccine vector and excipients, wherein the vaccine vector is a lipid nanoparticle.

[0082] Among them, the recombinant spike protein is derived from the new coronavirus spike protein and has an S-2P site mutation relative to the wild-type new coronavirus spike protein.

[0083] Specifically, in one embodiment, the recombinant spike protein has an S-2P site mutation relative to the spike protein of the new coronavirus OmicronEG.5.1 strain, and the recombinant spike protein includes an amino acid sequence as shown in SEQ ID NO.11, or an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence as shown in SEQ ID NO.11.

[0084] The ribonucleic acid molecule of the nucleotide sequence encoding the recombinant spike protein may include a nucleotide sequence as shown in SEQ ID NO.12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.12.

[0085] The technical solution of the present application is further described in detail below in conjunction with specific embodiments.

[0086] Example 1: Preparation of full-length mRNA vaccine for respiratory tract combined with viral prefusion F protein

[0087] Step 1: Construction of expression plasmid

[0088] The full-length gene encoding the pre-fusion F protein (SEQ ID NO.4, named AIMDNA005), 5'UTR sequence (SEQ ID NO.6), 3'UTR sequence (SEQ ID NO.7) and polyA tail sequence (SEQ ID NO.8) were synthesized and submitted to Nanjing GenScript Co., Ltd., which used the GenSmart codon optimization function to optimize human codons and then synthesized the gene to obtain the pUC57-AIMDNA005 plasmid;

[0089] The plasmid was double-digested with restriction endonucleases EcoRI / XbaI (NEB), and then electrophoresed on a 1% agarose gel at 160 V for 30 min to recover the target gene fragment.

[0090] pVAX1 was double-digested with EcoRI / XbaI restriction endonucleases and subjected to 1% agarose gel electrophoresis at 160V for 30 minutes to recover a 2956 bp fragment. The two recovered fragments were then ligated using a ligation cassette (TakaRaAMG1265A) and transformed into Escherichia coli competent STBL3.

[0091] After incubating the transformed E. coli at 37°C for 1 hour, spread it on the LB plate culture medium containing kanamycin resistance, and place the plate upside down in a 30°C culture medium incubator for overnight culture. The next day, use a sterile inoculation loop in the clean bench to pick single colonies, transfer them to LB liquid culture medium containing kanamycin resistance, and culture them overnight at 30°C and 220rpm.

[0092] The cells were collected by centrifugation and the plasmid was extracted. The correct pVAX1-preF plasmid was confirmed to be obtained after sequencing by Bio-Technology and named TSZ005.

[0093] Step 2: TSZ005 circular plasmid production

[0094] The TSZ005 plasmid was transformed into STBLE competent cells (NEB), and clones with stable plasmid yield were selected based on the results of subculture stability. The clones were inoculated into LB liquid medium and cultured overnight at 37°C. The plasmid was extracted using a large extraction kit (MN), linearized with XbaI, and the DNA fragments after enzyme digestion were recovered using a fragment recovery kit (QIAGEN, 28604) as an in vitro transcription (IVT) template.

[0095] Step 3: In vitro transcription and purification of mRNA stock solution

[0096] According to the instructions of the IVT kit (DD4101, Vazyme), a reaction system was established, including 10×Transcription buffer, ATP, GTP, CTP, N1-Me-pUTP (NMPUTP001, Synthgene), CAP GAG (CAP3011, Synthgene), in vitro transcription template, RNase Inhibitor, T7 RNA Polymerase, and RNase Free Water. The above reaction components were added to the EP tube according to the instructions, mixed evenly, and reacted at 37°C for 2-3 hours. DNA degrading enzyme was added to remove the DNA template, and then oligo dT affinity chromatography and TFF ultrafiltration were used to exchange the liquid to obtain a high-purity mRNA stock solution.

[0097] Step 4. mRNA-LNP encapsulation and purification

[0098] 4.1 Preparation of lipid phase working solution: Prepare lipid working solution according to the molar ratio of each lipid component (Dlin-MC3-DMA: DOPC: DMG-PEG2000: cholesterol = 50%: 20%: 1%: 29%), the total lipid concentration of the working solution is 30 mg / mL, and the solvent is anhydrous ethanol.

[0099] 4.2 Prepare aqueous working solution: Dissolve the mRNA stock solution in pH 4.0 citrate buffer to make the mRNA concentration 0.3 mg / mL.

[0100] 4.3 mRNA-LNP encapsulation: Using a microfluidic device (MPE-L2, Atson) and its supporting microfluidic chip, the aqueous phase working solution and the lipid phase working solution were mixed at a volume ratio of aqueous phase: lipid phase = 3:1 to prepare mRNA-LNP.

[0101] 4.4 Using TFF tangential flow filtration technology, the above mRNA-LNP solution was replaced with a PBS solution with a pH of 7.40 to remove ethanol and purify LNP, and finally an LNP solution containing mRNA encoding respiratory syncytial virus prefusion F protein was obtained and named TSZ005 vaccine group.

[0102] 4.5 Cryo-electron microscopy analysis of the microscopic morphology of the formed LNPs revealed Figure 1 , Figure 1 This is an electron micrograph of the mRNA vaccine prepared in Example 1 of the present application.

[0103] like Figure 1 As shown, the LNP morphology is a complete solid sphere, and the outermost layer has a clear shell structure with good uniformity.

[0104] Example 2: Preparation of respiratory combined virus prefusion F protein extracellular domain mRNA vaccine

[0105] The gene encoding the extracellular domain of the pre-fusion F protein (SEQ ID NO.5, named AIMDNA002), 5'UTR sequence (SEQ ID NO.6), 3'UTR sequence (SEQ ID NO.7) and polyA tail sequence (SEQ ID NO.8) were synthesized and submitted to Nanjing GenScript Co., Ltd. for human codon optimization using the GenSmart codon optimization function. After gene synthesis, the pUC57-AIMDNA002 plasmid was obtained.

[0106] In the same manner as in Example 1, expression plasmids were constructed, mRNA was purified by in vitro transcription, and LNP encapsulation and purification were performed in sequence to obtain an LNP solution containing mRNA encoding the extracellular domain of the prefusion F protein of respiratory syncytial virus, which was named TSZ002 vaccine group.

[0107] Example 3: Preparation of mRNA vaccine for the novel coronavirus Omicron EG.5.1 mutant

[0108] The gene encoding the spike protein of the novel coronavirus Omicron EG.5.1 strain (SEQ ID NO.12), 5'UTR sequence (SEQ ID NO.6), 3'UTR sequence (SEQ ID NO.7) and polyA tail sequence (SEQ ID NO.8) were synthesized and submitted to Nanjing GenScript Co., Ltd., which used the GenSmart codon optimization function to optimize the human codons and synthesize the gene to obtain the AIMDNA2023003 plasmid;

[0109] In the same manner as in Example 1, an expression plasmid was constructed, mRNA was purified by in vitro transcription, and LNP was encapsulated and purified to obtain an LNP solution containing mRNA encoding the S-2P protein of the novel coronavirus Omicron EG.5.1 strain, which was named the AIM2302 vaccine group.

[0110] Example 4: Combined mRNA vaccine of novel coronavirus and respiratory syncytial virus

[0111] The respiratory combined virus pre-fusion F protein full-length mRNA vaccine TSZ005 prepared in Example 1 and the new coronavirus Omicron EG.5.1 mutant strain mRNA vaccine AIM2302 prepared in Example 3 were mixed in a ratio of 1:1 to obtain a bivalent vaccine, which was labeled TSZ008.

[0112] The respiratory combined virus pre-fusion F protein extracellular domain mRNA vaccine TSZ002 prepared in Example 2 and the new coronavirus Omicron EG.5.1 mutant strain mRNA vaccine AIM2302 prepared in Example 3 were mixed in a ratio of 1:1 to obtain a bivalent vaccine, which was labeled TSZ007.

[0113] Example 5: Animal Immunization Experiment

[0114] 1. Mouse husbandry and grouping

[0115] All animals were housed in a specific pathogen-free facility. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee. Among them, 48 BALB / c (nu / nu) mice (female, 6-8 weeks old, weighing 18-22 g) were used. The mice were randomly divided into 6 groups with 8 mice in each group before inoculation.

[0116] 2. Experimental process

[0117] (1) Route of administration

[0118] The vaccine was injected into 6 groups of mice by intramuscular injection (lateral thigh muscles of mice, including but not limited to gluteus maximus and quadriceps femoris), with a volume of 0.05 mL or 0.1 mL per mouse.

[0119] (2) Time of administration

[0120] The drug was administered by intramuscular injection twice on the 0th and 14th days of culture, with one dose each time.

[0121] (3) Blood collection time

[0122] Blood was collected on day 0 (before immunization), day 14 (before immunization) and day 28 (full collection) to ensure that the blood volume was no less than 200 μl, and the serum was stored at -80°C for subsequent experimental testing.

[0123] Please refer to the table below for specific mouse grouping and injection conditions:

[0124]

[0125]

[0126] As shown in the table above, 5 groups of mice were injected with TSZ002, TSZ005, TSZ007, TSZ008, and AIM2302 vaccines prepared in Examples 1 to 4, respectively, and 1 group of mice was injected with LNP as a negative control group.

[0127] 3. Antibody testing

[0128] (1) ELISA was used to detect the IgG antibody titer against the pre-fusion F protein of respiratory syncytial virus in the serum of mice in groups 1 to 3 on days 14 and 28. Figure 2 .

[0129] See also Figure 2 , Figure 2 This is a graph showing the IgG antibody titer of mice immunized with mRNA vaccines in Example 5 of the present application. Figure 2 As shown in the figure, mice immunized with TSZ002 or TSZ005 mRNA vaccine produced high titer IgG antibodies against the prefusion F protein of respiratory syncytial virus, with a titer of 2*10 6 , indicating that the respiratory syncytial virus mRNA vaccine of the present application has high titer.

[0130] (2) The anti-RSV (A2 strain, Long strain, and 18537 strain) antibody titers in the sera of mice in groups 1 to 5 on day 28 were detected using the real virus neutralization method;

[0131] The pseudovirus neutralization method was used to detect the anti-EG.5.1 antibody titer in the serum of mice in groups 4 to 6 on the 28th day.

[0132] See also Figure 3 , Figure 3 This is a data graph of the neutralizing antibody titer of mice after immunization with mRNA vaccine in Example 5 of the present application.

[0133] like Figure 3 As shown, mice immunized with TSZ002 or TSZ005 mRNA vaccines can produce high titers of cross-neutralizing antibodies against RSV A2, Long, and B18537 strains;

[0134] Mice immunized with TSZ007 combined with mRNA vaccines (TSZ002 and AIM2302) produced high titers of cross-neutralizing antibodies against RSV A2, Long, and B18537 strains. The neutralizing antibody titers were slightly lower than those of TSZ002 and AIM2302 vaccines alone, but were still at the same order of magnitude.

[0135] Mice immunized with TSZ008 combined with mRNA vaccines (TSZ005 and AIM2302) produced high titers of cross-neutralizing antibodies against RSV A2, Long, and B18537 strains. The neutralizing antibody titers were slightly lower than those of TSZ005 and AIM2302 vaccines alone, but still at the same order of magnitude.

[0136] Therefore, the above data show that the combined vaccine formed by TSZ002 and AIM2302 vaccines and the combined vaccine formed by TSZ005 and AIM2302 vaccines have good antigenic compatibility and can still maintain high titers against respiratory syncytial virus.

[0137] Mice immunized with AIM2302 mRNA vaccine can produce high titer pseudovirus neutralizing antibodies against the new coronavirus mutant strain EG5.1;

[0138] Mice immunized with TSZ007 combined with mRNA vaccines (TSZ002 and AIM2302) can produce high titers of pseudovirus neutralizing antibodies against the new coronavirus mutant strain EG5.1. The neutralizing antibody titer is slightly lower than that of the AIM2302 vaccine alone, but still at the same order of magnitude;

[0139] Mice immunized with TSZ008 combined with mRNA vaccines (TSZ005 and AIM2302) can produce high titers of pseudovirus neutralizing antibodies against the new coronavirus mutant strain EG5.1. The neutralizing antibody titer is slightly lower than that of the AIM2302 vaccine alone, but still at the same order of magnitude;

[0140] Therefore, the above data show that the combined vaccine formed by TSZ002 and AIM2302 vaccines and the combined vaccine formed by TSZ005 and AIM2302 vaccines have good antigenic compatibility and can still maintain high titers against the new coronavirus mutant strain EG5.1.

[0141] In summary, the combined vaccine of the present application has good antigenic compatibility and can simultaneously achieve high titers against respiratory syncytial virus and the new coronavirus mutant strain EG5.1.

[0142] (3) The ELISPOT method was used to detect the levels of immune cytokine indexes in the blood spleen cells of groups 1 to 3 mice on day 28 based on the RSV peptide library. Figure 4 .

[0143] See also Figure 4 , Figure 4 This is a data chart of immune cytokine indicators of mice immunized with mRNA vaccines in Example 5 of the present application. Figure 4 As shown, mice immunized with TSZ002 and TSZ005 mRNA vaccines could produce extremely high RSV F protein-specific T cell immune responses.

[0144] Based on the experimental data of the above embodiment 5, it can be known that:

[0145] 1. The respiratory syncytial virus mRNA vaccine of the present application can effectively induce the production of neutralizing antibodies against RSV pre-fusion F protein. After immunization with the respiratory syncytial virus mRNA vaccine of the present application, the host can produce a very high respiratory syncytial virus F protein-specific T cell immune response;

[0146] 2. The combined mRNA vaccine of the new coronavirus and respiratory syncytial virus of the present application has good antigenic compatibility and can simultaneously achieve high titers against respiratory syncytial virus and the new coronavirus mutant strain EG5.1.

[0147] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0148] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A mutant of RSV prefusion F protein, characterized in that: The mutant is obtained by mutation of the full-length or partial region of the wild-type RSV pre-fusion F protein, and the mutation includes one or more of the following amino acid substitutions: S155C, S190F, V207L, S290C, L373R, A149C, Y458C, S46G, N67I, E92D, S215P, K465Q.

2. The mutant according to claim 1, characterized in that The mutations include the following amino acid substitutions: S155C, S190F, V207L, S290C, L373R, A149C, Y458C, S46G, N67I, E92D, S215P, K465Q.

3. The mutant according to claim 2, characterized in that The mutant is obtained by the mutation of the full length of the wild-type RSV pre-fusion F protein, and the mutant includes the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence shown in SEQ ID NO.

1.

4. The mutant according to claim 2, characterized in that The mutant is obtained by the mutation of the extracellular domain of the wild-type RSV pre-fusion F protein, and the mutant also includes a trimeric peptide, which includes the amino acid sequence as shown in SEQ ID NO.2, or an amino acid sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence as shown in SEQ ID NO.2; the mutant includes the amino acid sequence as shown in SEQ ID NO.3, or an amino acid sequence with at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence as shown in SEQ ID NO.

3.

5. A nucleic acid, characterized in that Comprising a nucleotide sequence for encoding the mutant according to claim 3, the nucleic acid comprises the nucleotide sequence as shown in SEQ ID NO.4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.

4.

6. A nucleic acid, characterized in that Comprising a nucleotide sequence for encoding the mutant according to claim 4, the nucleic acid comprises the nucleotide sequence as shown in SEQ ID NO.5, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.

5.

7. A ribonucleic acid molecule comprising the nucleic acid according to claim 5 or 6, characterized in that Also included are structural elements including one or more of a 5'UTR sequence, a 3'UTR sequence, and a polyA tail sequence.

8. The ribonucleic acid molecule according to claim 7, characterized in that The 5'UTR sequence includes the nucleotide sequence shown in SEQ ID NO.6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence shown in SEQ ID NO.

6.

9. The ribonucleic acid molecule according to claim 7, characterized in that The 3'UTR sequence includes the nucleotide sequence shown in SEQ ID NO.7, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence shown in SEQ ID NO.

7.

10. The ribonucleic acid molecule according to claim 7, characterized in that The polyA tail sequence includes at least 100 A nucleotides; preferably, the polyA tail sequence includes the nucleotide sequence shown in SEQ ID NO.8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence shown in SEQ ID NO.

8.

11. A DNA molecule, characterized in that Comprising a nucleotide sequence for transcribing the ribonucleic acid molecule according to any one of claims 7 to 10.

12. A recombinant plasmid, characterized in that: Containing the DNA molecule according to claim 11.

13. Use of the ribonucleic acid molecule according to any one of claims 7 to 10, the DNA molecule according to claim 11 or the recombinant plasmid according to claim 12 in the preparation of a respiratory syncytial virus mRNA vaccine.

14. A respiratory syncytial virus mRNA vaccine, characterized in that: It comprises the RNA molecules, vaccine carriers and adjuvants as described in any one of claims 7 to 10, wherein the vaccine carrier is a lipid nanoparticle.

15. A method for preparing a respiratory syncytial virus mRNA vaccine, characterized in that: include: Synthesize a DNA fragment encoding the mutant according to any one of claims 1 to 4 and a DNA fragment encoding the structural element according to any one of claims 7 to 10, and clone them into a first plasmid respectively, so as to connect them on the first plasmid to obtain a target gene fragment; Recovering the target gene fragment from the first plasmid, connecting it to the second plasmid, transforming the first competent cell for cultivation; Recovering the bacterial cells, extracting the second plasmid, transforming the second competent cells after sequencing, obtaining the expression plasmid based on the stability of the passage, extracting and linearizing the expression plasmid in sequence after culturing, and recovering the DNA fragments after enzyme digestion to obtain an in vitro transcription template; Performing in vitro transcription, and then removing the in vitro transcription template to obtain an mRNA stock solution; Dissolving the mRNA stock solution in citrate buffer to obtain an aqueous working solution; The aqueous phase working solution is mixed with the lipid phase working solution, and then the solvent is replaced by TFF tangential flow filtration to obtain the respiratory syncytial virus mRNA vaccine.

16. A combined mRNA vaccine of novel coronavirus and respiratory syncytial virus, characterized in that: include: The RNA molecule according to any one of claims 7 to 10; A ribonucleic acid molecule for a nucleotide sequence encoding a recombinant spike protein, wherein the recombinant spike protein is derived from a novel coronavirus spike protein and has an S-2P site mutation relative to a wild-type novel coronavirus spike protein; Vaccine carrier and excipients, wherein the vaccine carrier is lipid nanoparticles.

17. The combined mRNA vaccine according to claim 16, characterized in that The recombinant spike protein has an S-2P site mutation relative to the spike protein of the new coronavirus Omicron EG.5.1 strain, and the recombinant spike protein includes the amino acid sequence as shown in SEQ ID NO.11, or an amino acid sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the amino acid sequence as shown in SEQ ID NO.

11.

18. The combined mRNA vaccine according to claim 17, characterized in that The ribonucleic acid molecule of the nucleotide sequence encoding the recombinant spike protein includes a nucleotide sequence as shown in SEQ ID NO.12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% similarity to the nucleotide sequence as shown in SEQ ID NO.12.

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