Respiratory syncytial virus mRNA vaccine

Abstract

The invention relates to the field of prevention and treatment of respiratory diseases, and discloses a preparation method of a human respiratory syncytial virus preventive mRNA vaccine. The main components of the mRNA vaccine comprise mRNA for coding an antigen and lipid nanoparticles. According to the mRNA sequence provided by the invention, an independently researched and developed 5 'UTR / 3' UTR sequence and a codon optimization mode are adopted, and efficient translation of target protein can be realized. According to the mRNA vaccine provided by the invention, RSV F protein is selected as an antigen, and the RSV F protein sequence is optimally designed, so that stable pre-fusion conformation RSV F protein can be expressed, and after a mouse is immunized, the mouse can be induced to generate high-level RSV F protein specific binding antibody titer and neutralizing antibody titer.

Description

Technical Field

[0001] This specification relates to the field of respiratory disease treatment, and in particular to a respiratory syncytial virus mRNA vaccine. Background Art

[0002] Respiratory syncytial virus (RSV) is a common respiratory virus belonging to the Paramyxoviridae family. It is an important pathogen that causes lower respiratory tract infections (LRIs) in infants, the elderly, and immunocompromised patients. RSV has only one serotype, which is divided into two subtypes, A and B. The strains of the two subtypes can cause disseminated infections at the same time. RSV infection is widely prevalent worldwide and has obvious seasonality. Globally, RSV causes more than 336,000 elderly people to be hospitalized and 14,000 deaths each year. Studies have estimated that in 2015 alone, there were 33.1 million cases of RSV lower respiratory tract infections in infants under the age of 5 worldwide, resulting in 3.2 million hospitalizations and nearly 60,000 deaths. Among them, there were 1.4 million hospitalizations and more than 27,000 deaths in infants less than 6 months old.

[0003] RSV infection currently lacks specific treatment in the clinic, and prevention is the key to RSV management. RSV immunoprevention mainly relies on passive prevention (antibody drugs) or active prevention (preventive vaccines). The antigen selection of RSV vaccines and antibody drugs is mainly focused on the conserved F protein. The F protein mediates the fusion of the virus and cell membranes, thereby promoting the entry of the virus into the host cell. The coding region is conserved, and the neutralizing antibodies induced by it can simultaneously inhibit the infection of RSV subtypes A and B. The F protein is a type I transmembrane glycoprotein, which is first translated into an inactive single polypeptide precursor F0 in the cell, which is about 574 amino acids long. During the maturation of the F protein, the signal peptide and p27 polypeptide are removed, and F2 is connected to the F1 polypeptide through two disulfide bonds. Three F2-F1 heterodimers oligomerize to form a pre-fusion conformation F protein trimer (pre-F), which is anchored in the viral envelope through the transmembrane domain (TM). During the process of RSV entering cells, the pre-fusion conformation F protein (pre-F) is rearranged to the post-fusion conformation (post-F). The fusion peptide (FP) exposed in this conformation can bind to the cell membrane and promote the fusion of the viral envelope and the cell membrane.

[0004] When the F protein changes from preF to postF, the antigenic epitopes also change. Epitopes I, II, III and VI are common to preF and postF. and V are unique to pre-F. The experimental results show that the unique antigenic epitope of pre-F Neutralizing antibodies induced by V have higher neutralizing activity, especially epitopes The induced neutralizing antibody titer is 10-100 times that of epitope II, that is, pre-F can induce a stronger neutralizing immune response than post-F. Therefore, the pre-fusion conformation of the F protein (pre-F) is considered to be the preferred configuration as the desired vaccine antigen.

[0005] In recent years, with the in-depth study of the structure, function and stabilization strategy of the RSV virus F protein pre-fusion conformation, monoclonal antibodies and RSV vaccines targeting the RSV F pre-fusion conformation protein have continued to emerge, and some products have achieved good results. Compared with recombinant protein vaccines, the antigens produced by mRNA vaccines are translated in host cells, which can avoid the impact on the pre-fusion conformation of the antigen during the production process, and the mRNA production line is highly versatile. However, mRNA known in the art still has limitations in its intracellular stability and its in vitro transcription efficiency. Summary of the invention

[0006] In view of the above reasons, the present application provides an mRNA vaccine for preventing RSV infection, the main components of which include mRNA encoding RSV antigen protein and lipid nanoparticles. The mRNA vaccine obtained in the present application can produce a good immune effect after immunizing mice.

[0007] The present application provides an mRNA, including an mRNA of a pre-fusion conformation respiratory syncytial virus F protein variant, wherein the pre-fusion conformation respiratory syncytial virus F protein variant is selected from any of the following: a. Compared with the wild-type respiratory syncytial virus F protein, it includes the following mutations: S46G, E92D, P102A, deletion of N104-V144 to GS, A149C, S155C, S190F, V207L, S215P, S290C, L373R, I379V, M447V, Y458C, K465Q, deletion of C550-N574, and has the function of pre-fusion conformation respiratory syncytial virus F protein, and the amino acid sequence of the wild-type respiratory syncytial virus F protein is shown in SEQ ID NO.8; b. Compared with the wild-type, the homology is 80% to 90%, and compared with the variant described in a, it has more than 90% homology, and has the function of pre-fusion conformation respiratory syncytial virus F protein.

[0008] The present application also provides a pre-fusion conformation respiratory syncytial virus F protein variant, selected from any of the following: a. Compared with the wild-type respiratory syncytial virus F protein, it includes the following mutations: S46G, E92D, P102A, deletion of N104-V144 to GS, A149C, S155C, S190F, V207L, S215P, S290C, L373R, I379V, M447V, Y458C, K465Q, deletion of C550-N574, and has the function of pre-fusion conformation respiratory syncytial virus F protein, and the amino acid sequence of the wild-type respiratory syncytial virus F protein is shown in SEQ ID NO.8; b. Compared with the wild-type, the homology is 80% to 90%, and compared with the variant described in a, it has more than 90% homology, and has the function of pre-fusion conformation respiratory syncytial virus F protein.

[0009] The present application also provides a biological material selected from any of the following: a. a polynucleotide encoding the pre-fusion conformation respiratory syncytial virus F protein variant as described in claim 6 or 7; b. a nucleic acid construct comprising the polynucleotide described in a; c. a host cell comprising the nucleic acid construct described in b or having the polynucleotide described in a integrated into its genome.

[0010] The present application also provides a lipid nanoparticle-mRNA complex, wherein the lipid nanoparticle-mRNA complex comprises the above-mentioned mRNA and a lipid nanoparticle, and the mRNA is loaded in the lipid nanoparticle.

[0011] The present application also provides the use of the above-mentioned mRNA or the above-mentioned lipid nanoparticle-mRNA complex in the preparation of drugs for treating respiratory diseases or pre-fusion conformation respiratory syncytial virus F protein variants. Preferably, the respiratory disease is caused by respiratory syncytial virus.

[0012] The present application also provides the use of the above-mentioned mRNA, the above-mentioned pre-fusion conformation respiratory syncytial virus F protein variant, the above-mentioned biomaterial or the above-mentioned lipid nanoparticle-mRNA complex in the preparation of a drug. Preferably, the drug is a vaccine. More preferably, the vaccine is used to prevent respiratory diseases. Further preferably, the respiratory disease is caused by respiratory syncytial virus.

[0013] The present application also provides a pharmaceutical composition comprising an effective amount of the above-mentioned mRNA, the above-mentioned pre-fusion conformation respiratory syncytial virus F protein variant, the above-mentioned biological material or the above-mentioned lipid nanoparticle-mRNA complex.

[0014] The beneficial effects of the mRNA vaccine proposed in this specification include but are not limited to: (1) The mRNA sequence provided in this application adopts the independently developed 5'UTR / 3'UTR sequence and codon optimization method, which can achieve efficient translation of the target protein. (2) In order to induce high levels of neutralizing antibodies, this mRNA vaccine uses RSV F protein as an antigen and optimizes the protein sequence so that it can express RSV F protein in a stable pre-fusion conformation. After immunizing mice, it can induce mice to produce high levels of RSV F protein-specific binding antibody titers and neutralizing antibody titers. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present application will be further described in the form of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not restrictive, and include:

[0016] Figure 1 A is a schematic diagram of the structure of plasmid pNR-RSV-F according to some embodiments of the present application;

[0017] Figure 1 B is a schematic diagram of mRNA preparation according to some embodiments of the present application;

[0018] Figure 2 It is a graph of agarose gel electrophoresis detection of linearized plasmids according to some embodiments of the present application, wherein the circular plasmid pNR-2 (lane 1) or pNR-3 (lane 3) is linearized with BsaI and the recovered product pNR-2-BsaI (lane 2) or pNR-3-BsaI (lane 4) is analyzed by agarose gel electrophoresis, M1: DNA molecular weight standard 1kb DNA ladder III (near shore), M2: DNA molecular weight standard Trans15k DNA Marker (full gold);

[0019] Figure 3 The capped mRNA is analyzed by capillary electrophoresis according to some embodiments of the present application;

[0020] Figure 4 According to the analysis result of intracellular RSV F protein expression level shown in some embodiments of the present application, after HEK-293T cells were transfected with mRNA encoding different RSV F protein variant sequences for 24 hours, the expression of RSV F protein in different samples was detected by western blotting, the Mock group was not transfected with any mRNA, and α-Tubulin was used as the internal reference protein;

[0021] Figure 5This is a graph showing the analysis results of the expression level of RSV F protein (pre F) in the pre-fusion conformation on the cell membrane according to some embodiments of the present application. After HEK-293T cells were transfected with mRNA encoding different RSV F protein variant sequences for 72 hours, the expression levels of pre F proteins on the cell membranes of different samples were analyzed by flow cytometry using D25 antibody. The Mock group was not transfected with any mRNA.

[0022] Figure 6 This is a graph showing the results of detecting the specific IgG binding antibody titer against RSV F protein in the mouse serum after the mice were immunized with the candidate mRNA vaccine shown in some embodiments of the present application, PBS: PBS immunization group, NR-2: LNP-NR-2 immunization group, NR-3: LNP-NR-3 immunization group, MOD: LNP-MOD immunization group;

[0023] Figure 7 This is a graph showing the results of neutralizing antibody titers against RSV A2 virus in mouse serum after mice were immunized with the candidate mRNA vaccine shown in some embodiments of the present application, PBS: PBS immunization group, NR-2: LNP-NR-2 immunization group, NR-3: LNP-NR-3 immunization group, MOD: LNP-MOD immunization group;

[0024] Figure 8 The effect of the 5'UTR sequence (5'UTR-3) used in this application on the expression level of the new coronavirus spike protein (CoV-2S). HEK-293T cells were not transfected with mRNA (mock) or transfected with mRNA expressing S protein containing different 5'-UTR sequences (5'UTR-BNT (BNT162b2 (Fubitai) 5'UTR), 5'UTR-1, 5'UTR-2, 5'UTR-3 and 5'UTR-4). The cells were lysed 24 hours after transfection, and the expression levels of S protein and internal reference protein α-tubulin were detected by western blotting;

[0025] Fig. 9 The effect of the 3'-UTR sequence (3'UTR-4) used in this application on the expression level of the new coronavirus spike protein (CoV-2S). HEK-293T cells were not transfected (mock) or transfected with mRNA expressing S protein with different 3'-UTR sequences (3'UTR-BNT (BNT162b2 (Fubitai) 3'UTR), 3'UTR-2 and 3'UTR-4). The cells were lysed 24 hours after transfection, and the expression levels of S protein and internal reference protein α-tubulin were detected by protein immunoblotting. DETAILED DESCRIPTION

[0026] In order to more clearly illustrate the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, this specification can also be applied to other similar scenarios based on these drawings without creative work. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.

[0027] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprises" and "includes" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0028] Flowcharts are used in this specification to illustrate the operations performed by the system according to the embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed precisely in order. Instead, the steps may be processed in reverse order or simultaneously. At the same time, other operations may also be added to these processes, or one or more operations may be removed from these processes.

[0029] The present application provides an mRNA, including an mRNA of a pre-fusion conformation respiratory syncytial virus F protein variant, wherein the pre-fusion conformation respiratory syncytial virus F protein variant is selected from any of the following: a. Compared with the wild-type respiratory syncytial virus F protein, it includes the following mutations: S46G, E92D, P102A, deletion of N104-V144 to GS, A149C, S155C, S190F, V207L, S215P, S290C, L373R, I379V, M447V, Y458C, K465Q, deletion of C550-N574, and has the function of pre-fusion conformation respiratory syncytial virus F protein, and the amino acid sequence of the wild-type respiratory syncytial virus F protein is shown in SEQ ID NO.8; b. Compared with the wild-type, the homology is 80% to 90%, and compared with the variant described in a, it has more than 90% homology, and has the function of pre-fusion conformation respiratory syncytial virus F protein.

[0030] The "sequence" herein should generally be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the amino acid sequence, unless a more limited explanation is required herein.

[0031] "Homology" between two amino acid sequences or nucleotide sequences indicates the percentage of identical amino acids or identical nucleotides between the sequences. Methods for evaluating the degree of homology between amino acids or nucleotides are known to those skilled in the art. For example, amino acid homology is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine homology.

[0032] As used herein, the term "wild type" refers to a gene, protein, fungal cell or strain as found in nature.

[0033] In some embodiments, the mutation of the prefusion conformation respiratory syncytial virus F protein variant compared to the wild-type respiratory syncytial virus F protein may also include deletion of C550-N574 to QPRFAAA.

[0034] In some embodiments, the amino acid sequence of the pre-fusion conformational respiratory syncytial virus F protein variant is shown in SEQ ID NO.9 or SEQ ID NO.13. In some embodiments, preferably, the amino acid sequence of the pre-fusion conformational respiratory syncytial virus F protein variant is shown in SEQ ID NO.9. In some embodiments, the pre-fusion conformational respiratory syncytial virus F protein variant may have a homology of 86.1% compared to the wild type.

[0035] In some embodiments, the mRNA may include an RNA sequence corresponding to the nucleotide sequence SEQ ID NO.4 or SEQ ID NO.11. In some embodiments, preferably, the mRNA may include an RNA sequence corresponding to the nucleotide sequence SEQ ID NO.4.

[0036] In the present application, RNA sequences are represented by corresponding DNA sequences, and in RNA sequences, uracil is 1-methylpseudouracil (m1ψ).

[0037] It is known to those skilled in the art that stabilized nucleic acids generally exhibit resistance to in vivo degradation (e.g., degradation by exo- or endonucleases) and / or in vitro degradation (e.g., caused by manufacturing processes prior to vaccine administration, e.g., during preparation of the vaccine solution to be administered).

[0038] In order to obtain stabilized mRNA, in some embodiments, the mRNA may further include a 5' untranslated region; in some embodiments, conventional 5' untranslated regions may be applicable to the present invention; in order to obtain better protein expression effects, in a preferred embodiment, the nucleotide sequence corresponding to the 5' untranslated region shown in SEQ ID NO.3 of the present invention may be used.

[0039] The term "5' untranslated region" or "5'UTR element" refers to the portion located 5' (i.e., "upstream") of a coding sequence that is not translated into protein. The 5'-UTR is generally understood to be a specific portion of a messenger RNA (mRNA) that is located 5' to the mRNA coding sequence. Typically, the 5'-UTR starts at the transcription start site and ends one nucleotide before the start codon of the coding sequence. The 5'-UTR may contain elements for controlling gene expression, also referred to as regulatory elements. Such a regulatory element may be, for example, a ribosome binding site.

[0040] In order to obtain stabilized mRNA, in some embodiments, the mRNA may further include a 3' untranslated region; in some embodiments, conventional 3' untranslated regions may be applicable to the present invention; in order to obtain better protein expression effects, in a preferred embodiment, the nucleotide sequence corresponding to the 3' untranslated region shown in SEQ ID NO.5 of the present invention may be used.

[0041] The term "3' untranslated region" or "3'UTR element" refers to a portion located 3' (ie, "downstream") of a coding sequence and which is not usually translated into protein. Typically, a 3'-UTR is a portion of an mRNA that is located between the coding sequence and the polyadenylation sequence of the mRNA.

[0042] In some embodiments, the mRNA may further include a polyadenylic acid sequence, which may be 50-200 A; in some embodiments, preferably, the polyadenylic acid sequence is as shown in SEQ ID NO.6.

[0043] In some embodiments, the sequence corresponding to the mRNA may include a nucleotide sequence as shown in SEQ ID NO.10 or SEQ ID NO.12. In some embodiments, preferably, the sequence corresponding to the mRNA may include a nucleotide sequence as shown in SEQ ID NO.10; in some embodiments, preferably, the mRNA may further include a 5'-cap structure; in some embodiments, more preferably, the 5'-cap structure may be m7G(5')ppp(5')(2'-OMeA)pG.

[0044] The term "5'-cap structure" refers to a special structure at the 5' end of mRNA, also known as a methylguanosine cap, which can improve the stability of mRNA and enhance translation efficiency. In some embodiments, the 5'-cap structure can be co-transcriptionally capped by adding a cap analog during in vitro transcription of mRNA. The preferred 5'-cap analog is m7G(5')ppp(5')(2'-OMeA)pG. In some embodiments, the 5'-cap structure can be post-transcriptionally capped by a capping enzyme after mRNA transcription is completed. Commonly used ones are vaccinia virus capping enzyme and 2'-O-methyltransferase.

[0045] In some embodiments, the mRNA contains chemically modified nucleotides to reduce the immunogenicity of the mRNA vaccine, enhance the stability of the mRNA, and improve the expression capacity of the encoded protein. The chemically modified nucleotides include: 1-methylpseudouridine (m1ψ), pseudouridine (ψ), 5-methoxyuridine (5moU) or 5-methylcytosine nucleoside (m5C). Preferably, all uracil nucleotides in the mRNA are replaced with 1-methylpseudouridine (m1ψ).

[0046] The present application also provides a pre-fusion conformation respiratory syncytial virus F protein variant, selected from any of the following: a. Compared with the wild-type respiratory syncytial virus F protein, it includes the following mutations: S46G, E92D, P102A, deletion of N104-V144 to GS, A149C, S155C, S190F, V207L, S215P, S290C, L373R, I379V, M447V, Y458C, K465Q, deletion of C550-N574, and has the function of pre-fusion conformation respiratory syncytial virus F protein, and the amino acid sequence of the wild-type respiratory syncytial virus F protein is shown in SEQ ID NO.8; b. Compared with the wild-type, the homology is 80% to 90%, and compared with the variant described in a, it has more than 90% homology, and has the function of pre-fusion conformation respiratory syncytial virus F protein.

[0047] In some embodiments, compared to the wild-type respiratory syncytial virus F protein, the mutation may also include deleting C550-N574 to become QPRFAAA.

[0048] In some embodiments, the amino acid sequence of the pre-fusion conformational respiratory syncytial virus F protein variant is shown in SEQ ID NO.9 or SEQ ID NO.13. In some embodiments, preferably, the amino acid sequence of the pre-fusion conformational respiratory syncytial virus F protein variant is shown in SEQ ID NO.9. In some embodiments, the pre-fusion conformational respiratory syncytial virus F protein variant may have a homology of 86.1% compared to the wild type.

[0049] The present application also provides a biological material selected from any of the following: a. a polynucleotide encoding the pre-fusion conformation respiratory syncytial virus F protein variant as described in claim 6 or 7; b. a nucleic acid construct comprising the polynucleotide described in a; c. a host cell comprising the nucleic acid construct described in b or having the polynucleotide described in a integrated into its genome.

[0050] Due to their own properties, such as negative charge and susceptibility to degradation by nucleases, nucleic acid drugs cannot effectively penetrate the cell membrane and enter cells and are quickly degraded in the body. Therefore, a good delivery system is needed to stably deliver the nucleic acid to the target location and make it effective.

[0051] The present application also provides a lipid nanoparticle-mRNA complex, wherein the lipid nanoparticle-mRNA complex comprises the above-mentioned mRNA and a lipid nanoparticle, and the mRNA is loaded in the lipid nanoparticle.

[0052] In some embodiments, the raw materials of the lipid nanoparticles include ionizable cationic lipids, neutral lipids, sterols and PEGylated lipids.

[0053] In some embodiments, the molar ratio of the ionizable cationic lipid: neutral lipid: sterol: PEGylated lipid may be (20-65): (5-25): (30-50): (0.5-3). Preferably, the molar ratio may be 47.4: 10: 40.8: 1.8.

[0054] In some embodiments, the ionizable cationic lipid can be selected from any one or more of the following: ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate (SM-102), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA), 1,2-dioleyl-3-dimethylamino-propane (DODMA) , 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecan-2-ol) (C12-200) or 1,2-dialeneoxy-3-dimethylaminopropane (DLinDMA); In some embodiments, preferably, the ionizable cationic lipid can be ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0055] In some embodiments, the neutral lipid is selected from any one or more of the following: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), preferably, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0056] In some embodiments, the sterol is cholesterol or a naturally occurring derivative thereof, preferably cholesterol.

[0057] In some embodiments, the pegylated lipid can be selected from any one or more of the following: methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159), 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), polyethylene glycol phospholipids (PEG-DSPE), polyethylene glycol-distearoylphosphatidylcholine (PEG-DSPC), polyethylene glycol diacylglycerol (PEG-DAG), methoxypolyethylene glycol-tetradecylpropylamine (PEG-DTDA), polyethylene glycol phosphatidylethanolamine (PEG-PE) or PEG succinic acid diacylglycerol (PEG-S-DAG); in some embodiments, preferably, the pegylated lipid can be methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159).

[0058] In some embodiments, in the lipid nanoparticle-mRNA complex, the mass ratio of the mRNA to the lipid nanoparticle is 1:5 to 1:50, and the preferred mass ratio is 1:10 to 1:35.

[0059] In some embodiments, in the lipid nanoparticle-mRNA complex, the nitrogen-phosphorus ratio of the ionizable cationic lipid compound in the lipid nanoparticle to the mRNA is 1:1 to 12:1, and a more preferred nitrogen-phosphorus ratio is 3:1 to 9:1.

[0060] In some embodiments, the particle diameter of the lipid nanoparticle-mRNA complex is 50 nm to 300 nm, preferably 70-120 nm.

[0061] The present application also provides the use of the above-mentioned mRNA, the above-mentioned pre-fusion conformation respiratory syncytial virus F protein variant, the above-mentioned biomaterial or the above-mentioned lipid nanoparticle-mRNA complex in the preparation of a drug. Preferably, the drug is a vaccine. More preferably, the vaccine is used to prevent respiratory diseases. Further preferably, the respiratory disease is caused by respiratory syncytial virus.

[0062] "Prevention" is treatment administered to a subject who does not show signs of a disease or who shows only early signs of the disease, for the purpose of reducing the risk of developing the disease.

[0063] The present application also provides a pharmaceutical composition comprising an effective amount of the above-mentioned mRNA, the above-mentioned pre-fusion conformation respiratory syncytial virus F protein variant, the above-mentioned biological material or the above-mentioned lipid nanoparticle-mRNA complex.

[0064] The term "pharmaceutical composition" refers to a preparation which is in such form that the biological activity of the active ingredient contained therein is effective, and which contains no additional ingredients which would be unacceptably toxic to a subject to which the composition would be administered.

[0065] The term "effective" means sufficient to achieve a desired, expected or intended result. When used in the context of preventing / treating a patient or subject with a compound, "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means an amount of the compound that, when administered to a subject or patient to prevent / treat a disease, is sufficient to achieve such treatment of the disease.

[0066] In some embodiments, the pharmaceutical composition may further include a pharmaceutically acceptable carrier.

[0067] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than the active ingredient, which is non-toxic to an individual. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. The pharmaceutically acceptable carrier can be a sterile liquid, such as water, saline solution, aqueous dextrose, aqueous glycerol, and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.

[0068] In some embodiments, the pharmaceutical composition may be a vaccine.

[0069] "Vaccine" refers to a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, vaccines induce an antigen-specific immune response to an antigen of a pathogen (e.g., a viral pathogen) or to a cellular component associated with a pathological condition. A vaccine may include a polynucleotide (e.g., a nucleic acid encoding a disclosed prefusion conformation respiratory syncytial virus F protein variant), a peptide or polypeptide (e.g., a disclosed prefusion conformation respiratory syncytial virus F protein variant), a virus, a cell, or one or more cellular components.

[0070] The present application also provides a method for preventing / treating respiratory diseases, the method comprising administering to an individual with respiratory diseases an effective amount of the above mRNA, prefusion conformation respiratory syncytial virus F protein variant, lipid nanoparticle-mRNA complex or pharmaceutical composition for prevention / treatment. In some embodiments, the respiratory disease is caused by respiratory syncytial virus.

[0071] The term "effective amount" refers to the quantity or dosage of the above-mentioned mRNA, prefusion conformation respiratory syncytial virus F protein variant, lipid nanoparticle-mRNA complex or pharmaceutical composition of the present application, which produces the expected effect in the individual treated after being administered in a single or multiple doses. "Preventive / therapeutic effective amount" refers to the amount that effectively achieves the desired preventive / therapeutic result at the required dosage and for the required time period. The preventive / therapeutic effective amount of the above-mentioned mRNA, prefusion conformation respiratory syncytial virus F protein variant, lipid nanoparticle-mRNA complex or pharmaceutical composition in the present application can vary according to various factors such as disease state, age, sex and weight of the individual. The preventive / therapeutic effective amount is also such an amount, wherein any toxic or harmful effects of the above-mentioned mRNA, prefusion conformation respiratory syncytial virus F protein variant, lipid nanoparticle-mRNA complex or pharmaceutical composition are inferior to the beneficial effects of prevention / treatment.

[0072] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0073] Example 1 - Preparation of mRNA

[0074] The mRNA of the present application is prepared by in vitro transcription (1-methyl pseudouracil (m1ψ) is used instead of uracil during transcription). The preparation process is shown in Figure 1 B. The template plasmid used was pUC57-kan (GenScript) as the vector, and all the sequences required for the transcription of the mRNA sequence to be protected were inserted, including:

[0075] A suitable promoter, such as T7 or SP6 promoter, for in vitro transcription. The present application uses T7 promoter (SEQ ID NO. 2: TAATACGACTCACTATA);

[0076] 5'-cap structure, the cap structure can be added by enzymatic reaction, or a cap analog (preferably m7G(5')ppp(5')(2'-OMeA)pG) can be added by co-transcription. The present application adopts enzymatic reaction;

[0077] 5'UTR element (SEQ ID NO.3: GGGTCCGGATTAACCCTGAGCTAAAGTAGCTTACCGCGCAAACCTGCGAGGACGGTTT ACGGTGCTACGACCGCCCGCCACC). The 5'UTR element used in this embodiment is preferred. Previous studies (see patent application CN202211162547.2 for details) have proved that the 5'UTR can be used to increase the expression of protein to prepare protein, and can be used to prepare mRNA vaccines. Specifically, in the protein expression level experiment, it was proved that after using this 5'UTR, the expression of firefly luciferase and green fluorescent protein (Fluc-GFP) in eukaryotic HEK293-T cells was better than that of the alpha globin 5'UTR sequence (5'UTR-AG+G) reported in the existing literature (reference: AdvMater.2020October;32(40):e2004452.doi:10.1002 / adma.202004452). After using this 5'UTR, the expression level of the new coronavirus spike protein in eukaryotic HEK293-T cells was better than that of other randomly synthesized 5'UTRs and BNT162b2 (Fubitai) 5'UTR (the results are shown in Figure 2). Figure 8 The purpose of using the 5'UTR in this embodiment is also to increase the expression level of the downstream target protein;

[0078] Kozak sequence: translation initiation signal (GCCACCATG);

[0079] A coding region, wherein the coding region encodes a RSV F protein variant (NR-2 or NR-3), wherein the variant is based on the F protein of the RSV A2 strain (NCBI sequence number: P03420) with some mutations;

[0080] SEQ ID NO.11 (sequence encoding RSV F protein variant NR-2):

[0081]

[0082] SEQ ID NO.4 (sequence encoding RSV F protein variant NR-3):

[0083] 6 / 2.20x10 6 ), after using this 3'UTR, the expression level of the new coronavirus spike protein in eukaryotic HEK293-T cells was better than that of other randomly synthesized 3'UTRs and BNT162b2 (Fubitai) 3'UTR (the results are shown in Fig. 9 The purpose of using the 3'UTR in this embodiment is also to increase the expression level of the upstream target protein;

[0084] Polyadenylic acid (poly (A)) sequence (SEQ ID NO. 6: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA);

[0085] As the linearization restriction site, Class II restriction sites are preferred, such as BsaI, BspQI, etc. The present application uses the BsaI enzyme site (SEQ ID NO.7: GGTCTC).

[0086] Plasmids pNR-RSV F: pNR-2 (sequence is SEQ ID NO.1 excluding 2060-2080) and pNR-3 (sequence is SEQ ID NO.1) containing RSV F protein variant transcription regions were commissioned to be synthesized by GenScript. The map of plasmid pNR-RSV F can be found at Figure 1 A, the prepared circular plasmid was digested with BsaI enzyme (nearshore) (incubated at 37°C overnight) and then The enzyme digestion product was purified using the SV Geland PCR Clean-Up System Kit (Promega) to obtain a linearized plasmid. The linearized plasmid was analyzed by 1% agarose gel electrophoresis. Figure 2 It shows that the main configuration of pNR-2 plasmid before restriction digestion (lane 1) and pNR-3 plasmid before restriction digestion (lane 3) is supercoiled plasmid; after BsaI digestion, pNR-2-BsaI (lane 2) and pNR-3-BsaI (lane 4) can be seen with a single band consistent with the theoretical size of the plasmid (4415bp), indicating that almost all plasmids have been successfully linearized.

[0087] SEQ ID NO.1:

[0088]

[0089] The above linearized plasmid pNR-2-BsaI or pNR-3-BsaI was used as a template, and an in vitro transcription reaction was performed according to the instructions of the T7 High Yield RNA Transcription kit (Near Shore, E131), wherein 1-methyl pseudouracil (m1ψ) was used instead of uracil, and the reaction was carried out at 37°C for 3 hours. After the reaction was completed, DNase I (Near Shore) was added and treated at 37°C for 15 minutes to remove the residual DNA template. After the transcription product was purified by lithium chloride precipitation, an uncapped mRNA product was obtained: mRNA-NR-2 (SEQID NO.12) or mRNA-NR-3 (SEQ ID NO.10). The specific steps are as follows: add 1.5 times the volume of lithium chloride solution (7.5M, Thermo, AM9480) to the transcription product, mix well and let stand at -20°C for at least 30 minutes, centrifuge at 12,000rpm, 4°C for 15 minutes, remove the supernatant, collect the precipitate, wash the precipitate twice with pre-cooled 70% ethanol, and then re-dissolve the precipitate with enzyme-free water.

[0090] SEQ ID NO.12: (mRNA-NR-2)

[0091]

[0092] SEQ ID NO.10:(mRNA-NR-3)

[0093]

[0094] The above mRNA products mRNA-NR-2 (SEQ ID NO.12) or mRNA-NR-3 (SEQ ID NO.10) also need to add a cap structure at 5' to translate the target protein normally. There are two main methods for adding a cap structure to mRNA. One is to add a cap analog to the in vitro transcription reaction system and add a 5' cap structure during transcription; the other is to cap the uncapped mRNA product by a capping enzyme after the in vitro transcription reaction is completed. This embodiment adopts the second capping method, and the specific steps are as follows: After the above uncapped mRNA is purified by lithium chloride precipitation, a capping reaction is performed according to the instructions of the capping kit Cap1Capping System (nearshore), and the reaction is carried out at 37°C for 1 hour. After the reaction is completed, the capped product is purified by lithium chloride precipitation or oligo dT affinity chromatography. The specific steps of purification using Oligo dT affinity chromatography are as follows: the Oligo dT chromatography column (BIA, Cat NO.: 311.1219-2) is washed with 0.1M NaOH aqueous solution, then rinsed with ultrapure water, and then balanced with equilibration buffer (50mM PB, 250mM NaCl, pH8.0), and then the sample to be purified (capping reaction product) is loaded onto the column and balanced with equilibration buffer, and then washed with washing buffer (50mM PB, pH8.0), and finally the bound components are eluted with ultrapure water and collected (i.e., the purified capped mRNA).

[0095] In order to analyze the quality of the capped mRNA product, the inventors analyzed the integrity of the RNA by capillary electrophoresis technology, that is, the percentage of the target RNA product in the total transcription product was analyzed by the Qsep1 fully automatic nucleic acid protein analysis system (BiOptic). The specific method is as follows: The RNA product was diluted to 50-60ng / μL with 1×Dilution Buffer (BiOptic), heated at 70°C for 2min, quickly transferred to ice for 5min, and detected with the matching R1 card holder (BiOptic). The molecular weight of each peak in the test results was calibrated according to the RNA6000 molecular weight standard (invitrogen). Finally, the integrity data of the target RNA was obtained by analyzing the proportion of peaks consistent with the expected RNA molecular weight. The capillary electrophoresis analysis results of the capped mRNA products mRNA-NR-2 and mRNA-NR-3 showed that the peak area of ​​the main peak accounted for 93.1% and 92.2%, respectively, indicating that this mRNA has a high integrity ( Figure 3 ).

[0096] Example 2-In vitro activity detection of mRNA

[0097] To verify the ability of RSV F protein variant mRNA expressed in Example 1 to express RSV pre-fusion conformation F protein, this example analyzes the expression of RSV F protein after transfection of HEK-293T cells with mRNA encoding RSV F protein variants. In this example, three candidate RSV F protein variant sequences (NR-1, NR-2, and NR-3) were compared, and the mRNA sequence (MOD) in the moderna patent was selected as a control; the mutation sites of different RSV F protein variant sequences compared with wild-type RSV F protein are shown in Table 1.

[0098] Table 1. Mutation sites of different RSV F protein variant sequences compared with wild-type RSV F protein

[0099]

[0100] The mRNA sequences encoding NR-1, NR-2 and NR-3 protein sequences use the same 5'UTR (SEQ ID NO.3), 3'UTR (SEQ ID NO.5) and poly (A) sequence (SEQ ID NO.6), as well as the same codon optimization method; the mRNA sequence encoding the MOD sequence uses a sequence consistent with the moderna patent (SEQ ID NO.15 in application number CN115103682A). In this embodiment, the NR-2 protein sequence (SEQ ID NO.13) or the NR-3 protein sequence (SEQ ID NO.9) has a homology of 86.1% with the wild-type RSV F protein (A2, NCBI sequence number: P03420) sequence (SEQ ID NO.8). The mRNA sequence encoding NR-2, mRNA-NR-2 (SEQ ID NO.12), has a homology of 77.4% with the mRNA sequence of MOD. The mRNA sequence encoding NR-3, mRNA-NR-3 (SEQ ID NO. 10), has a similarity of 77.5% with the mRNA sequence of MOD.

[0101] SEQ ID NO.8 (wild-type RSV A2 F protein sequence):

[0102] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0103] SEQ ID NO.13 (NR-2 RSV F protein variant sequence):

[0104] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLY***

[0105] SEQ ID NO.9 (NR-3 RSV F protein variant sequence):

[0106] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKS ALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAY VVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNR GIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYQPRFAAA***

[0107] In this example, the expression level of RSV F protein was first detected by Western blotting. The specific method was as follows: HEK-293T cells grown in a dense monolayer were obtained by trypsin digestion to obtain a cell suspension. 5 The cells were seeded in 6-well plates at a density of 100 cells / mL. The cell culture medium was high-glucose DMEM (Hyclone) containing 10% fetal bovine serum (Capricorn Scientific), 1% penicillin-streptomycin (Gibco), 1% non-essential amino acids (Gibco), and 1% sodium pyruvate (Gibco). The percentages in the culture medium are all volume percentages. The next day, 300 ng of mRNA of different sequences were added by MessengerMAX TM(Thermo) transfected HEK-293T cells. 24 hours after cell transfection, rinse the cells in the 6-well plate with PBS, then add 1 mL of PBS to each well, collect the cells into a 1.5 mL EP tube with a cell scraper, centrifuge at 4000 rpm, 4°C for 5 minutes, collect the precipitate, add 100 μL of lysis buffer (RIPA with 1x protease inhibitor and 1% nuclease) to the precipitate, and lyse on ice for 15 minutes. Centrifuge at 13200 rpm, 4°C for 5 minutes, and collect the supernatant. Add 25 μL of 5xSDS protein loading buffer (New Cyme) to each 100 μL cell lysate tube and heat in a metal bath at 100°C for 5 minutes. Take 4-12% SurePAGE TM Protein precast gel (GenScript), 160V constant voltage electrophoresis for 40min, then 400mA constant current transfer for 30min. The transferred membrane was placed in blocking solution (5% milk) for 1h at room temperature. The blocking solution was discarded and the primary antibody was incubated at room temperature for 2h. The antibodies were RSV-F rabbit anti-antibody (Sino Biological, 1:200 dilution in blocking solution); α-Tubulin mouse anti-antibody (Proteintech, 1:10000 dilution in blocking solution). The primary antibody was discarded, the membrane was washed 3 times with TBST, 5min each time, and incubated with HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) or HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Proteintech, 1:5000 dilution in blocking solution) at room temperature for 1h. The secondary antibody was discarded, and the membrane was washed 5 times with TBST, 5min each time. The ultrasensitive ECL chemiluminescent substrate (Syntheme) was mixed 1:1 and dropped on the membrane for 1 minute. The membrane was placed in a 4600SF chemiluminescence image analysis system (Tianneng) for imaging.

[0108] The results are as follows Figure 4 As shown, the negative control Mock group (HEK-293T cells not transfected with mRNA) did not detect the expression of RSV F protein. The cells transfected with RSV F protein variant mRNA were able to detect the expression of F protein consistent with the predicted size. Among them, the protein expression level of NR-1 group was the lowest, and the protein expression levels of NR-2 and NR-3 groups were high.

[0109] To further analyze the expression level of RSV F protein (pre F) in the prefusion conformation on the cell membrane after transfection with different mRNAs, we used D25 antibody (which recognizes the unique antigenic epitope of pre F) The expression level of cell membrane pre F protein after transfection of different RSV F protein variant mRNA was analyzed by flow cytometry. The specific method is as follows: HEK-293T cells grown in a dense monolayer attached to the wall were obtained by trypsin digestion and cell suspension was obtained. 1.5×10 5 The cells were seeded in a 24-well plate at a density of 10 cells / mL. The next day, 300 ng of mRNA of different sequences were added by MessengerMAX TM (Thermo) transfected HEK-293T cells. 72 hours after transfection, the HEK-293T cells were rinsed with PBS, and then 200μL Trypsin (Gibco) was added to digest the cells and incubated at 37℃ for 2min. 200μL complete medium was added to the dissociated cells to neutralize Trypsin, centrifuged at 1000rpm for 5min at room temperature, and the supernatant was discarded. 500μL complete medium was added to resuspend the cells, and 60μL was taken into a new 1.5mL EP tube. 1.33μL D25 (Biointron) antibody was added respectively, and incubated at 4℃ for 30min. Centrifuged at 1500rpm for 5min, discarded the supernatant, and washed twice with 500μL PBS. 100μL PE (1:200, Jackson) secondary antibody was added, and incubated at 4℃ for 30min in the dark. Centrifuge at 1500 rpm for 5 min, discard the supernatant, wash once with PBS, add 200 μL PBS and collect on NovoCyte2000R flow cytometer (Agilent). Collection conditions: PE channel, 10,000 cells collected, volume 50 μL, sample flow rate is medium speed.

[0110] The results are shown in Table 2 and Figure 5 As shown in the figure, the expression of RSV F protein was almost not detected on the cell membrane surface of HEK-293T cells not transfected with mRNA (Mock group), while the expression of RSV F protein with prefusion conformation (pre F) could be detected in cells transfected with RSV F protein variant mRNA. Among them, the expression of pre F in NR-1 group was the lowest and the most unstable; the expression of pre F in NR-2 and NR-3 groups was high and stable.

[0111] Table 2. Analysis results of expression level of pre-fusion conformation RSV F protein (pre F) on cell membrane.

[0112]

[0113]

[0114] In summary, the mRNA of NR-2 and NR-3 sequences has a strong ability to express RSV F pre-fusion conformation protein and is expected to serve as candidate sequences for RSV vaccine.

[0115] Example 3 - Preparation and characterization of lipid nanoparticles

[0116] In order to prevent mRNA degradation and effectively deliver it to animals, lipid nanoparticles (LNP) are often used as carriers. The specific steps of preparing LNP of the present invention are as follows:

[0117] Prepare the aqueous phase: dilute the capped mRNA product in 25 mM citrate buffer at a final concentration of 100 ng / μL;

[0118] Preparation of organic phase: Dissolve each lipid component (ionizable cationic lipid, DSPC, cholesterol, PEGylated lipid) in ethanol according to the molar ratio shown in Table 3.

[0119] The aqueous solution and the organic solution were loaded into the BD syringes respectively, and the two syringes were connected to the chip of the nanodrug preparation system. The appropriate injection volume, injection flow rate (6mL / min), and aqueous phase to organic phase flow rate ratio (3:1) were set on the display screen, and the start button was clicked to inject the feed solution into the chip. Observe the product color at the outlet of the chip, discard the appropriate amount of milky white droplets at the front and end, and collect the middle end sample into the EP tube. The collected product was placed in a dialysis bag (molecular weight cutoff: 10KDa) and dialyzed in Tris buffer for 24 hours. The lipid nanoparticles were concentrated by centrifugation using an ultrafiltration centrifuge tube, and then sterile filtered after adding sucrose solution to obtain the finished product. The specific formulation of mRNA-LNP is shown in Table 3.

[0120] Table 3. mRNA-LNP preparation formula

[0121]

[0122] The particle size and PDI were tested using standard detection methods on Malvern's Zetasizer instrument. The encapsulation efficiency and content of the finished product were tested and calculated according to the instructions of the Ribogreen kit. The mRNA in the lipid nanoparticles was extracted using isopropanol, and the mRNA integrity was analyzed using standard detection methods on Bioptic's Qsep1 instrument.

[0123] The test results of particle size, PDI, encapsulation efficiency and mRNA integrity of the mRNA-loaded lipid nanoparticles (LNPs) prepared in this example are shown in Table 4.

[0124] Table 4. mRNA-LNP characterization results

[0125] LNP Sample No. Particle size (nm) PDI Encapsulation rate (%) mRNA integrity (%) LNP-NR-2 71.6 0.1019 97.82％ 92.1％ LNP-NR-3 72.88 0.09333 97.19％ 89.8％ LNP-MOD 83.39 0.09593 95.46％ 89.9％

[0126] Example 4 - In vivo immunogenicity study in mice

[0127] Example 3 The prepared lipid nanoparticles LNP-NR-2, LNP-NR-3 or control LNP-MOD containing candidate mRNA were immunized with BALB / C mice (18-24g, female) by intramuscular (IM) injection, n=8 per group, each mouse was immunized with 50μL of lipid nanoparticles (containing 1μg mRNA), and the negative control group was immunized with PBS. Immunization was performed twice (day 0 and day 21), and mouse serum was collected 14 days after the second immunization (i.e., day 35 after the first administration).

[0128] The antibody titer that specifically binds to RSV F protein in mouse serum was determined by enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: 100 μL of RSV F protein (Sino-Biotech, Cat. No.: 11049-V08B) at a concentration of 1 μg / mL was added to a 96-well plate and placed at 4°C for overnight coating. After coating, wash three times with PBS containing 0.05% Tween-20 (PBST). Add 250 μL of PBST containing 5% skim milk to each well and place at 37°C for blocking for 1 hour. After washing, add 100 μL of mouse serum diluted in multiple ratios to each well, set two replicate wells for each dilution gradient, and incubate at 37°C for 1 hour. After washing, add 100 μL of HRP-labeled goat anti-mouse secondary antibody (Southern Biotech, Cat. No.: 1036-05) to each well and incubate at 37°C for 1 hour. After washing, 100 μL TMB colorimetric solution (Invitrogen, catalog number: 00-2023) was added to each well, and color was developed for 15 min at room temperature. 50 μL stop solution (NCM Biotech, E40500) was added to each well to stop color development, and the absorbance value at OD450 nm was measured by an ELISA reader.

[0129] Calculation method: If the OD450 of the test well is greater than 0.1 and is greater than 2.1 times that of the negative control well (P / N>2.1, where P is the OD value of the test serum measured at a certain dilution multiple, and N is the OD value of the negative serum measured at the corresponding dilution multiple), it is judged as positive, and the highest dilution multiple of the serum judged as positive is the serum antibody titer.

[0130] like Figure 6 As shown, after mice were immunized with the candidate mRNA vaccine (NR-2 or NR-3), higher titers of RSV F protein-specific IgG binding antibodies were induced, which was slightly better than that of the control mRNA vaccine (MOD) immunization group.

[0131] RSV-specific neutralizing antibodies in mouse serum were determined by a micro-neutralizing antibody detection method. The specific steps are as follows: the serum samples to be tested were inactivated at 56°C for 30 minutes before the experiment. Then, the samples were diluted in multiples using culture medium, with the serum in the PBS group starting from a dilution of 1:10, diluted 3 times, and the highest dilution being 2430. The serum in the NR-2 group, NR-3 group, or MOD group started from a dilution of 1:100, diluted 3 times, and the highest dilution being 24300. The diluted samples were incubated with 400TCID50 / mL of virus (RSV A2) at 37°C and 5% CO2 for 1 hour. Subsequently, Hep-2 cells were cultured at a certain density (2.5x10 4 Cells / well) were inoculated into the test wells, and a cell control group (cells, no virus infection) and a virus control group (cells infected with virus, no other treatment) were set up at the same time. The cells were cultured at 37°C and 5% CO2 for 5 days. The cell culture supernatant was discarded, and after fixing the cells, the wells were blocked with 5% BSA at room temperature for 1 hour, and then washed 3 times with TBST solution. After washing the plate, the primary antibody solution (RSV-F antibody (Sino Biological, 1:5000 diluted with TBST)) was added, incubated at 37°C for 1 hour, washed 3 times with TBST solution, and then the secondary antibody solution (HRP-labeled IgG antibody, 1:2000 diluted with TBST) was added, incubated at 37°C for 1 hour, washed 3 times with TBST solution, and TMB substrate (Sera care) was added and placed at room temperature for 10 minutes. After the incubation, the absorbance was read at 450nm using an enzyme reader. The raw data was used to calculate the neutralization activity of the samples at different concentrations. The maximum dilution factor at which the antibody antiviral activity was >50% was taken as the neutralization titer.

[0132] Y=100-(ac) / (bc)*100

[0133] Where: Y = % antibody activity; a = OD450 reading of sample; b = OD450 reading of virus control group; c = OD450 reading of cell control group;

[0134] like Figure 7 As shown, after mice were immunized with the candidate mRNA vaccine (NR-2 or NR-3), higher titers of RSV neutralizing antibodies were induced, which were better than those of the control mRNA vaccine (MOD) immunization group and had good stability.

[0135] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0136] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0137] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0138] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. An mRNA comprising an mRNA of a prefusion conformation respiratory syncytial virus F protein variant, wherein the prefusion conformation respiratory syncytial virus F protein variant is selected from any one of the following: a. Compared with the wild-type respiratory syncytial virus F protein, it includes the following mutations: S46G, E92D, P102A, deletion of N104-V144 to GS, A149C, S155C, S190F, V207L, S215P, S290C, L373R, I379V, M447V, Y458C, K465Q, deletion of C550-N574, and has the function of the pre-fusion conformation respiratory syncytial virus F protein, and the amino acid sequence of the wild-type respiratory syncytial virus F protein is shown in SEQ ID NO.8; b. The homology is 80% to 90% compared to the wild type, and more than 90% homology compared to the variant described in a, and possesses the function of the pre-fusion conformation respiratory syncytial virus F protein.

2. The mRNA according to claim 1, characterized in that The mutation of the pre-fusion conformation respiratory syncytial virus F protein variant compared to the wild-type respiratory syncytial virus F protein also includes deletion of C550-N574 to QPRFAAA; And / or, the amino acid sequence of the pre-fusion conformation respiratory syncytial virus F protein variant is shown in SEQ ID NO.9 or SEQ ID NO.13; And / or, the pre-fusion conformation respiratory syncytial virus F protein variant has a homology of 86.1% compared to the wild type.

3. The mRNA according to claim 1, characterized in that The mRNA includes an RNA sequence corresponding to the nucleotide sequence SEQ ID NO.4 or SEQ ID NO.

11.

4. The mRNA according to claim 1, characterized in that The mRNA further includes a 5' untranslated region. Preferably, the nucleotide sequence corresponding to the 5' untranslated region is shown in SEQ ID NO.3; And / or, the mRNA further comprises a 3' untranslated region, and preferably, the nucleotide sequence corresponding to the 3' untranslated region is shown as SEQ ID NO.5; And / or, the mRNA further comprises a polyadenylic acid sequence, preferably, the polyadenylic acid sequence is as shown in SEQ ID NO.6; And / or, the mRNA further comprises a 5'-cap structure, preferably, the 5'-cap structure is added by a capping enzyme or the 5'-cap structure is m7G(5')ppp(5')(2'-OMeA)pG, more preferably, the capping enzyme is a vaccinia virus capping enzyme or a 2'-O-methyltransferase.

5. The mRNA according to claim 1, characterized in that The sequence corresponding to the mRNA includes the nucleotide sequence shown in SEQ ID NO.10 or SEQ ID NO.

12. Preferably, the mRNA also includes a 5'-cap structure. More preferably, the 5'-cap structure is added by a capping enzyme or the 5'-cap structure is m7G(5')ppp(5')(2'-OMeA)pG. Further preferably, the capping enzyme is a vaccinia virus capping enzyme or a 2'-O-methyltransferase. And / or, the mRNA includes chemical modifications, preferably, all uracil nucleotides in the mRNA are 1-methylpseudouracil (m1ψ).

6. A pre-fusion conformation respiratory syncytial virus F protein variant selected from any of the following: a. Compared with the wild-type respiratory syncytial virus F protein, it includes the following mutations: S46G, E92D, P102A, deletion of N104-V144 to GS, A149C, S155C, S190F, V207L, S215P, S290C, L373R, I379V, M447V, Y458C, K465Q, deletion of C550-N574, and has the function of the pre-fusion conformation respiratory syncytial virus F protein, and the amino acid sequence of the wild-type respiratory syncytial virus F protein is shown in SEQ ID NO.8; b. The homology is 80% to 90% compared to the wild type, and more than 90% homology compared to the variant described in a, and possesses the function of the pre-fusion conformation respiratory syncytial virus F protein.

7. The prefusion conformation respiratory syncytial virus F protein variant according to claim 6, characterized in that Compared with the wild-type respiratory syncytial virus F protein, the mutation also includes deletion of C550-N574 to QPRFAAA; And / or, the amino acid sequence of the pre-fusion conformation respiratory syncytial virus F protein variant is shown in SEQ ID NO.9 or SEQ ID NO.13; And / or, the pre-fusion conformation respiratory syncytial virus F protein variant has a homology of 86.1% compared to the wild type.

8. A biological material selected from any of the following: a. A polynucleotide encoding the pre-fusion conformation respiratory syncytial virus F protein variant according to claim 6 or 7; b. a nucleic acid construct comprising the polynucleotide described in a; c. A host cell comprising the nucleic acid construct described in b or having the polynucleotide described in a integrated into its genome.

9. A lipid nanoparticle-mRNA complex, characterized in that: The lipid nanoparticle-mRNA complex comprises the mRNA according to any one of claims 1 to 5 and a lipid nanoparticle, wherein the mRNA is loaded in the lipid nanoparticle.

10. The lipid nanoparticle-mRNA complex according to claim 9, characterized in that The raw materials of lipid nanoparticles include ionizable cationic lipids, neutral lipids, sterols and PEGylated lipids.

11. The lipid nanoparticle-mRNA complex according to claim 10, characterized in that In the lipid nanoparticle-mRNA complex, the mass ratio of the mRNA to the lipid nanoparticle is 1:5 to 1:50, and the preferred mass ratio is 1:10 to 1:35; And / or, the molar ratio of the ionizable cationic lipid: neutral lipid: sterol: PEGylated lipid is (20-65): (5-25): (30-50): (0.5-3), preferably, the molar ratio of the cationic lipid: neutral lipid: sterol: PEGylated lipid is 47.4: 10: 40.8: 1.8; And / or, the ionizable cationic lipid is selected from any one or more of the following: ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 1,2-dioleyl-3-dimethylamino-propane, 1, 1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecan-2-ol) or 1,2-dilinoleyloxy-3-dimethylaminopropane, preferably, the ionizable cationic lipid is ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); And / or, the neutral lipid is selected from any one or more of the following: 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dipalmitoylphosphatidylcholine, preferably, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine; and / or, the sterol is cholesterol or a naturally occurring derivative thereof, preferably cholesterol; And / or, the PEGylated lipid is selected from any one or more of the following: methoxypolyethylene glycol ditetradecyl acetamide, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol, polyethylene glycol phospholipids, polyethylene glycol-distearoylphosphatidylcholine, PEGylated diacylglycerol, methoxypolyethylene glycol-tetradecylpropylamine, PEGylated phosphatidylethanolamine or PEG succinic acid diacylglycerol, preferably, the PEGylated lipid is methoxypolyethylene glycol ditetradecyl acetamide.

12. Use of the mRNA according to any one of claims 1 to 5, the prefusion conformation respiratory syncytial virus F protein variant according to claim 6 or 7, the biomaterial according to claim 8 or the lipid nanoparticle-mRNA complex according to any one of claims 9 to 11 in the preparation of a drug, preferably, the drug is a vaccine, more preferably, the vaccine is used to prevent respiratory diseases, and further preferably, the respiratory diseases are caused by respiratory syncytial virus.

13. A pharmaceutical composition comprising an effective amount of the mRNA according to any one of claims 1 to 5, the prefusion conformation respiratory syncytial virus F protein variant according to claim 6 or 7, the biomaterial according to claim 8, or the lipid nanoparticle-mRNA complex according to any one of claims 9 to 11.

14. The pharmaceutical composition according to claim 13, characterized in that The pharmaceutical composition is a vaccine.

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