WO2024131862A1 - 一种rsv疫苗及其制备方法与应用 - Google Patents

一种rsv疫苗及其制备方法与应用 Download PDF

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WO2024131862A1
WO2024131862A1 PCT/CN2023/140371 CN2023140371W WO2024131862A1 WO 2024131862 A1 WO2024131862 A1 WO 2024131862A1 CN 2023140371 W CN2023140371 W CN 2023140371W WO 2024131862 A1 WO2024131862 A1 WO 2024131862A1
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Prior art keywords
protein
rsv
pref
amino acid
seq
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PCT/CN2023/140371
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English (en)
French (fr)
Inventor
王希良
张金灿
程晋霞
王莉
李世崇
王立博
Original Assignee
北京吉诺卫生物科技有限公司
吉诺卫(廊坊临空自贸区)生物制品有限公司
吉诺卫（上海）生物制品有限公司
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Priority claimed from CN202211654193.3A external-priority patent/CN115850396B/zh
Priority claimed from CN202311083641.3A external-priority patent/CN117106809A/zh
Priority claimed from CN202311686080.6A external-priority patent/CN117771359A/zh
Application filed by 北京吉诺卫生物科技有限公司, 吉诺卫(廊坊临空自贸区)生物制品有限公司, 吉诺卫（上海）生物制品有限公司 filed Critical 北京吉诺卫生物科技有限公司
Publication of WO2024131862A1 publication Critical patent/WO2024131862A1/zh

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  • the present invention claims priority to three applications filed in China with application number 2022116541933 and application date December 22, 2022, in China with application number 2023110836413 and application date August 25, 2023, and in China with application number 2023116860806, which are incorporated into the present invention in their entirety and serve as a part of the present invention.
  • the invention belongs to the field of biotechnology, and specifically relates to an RSV vaccine and a preparation method and application thereof.
  • Respiratory syncytial virus was first discovered in 1955. It belongs to the family Paramyxoviridae, subfamily Pneumovirinae, genus Pneumovirus. It can be divided into two subtypes, A and B, based on the sequence of the G protein.
  • RSV is a non-segmented negative-strand RNA virus with a genome length of 15.2kb and 10 genes encoding a total of 11 proteins, including non-structural proteins (NS1, NS2), nucleoprotein (N), phosphoprotein (P), matrix protein (M), RNA-dependent RNA polymerase (L), transcription elongation factor (M2-1), regulatory factor (M2-2) and three envelope glycoproteins (adhesion protein (G), fusion protein (F) and small hydrophobic protein (SH)).
  • NS1, NS2 non-structural proteins
  • N nucleoprotein
  • P phosphoprotein
  • M matrix protein
  • M2-1 RNA-dependent RNA polymerase
  • M2-1 RNA-dependent RNA polymerase
  • M2-1 transcription elongation factor
  • M2-2 transcription elongation factor
  • G fusion protein
  • SH small hydrophobic protein
  • RSV is a viral pathogen that causes respiratory tract infection (RTI).
  • the infection mainly causes symptoms of lower respiratory tract infection, among which a considerable proportion of patients have severe symptoms (such as bronchiolitis and pneumonia), requiring hospitalization and having a high mortality rate.
  • RSV can be transmitted through contact between people, or inhaled through coughing or sneezing, or through contact with pollutants. It mainly infects epithelial cells of the nasal cavity and large and small airways of the lungs. It may also infect alveolar macrophages and other types of cells in the lungs, causing cells to fuse together to form syncytia.
  • Vaccine research is currently the most concentrated area for the prevention and treatment of RSV.
  • formalin-inactivated RSV vaccine was developed for clinical research. This was also the first RSV vaccine to enter clinical trials.
  • the vaccine not only failed to produce a protective effect against RSV disease, but in subsequent natural infections, vaccinated children developed severe enhanced disease (ERD), significantly increased hospitalization rates, and even caused death. Therefore, the vaccine ultimately failed to enter clinical application.
  • ELD severe enhanced disease
  • RSV vaccine research has also made major breakthroughs.
  • Many types of RSV vaccines such as live attenuated vaccines, inactivated vaccines, chimeric vector vaccines, subunit vaccines, virus-like particle vaccines, replication-defective virus vector vaccines, nucleic acid vaccines, etc., have shown clinical application potential in the clinical research trial stage.
  • RSV vaccines have shown clinical application potential in the clinical research trial stage.
  • the purpose of the present invention is to provide an RSV vaccine with high immune titer, high stability and good safety.
  • the present invention first provides a protein.
  • the protein provided by the present invention comprises mutations in one or more sites of positions 67, 88, 110, 144, 159, 173, 202, 227, 236, 248, 289, 309, 334, 344, 370, 389, 419 and/or 468 of the amino acid sequence of the Pre-F protein.
  • the protein provided by the present invention is a protein obtained by subjecting the amino acid sequence of the Pre-F protein to at least one of the following mutations a1) to a18):
  • the mutation of the pre-F protein includes
  • the mutation includes:
  • the Pre-F protein is any of the following:
  • (A2) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the protein described in (A1);
  • (A3) a protein having the same function obtained by replacing and/or deleting and/or adding one or more amino acid residues of any one of (A1)-(A2);
  • (A4) A protein having 98% or more identity with any one of (A1) to (A2) and having the same function.
  • the protein (or a mutant protein called Pre-F protein) is any one of the following:
  • M1 the protein represented by SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 or SEQ ID No.5 or SEQ ID No.6 or SEQ ID No.7 or SEQ ID No.8 or SEQ ID No.9;
  • (M2) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the protein described in (M1);
  • (M3) a protein having the same function obtained by replacing and/or deleting and/or adding one or more amino acid residues of any one of (M1)-(M2);
  • (M4) A protein that is more than 98% identical to any one of (M1)-(M2) and has the same function.
  • the present invention further provides a fusion protein.
  • the fusion protein provided by the present invention comprises the above protein and a ferritin mutant
  • the ferritin mutant is a protein obtained by subjecting the ferritin amino acid sequence to at least one of the following mutations b1) to b3):
  • the ferritin is any of the following:
  • (B2) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the protein described in (B1);
  • (B4) A protein having 98% or more identity with any one of (B1) to (B2) and having the same function.
  • ferritin mutant is any one of the following:
  • (N2) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the protein described in (N1);
  • N3 A protein having the same function obtained by replacing and/or deleting and/or adding one or more amino acid residues of any one of (N1)-(N2);
  • (N4) A protein that is 98% or more identical to any one of (N1) to (N2) and has the same function.
  • fusion protein is any one of the following:
  • (C1) the protein represented by SEQ ID No.12 or SEQ ID No.13 or SEQ ID No.14 or SEQ ID No.15 or SEQ ID No.16 or SEQ ID No.17 or SEQ ID No.18 or SEQ ID No.19;
  • (C2) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the protein described in (C1);
  • (C3) a protein having the same function obtained by replacing and/or deleting and/or adding one or more amino acid residues of any of (C1)-(C2);
  • (C4) A protein having 98% or more identity with any one of (C1) to (C2) and having the same function.
  • the tag refers to a polypeptide or protein fused and expressed together with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein.
  • the tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and/or a SUMO tag, etc.
  • substitution and/or deletion and/or addition of one or several amino acid residues is the substitution and/or deletion and/or addition of no more than 10 amino acid residues outside the amino acid mutation sites described in a1)-a18) or b1)-b3) above.
  • any of the above-mentioned proteins or fusion proteins can be artificially synthesized, or the encoding gene can be synthesized first and then expressed biologically.
  • the invention also provides a biomaterial.
  • the biomaterial provided by the present invention is at least one of the following D1)-D5):
  • D2 an expression cassette containing the nucleic acid molecule described in D1);
  • D3 a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);
  • D4 a recombinant microorganism containing the nucleic acid molecule described in D1), a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in B3);
  • D5 A recombinant cell line containing the nucleic acid molecule described in D1), a recombinant cell line containing the expression cassette described in D2), or a recombinant cell line containing the recombinant vector described in D3).
  • nucleic acid molecule encoding the protein is E1) or E2):
  • E2 A DNA molecule that has more than 75% identity with the DNA molecule defined in E1) and encodes the fusion protein.
  • the nucleic acid molecule encoding the fusion protein is F1) or F2):
  • F2 A DNA molecule that has more than 75% identity with the DNA molecule defined in F1) and encodes the fusion protein.
  • the nucleic acid molecule may be DNA, such as recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA.
  • the mRNA comprises
  • G1 mRNA sequence obtained by replacing T with U in position 1-1422 or the full-length sequence of any of SEQ ID No. 20-27;
  • G2 a degenerate or complementary sequence of G1)
  • G3 An mRNA molecule that has more than 75% identity with the mRNA molecule defined in G1) or G2) and encodes a fusion protein with the same function.
  • the mRNA includes, in addition to the above-mentioned coding region, a 5' cap structure, a 5' non-coding region, a 3' non-coding region and/or an mRNA with a polyadenylic acid tail.
  • the mRNA sequence is a natural or modified RNA, wherein the modified RNA comprises modifying the RNA by partially or completely replacing natural uridine with modified uridine.
  • the modified RNA is formed by replacing all natural uridine with 1-methyl-pseudouridine.
  • the present invention provides an mRNA, which comprises a first open reading frame, which comprises a nucleic acid encoding a monomeric subunit protein and at least one immunogenic portion from RSV: the monomeric subunit protein is selected from the group consisting of a monomeric ferritin subunit, a monomeric encapsulin protein, a monomeric 03-33 protein, a monomeric sulfur oxygenase reductase protein, a monomeric dioxotetrahydropteridine synthase protein, and/or a monomeric pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase protein, the monomeric subunit protein expressed by the first open reading frame self-assembles into nanoparticles, and displays the at least one immunogenic portion on the surface of the nanoparticles.
  • the monomeric subunit protein is selected from the group consisting of a monomeric ferritin subunit, a monomeric encapsulin protein, a monomeric 03-33 protein, a monomeric
  • the present invention provides an mRNA, which comprises at least two open reading frames, wherein the first open reading frame comprises a nucleic acid encoding a monomeric subunit protein selected from the following group: a monomeric ferritin subunit, a monomeric encapsulin protein, a monomeric 03-33 protein, a monomeric sulfur oxygenase reductase protein, a monomeric dioxotetrahydropteridine synthase protein, and/or a monomeric pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase protein, and the second open reading frame comprises a nucleic acid encoding at least one immunogenic portion from RSV, the monomeric subunit protein expressed by the first open reading frame can self-assemble into nanoparticles, and the at least one immunogenic portion expressed by the second open reading frame can bind to the nanoparticles expressed by the first open reading frame.
  • the first open reading frame comprises a nucleic acid encoding a monomeric subunit
  • the at least one immunogenic portion is selected from a mutant protein of the pre-F protein of RSV, and the mutant protein of the pre-F protein is as defined above.
  • the protein encoded by the mRNA is a fusion protein consisting of a Pre-F mutant protein, a linker and a ferritin mutant in sequence from the N-terminus to the C-terminus.
  • the fusion protein encoded by the mRNA may further include a tag, which refers to a polypeptide or protein fused and expressed together with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein.
  • the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and/or a SUMO tag, etc.
  • the protein encoded by the mRNA is a fusion protein consisting of a Pre-F mutant protein, a linker and a ferritin mutant in sequence from the N-terminus to the C-terminus.
  • the fusion protein encoded by the mRNA may further include a tag, which refers to a polypeptide or protein fused and expressed together with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein.
  • the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and/or a SUMO tag, etc.
  • the mRNA includes any one of the following:
  • (H3) An mRNA that is 80% or more identical to (H1) or (H2) and encodes a protein with the same function.
  • the first open reading frame comprises a portion of the mRNA shown in SEQ ID No. 30-33, and the portion of the mRNA shown in SEQ ID No. 4-7 comprises at least the coding region of the Pre-F mutant protein and/or the coding region of the ferritin mutant shown in Table 8.
  • the portion of the mRNA shown in SEQ ID No. 4-7 comprises at least the coding region of the Pre-F mutant protein and/or the coding region of the ferritin mutant shown in Table 8.
  • the first open reading frame includes the mRNA of the Pre-F mutant protein coding region in SEQ ID No.30-33 or the sequence defined by (C2) and (C3) above
  • the second open reading frame includes the mRNA of the ferritin mutant coding region in SEQ ID No.30-33 or the sequence defined by (C2) and (C3) above.
  • the first open reading frame and/or the second open reading frame comprises mRNA encoding a tag protein.
  • the tag can bind the protein expressed by the first open reading frame and/or the second open reading frame.
  • the immunogenic part binds to the monomer subunit protein to form a nanoparticle to promote multivalent display of the antigen on the nanoparticle.
  • the tag is preferably a motif tag.
  • the motif tag protein is SpyTag and/or SpyCatcher as defined above.
  • the monomeric subunit protein comprises a monomeric encapsulin protein, a monomeric 03-33 protein, a monomeric sulfur oxygenase reductase protein, a monomeric dioxotetrahydropteridine synthase protein, and/or a monomeric pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase protein.
  • the mRNA includes, in addition to the above-mentioned coding region, a 5' cap structure, a 5' non-coding region, a 3' non-coding region and/or an mRNA with a polyadenylated tail.
  • the mRNA sequence is a natural or modified RNA, wherein the modified RNA comprises modifying the RNA by partially or completely replacing natural uridine with modified uridine.
  • nucleotide sequence encoding the above-mentioned protein or fusion protein of the present invention can easily mutate by using known methods, such as directed evolution and point mutation.
  • Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence encoding the above-mentioned protein or fusion protein are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the above-mentioned protein or fusion protein and have the same function.
  • the identity refers to sequence similarity with natural amino acid or nucleic acid sequences. "Identity" includes nucleotide sequences that have 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein encoding the amino acid sequence shown in SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 or SEQ ID No.5 or SEQ ID No.6 or SEQ ID No.7 or SEQ ID No.8 or SEQ ID No.9 or SEQ ID No.12 or SEQ ID No.13 or SEQ ID No.14 or SEQ ID No.15 or SEQ ID No.16 or SEQ ID No.17 or SEQ ID No.18 or SEQ ID No.19 of the present invention. Identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.
  • the aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.
  • the expression cassette refers to a DNA capable of expressing the above protein or fusion protein in a host cell
  • the DNA may include not only a promoter for initiating transcription of the gene sequence encoding the above protein or fusion protein, but also a terminator for terminating transcription of the gene sequence encoding the above protein or fusion protein.
  • the expression cassette may also include an enhancer sequence.
  • the vector may be a plasmid, a cosmid, a phage or a virus vector.
  • the microorganism may be yeast, bacteria, algae or fungi.
  • the cell may be a prokaryotic cell or a eukaryotic cell.
  • the present invention also provides a method for preparing the above protein or fusion protein.
  • the method for preparing the above protein or fusion protein provided by the present invention comprises the following steps: expressing the nucleic acid molecule encoding the above protein or fusion protein in an organism or biological cell to obtain the fusion protein.
  • the method includes the following steps: introducing a nucleic acid molecule encoding the protein or fusion protein into CHO K1Q cells to obtain recombinant cells; and culturing the recombinant cells to obtain the protein or fusion protein.
  • nucleic acid molecule of the protein or fusion protein is introduced into CHO K1Q cells via a recombinant plasmid.
  • the recombinant plasmid is a plasmid obtained by inserting the nucleic acid molecule of the protein or fusion protein into a vector plasmid.
  • the vector plasmid is a pKS001 vector plasmid.
  • the recombinant plasmid is a recombinant plasmid pKS001-RSV-PreF-A-NP, pKS001-RSV-PreF-B-NP, pKS001-RSV-PreF-C-NP, pKS001-RSV-PreF-D-NP, pKS001-RSV-PreF-E-NP, pKS001-RSV-PreF-F-NP, pKS001-RSV-PreF-G-NP or pKS001-RSV-PreF-H-NP.
  • the present invention also provides a method for preparing the above-mentioned biological material, which comprises designing a DNA sequence according to the encoded fusion protein and constructing a vector containing the DNA.
  • the preparation method further comprises transcribing the vector to express mRNA.
  • the present invention provides a method for preparing the above-mentioned mRNA, which comprises designing a DNA sequence according to the monomeric subunit protein encoded by the mRNA and at least one immunogenic portion from RSV, constructing a vector containing the DNA, and transcribing the vector to express the mRNA.
  • the at least one immunogenic portion from RSV includes a mutant protein of the pre-F protein, the monomeric subunit protein includes a ferritin mutant, and the mutant protein of the pre-F protein is expressed by fusion or self-assembled with the ferritin mutant.
  • the mutant protein of the pre-F protein is expressed by fusion or self-assembled with the ferritin mutant.
  • the fusion expression is connected via a connecting peptide or the self-assembly is achieved via a tag.
  • the tags are Spy and Catcher.
  • the sequences of Spy and Catcher are shown as SEQ ID No. 28 and 29 respectively.
  • the present invention also provides the above protein or the above fusion protein or the above biological material or the protein or fusion protein prepared according to the above method. New uses for white.
  • the present invention provides the use of the above protein or the above fusion protein or the above biological material or the protein or fusion protein prepared according to the above method in any one of the following Y1)-Y4);
  • the present invention also provides a vaccine.
  • the active ingredient of the vaccine provided by the present invention is the above protein or the above fusion protein or the above biological material or the protein or fusion protein prepared according to the above method.
  • the biological material comprises mRNA.
  • the vaccine is a fusion protein vaccine.
  • the vaccine is an mRNA vaccine.
  • the vaccine also includes an adjuvant.
  • the adjuvant may be an aluminum adjuvant.
  • the aluminum adjuvant may be one or more of aluminum hydroxide and aluminum phosphate.
  • the mass ratio of the protein to the aluminum adjuvant can be 1:(0.5-300), which can be any value or range of the above ratio, for example, 1:(1-275), 1:(10-200), 1:(30-180), 1:(40-100), 1:(10-80), 1:(25-75), 1:(50-75), 1:50, and the like.
  • the vaccine comprises a double adjuvant, wherein the double adjuvant comprises the aluminum adjuvant and the CpG adjuvant defined above.
  • the CpG adjuvant includes any CpG adjuvant, such as CpG1018, CpG-cjx, and the like.
  • the mass ratio of the protein to the CpG adjuvant may be 1:(0.5-300), which may be any value or range of the above ratio, for example, 1:(1-275), 1:(10-200), 1:(30-180), 1:(40-100), 1:(10-80), 1:(25-75), 1:(50-75), 1:50, and the like.
  • the mass ratio of aluminum adjuvant to CpG adjuvant in the dual adjuvant is (1-5):(5-1), which can be any value or range of the above ratio, such as 1:2, 1:1, 2:1, etc.
  • the mass ratio of protein:aluminum adjuvant:CpG adjuvant is 1:50:50.
  • the mRNA vaccine comprises any of the mRNA defined above and lipid nanoparticles, wherein the mRNA is encapsulated in the lipid nanoparticles.
  • lipid nanoparticles in the present invention refers to particles with at least one nanometer size, which contain at least one lipid.
  • the lipid nanoparticles include cationic lipids, neutral phospholipids, steroidal lipids and polyethylene glycol-lipids.
  • neutral phospholipid herein refers to uncharged, non-phosphoglyceride phospholipid molecules.
  • polyethylene glycol-lipid refers to a molecule comprising a lipid portion and a polyethylene glycol (PEG) portion.
  • the cationic lipid compound structure is: DLin-MC3-DMA, ALC-0315, or SM-102.
  • the neutral phospholipid is selected from: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) or one or more combinations thereof, preferably DSPC.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • the steroidal lipids are selected from avenasterol, ⁇ -sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol and cholesterol modified by polypeptide; one or more combinations of lanosterol, luminosterol, alginasterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, preferably cholesterol.
  • the polyethylene glycol (PEG)-lipid is selected from: 2-[(polyethylene glycol)-2000]-N,N-tetracosyl acetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxy polyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterol glycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, One or more combinations of PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) or DMG-PEG2000, preferably DMG
  • the molar ratio of cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid is (40-50):(5-15):(35-45):(1-5), and more preferably, the ratio is 45:10:43:2.
  • the mass ratio of the mRNA solution to lipid nanoparticles is (2-8):1, more preferably 3:1.
  • the vaccine is a liquid preparation or a lyophilized powder. More preferably, the vaccine is an oral preparation, an intramuscular injection preparation, an intravenous injection preparation or an inhalation preparation. Further preferably, the vaccine is an aerosol inhalation preparation or a dry powder inhalation preparation.
  • the vaccine of the present invention may further comprise a pharmaceutically acceptable excipient, which may be a carrier, a diluent, an adjuvant or a nucleotide sequence encoding an adjuvant, a solubilizer, an adhesive, a lubricant, a suspending agent, a transfection accelerator, and the like.
  • a pharmaceutically acceptable excipient which may be a carrier, a diluent, an adjuvant or a nucleotide sequence encoding an adjuvant, a solubilizer, an adhesive, a lubricant, a suspending agent, a transfection accelerator, and the like.
  • the transfection facilitating agent includes, but is not limited to, surfactants such as immunostimulating complexes, Freunds incomplete adjuvant, LPS analogs (e.g., monophosphoryl ester A), cell wall peptides, benzoquinone analogs, squalene, hyaluronic acid, lipids, lipids, calcium ions, viral proteins, cations, polycations (e.g., poly-L-glutamic acid (LGS)) or nanoparticles or other known transfection facilitating agents.
  • surfactants such as immunostimulating complexes, Freunds incomplete adjuvant, LPS analogs (e.g., monophosphoryl ester A), cell wall peptides, benzoquinone analogs, squalene, hyaluronic acid, lipids, lipids, calcium ions, viral proteins, cations, polycations (e.g., poly-L-glutamic acid (LGS)) or nanoparticles or other known transfection
  • the nucleotide sequence encoding the adjuvant is a nucleotide sequence encoding at least one of the following adjuvants: GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD1/2, lactoferrin, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF- ⁇ , INF- ⁇ , Lymphotoxin- ⁇ , hGH, MCP- 1.
  • the product may be a vaccine.
  • the present invention provides a pharmaceutical composition, which comprises the fusion protein, biological material or mRNA vaccine defined above.
  • the pharmaceutical composition is used to prevent respiratory syncytial virus infectious diseases.
  • the present invention provides a method for preparing the above-mentioned pharmaceutical composition, which comprises the step of preparing the above-mentioned fusion protein, biological material or mRNA.
  • the preparation method comprises dissolving lipid nanoparticles in a solvent and then mixing and encapsulating the lipid nanoparticles with mRNA to obtain an mRNA pharmaceutical composition.
  • the lipid nanoparticles are as defined above.
  • the present invention also provides a method for producing an immune response, which may include administering any of the above-mentioned pharmaceutical compositions to a subject.
  • the present invention also provides a method for preventing and/or treating RSV infectious diseases, which may include administering the pharmaceutical composition to a subject.
  • the RSV infectious disease may include respiratory system, digestive system, cardiovascular system and/or nervous system infection.
  • the respiratory system infection may include respiratory tract infection and/or lung infection.
  • the respiratory tract infection may include severe acute respiratory syndrome, hypoxic respiratory failure, sepsis, septic shock, nasopharyngitis, rhinitis, pharyngitis, tracheitis and/or bronchitis
  • the lung infection may include pneumonia and/or lung injury.
  • the digestive system infection may include intestinal diseases, poor appetite, nausea, vomiting, abdominal pain and/or diarrhea.
  • an immune response against RSV can be induced in the subject.
  • the immune response can be a cellular immune response, a humoral immune response, or a cellular immune response and a humoral immune response.
  • the cellular immune response may include a B cell immune response and a T cell immune response.
  • the subject described herein can be a human or a non-human animal.
  • non-human animal may be a non-human mammal.
  • the non-human mammal may be any one of mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cows, and horses, but is not limited thereto.
  • the subjects described herein include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after infection).
  • the administration described herein includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral and nasal spraying, or aerosol inhalation.
  • the present invention enhances the effective immunogenicity, stability and safety of Pre-F protein through specific antigen mutation design, and further enhances immunogenicity by exposing the required epitopes on the surface of nanoparticles.
  • the vaccine prepared by the present invention can achieve good immune effects at low doses, among which the neutralizing titer of RSV-PreF-C-NP group can reach 19836.
  • the present invention solves the problem of poor stability of wild antigens.
  • the ferritin-PreF fusion protein prepared by the present invention can induce respiratory syncytial virus antibodies with neutralizing activity after entering the body, thereby giving the body corresponding immune protection.
  • the ferritin-PreF fusion protein prepared by the present invention can effectively stimulate the body's cellular immune mechanism, and the immune response induced is Th1/Th2 balanced, which can avoid immune overreaction caused by Th2 extremism and has good safety.
  • the mRNA vaccine of the present invention can induce antibodies with neutralizing activity against respiratory syncytial virus, thereby giving the body corresponding humoral immune protection.
  • the mRNA vaccine can effectively stimulate the body's cellular immune mechanism, increase the frequency of IFN- ⁇ secretion by spleen cells, and effectively induce cellular immunity, thereby bringing longer-lasting and comprehensive protection.
  • the present invention obtains a suitable adjuvant combination and an effective concentration range by screening adjuvants.
  • the two adjuvants have a synergistic effect and can significantly improve the immune effect of RSV vaccine.
  • the present invention performs mutation design on RSV Pre-F related sequences and ferritin nanoparticles, and fuses and expresses Pre-F mutant proteins and ferritin mutant particles in eukaryotic cells to obtain ferritin-PreF fusion protein nanoparticles with multiple Pre-Fs concentrated on the surface.
  • the ferritin-PreF fusion protein nanoparticles stabilize and expose the antigenic epitopes required to be displayed, destroy or hide unnecessary antigenic epitopes, and effectively improve the immunogenicity, production stability and safety of the antigen.
  • serum with high protective titer can be obtained, and mouse serum can produce a high neutralizing titer against real viruses.
  • ferritin-PreF fusion protein prepared by the present invention also has sufficient physical stability and good safety.
  • the obtained mRNA vaccine can effectively stimulate the body's cellular immune mechanism, increase the frequency of splenocytes secreting IFN- ⁇ , and effectively induce cellular immunity, thereby bringing longer-lasting and comprehensive protection.
  • FIG1 is a schematic diagram of the structure of the pKS001 vector.
  • Figure 2 shows the results of SDS-PAGE electrophoresis of ferritin-PreF fusion protein.
  • Lane 1 is the flow-through after RSV-PreF-C-NP purification
  • lane 2 is the elution product after RSV-PreF-C-NP purification
  • lanes 3 and 4 are the elution products after RSV-PreF-D-NP and RSV-PreF-E-NP purification, respectively.
  • D Lane 1 is the elution product after RSV-PreF-F-NP purification
  • lane 2 is a molecular weight marker.
  • Lane 1 and lane 2 are the flow-through and elution products after RSV-PreF-G-NP purification, respectively.
  • Lane 1 is the molecular weight marker
  • lane 2 is the flow-through after RSV-PreF-H-NP purification
  • lane 3 is the interval blank lane
  • lane 4 is the elution product after RSV-PreF-H-NP purification.
  • Figure 3 shows the WB detection results of the purified ferritin-PreF fusion protein. From left to right, they are the WB detection results of the purified products of RSV-PreF-A-NP, RSV-PreF-B-NP, RSV-PreF-C-NP, RSV-PreF-D-NP, RSV-PreF-E-NP, RSV-PreF-F-NP, RSV-PreF-G-NP, and RSV-PreF-H-NP.
  • Figure 4 is a morphology diagram of the nanoparticles of ferritin-PreF fusion protein.
  • A-H are electron micrographs of the purified products of RSV-PreF-A-NP, RSV-PreF-B-NP, RSV-PreF-C-NP, RSV-PreF-D-NP, RSV-PreF-E-NP, RSV-PreF-F-NP, RSV-PreF-G-NP, and RSV-PreF-H-NP, respectively.
  • FIG. 5 shows the results of immunogenicity studies of ferritin-PreF fusion protein vaccines.
  • FIG6 shows the Log2 of the neutralization efficacy value after immunization of mice with ferritin-PreF fusion protein.
  • Figure 7 shows the ELISA titers and history of IgG1 and IgG2a in the serum of mice immunized with the ferritin-PreF fusion protein RSV-PreF-C-NP Control titers of formalin-inactivated vaccines.
  • Figure 8 shows the frequency of IFN- ⁇ secretion by spleen cells after immune stimulation with different mRNA vaccines.
  • the RSV virus type A Long strain in the following examples is recorded in the literature “Cultures of HEp-2 cells persistently infected by human respiratory syncytial virus differ in chemokine expression and resistance to apoptosis as compared to lytic infections of the same cell type”.
  • the RSV virus type B BA9 strain in the following examples is recorded in the document “Genetic Diversity and Molecular Epidemiology of Circulating Respiratory Syncytial Virus in Central Taiwan, 2008-2017”.
  • the formalin inactivated vaccine FI-RSV in the following embodiment is recorded in the document “Enhanced pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin-4 (IL-4) and IL-10”.
  • IL-4 interleukin-4
  • the RSV Pre-F related sequence is mutated and designed to obtain the Pre-F mutant protein, which is then fused with the ferritin related sequence to form a ferritin-PreF integrated subunit, and then the self-assembly property of ferritin is utilized to form nanoparticles with good Pre-F antigen display effect.
  • the specific steps are as follows:
  • the RSV Pre-F protein amino acid sequence (the Pre-F protein amino acid sequence is shown in SEQ ID No. 1) is mutated by at least one of the following 1)-18) to obtain a Pre-F mutant protein:
  • the Pre-F mutant proteins are RSV-PreF-A, RSV-PreF-B, RSV-PreF-C, RSV-PreF-D, RSV-PreF-E, RSV-PreF-F, RSV-PreF-G and RSV-PreF-H.
  • amino acid sequences of Pre-F mutant proteins RSV-PreF-A, RSV-PreF-B, RSV-PreF-C, RSV-PreF-D, RSV-PreF-E, RSV-PreF-F, RSV-PreF-G and RSV-PreF-H are shown in SEQ ID No.2 to SEQ ID No.9, respectively.
  • ferritin amino acid sequence (the ferritin amino acid sequence is shown in SEQ ID No. 10) is mutated by at least one of the following 1)-3) to obtain a ferritin mutant:
  • amino acid sequence of the ferritin mutant is shown as SEQ ID No.11.
  • the Pre-F mutant protein and the ferritin mutant were fused via a linker (SGSGGGSG) to prepare a ferritin-PreF fusion protein, which consisted of a Pre-F mutant protein, a linker (SGSGGGSG) and a ferritin mutant from the N-terminus to the C-terminus.
  • a linker SGSGGGSG
  • ferritin-PreF fusion proteins are RSV-PreF-A-NP, RSV-PreF-B-NP, RSV-PreF-C-NP, RSV-PreF-D-NP, RSV-PreF-E-NP, RSV-PreF-F-NP, RSV-PreF-G-NP and RSV-PreF-H-NP, and their amino acid sequences are shown in SEQ ID No.12 to SEQ ID No.19, and their coding gene sequences are shown in SEQ ID No.20 to SEQ ID No.27, respectively.
  • Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize plasmids containing the gene sequences encoding the above-mentioned ferritin-PreF fusion proteins, and they were named plasmids RSV-PreF-A-NP, RSV-PreF-B-NP, RSV-PreF-C-NP, RSV-PreF-D-NP, RSV-PreF-E-NP, RSV-PreF-F-NP, RSV-PreF-G-NP and RSV-PreF-H-NP, respectively.
  • the pKS001 vector plasmid (Zhongshan Kangtian Shenghe Biotechnology Co., Ltd., catalog number A14101) was double-digested with restriction endonucleases Hind III-HF and Not I-HF (NEB, catalog numbers R3104V and R3189L, respectively) to obtain the backbone vector.
  • the schematic diagram of the pKS001 vector plasmid structure is shown in Figure 1.
  • Plasmids RSV-PreF-A-NP, RSV-PreF-B-NP, RSV-PreF-C-NP, RSV-PreF-D-NP, RSV-PreF-E-NP, RSV-PreF-F-NP, RSV-PreF-G-NP and RSV-PreF-H-NP were double-digested with restriction endonucleases Hind III-HF and Not I-HF to obtain the target fragments respectively.
  • the backbone vector was ligated to each target fragment using Quick ligase (NEB, catalog number M2200L) and then transformed into competent Escherichia coli Trans10 (Beijing Quanshijin Biotechnology Co., Ltd., catalog number CD101). Positive clones were screened and plasmids were extracted for sequencing verification.
  • the plasmids verified correctly by sequencing were named recombinant plasmids pKS001-RSV-PreF-A-NP, pKS001-RSV-PreF-B-NP, pKS001-RSV-PreF-C-NP, pKS001-RSV-PreF-D-NP, pKS001-RSV-PreF-E-NP, pKS001-RSV-PreF-F-NP, pKS001-RSV-PreF-G-NP and pKS001-RSV-PreF-H-NP.
  • the above-mentioned recombinant plasmids pKS001-RSV-PreF-A-NP, pKS001-RSV-PreF-B-NP, pKS001-RSV-PreF-C-NP, pKS001-RSV-PreF-D-NP, pKS001-RSV-PreF-E-NP, pKS001-RSV-PreF-F-NP, pKS001-RSV-PreF-G-NP and pKS001-RSV-PreF-H-NP were electroporated and expressed in CHO K1Q cells (Kangsheng Biotechnology Co., Ltd., catalog number A14101), and cell lines with high expression were screened.
  • DPBS washing discard the supernatant culture medium, obtain the desired cells, add 1 mL D-PBS (Thermo Fisher Scientific Gibco, Catalog No.: 2334304) to resuspend the cells, and centrifuge at 1000 rpm for 5 minutes.
  • Electroporation Add the cell suspension mixed with plasmid into the H1 electroporation cup (Suzhou Yida Biotechnology Co., Ltd., catalog number: H10201) at a volume of 200ul+DNA/cup, insert the electroporation cup into the base, and perform electroporation according to the electroporation conditions shown in Table 1.
  • the specific steps of culturing and screening of cell clones are as follows: the cells in the above T25 square flask are sampled and monitored for viability using a cell counter (Sanofi, model: Countess II FL). When the viability is higher than 70%, 96-well plates are plated at 10,000 cells per well, cultured in CD04 culture medium containing 25 mM MSX (Sigma, catalog number: M5379-1G), positive clones are selected by ELISA, and culture is continued to be expanded to 125 mL (Wuxi Nice Life Sciences Co., Ltd., catalog number: 781011) shake flasks, cultured in 125 mL shake flasks, and after about 5-7 days, the viability is detected by a counter and drops to between 50-80%, and the supernatant is taken for ELISA detection.
  • the ELISA detection method is as follows: the supernatant was diluted 10 times, 100 times, 1000 times, and 10,000 times, and coated, and a 1500-fold diluted F protein antibody (Pujian Biotechnology (Wuhan) Technology Co., Ltd., catalog number: 62814) was used as the primary antibody, and goat anti-human IgG-HRP (Solebo, catalog number: SE101-1ml) was used as the secondary antibody.
  • the signal was read using an enzyme reader (Shanghai Kehua, catalog number: RD-SH-012), and the sample with the strongest signal was selected as the highest expression sample.
  • the supernatant of the highest expression sample was harvested for the next step of purification.
  • the selected cell supernatant culture medium was centrifuged at 8000r/min for 20 minutes, filtered using a 0.45um filter membrane (Jin Teng, catalog number: JTSF025013/014) to obtain about 100mL of solution, and the balance solution was added to 200mL.
  • the QFF column was balanced with the balance solution, and the sample was loaded with the A1 pump at a flow rate of 1.5mL/min. After the loading was completed, it was rinsed with the balance solution until the absorption value returned to the value before loading and stabilized.
  • the eluent (20mM Tris, 0.5M NaCl, pH8.5) was gradient eluted at a flow rate of 2mL/min, 0-100% B, 50min. The elution peak was collected.
  • the supernatant was concentrated 5-10 times, passed through a Superose 6 prep grade column at a flow rate of 1mL/min, and the sample of the absorption peak was collected to obtain a ferritin-PreF fusion protein solution, which was concentrated for SDS-PAGE and western blot analysis.
  • SDS-PAGE analysis The specific steps of SDS-PAGE analysis are as follows: add 20uL of 5 ⁇ protein loading to 80uL of ferritin-PreF fusion protein solution, treat at 95°C for 10min and centrifuge. Take 15uL of supernatant for SDS-PAGE analysis and stain to observe protein expression.
  • SDS-PAGE electrophoresis Prepare 10% SDS-PAGE gel with a thickness of 1.0 mm, perform gel electrophoresis in 1 ⁇ SDS electrophoresis buffer, load 20 ul of protein sample, use 80 V voltage, and switch to 130 V after the sample enters the separation gel.
  • Blocking Infiltrate the membrane with PBST blocking buffer containing 5% skimmed milk powder and block at 37°C for 1 h.
  • Secondary antibody incubation Add secondary antibody (Bioworld, Goat Anti-Rabbit IgG (H+L) HRP, Catalog No.: AA092030) diluted in 1 ⁇ PBST and incubate at 37°C for 45min. After incubation, wash the membrane three times with 1 ⁇ PBST on a shaker at 70rpm, 10min each time.
  • secondary antibody Bioworld, Goat Anti-Rabbit IgG (H+L) HRP, Catalog No.: AA092030
  • Color development Use DAB color development kit (Solarbio, catalog number: DA1016) for color development.
  • the purified products of the ferritin-PreF fusion proteins RSV-PreF-A-NP, RSV-PreF-B-NP, RSV-PreF-C-NP, RSV-PreF-D-NP, RSV-PreF-E-NP, RSV-PreF-F-NP, RSV-PreF-G-NP and RSV-PreF-H-NP prepared in Example 1 were negatively stained respectively.
  • the specific negative staining operation is as follows:
  • the ultra-thin carbon film was pre-evacuated for 3 minutes using a Harrick Basic Plasma Cleaner PDC-32G-2 instrument, then glow-discharged for 30 seconds in the medium position and removed.
  • a 4um sample was taken with a pipette and dropped onto the carbon film. After placing it horizontally for 1 minute, it was blotted dry with filter paper. Then 7um 2% uranyl acetate was added and placed for 1 minute. It was blotted dry with filter paper. After placing it for several minutes, the negatively stained purified sample was observed by electron microscopy using a FEI Tecnai Arctica TEM D683 transmission electron microscope.
  • mice 64 6-8 week old Balb/c mice were randomly divided into 8 groups, 8 mice in each group.
  • the treatment method of each group was as follows:
  • RSV-PreF-A-NP Two thigh muscle injections were performed on days 0 and 21, respectively, with each injection of 1ug of ferritin-PreF fusion protein RSV-PreF-A-NP, 50ug of aluminum hydroxide adjuvant (Changchun Institute of Biological Products Co., Ltd., batch number: ZP18-003-202106) and 100ul PBS buffer (Solarbio, product number P1020).
  • RSV-PreF-B-NP Two thigh muscle injections were performed on days 0 and 21, respectively, with each injection of 1ug of ferritin-PreF fusion protein RSV-PreF-B-NP, 50ug of aluminum hydroxide adjuvant (Changchun Institute of Biological Products Co., Ltd., batch number: ZP18-003-202106) and 100ul PBS buffer (Solarbio, product number P1020).
  • RSV-PreF-C-NP Two thigh muscle injections were performed on days 0 and 21, respectively, with each injection of 1ug of ferritin-PreF fusion protein RSV-PreF-C-NP, 50ug of aluminum hydroxide adjuvant (Changchun Institute of Biological Products Co., Ltd., batch number: ZP18-003-202106) and 100ul PBS buffer (Solarbio, product number P1020).
  • RSV-PreF-D-NP Two thigh muscle injections were performed on days 0 and 21, respectively, with each injection of 1ug of ferritin-PreF fusion protein RSV-PreF-D-NP, 50ug of aluminum hydroxide adjuvant (Changchun Institute of Biological Products Co., Ltd., batch number: ZP18-003-202106) and 100ul PBS buffer (Solarbio, product number P1020).
  • RSV-PreF-E-NP Two thigh muscle injections were performed on days 0 and 21, respectively, with each injection of 1ug of ferritin-PreF fusion protein RSV-PreF-E-NP, 50ug of aluminum hydroxide adjuvant (Changchun Institute of Biological Products Co., Ltd., batch number: ZP18-003-202106) and 100ul PBS buffer (Solarbio, product number P1020).
  • RSV-PreF-F-NP Two thigh muscle injections were performed on days 0 and 21, respectively, with 1ug of ferritin-PreF fusion protein RSV-PreF-F-NP and 50ug of aluminum hydroxide adjuvant (Changchun Institute of Biological Products Co., Ltd., batch number: ZP18-003-202106) injected each time. and 100ul PBS buffer (Solarbio, Cat. No. P1020).
  • RSV-PreF-G-NP Two thigh muscle injections were performed on days 0 and 21, respectively, with each injection of 1ug of ferritin-PreF fusion protein RSV-PreF-G-NP, 50ug of aluminum hydroxide adjuvant (Changchun Institute of Biological Products Co., Ltd., batch number: ZP18-003-202106) and 100ul PBS buffer (Solarbio, product number P1020).
  • RSV-PreF-H-NP Two thigh muscle injections were performed on days 0 and 21, respectively, with each injection of 1ug of ferritin-PreF fusion protein RSV-PreF-H-NP, 50ug of aluminum hydroxide adjuvant (Changchun Institute of Biological Products Co., Ltd., batch number: ZP18-003-202106) and 100ul PBS buffer (Solarbio, product number P1020).
  • mouse serum was collected for ELISA analysis.
  • the specific steps of ELISA analysis were as follows: 200 ng of RSV F protein (Sino Biological, Catalog No.: 11049-V08B) was used for coating per well, mouse serum was used as the primary antibody, and the dilution was performed in a gradient manner according to 250 times, 1250 times, 6250 times, 31250 times, 156250 times, 781250 times, and 3906250 times.
  • Mouse secondary antibody Cell Signaling Technology, Catalog No.: 7076S
  • a microplate reader Shanghai Kehua, Catalog No.: RD-SH-012
  • ferritin-PreF fusion proteins RSV-PreF-D-NP, RSV-PreF-C-NP, RSV-PreF-B-NP, RSV-PreF-E-NP, RSV-PreF-G-NP, and RSV-PreF-H-NP still have ELISA signals detected.
  • ferritin-PreF fusion protein RSV-PreF-C-NP has the highest ELISA titer.
  • the TCID50 of RSV virus type A Long strain cultured in Hep-2 cells in DMEM medium containing 10% bovine serum is 2.81E+07. Select 8 portions of mouse sera from each group and dilute them in DMEM medium containing 2% bovine serum. Starting from 40-fold dilution, dilute to 29160 times in a 3-fold dilution gradient, then mix with an equal volume of 200TCID50 virus solution, place at 37°C for 1 hour, spread 200ul per well on the Hep-2 cell plate, set 3 replicates for each mouse serum, culture at 37°C for 5-7 days, and observe the cytopathic condition.
  • the neutralization titers of RSV-PreF-A-NP, RSV-PreF-D-NP, RSV-PreF-C-NP, and RSV-PreF-E-NP in the present invention all exceed the Log2 of the highest efficacy value against type A strains in the sixth week after immunization of similar vaccines developed by Johnson & Johnson (the Log2 of the highest efficacy value against type A strains in the sixth week after immunization of similar vaccines developed by Johnson & Johnson is about 11, and the converted titer is about 2100).
  • RSV virus type A Long strain was replaced with RSV virus type B BA9 strain, and the same titer analysis procedure was performed using the above-mentioned mouse sera.
  • results are shown in Table 3.
  • the results show that the mean neutralization titer of RSV-PreF-C-NP in mouse serum is 10568, and the log2 value is 13.4.
  • RSV-PreF-A-NP, RSV-PreF-D-NP, RSV-PreF-C-NP, and RSV-PreF-E-NP in the present invention are close to or exceed the titer of 2100.
  • each group of purified ferritin-PreF fusion proteins was subjected to a physical stability (physical environmental challenge) test, and the specific steps are as follows:
  • results in the table are the average values of 4 dilution gradients of 10, 100, 1000 and 10000 times.
  • the serum of mice immunized with the ferritin-PreF fusion protein RSV-PreF-C-NP was selected for ELISA analysis of IgG1 and IgG2a.
  • the ELISA analysis adopted the same settings as the serum ELISA test, and the secondary antibodies used were IgG1-specific secondary antibodies (abcam, catalog number: GR3395386-5) and IgG2a-specific secondary antibodies (abcam, catalog number: GR3413688-1).
  • formalin-inactivated vaccine FI-RSV and similar vaccines developed by Johnson & Johnson were used as controls.
  • FIG7 show that the titers of IgG1 and IgG2a in the serum reached about 6 (see RSVNP IgG1 and RSVNP IgG2a in FIG7 ), and the ratio was approximately equal to 1, which was significantly better than the formalin inactivated vaccine (see FI-RSVNP IgG1 and FI-RSV IgG2a in FIG7 ), and even better than the similar vaccine developed by Johnson & Johnson (IgG2a about 5.8 and IgG1 about 5).
  • the immune response caused by the ferritin-PreF fusion protein prepared by the present invention after injection is Th1/Th2 balanced, which can avoid the immune overreaction (VED for short) caused by Th2 extremism, and has good safety.
  • Tags are added to the sequences of ferritin and PreF mutant protein, respectively, and self-assembled by tags in a 1:1 ratio.
  • Catcher GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT, SEQ ID No. 28
  • Spy RAVPHIVMVDAYKRYK, SEQ ID No. 29
  • the two are assembled into nanoparticles through Spy-Catcher interaction.
  • pVAX1 Provided by Nanjing GenScript Biotechnology Co., Ltd., the plasmid map is shown in the figure
  • HindIII and XbaI The relevant plasmids were synthesized and provided by Nanjing GenScript Biotechnology Co., Ltd.
  • mice 64 6-8 week old Balb/c mice were randomly divided into 8 groups, 8 mice in each group.
  • the treatment method of each group was as follows:
  • Plasmids containing the above 8 groups of DNA sequences were electroporated into BALB/c mice. 50 ⁇ g of the vector plasmid was injected into the posterior thigh muscle, and then six electric pulses (60 V, 20 ms) were applied using a device (Terasa Healthcare Sci-Tech, China). Mice inoculated with 50 ⁇ g pKS by EP were used as negative controls. The mice were immunized twice every two weeks. Serum from the two immunizations was used for neutralization experiments to compare the neutralization protection efficiency.
  • RSV virus type A Long strain (RSV virus type A Long strain is recorded in the literature: Cultures of HEp-2 cells persistently infected by human respiratory syncytial virus differ in chemokine expression and resistance to apoptosis as compared to lytic infections of the same cell type) was cultured in Hep-2 cells in DMEM medium with 10% bovine serum. The TCID50 was 2.81E+07. Eight portions of mouse sera from each group were selected and diluted in DMEM medium with 2% bovine serum.
  • the eight DNA sequences can all produce neutralization titers, and their values are all greater than 100, while the effects of DNA-A, DNA-B, DNA-C, and DNA-D are better (encoding RSV-PreF-A-NP, RSV-PreF-B-NP, RSV-PreF-C-NP, and RSV-PreF-D-NP, respectively), with values greater than 700, which may be related to the mutations in the fusion proteins they encode, and their mutations all have mutations at position 88.
  • the mRNA sequences encoding RSV-PreF-A-NP, RSV-PreF-B-NP, RSV-PreF-C-NP, and RSV-PreF-D-NP were obtained by replacing T in SEQ ID No. 20-23 with U.
  • the coding region of the Pre-F mutant protein of SEQ ID No. 20-23 and the coding region of the ferritin mutant are defined in Table 8.
  • the mRNA sequences encoding RSV-PreF-E-NP, RSV-PreF-F-NP, RSV-PreF-G-NP, and RSV-PreF-H-NP can be obtained by replacing T in SEQ ID No.24-27 with U.
  • the DNA sequence contains elements related to RNA transcription.
  • the plasmid is transformed into Escherichia coli for amplification.
  • the plasmid after fermentation and purification is linearized with the restriction endonuclease BspQ1. Transcription is performed using a T7 in vitro transcription kit to obtain uncapped mRNA.
  • the transcription template is digested with DNaseI, and the mRNA is purified by precipitation.
  • the mRNA is capped with a Cap1 capping kit, and the capped mRNA is purified with an mRNA purification kit (respectively encoding the mRNA sequences of RSV-PreF-A-NP, RSV-PreF-B-NP, RSV-PreF-C-NP, and RSV-PreF-D-NP).
  • the purified mRNA is dissolved in an acidic sodium citrate buffer for standby use.
  • mRNA-LNP mRNA vaccine
  • DLin-MC3-DMA DSPC: cholesterol: PEG2000-DMG
  • the total flow rate of the nanomedicine manufacturing equipment is set to 12ml/min.
  • the mRNA solution and the lipid nanoparticle mixed solution are encapsulated at a flow rate ratio of 3:1.
  • the tangential flow filtration system is ultrafiltered to exchange the liquid and collect the sample, and a sucrose solution is added to obtain an mRNA vaccine (mRNA-LNP).
  • the sampling and measurement of the encapsulation rate are all >91%, the average particle size is about 82nm, PDI ⁇ 0.1, and the Zeta potential is about -8.20mV.
  • mice On day 0, BALB/c mice were injected intradermally (i.d.) with the mRNA vaccine prepared in Example 8.
  • Neutralization titer detection The detection was carried out using the same method as in Example 7.
  • the neutralizing protective antibody titer induced by the nanoparticle-shaped mRNA of the present invention in mice exceeds that of MRK-12 (the sequence and method are from SEQ ID No. 21 of Moderna RSV mRNA patent US20230114180A1) produced simultaneously, and the mRNA vaccine encoding RSV-PreF-D-NP, RSV-PreF-B-NP, and RSV-PreF-C-NP is particularly effective.
  • the frequency of IFN- ⁇ secreting cells after mRNA vaccine stimulation (5 samples in each group) was detected by intracellular cytokine staining flow cytometry (ICS).
  • the average frequency of IFN- ⁇ secretion per 2 ⁇ 10 5 spleen cells can reach 73.4 (5 samples were 58.33, 51, 79, 97.7 and 81, respectively), while the control group MRK-12 was only 6.4 (5 samples were 0, 0.66, 27, 2.33 and 2, respectively), and there was a significant difference between the two.
  • the above Elispot cell immunity results show that the cell immunity induced by the mRNA vaccine of the present invention is significantly higher than that of MRK-12, indicating that there is a significant cell immunity protection effect, and cell immunity can bring more long-lasting and comprehensive protection.
  • mice On day 0, BALB/c mice were injected intradermally (i.d.) with the mRNA vaccine prepared in Example 8.
  • Neutralization titer detection The detection was carried out using the same method as in Example 7.
  • the ferritin-PreF (RSV-PreF-D-NP, SEQ ID No. 15) fusion protein (represented by RSV-NP in the following table) purified in Example 1 was used.
  • the solvent was selected to be 20 mM phosphate buffer, 0.15 M NaCl, pH 4.5) to obtain RSV ferritin-PreF fusion protein antigen.
  • diluted antigen and aluminum hydroxide adjuvant suspension or aluminum phosphate adjuvant suspension (wherein the aluminum content is 10 mg/ml, the solute is aluminum hydroxide or aluminum phosphate, and the solvent is PBS; purchased from Changchun Institute of Biological Products) were placed in a glass bottle equipped with a rotor at different volumes and mixed evenly at 80 rpm/min to obtain different aluminum salt adjuvant vaccines, wherein the mass ratio of the antigen to the adjuvant is shown in Table 9 or Table 10.
  • the vaccine prepared above is aseptically dispensed into 2 ml vials (or pre-filled glass syringes), 0.5 ml (or 1.0 ml) per vial, and stored at 2-8°C away from light after sealing.
  • PBS control group Take out PBS powder (purchased from Solebow, product number: P1003) and dissolve each bag with 2L of sterile distilled water.
  • mice 6-8 week old C57BL/6 mice were randomly divided into groups, with 8 mice in each group.
  • the above-prepared groups were injected intramuscularly into the thigh muscles, and vaccine group, adjuvant-free group and adjuvant group were set up.
  • the secondary immunization (immunization dose and method are shown in the following four) was performed on 0d and 21d (the day of the first immunization was recorded as day 0, and the week from the day of immunization was recorded as week 1 of immunization), with an interval of 21 days between the second immunization.
  • Blood samples were collected at 3 and 5 weeks after immunization, and spleen samples were collected at 5 weeks.
  • mouse serum was collected for ELISA analysis.
  • the specific steps of ELISA analysis were as follows: 200 ng of RSV F protein (Sino Biological, Catalog No.: 11049-V08B) was used for coating per well, mouse serum was used as the primary antibody, and the dilution was performed in a gradient manner according to 250 times, 1250 times, 6250 times, 31250 times, 156250 times, 781250 times, and 3906250 times.
  • the secondary antibody was mouse secondary antibody (Cell Signaling Technology, Catalog No.: 7076S), and the signal was read using an enzyme reader (Shanghai Kehua, Catalog No.: RD-SH-012).
  • the two commonly used aluminum adjuvants can significantly increase the antibody titer, and the combination of aluminum hydroxide adjuvant and RSV-NP produces higher serum ELISA.
  • the vaccine formulation test found that the best antigen content was 1 ⁇ g; the best aluminum hydroxide adjuvant was 50 ⁇ g, and the best CpG adjuvant was 50 ⁇ g.
  • the TCID50 of RSV virus type A Long strain (RSV virus type A Long strain is recorded in the literature: Cultures of HEp-2 cells persistently infected by human respiratory syncytial virus differ in chemokine expression and resistance to apoptosis as compared to lytic infections of the same cell type) cultured in Hep-2 cells in DMEM medium containing 10% bovine serum was 2.81E+07. Group 24 in Table 13 was selected, and the corresponding components were deleted with reference to Table 14 to prepare each experimental group. Eight portions of mouse serum were diluted with 2% bovine serum DMEM medium.
  • the serum was diluted 3-fold to 174960-fold, mixed with an equal volume of 200 TCID50 virus solution, placed at 37°C for 1 hour, and 200ul per well was plated on Hep-2 cell plates. Three replicate wells were set for each mouse serum, and the cells were cultured at 37°C for 5-7 days to observe the cytopathic effect.
  • the main epidemic strains of RSV virus are divided into type A and type B.
  • the RSV virus type A Long strain was replaced with the RSV virus type B strain (recorded in the literature: Genetic Diversity and Molecular Epidemiology of Circulating Respiratory Syncytial Virus in Central Taiwan, 2008-2017), and the same titer analysis procedure was performed using the above-mentioned groups of mouse sera to obtain the data in Table 15.
  • RSV-PreF-A-NP SEQ ID No.12
  • RSV-PreF-B-NP SEQ ID No.13
  • RSV-PreF-C-NP SEQ ID No.14
  • RSV-PreF-E-NP SEQ ID No.16

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Abstract

提供一种RSV疫苗及其制备方法与应用。通过将RSV的Pre-F相关序列及铁蛋白纳米颗粒进行突变设计，并将Pre-F突变蛋白和铁蛋白突变体在真核细胞中进行融合表达，得到具备多个Pre-F在表面集中展示的铁蛋白-PreF融合蛋白纳米颗粒，该铁蛋白-PreF融合蛋白纳米颗粒的氨基酸序列为SEQ ID No.20-27中任一种。通过实验表明：将本发明制备的铁蛋白-PreF融合蛋白注射至小鼠，可以获取到高保护效价的血清，且小鼠血清针对真病毒可产生较高的中和效价，同时通过稳定性实验和安全性实验证明：制备的铁蛋白-PreF融合蛋白还具备足够的物理稳定性和较好的安全性。同时包含编码上述铁蛋白-PreF融合蛋白的mRNA疫苗可赋予个体有效的体液和细胞免疫保护。

Description

一种RSV疫苗及其制备方法与应用
交叉引用
本发明要求在中国的、申请号为2022116541933、申请日为2022年12月22日，在中国的、申请号为2023110836413、申请日为2023年08月25日，以及在中国的、申请号为2023116860806的三个申请为优先权，该申请整体引入到本发明中，并作为本发明的一部分。
技术领域
本发明属于生物技术领域，具体涉及一种RSV疫苗及其制备方法与应用。
背景技术
呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)于1955年首次被发现，属于副黏病毒科(Paramyxoviridae)，肺炎病毒亚科(Pneumovirinae)，肺炎病毒属(Pneumovirus)，依据G蛋白的序列可分为A和B两个亚型。RSV为非节段性的负链RNA病毒，其基因组长度为15.2kb，有10个基因，共编码11种蛋白，包括非结构蛋白(NS1，NS2)、核蛋白(N)、磷蛋白(P)、基质蛋白(M)、RNA依赖RNA聚合酶(L)、转录延伸因子(M2-1)、调控因子(M2-2)和3种包膜糖蛋白(黏附蛋白(G)、融合蛋白(F)和小疏水蛋白(SH))。
RSV是引起呼吸道感染(Respiratory tract infection，RTI)的病毒性病原体，感染主要引起下呼吸道感染症状，其中严重症状的患者占有相当高的比例(如毛细支气管炎和肺炎)，需要住院治疗，具有较高的死亡率。RSV可以通过人与人之间接触传播，或者通过咳嗽或打喷嚏而吸入传播，也可通过接触污染物而获得感染，主要感染鼻腔以及肺部大、小气道的上皮细胞，也可能感染肺泡巨噬细胞和肺部其它类型的细胞，可引起细胞融合在一起形成合胞体。
疫苗研究是目前防治RSV最为集中的领域，早在20世纪60年代，福尔马林灭活RSV疫苗被研制出来进行临床研究，这也是首个进入临床试验的RSV疫苗，但该疫苗不仅没有产生对RSV疾病的保护作用，且在后来的自然感染中，接种过疫苗的儿童发生了严重的疾病增强(ERD)现象，住院率明显增加，甚至引起死亡，因此，该疫苗最终未能进入临床应用。
近些年，随着反向遗传学、疫苗学、分子病毒学、基因组学、免疫学等技术水平的不断提升和发展，RSV疫苗的研究也取得了重大的突破，许多种类的RSV疫苗，如减毒活疫苗、灭活疫苗、嵌合载体疫苗、亚单位疫苗、病毒样颗粒疫苗、复制缺陷型病毒载体疫苗、核酸疫苗等在临床研究试验阶段表现出了临床应用潜力。然而截止目前，世界范围内还没有有效的RSV疫苗上市，其中最主要的问题是RSV疫苗的免疫效价低和生产稳定性低。
发明内容
本发明的目的是提供一种免疫效价高、稳定性高且安全性好的RSV疫苗。
为了实现上述目的，本发明首先提供了一种蛋白。
本发明提供的蛋白包括将Pre-F蛋白氨基酸序列第67、88、110、144、159、173、202、227、236、248、289、309、334、344、370、389、419和/或468位中一个或者多个位点的突变。
优选的，本发明提供的蛋白为将Pre-F蛋白氨基酸序列进行如下a1)-a18)中至少一种突变后得到的蛋白：
a1)将Pre-F蛋白氨基酸序列第67位的异亮氨酸突变为天冬酰胺，I67N；
a2)将Pre-F蛋白氨基酸序列第88位的丝氨酸突变为天冬酰胺，S88N；
a3)将Pre-F蛋白氨基酸序列第110位的半胱氨酸突变为丙氨酸，C110A；
a4)将Pre-F蛋白氨基酸序列第144位的天冬酰胺突变为甘氨酸，且在第144位和第145位氨基酸之间插入半胱氨酸，N144C；
a5)将Pre-F蛋白氨基酸序列第159位的酪氨酸突变为半胱氨酸，Y159C；
a6)将Pre-F蛋白氨基酸序列第173位的半胱氨酸缺失，ΔC173；
a7)将Pre-F蛋白氨基酸序列第202位的丙氨酸突变为半胱氨酸，A202C；
a8)将Pre-F蛋白氨基酸序列第227位的异亮氨酸突变为天冬酰胺，I227N；
a9)将Pre-F蛋白氨基酸序列第236位的丝氨酸突变为精氨酸，S236R；
a10)将Pre-F蛋白氨基酸序列第248位的丝氨酸突变为半胱氨酸，S248C；
a11)将Pre-F蛋白氨基酸序列第289位的谷氨酸突变为天冬酰胺，E289N；
a12)将Pre-F蛋白氨基酸序列第309位的丝氨酸突变为天冬酰胺，S309N；
a13)将Pre-F蛋白氨基酸序列第334位的精氨酸突变为酪氨酸，R334Y；
a14)将Pre-F蛋白氨基酸序列第344位的天冬酰胺突变为谷氨酸，N344E；
a15)将Pre-F蛋白氨基酸序列第370位的丝氨酸突变为甘氨酸，S370G；
a16)将Pre-F蛋白氨基酸序列第389位的天冬酰胺突变为半胱氨酸，N389C；
a17)将Pre-F蛋白氨基酸序列第419位的半胱氨酸突变为酪氨酸，C419Y；
a18)将Pre-F蛋白氨基酸序列第468位的精氨酸突变为天冬酰胺，R468N；
更优选的，所述pre-F蛋白的突变包括在
1第88位的突变，
2第88和289位的突变，
3第88和389位的突变，
4第88、289、309和468位的突变，
5第67、88、144和389位的突变，或者，
6第67、88、110、144、289、309、389和468位的突变。
进一步优选的，所述突变包括：
1)S88N，
2)S88N和E289N，
3)S88N和N389C，
4)S88N、E289N、S309N和R468N，
5)I67N、S88N、N144C和N389C，
6)I67N、S88N、C110A、N144C、E289N、S309N、N389C和R468N，
7)I67N、S88N、143与144位之间插入甘氨酸、N144C、N389C。
所述Pre-F蛋白为如下任一种：
(A1)SEQ ID No.1所示的蛋白；
(A2)在(A1)所述蛋白的N端或/和C端连接标签得到的融合蛋白；
(A3)将(A1)-(A2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白；
(A4)与(A1)-(A2)任一种具有98％以上同一性且具有相同功能的蛋白。
进一步的，所述蛋白(或称之为Pre-F蛋白的突变蛋白)为如下任一种：
(M1)SEQ ID No.2或SEQ ID No.3或SEQ ID No.4或SEQ ID No.5或SEQ ID No.6或SEQ ID No.7或SEQ ID No.8或SEQ ID No.9所示的蛋白；
(M2)在(M1)所述蛋白的N端或/和C端连接标签得到的融合蛋白；
(M3)将(M1)-(M2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白；
(M4)与(M1)-(M2)任一种具有98％以上同一性且具有相同功能的蛋白。
为了实现上述目的，本发明又提供了一种融合蛋白。
本发明提供的融合蛋白包括上述蛋白和铁蛋白突变体；
所述铁蛋白突变体为将铁蛋白氨基酸序列进行如下b1)-b3)中至少一种突变后得到的蛋白：
b1)将铁蛋白氨基酸序列第15位的天冬酰胺突变为谷氨酰胺；，D15Q
b2)将铁蛋白氨基酸序列第96位的丝氨酸突变为天冬酰胺，S96D；
b3)将铁蛋白氨基酸序列第119位的酪氨酸突变为精氨酸，Y119R；
所述铁蛋白为如下任一种：
(B1)SEQ ID No.10所示的蛋白；
(B2)在(B1)所述蛋白的N端或/和C端连接标签得到的融合蛋白；
(B3)将(B1)-(B2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相 同功能的蛋白；
(B4)与(B1)-(B2)任一种具有98％以上同一性且具有相同功能的蛋白。
进一步的，所述铁蛋白突变体为如下任一种：
(N1)SEQ ID No.11所示的蛋白；
(N2)在(N1)所述蛋白的N端或/和C端连接标签得到的融合蛋白；
(N3)将(N1)-(N2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白；
(N4)与(N1)-(N2)任一种具有98％以上同一性且具有相同功能的蛋白。
更进一步的，所述融合蛋白为如下任一种：
(C1)SEQ ID No.12或SEQ ID No.13或SEQ ID No.14或SEQ ID No.15或SEQ ID No.16或SEQ ID No.17或SEQ ID No.18或SEQ ID No.19所示的蛋白；
(C2)在(C1)所述蛋白的N端或/和C端连接标签得到的融合蛋白；
(C3)将(C1)-(C2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白；
(C4)与(C1)-(C2)任一种具有98％以上同一性且具有相同功能的蛋白。
上述(A2)或(M2)或(B2)或(N2)或(C2)所述的融合蛋白中，所述标签是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
上述(A3)或(M3)或(B3)或(N3)或(C3)所述的融合蛋白中，所述一个或几个氨基酸残基的取代和/或缺失和/或添加为在上述a1)-a18)或b1)-b3)所述氨基酸突变位点以外进行不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述任一所述蛋白或融合蛋白可人工合成，也可先合成其编码基因，再进行生物表达得到。
本发明还提供了一种生物材料。
本发明提供的生物材料为下述D1)-D5)中至少一种：
D1)编码上述蛋白或融合蛋白的核酸分子；
D2)含有D1)所述核酸分子的表达盒；
D3)含有D1)所述核酸分子的重组载体、或含有D2)所述表达盒的重组载体；
D4)含有D1)所述核酸分子的重组微生物、含有D2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
D5)含有D1)所述核酸分子的重组细胞系、含有D2)所述表达盒的重组细胞系、或含有D3)所述重组载体的重组细胞系。
上述生物材料中，编码所述蛋白的核酸分子为E1)或E2)：
E1)或SEQ ID No.21第1-1422位或SEQ ID No.22第1-1422位或SEQ ID No.23第1-1422位或SEQ IDSEQ ID No.20第1-1422位No.24第1-1422位或SEQ ID No.25第1-1422位或SEQ ID No.26第1-1422位或SEQ ID No.27第1-1422位所示的DNA分子；
E2)与E1)限定的DNA分子具有75％以上的同一性且编码所述融合蛋白的DNA分子。
编码所述融合蛋白的核酸分子为F1)或F2)：
F1)SEQ ID No.20或SEQ ID No.21或SEQ ID No.22或SEQ ID No.23或SEQ ID No.24或SEQ ID No.25或SEQ ID No.26或SEQ ID No.27所示的DNA分子；
F2)与F1)限定的DNA分子具有75％以上的同一性且编码所述融合蛋白的DNA分子。
其中，所述核酸分子可以是DNA，如重组DNA；所述核酸分子也可以是RNA，如mRNA。
优选的，所述mRNA包括
G1)SEQ ID No.20-27任一序列的第1-1422位或者全长序列中将T替换为U所获得的mRNA序列；
G2)G1)的简并或者互补序列；
G3)与G1)或G2)限定的mRNA分子具有75％以上的同一性且编码具有相同功能的融合蛋白的mRNA分子。
优选的，所述mRNA包括上述编码区外，还包括5’帽子结构，5’非编码区，3’非编码区和/或多聚腺苷酸尾的mRNA。
优选的，所述mRNA序列是天然或修饰的RNA，所述修饰的RNA包括通过用修饰的尿苷部分或全部取代天然尿苷对RNA进行修饰。
例如，所述修饰的RNA为通过用1-甲基-假尿苷对天然尿苷进行全部替换。
本发明另一方面，提供一种mRNA，所述mRNA包含第一开放阅读框，所述第一开放阅读框包含编码单体亚基蛋白和来自RSV的至少一个免疫原性部分的核酸：所述单体亚基蛋白选自下组的单体铁蛋白亚基，单体encapsulin蛋白，单体03-33蛋白，单体硫加氧酶还原酶蛋白，单体二氧四氢蝶啶合酶蛋白，和/或单体丙酮酸脱氢酶复合物二氢硫辛酰胺乙酰转移酶蛋白，所述第一开放阅读框表达的单体亚基蛋白自组装为纳米颗粒，且在所述纳米颗粒的表面上展示所述至少一个免疫原性部分。
本发明另一方面，提供一种mRNA，所述mRNA至少包含两个开放阅读框，其中，第一开放阅读框包括编码选自下组的单体亚基蛋白的核酸：单体铁蛋白亚基，单体encapsulin蛋白，单体03-33蛋白，单体硫加氧酶还原酶蛋白，单体二氧四氢蝶啶合酶蛋白，和/或单体丙酮酸脱氢酶复合物二氢硫辛酰胺乙酰转移酶蛋白，第二开放阅读框包括编码来自RSV的至少一个免疫原性部分的核酸，所述第一开放阅读框表达的单体亚基蛋白能自组装为纳米颗粒，且第二开放阅读框表达的所述至少一个免疫原性部分能够与第一开放阅读框表达的所述纳米颗粒结合。
优选的，所述至少一个免疫原性部分选自RSV的pre-F蛋白的突变蛋白，所述pre-F蛋白的突变蛋白如上所定义。
进一步优选的，所述mRNA编码的蛋白自N端至C端依次由Pre-F突变蛋白、linker和铁蛋白突变体组成的融合蛋白。
优选的，所述mRNA编码的融合蛋白还可以包括标签，所述标签是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
进一步优选的，所述mRNA编码的蛋白自N端至C端依次由Pre-F突变蛋白、linker和铁蛋白突变体组成的融合蛋白。
优选的，所述mRNA编码的融合蛋白还可以包括标签，所述标签是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
优选的，所述mRNA包括如下任一种：
(H1)、SEQ ID No.30-33所示的mRNA的部分或者全部；
(H2)、(H1)的简并或者互补序列；
(H3)、与(H1)或(H2)具有80％以上同一性且具有编码相同功能的蛋白的mRNA。
其中，SEQ ID No.30-33中对应的编码区如表8所示。
表8：SEQ ID No.30-33中对应的编码区
更优选的，所述第一开放阅读框包含SEQ ID No.30-33所示的mRNA的部分，所述SEQ ID No.4-7所示的mRNA的部分至少包括表8所示的Pre-F突变蛋白的编码区和/或铁蛋白突变体的编码区。或者，
更优选的，所述第一开放阅读框包括SEQ ID No.30-33中Pre-F突变蛋白编码区的mRNA或者上述(C2)和(C3)所限定的序列，所述第二开放阅读框包括SEQ ID No.30-33中铁蛋白突变体编码区的mRNA或者上述(C2)和(C3)所限定的序列。
进一步优选的，所述第一开放阅读框和/或第二开放阅读框包括编码标签蛋白的mRNA。
所述标签可以将第一开放阅读框和/或第二开放阅读框表达的蛋白结合。所述免疫原性部分与所述单体亚基蛋白之间的结合后形成纳米颗粒，以促进抗原在纳米颗粒上的多价显示。
所述标签优选为基序标签。
更优选的，所述基序标签蛋白为上述定义SpyTag和/或SpyCatcher。
优选的，所述单体亚基蛋白包括单体encapsulin蛋白，单体03-33蛋白，单体硫加氧酶还原酶蛋白，单体二氧四氢蝶啶合酶蛋白，和/或单体丙酮酸脱氢酶复合物二氢硫辛酰胺乙酰转移酶蛋白。
优选的，所述mRNA包括上述编码区外，还包括5’帽子结构，5’非编码区，3’非编码区和/或多聚腺苷酸尾的mRNA。
优选的，所述mRNA序列是天然或修饰的RNA，所述修饰的RNA包括通过用修饰的尿苷部分或全部取代天然尿苷对RNA进行修饰。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码上述蛋白或融合蛋白的核苷酸序列进行突变。那些经过人工修饰的，具有编码上述蛋白或融合蛋白的核苷酸序列75％或者更高同一性的核苷酸，只要编码上述蛋白或融合蛋白且具有相同功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
所述同一性是指与天然氨基酸或者核酸序列的序列相似性。“同一性”包括与本发明的编码SEQ ID No.2或SEQ ID No.3或SEQ ID No.4或SEQ ID No.5或SEQ ID No.6或SEQ ID No.7或SEQ ID No.8或SEQ ID No.9或SEQ ID No.12或SEQ ID No.13或SEQ ID No.14或SEQ ID No.15或SEQ ID No.16或SEQ ID No.17或SEQ ID No.18或SEQ ID No.19所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，具有80％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
上述生物材料中，所述表达盒是指能够在宿主细胞中表达上述蛋白或融合蛋白的DNA，该DNA不但可包括启动上述蛋白或融合蛋白编码基因序列转录的启动子，还可包括终止上述蛋白或融合蛋白编码基因序列转录的终止子。进一步，所述表达盒还可包括增强子序列。
上述生物材料中，所述载体可为质粒、黏粒、噬菌体或病毒载体。
上述生物材料中，所述微生物可为酵母、细菌、藻或真菌。
上述生物材料中，所述细胞可为原核生物细胞或真核生物细胞。
为了实现上述目的，本发明还提供了上述蛋白或融合蛋白的制备方法。
本发明提供的上述蛋白或融合蛋白的制备方法包括如下步骤：使编码上述蛋白或融合蛋白的核酸分子在生物或生物细胞中进行表达，得到所述融合蛋白。
进一步的，所述方法包括如下步骤：将编码所述蛋白或融合蛋白的核酸分子导入CHO K1Q细胞，得到重组细胞；培养所述重组细胞，得到所述蛋白或融合蛋白。
更进一步的，所述蛋白或融合蛋白的核酸分子通过重组质粒导入CHO K1Q细胞。
所述重组质粒为将所述蛋白或融合蛋白的核酸分子插入载体质粒后得到的质粒。
在本发明的具体实施例中，所述载体质粒为pKS001载体质粒。所述重组质粒为重组质粒pKS001-RSV-PreF-A-NP、pKS001-RSV-PreF-B-NP、pKS001-RSV-PreF-C-NP、pKS001-RSV-PreF-D-NP、pKS001-RSV-PreF-E-NP、pKS001-RSV-PreF-F-NP、pKS001-RSV-PreF-G-NP或pKS001-RSV-PreF-H-NP。
本发明还提供上述生物材料的制备方法，所述制备方法包括根据编码的融合蛋白设计DNA序列，构建包含所述DNA的载体。
优选的，所述制备方法还包括将载体转录表达mRNA。
更优选的，本发明提供一种上述mRNA的制备方法，所述制备方法包括，根据mRNA编码的单体亚基蛋白和来自RSV的至少一个免疫原性部分设计DNA序列，构建包含所述DNA的载体，将载体转录表达mRNA。
优选的，所述来自RSV的至少一个免疫原性部分包括pre-F蛋白的突变蛋白，所述单体亚基蛋白包括铁蛋白突变体，所述pre-F蛋白的突变蛋白与铁蛋白突变体融合表达或自组装。
优选的，所述pre-F蛋白的突变蛋白与铁蛋白突变体融合表达或自组装。
更优选的，所述融合表达是通过连接肽进行连接或所述自组装是通过标签实现自组装。
进一步优选的，所述标签为Spy和Catcher，在一个具体的实施方式分别中Spy和Catcher的序列如SEQ ID No.28和29所示。
本发明还提供了上述蛋白或上述融合蛋白或上述生物材料或按照上述方法制备得到的蛋白或融合蛋 白的新用途。
本发明提供了上述蛋白或上述融合蛋白或上述生物材料或按照上述方法制备得到的蛋白或融合蛋白在如下Y1)-Y4)任一种中的应用；
Y1)作为免疫原；
Y2)制备抗呼吸道合胞病毒的产品；
Y3)制备预防和/或治疗呼吸道合胞病毒感染的产品；
Y4)制备预防和/或治疗呼吸道合胞病毒所致疾病的产品。
为了实现上述目的，本发明还提供了一种疫苗。
本发明提供的疫苗的活性成分为上述蛋白或上述融合蛋白或上述生物材料或按照上述方法制备得到的蛋白或融合蛋白。
优选的，所述生物材料包括mRNA。
优选的，所述疫苗为融合蛋白疫苗。
优选的，所述疫苗为mRNA疫苗。
进一步的，所述疫苗还包括佐剂。
再进一步的，所述佐剂可为铝佐剂。所述铝佐剂可为氢氧化铝、磷酸铝中的一种或多种。
更进一步的，所述蛋白与所述铝佐剂的质量比可为1：(0.5-300)，可以为上述比例的任意值或者范围，例如1：(1-275)、1：(10-200)、1：(30-180)、1：(40-100)、1：(10-80)、1：(25-75)、1：(50-75)、1：50等等。
优选的，所述疫苗包括双佐剂，所述双佐剂包括上述定义的铝佐剂和CpG佐剂。
在一些实施方案中，所述CpG佐剂包括任一的CpG佐剂，例如CpG1018、CpG-cjx等等。
在一些实施方案中，所述蛋白与所述CpG佐剂的质量比可为1：(0.5-300)，可以为上述比例的任意值或者范围，例如1：(1-275)、1：(10-200)、1：(30-180)、1：(40-100)、1：(10-80)、1：(25-75)、1：(50-75)、1：50等等。
在一些实施方案中，所述双佐剂中铝佐剂与CpG佐剂的质量比为(1-5)：(5-1)，可以为上述比例的任意值或者范围，例如1：2、1：1、2：1等等。
在一些实施方案中，所述蛋白：铝佐剂：CpG佐剂的质量比为1:50:50。
更进一步的，所述mRNA疫苗上述任一定义的mRNA和脂质纳米颗粒，所述mRNA包封于所述脂质纳米颗粒中。
本发明术语“脂质纳米颗粒”(Lipid nanoparticles，LNP)是指具有至少一个纳米量级尺寸的颗粒，其包含至少一种脂质。
更优选的，所述脂质纳米颗粒包括阳离子脂质、中性磷脂、甾族脂质和聚乙二醇-脂质。
本发明术语“中性磷脂”术语是指不带电荷的、非磷酸甘油酯的磷脂分子。
本发明术语“聚乙二醇-脂质”是指包含脂质部分和聚乙二醇(PEG)部分的分子。
优选的，阳离子脂质化合物结构：DLin-MC3-DMA，ALC-0315，或SM-102。
优选的，所述的中性磷脂选自：1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)中的一种或多种组合，优选的为DSPC。
优选的，所述的甾族脂质选自燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇；羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合，优选的为胆固醇。
优选的，所述的聚乙二醇(PEG)-脂质选自：2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、 PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)或DMG-PEG2000中的一种或多种组合，优选的为DMG-PEG2000。
优选的，所述脂质纳米颗粒中，阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质按摩尔比为(40-50):(5-15):(35-45):(1-5)，更优选的，所述比例为45:10:43:2。
优选的，所述mRNA溶液：脂质纳米颗粒的质量比为(2-8):1。更优选为3:1。
更优选的，所述疫苗为液体制剂或冻干粉剂。更优选的，所述疫苗为口服制剂、肌肉注射制剂、静脉注射制剂或吸入制剂。进一步优选的，所述疫苗为雾化吸入剂或干粉吸入剂。
本发明所述的疫苗还可以包含药学上可接受的赋形剂。所述药学上可接受的赋形剂可以是载体、稀释剂、佐剂或编码佐剂核苷酸序列、增溶剂、粘合剂、润滑剂、助悬剂、转染促进剂等。
所述转染促进剂包括但不限于表面活性剂如免疫刺激复合物、费氏(Freunds)不完全佐剂、LPS类似物(例如单磷酰酯A)、胞壁肽、苯醌类似物、角鲨烯、透明质酸、脂质、脂质、钙离子、病毒蛋白质、阳离子、聚阳离子(例如聚-L-谷氨酸(LGS))或纳米粒子或其他已知的转染促进剂。
所述的编码佐剂的核苷酸序列为编码如下至少一种佐剂的核苷酸序列：GM-CSF、IL-17、IFNg、IL-15、IL-21、抗PD1/2、乳铁蛋白、鱼精蛋白、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12、INF-α、INF-γ、Lymphotoxin-α、hGH、MCP-1、MIP-1a、MIP-1p、IL-8、RANTES、L-选择蛋白、P-选择蛋白、E-选择蛋白、CD34、GlyCAM-1、MadCAM-1、LFA-1、VLA-1、Mac-1、pl50.95、PECAM、ICAM-1、ICAM-2、ICAM-3、CD2、LFA-3、M-CSF、、CD40、CD40L、血管生长因子、成纤维细胞生长因子、神经生长因子、血管内皮生长因子、Apo-1、p55、WSL-1、DR3、TRAMP、Apo-3、AIR、LARD、NGRF、DR4、DR5、KILLER、TRAIL-R2、TRICK2、DR6、半胱天冬酶ICE、Fos、c-jun、Sp-1、Ap-1、Ap-2、p38、p65Rel、MyD88、IRAK、TRAF6、IkB、无活性的NIK、SAP K、SAP-1、JNK、NFkB、Bax、TRAIL、TRAILrec、TRAILrecDRC5、TRAIL-R3、TRAIL-R4、RANK、RANK LIGAND、Ox40、Ox40LIGAND、NKG2D、MICA、MICB、NKG2A、NKG2B、NKG2C、NKG2E、NKG2F、TAP1、TAP2以及其功能性片段。
上述疫苗在如下Y1)-Y3)任一种中的应用也属于本发明的保护范围；
Y1)制备抗呼吸道合胞病毒的产品；
Y2)制备预防和/或治疗呼吸道合胞病毒感染的产品；
Y3)制备预防和/或治疗呼吸道合胞病毒所致疾病的产品。
上述任一所述应用或产品或方法中，所述产品可为疫苗。
本发明另一方面，提供一种药物组合物，所述药物组合物包括上述定义的融合蛋白、生物材料或mRNA疫苗。
优选的，所述药物组合物用于预防呼吸道合胞病毒感染性疾病。
本发明另一方面，提供一种上述药物组合物的制备方法，所述制备方法包括制备上述融合蛋白、生物材料或mRNA的步骤。
优选的，所述制备方法包括将脂质纳米颗粒溶解至溶剂后与mRNA混合包封后制得mRNA药物组合物。
所述脂质纳米颗粒如上所定义。
本发明另一方面，还提供了一种产生免疫应答的方法，所述方法可包括给受试者施用上述任一的药物组合物。
本发明另一方面，还提供一种预防和/或治疗RSV感染性疾病的方法，所述方法可包括给受试者施用所述药物组合物。
上述方法中，所述RSV感染性疾病可包括呼吸系统、消化系统、心血管系统和/或神经系统感染。
上述方法中，所述呼吸系统感染可包括呼吸道感染和/或肺部感染。
上述方法中，所述呼吸道感染可包括严重急性呼吸道综合征、低氧性呼吸衰竭、脓毒症、脓毒性休克、鼻咽炎、鼻炎、咽喉炎、气管炎和/或支气管炎，所述肺部感染可包括肺炎和/或肺损伤。
上述方法中，所述消化系统感染可包括肠道疾病、纳差、恶心、呕吐、腹痛和/或腹泻。
上述方法中，给受试者施用所述药物组合物后，可在受试者中引发针对RSV的免疫反应。所述免疫反应可为细胞免疫反应，或体液免疫反应，或细胞免疫反应和体液免疫反应。
所述细胞免疫反应可包括B细胞免疫反应和T细胞免疫反应。
本文所述受试者可为人类或非人类动物。
进一步地，所述非人类动物可为非人类哺乳动物。
所述非人类哺乳动物可为小鼠、大鼠、豚鼠、仓鼠、猪，犬，羊，猴，兔、猫、牛、马中的任意一种但不限于此。
本文所述受试者包括但不限于健康受试者、有症状感染受试者、无症状感染受试者或康复受试者(感染后恢复的受试者)。
本文所述施用包括但不限于肌肉注射、皮下注射、皮内注射、静脉注射、动脉注射、腹腔注射、微针注射、粘膜给药、口服、口鼻腔喷入或雾化吸入。
本发明的有益效果如下：
1、本发明通过特异性的抗原突变设计增强了Pre-F蛋白的有效免疫原性、稳定性和安全性，并通过在纳米颗粒表面的展示将所需要的表位外露，进一步增强了免疫原性。通过实验证明：本发明制备的疫苗可以在低剂量时获得较好的免疫效果，其中，RSV-PreF-C-NP组中和效价可达到19836。
2、本发明解决了野生抗原稳定性差的问题，本发明制备的铁蛋白-PreF融合蛋白在进入生物体内之后能够诱导出具备中和活性的呼吸道合胞病毒抗体，从而赋予该机体相应的免疫保护。
3、本发明制备的铁蛋白-PreF融合蛋白能够有效激发机体的细胞免疫机制，并且引起的免疫反应是Th1/Th2平衡的，可以避免Th2偏激所导致的免疫过激反应，具备较好的安全性。
4、本发明的mRNA疫苗进入生物体内之后能够诱导出具备中和活性的呼吸道合胞病毒抗体，从而赋予该机体相应的体液免疫保护。同时，mRNA疫苗能够有效激发机体的细胞免疫机制，提高脾细胞分泌IFN-γ的频率，有效诱导细胞免疫，从而带来更长效和全面的保护。
5、本发明通过对佐剂的筛选，获得适合的佐剂组合以及有效的浓度范围，双佐剂之间具有协同作用，可以显著性提高RSV疫苗的免疫效果。
本发明通过将RSV的Pre-F相关序列及铁蛋白纳米颗粒进行突变设计，并将Pre-F突变蛋白和铁蛋白突变体颗粒在真核细胞中进行融合表达，得到具备多个Pre-F在表面集中展示的铁蛋白-PreF融合蛋白纳米颗粒，该铁蛋白-PreF融合蛋白纳米颗粒通过稳定和暴露所需要展现的抗原表位，破坏或隐藏不需要的抗原表位，有效的提高了抗原的免疫原性、生产稳定性和安全性。通过实验表明：将本发明制备的铁蛋白-PreF融合蛋白注射至小鼠，可以获取到高保护效价的血清，且小鼠血清针对真病毒可产生较高的中和效价，同时通过稳定性实验和安全性实验证明：本发明制备的铁蛋白-PreF融合蛋白还具备足够的物理稳定性和较好的安全性。获得的mRNA疫苗能够有效激发机体的细胞免疫机制，提高脾细胞分泌IFN-γ的频率，有效诱导细胞免疫，从而带来更长效和全面的保护。
附图说明
图1为pKS001载体的结构示意图。
图2为铁蛋白-PreF融合蛋白的SDS-PAGE电泳检测结果。A：泳道1是RSV-PreF-A-NP纯化后的洗脱产物，泳道2、泳道3分别是流穿和上清，泳道4是分子量标记物(Solarbio，货号：PR1910，下同)。B：泳道1是RSV-PreF-B-NP纯化后的洗脱产物，泳道2、泳道3分别是流穿和分子量标记物。C：泳道1是RSV-PreF-C-NP纯化后的流穿，泳道2是RSV-PreF-C-NP纯化后的洗脱产物，泳道3、泳道4分别是RSV-PreF-D-NP、RSV-PreF-E-NP纯化后的洗脱产物。D：泳道1是RSV-PreF-F-NP纯化后的洗脱产物，泳道2是分子量标记物。E：泳道1、泳道2分别是RSV-PreF-G-NP纯化后的流穿、洗脱产物。F：泳道1是分子量标记物，泳道2是RSV-PreF-H-NP纯化后的流穿，泳道3是间隔空白泳道，泳道4是RSV-PreF-H-NP纯化后的洗脱产物。
图3为纯化后铁蛋白-PreF融合蛋白的WB检测结果。从左往右依次为RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP、RSV-PreF-E-NP、RSV-PreF-F-NP、RSV-PreF-G-NP、RSV-PreF-H-NP纯化产物的WB检测结果。
图4为铁蛋白-PreF融合蛋白的纳米颗粒形态图。A-H分别是RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP、RSV-PreF-E-NP、RSV-PreF-F-NP、RSV-PreF-G-NP、RSV-PreF-H-NP纯化产物对应的电镜照片。
图5为铁蛋白-PreF融合蛋白疫苗的免疫原性研究结果。
图6为铁蛋白-PreF融合蛋白免疫小鼠后的中和效价值的Log2。
图7为铁蛋白-PreF融合蛋白RSV-PreF-C-NP免疫后的小鼠血清中IgG1和IgG2a的ELISA效价及历史 福尔马林灭活疫苗的对照效价。
图8为不同mRNA疫苗免疫刺激后脾细胞分泌IFN-γ的频率。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到或者可通过已知方法制备得到。
下述实施例中的RSV病毒A型Long株记载于文献“Cultures of HEp-2 cells persistently infected by human respiratory syncytial virus differ in chemokine expression and resistance to apoptosis as compared to lytic infections of the same cell type”中。
下述实施例中的RSV病毒B型BA9株记载于文献“Genetic Diversity and Molecular Epidemiology of Circulating Respiratory Syncytial Virus in Central Taiwan,2008-2017”中。
下述实施例中的强生公司开发的同类疫苗记载于文献“preF mmunogenicity and protective efficacy of adenoviral and subunit RSVvaccines based on stabilized prefusion F protein in preclinical models”中。
下述实施例中的福尔马林灭活疫苗FI-RSV记载于文献“Enhanced pulmonary histopathology induced by respiratory syncytial virus(RSV)challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin-4(IL-4)and IL-10”中。
实施例1、铁蛋白-PreF融合蛋白的设计、制备与纯化
将RSV的Pre-F相关序列进行突变与设计，得到Pre-F突变蛋白，并使其与铁蛋白相关序列进行融合，形成铁蛋白-PreF一体的亚单位，然后利用铁蛋白的自组装性能，成为具备良好Pre-F抗原展示效果的纳米颗粒。具体步骤如下：
一、铁蛋白-PreF融合蛋白的设计
1、RSV的Pre-F相关序列的设计
为了展示和稳定需要的表位，破坏或掩盖不需要的表位，将RSV的Pre-F蛋白氨基酸序列(Pre-F蛋白氨基酸序列如SEQ ID No.1所示)进行如下1)-18)中至少一种突变，得到Pre-F突变蛋白：
1)将Pre-F蛋白氨基酸序列第67位的异亮氨酸(I)突变为天冬酰胺(N)。
2)将Pre-F蛋白氨基酸序列第88位的丝氨酸(S)突变为天冬酰胺(N)。
3)将Pre-F蛋白氨基酸序列第110位的半胱氨酸(C)突变为丙氨酸(A)。
4)将Pre-F蛋白氨基酸序列第144位的天冬酰胺(N)突变为甘氨酸(G)，且在第144位和第145位氨基酸之间插入半胱氨酸(C)。
5)将Pre-F蛋白氨基酸序列第159位的酪氨酸(Y)突变为半胱氨酸(C)。
6)将Pre-F蛋白氨基酸序列第173位的半胱氨酸(C)缺失。
7)将Pre-F蛋白氨基酸序列第202位的丙氨酸(A)突变为半胱氨酸(C)。
8)将Pre-F蛋白氨基酸序列第227位的异亮氨酸(I)突变为天冬酰胺(N)。
9)将Pre-F蛋白氨基酸序列第236位的丝氨酸(S)突变为精氨酸(R)。
10)将Pre-F蛋白氨基酸序列第248位的丝氨酸(S)突变为半胱氨酸(C)。
11)将Pre-F蛋白氨基酸序列第289位的谷氨酸(E)突变为天冬酰胺(N)。
12)将Pre-F蛋白氨基酸序列第309位的丝氨酸(S)突变为天冬酰胺(N)。
13)将Pre-F蛋白氨基酸序列第334位的精氨酸(R)突变为酪氨酸(Y)。
14)将Pre-F蛋白氨基酸序列第344位的天冬酰胺(N)突变为谷氨酸(E)。
15)将Pre-F蛋白氨基酸序列第370位的丝氨酸(S)突变为甘氨酸(G)。
16)将Pre-F蛋白氨基酸序列第389位的天冬酰胺(N)突变为半胱氨酸(C)。
17)将Pre-F蛋白氨基酸序列第419位的半胱氨酸(C)突变为酪氨酸(Y)。
18)将Pre-F蛋白氨基酸序列第468位的精氨酸(R)突变为天冬酰胺(N)。
在本发明中，所述Pre-F突变蛋白分别为RSV-PreF-A、RSV-PreF-B、RSV-PreF-C、RSV-PreF-D、 RSV-PreF-E、RSV-PreF-F、RSV-PreF-G和RSV-PreF-H。
Pre-F突变蛋白RSV-PreF-A、RSV-PreF-B、RSV-PreF-C、RSV-PreF-D、RSV-PreF-E、RSV-PreF-F、RSV-PreF-G和RSV-PreF-H的氨基酸序列分别如SEQ ID No.2-SEQ ID No.9所示。
2、纳米颗粒序列的设计
为了提升颗粒的稳定性和完整度，将铁蛋白氨基酸序列(铁蛋白氨基酸序列如SEQ ID No.10所示)进行如下1)-3)中至少一种突变，得到铁蛋白突变体：
1)将铁蛋白氨基酸序列第15位的天冬酰胺(N)突变为谷氨酰胺(Q)。
2)将铁蛋白氨基酸序列第96位的丝氨酸(S)突变为天冬酰胺(N)。
3)将铁蛋白氨基酸序列第119位的酪氨酸(Y)突变为精氨酸(R)。
在本发明中，铁蛋白突变体的氨基酸序列如SEQ ID No.11所示。
二、铁蛋白-PreF融合蛋白的制备
1、重组质粒的构建
1)铁蛋白-PreF的基因融合设计
将Pre-F突变蛋白和铁蛋白突变体通过linker(SGSGGGSG)进行融合，制备铁蛋白-PreF融合蛋白，该铁蛋白-PreF融合蛋白自N端至C端依次由Pre-F突变蛋白、linker(SGSGGGSG)和铁蛋白突变体组成。
铁蛋白-PreF融合蛋白分别为RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP、RSV-PreF-E-NP、RSV-PreF-F-NP、RSV-PreF-G-NP和RSV-PreF-H-NP，其氨基酸序列分别如SEQ ID No.12-SEQ ID No.19所示，编码基因序列分别如SEQ ID No.20-SEQ ID No.27所示。
委托南京金斯瑞生物科技有限公司分别合成含有上述各铁蛋白-PreF融合蛋白编码基因序列的质粒，并将其分别命名为质粒RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP、RSV-PreF-E-NP、RSV-PreF-F-NP、RSV-PreF-G-NP和RSV-PreF-H-NP。
2)重组质粒的构建
用限制性内切酶Hind III-HF和Not I-HF(NEB，货号分别为R3104V和R3189L)对pKS001载体质粒(中山康天晟合生物技术有限公司，货号为A14101)进行双酶切，得到骨架载体。pKS001载体质粒结构示意图如图1所示。
用限制性内切酶Hind III-HF和Not I-HF分别对质粒RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP、RSV-PreF-E-NP、RSV-PreF-F-NP、RSV-PreF-G-NP和RSV-PreF-H-NP进行双酶切，分别得到目的片段。
用Quick连接酶(NEB，货号为M2200L)分别将骨架载体与各个目的片段连接后转化大肠杆菌感受态Trans10(北京全式金生物技术股份有限公司，货号为CD101)，筛选阳性克隆并提取质粒进行测序验证，将测序验证正确的质粒分别命名为重组质粒pKS001-RSV-PreF-A-NP、pKS001-RSV-PreF-B-NP、pKS001-RSV-PreF-C-NP、pKS001-RSV-PreF-D-NP、pKS001-RSV-PreF-E-NP、pKS001-RSV-PreF-F-NP、pKS001-RSV-PreF-G-NP和pKS001-RSV-PreF-H-NP。
测序结果表明：重组质粒pKS001-RSV-PreF-A-NP、pKS001-RSV-PreF-B-NP、pKS001-RSV-PreF-C-NP、pKS001-RSV-PreF-D-NP、pKS001-RSV-PreF-E-NP、pKS001-RSV-PreF-F-NP、pKS001-RSV-PreF-G-NP和pKS001-RSV-PreF-H-NP分别为将pKS001载体的Hind III-HF和Not I-HF酶切位点间的DNA片段替换为SEQ ID No.20、SEQ ID No.21、SEQ ID No.22、SEQ ID No.23、SEQ ID No.24、SEQ ID No.25、SEQ ID No.26和SEQ ID No.27所示的DNA分子，且保持pKS001载体其他序列不变后得到的质粒。
2、铁蛋白-PreF融合蛋白的表达
将上述重组质粒pKS001-RSV-PreF-A-NP、pKS001-RSV-PreF-B-NP、pKS001-RSV-PreF-C-NP、pKS001-RSV-PreF-D-NP、pKS001-RSV-PreF-E-NP、pKS001-RSV-PreF-F-NP、pKS001-RSV-PreF-G-NP和pKS001-RSV-PreF-H-NP分别在CHO K1Q细胞(康晟生物医药有限公司，货号为A14101)中进行电转和表达，并筛选高表达的细胞株。
使用苏州壹达生物科技有限公司的电转仪EBXP-F1进行电转，具体电转步骤如下：
1)电转前准备：电转前30min将Buffer、细胞培养液、D-PBS提前取出恢复至室温。
2)收集细胞、计数：细胞混悬均匀后置于离心管，进行计数。
3)离心：取所需培养液细胞置于一新的离心管内，在离心机(苏州市国飞实验室仪器有限公司，货号：TDL-5A)中1000rpm离心5min。
4)DPBS清洗：弃去上清培养液，获得所需细胞，加入1mL D-PBS(赛默飞Gibco，货号：2334304)重悬细胞，1000rpm离心5min。
5)DNA、细胞、buffer混合：弃去D-PBS，加入所需量电转缓冲液(苏州壹达生物科技有限公司，货号：H10305)和10ug质粒，轻轻吹打混匀。
6)电转：将混有质粒的细胞悬液按照200ul+DNA体积/杯，加入到H1电转杯(苏州壹达生物科技有限公司，货号：H10201)中，将电转杯插入基座内，按照表1所示电转条件进行电转。
表1电转条件
7)培养：将电转后的细胞加入含有10mL CDO4培养基(康晟生物医药有限公司，货号：A11004)的T25方瓶(无锡耐思生命科技股份有限公司，货号：707003)培养48小时。
细胞克隆的培养与筛选的具体如下步骤：将上述T25方瓶中的细胞取样，使用细胞计数仪(赛诺菲，型号：Countess II FL)进行活率监控。在活率高于70％的情况下，按照每孔10000个细胞进行96孔板的铺板，在含有25mM MSX(Sigma，货号：M5379-1G)的CD04培养液中培养，通过ELISA方法选取阳性克隆，继续进行扩大培养至125mL(无锡耐思生命科技股份有限公司，货号：781011)摇瓶，在125mL摇瓶中培养，约5-7天后，使用计数仪检测活率下降至50-80％之间时，取上清液进行ELISA检测。
ELISA检测方法如下：上清液按照10倍、100倍、1000倍、10000倍进行稀释、包被，使用1500倍稀释的F蛋白抗体(普健生物(武汉)科技有限公司，货号：62814)作为一抗，羊抗人IgG-HRP(索莱宝，货号SE101-1ml)作为二抗，使用酶标仪(上海科华，货号：RD-SH-012)进行信号读取，选出信号最强的作为最高表达样品。收获最高表达样品的上清液进行下一步纯化。
三、铁蛋白-PreF融合蛋白的纯化
按照文献Flexible RSV Prefusogenic Fusion Glycoprotein Exposes Multiple Neutralizing Epitopes that May Collectively Contribute to Protective Immunity中的方法，通过Capto Lentil Lectin(Cytiva，货号：17548902)、Q Sepharose FF(Cytiva，货号：17051060)、Capto Core 400(Cytiva，货号：17372402)、Superose 6 prep grade(Cytiva，货号：10321079)，对表达细胞株培养液上清进行纯化。具体纯化步骤如下：
将选定的细胞上清培养液进行8000r/min高速离心20分钟，使用0.45um滤膜(津腾，货号：JTSF025013/014)进行过滤，得到约100mL溶液，补平衡液至200mL。使用平衡液平衡QFF柱，A1泵上样，流速1.5mL/min。上样完成后，用平衡液冲洗至吸收值回落至上样前并稳定。洗脱液(20mM Tris，0.5M NaCl，pH8.5)梯度洗脱，流速2mL/min，0-100％B，50min。收集洗脱峰。将上清液浓缩5-10倍，在流速1mL/min的流速下过Superose 6 prep grade柱子，收集吸收峰的样品，得到铁蛋白-PreF融合蛋白溶液，浓缩后用于SDS-PAGE和western blot分析。
SDS-PAGE分析具体步骤如下：向80uL铁蛋白-PreF融合蛋白溶液中加入5×蛋白上样loading 20uL，95℃处理10min后离心。取15uL上清用于SDS-PAGE分析，染色处理观察蛋白表达情况。
Western blot(WB)分析具体步骤如下：
1、SDS-PAGE电泳：制备10％厚度为1.0mm的SDS-PAGE胶，在1×SDS电泳缓冲液中进行凝胶电泳，取20ul蛋白样品上样，使用80V电压待样品进入分离胶后，切换到130V。
2、半干法转膜：使用转移电泳槽(君意，货号：JY-ZY3)。准备1张与分离胶大小一致的PVDF膜和6张滤纸，并用1×膜转移缓冲液(39mM甘氨酸，48mM Tris，0.037％SDS，20％甲醇)浸湿，电泳结束后切掉多余浓缩胶和分离胶，按照阳极电极-三层浸湿滤纸-PVDF膜-蛋白胶-三层浸湿滤纸-阴极电极的方向搭建石墨电极-转移膜胶复合体，接通电源，根据凝胶面积按1.0mA/cm2恒流转膜60min。
3、封闭：用含有5％脱脂奶粉的PBST封闭缓冲液浸润膜，在37℃封闭1h。
4、一抗孵育：将封闭好的膜浸润到加有一抗(Invitrogen，RSV Fusion Protein Polyclonal Antibody，货号：XD3556234B)的1×PBST缓冲液，置于37℃孵育60min。孵育结束后，使用1×PBST在70rpm摇床清洗膜三次，每次10min。
5、二抗孵育：加入1×PBST稀释的二抗(Bioworld，Goat Anti-Rabbit IgG(H+L)HRP，货号：AA092030)，37℃孵育45min。孵育结束后，使用1×PBST在70rpm摇床清洗膜三次，每次10min。
6、显色：使用DAB显色试剂盒(Solarbio，货号：DA1016)进行显色。
铁蛋白-PreF融合蛋白溶液的SDS-PAGE电泳检测结果如图2所示。WB检测结果如图3所示。结果表明：上述重组质粒pKS001-RSV-PreF-A-NP、pKS001-RSV-PreF-B-NP、pKS001-RSV-PreF-C-NP、pKS001-RSV-PreF-D-NP、pKS001-RSV-PreF-E-NP、pKS001-RSV-PreF-F-NP、pKS001-RSV-PreF-G-NP和pKS001-RSV-PreF-H-NP均分别在CHO K1Q细胞中成功表达得到大小约为74KD的目的蛋白(铁蛋白-PreF融合蛋白RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP、RSV-PreF-E-NP、RSV-PreF-F-NP、RSV-PreF-G-NP和RSV-PreF-H-NP)。
实施例2、铁蛋白-PreF融合蛋白的纳米颗粒形态分析
分别将实施例1中制备的铁蛋白-PreF融合蛋白RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP、RSV-PreF-E-NP、RSV-PreF-F-NP、RSV-PreF-G-NP和RSV-PreF-H-NP的纯化产物进行负染。具体负染操作如下：
将超薄碳膜利用Harrick Basic Plasma Cleaner PDC-32G-2仪器进行预抽真空3min，然后medium挡位辉光放电30s，取出。移液枪取4um样品滴到碳膜上，水平放置1min后，用滤纸吸干，然后滴加7um 2％的醋酸铀，放置1min，用滤纸吸干，放置数分钟后使用FEI Tecnai Arctica TEM D683透射电镜对负染后的纯化样品进行电镜观察。
结果如图4所示，结果表明：铁蛋白-PreF融合蛋白RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP、RSV-PreF-E-NP、RSV-PreF-F-NP、RSV-PreF-G-NP和RSV-PreF-H-NP的纯化产物样品在电镜中均可以观察到规则的纳米颗粒，并经过透射电镜分析，可以看到清晰的纳米颗粒形态，颗粒完整性较好。
实施例3、铁蛋白-PreF融合蛋白的免疫原性研究
一、免疫
1、实验材料与方法
实验材料：6-8周雌性Balb/c小鼠(斯贝福(北京)生物技术有限公司，货号：B201-02)。
实验方法：选取64只6-8周Balb/c小鼠，随机分成8组，每组8只，每组处理方法如下：
RSV-PreF-A-NP：分别在第0天、21天进行两次大腿肌肉注射，每次注射1ug的铁蛋白-PreF融合蛋白RSV-PreF-A-NP、50ug氢氧化铝佐剂(长春生物制品研究所有限责任公司，批号：ZP18-003-202106)和100ul PBS缓冲液(Solarbio，货号P1020)。
RSV-PreF-B-NP：分别在第0天、21天进行两次大腿肌肉注射，每次注射1ug的铁蛋白-PreF融合蛋白RSV-PreF-B-NP、50ug氢氧化铝佐剂(长春生物制品研究所有限责任公司，批号：ZP18-003-202106)和100ul PBS缓冲液(Solarbio，货号P1020)。
RSV-PreF-C-NP：分别在第0天、21天进行两次大腿肌肉注射，每次注射1ug的铁蛋白-PreF融合蛋白RSV-PreF-C-NP、50ug氢氧化铝佐剂(长春生物制品研究所有限责任公司，批号：ZP18-003-202106)和100ul PBS缓冲液(Solarbio，货号P1020)。
RSV-PreF-D-NP：分别在第0天、21天进行两次大腿肌肉注射，每次注射1ug的铁蛋白-PreF融合蛋白RSV-PreF-D-NP、50ug氢氧化铝佐剂(长春生物制品研究所有限责任公司，批号：ZP18-003-202106)和100ul PBS缓冲液(Solarbio，货号P1020)。
RSV-PreF-E-NP：分别在第0天、21天进行两次大腿肌肉注射，每次注射1ug的铁蛋白-PreF融合蛋白RSV-PreF-E-NP、50ug氢氧化铝佐剂(长春生物制品研究所有限责任公司，批号：ZP18-003-202106)和100ul PBS缓冲液(Solarbio，货号P1020)。
RSV-PreF-F-NP：分别在第0天、21天进行两次大腿肌肉注射，每次注射1ug的铁蛋白-PreF融合蛋白RSV-PreF-F-NP、50ug氢氧化铝佐剂(长春生物制品研究所有限责任公司，批号：ZP18-003-202106) 和100ul PBS缓冲液(Solarbio，货号P1020)。
RSV-PreF-G-NP：分别在第0天、21天进行两次大腿肌肉注射，每次注射1ug的铁蛋白-PreF融合蛋白RSV-PreF-G-NP、50ug氢氧化铝佐剂(长春生物制品研究所有限责任公司，批号：ZP18-003-202106)和100ul PBS缓冲液(Solarbio，货号P1020)。
RSV-PreF-H-NP：分别在第0天、21天进行两次大腿肌肉注射，每次注射1ug的铁蛋白-PreF融合蛋白RSV-PreF-H-NP、50ug氢氧化铝佐剂(长春生物制品研究所有限责任公司，批号：ZP18-003-202106)和100ul PBS缓冲液(Solarbio，货号P1020)。
二、ELISA法检测血清中的抗体
免疫后第28天(约6周)采取小鼠血清用于ELISA分析，ELISA分析具体步骤如下：使用每孔200ng的RSV的F蛋白(义翘神州，货号：11049-V08B)进行包被，小鼠血清作为一抗，按照250倍、1250倍、6250倍、31250倍、156250倍、781250倍、3906250倍进行梯度稀释，鼠二抗(Cell Signaling Technology，货号：7076S)作为二抗，使用酶标仪(上海科华，货号：RD-SH-012)进行信号读取。
经过2次免疫后的小鼠血清在ELISA中产生的效价检测结果如图5所示。结果表明：在稀释至781250倍时，铁蛋白-PreF融合蛋白RSV-PreF-D-NP、RSV-PreF-C-NP、RSV-PreF-B-NP、RSV-PreF-E-NP、RSV-PreF-G-NP、RSV-PreF-H-NP仍然有检测到ELISA信号。其中，铁蛋白-PreF融合蛋白RSV-PreF-C-NP的ELISA效价最高。
三、小鼠血清CPE中和试验
使用Hep-2细胞在含有10％牛血清的DMEM培养基中培养RSV病毒A型Long株的TCID50为2.81E+07。选取上述各组小鼠血清各8份，使用含有2％牛血清的DMEM培养基进行稀释。从40倍稀释开始，按照3倍稀释梯度稀释至29160倍，然后与等体积200TCID50的病毒液混合，37℃放置1小时，每孔200ul铺到Hep-2细胞板，每只小鼠血清设置3个复孔，37℃培养5-7天，观察细胞病变情况。
结果如表2和图6所示。结果表明：RSV-PreF-C-NP组中和效价均值最高，达到19836，RSV-PreF-C-NP组中和效价均值的Log2达到14.3。而且本发明中的RSV-PreF-A-NP、RSV-PreF-D-NP、RSV-PreF-C-NP、RSV-PreF-E-NP中和效价均超出了强生公司开发的同类疫苗免疫后第六周的针对A型毒株的最高效值的Log2(强生公司开发的同类疫苗免疫后第六周的针对A型毒株的最高效值的Log2约为11，换算效价约2100)。
表2、中和效价均值
按照上述实验方法，将RSV病毒A型Long株替换为RSV病毒B型BA9株，使用上述各组小鼠血清进行同样程序的效价分析。
结果如表3所示。结果表明：RSV-PreF-C-NP小鼠血清中和效价均值为10568，log2值为13.4。本发明中的RSV-PreF-A-NP、RSV-PreF-D-NP、RSV-PreF-C-NP、RSV-PreF-E-NP均接近或超过效价2100。
以上结果表明：将本发明制备的铁蛋白-PreF融合蛋白注射至小鼠，可以获取到高保护效价的血清，且小鼠血清针对RSV主要流行毒株A型和B型均可产生较高的中和效价。
表3中和效价均值

实施例4、铁蛋白-PreF融合蛋白的稳定性试验
为了验证本发明制备的铁蛋白-PreF融合蛋白的稳定性，将纯化后的各组铁蛋白-PreF融合蛋白进行物理稳定性(物理环境挑战)试验，具体步骤如下：
将5份40ug/uL的铁蛋白-PreF融合蛋白溶液以及梯度稀释后的铁蛋白-PreF融合蛋白溶液分别放置在pH7.4(25℃)、pH3.8(25℃)、pH10(25℃)、50℃(pH7.4)、70℃(pH7.4)的环境中1小时(分别对应图中从左往右1-5列)，采用与细胞克隆筛选相同的方法进行ELISA分析。
结果表明：各组融合蛋白在不同pH和温度处理后的抗原结合活性均保持在原始未处理蛋白的75％以上。说明本发明制备的铁蛋白-PreF融合蛋白均具备足够的物理稳定性。如表4所示为RSV-PreF-C-NP蛋白的稳定性结果，其他各组的结果类似。
表4物理环境挑战后ELISA信号强度保留百分比
注：表中结果为10、100、1000、10000倍共4个稀释梯度的平均值。
实施例5、铁蛋白-PreF融合蛋白的安全性实验
为了验证本发明制备的铁蛋白-PreF融合蛋白免疫后所引发的免疫反应中Th1\Th2的平衡性(参考文献：Immunological Lessons from Respiratory Syncytial Virus Vaccine Development)，选择铁蛋白-PreF融合蛋白RSV-PreF-C-NP免疫后的小鼠血清进行了IgG1和IgG2a的ELISA分析，ELISA分析采用与血清ELISA检测相同的设置，二抗分别使用IgG1特异性二抗(abcam，货号：GR3395386-5)和IgG2a特异性二抗(abcam，货号：GR3413688-1)。同时以福尔马林灭活疫苗FI-RSV以及强生公司开发的同类疫苗作为对照。
结果如图7所示，结果表明：血清中IgG1和IgG2a的滴度达到了约6(见图7中的RSVNP IgG1和RSVNP IgG2a)，而且其比例约等于1，明显优于福尔马林灭活疫苗(见图7中的FI-RSVNP IgG1和FI-RSV IgG2a)，甚至优于强生公司开发的同类疫苗(IgG2a约5.8和IgG1约5)。说明本发明制备的铁蛋白-PreF融合蛋白注射后引起的免疫反应是Th1/Th2平衡的，可以避免Th2偏激所导致的免疫过激反应(简称VED)，具备较好的安全性。
实施例6铁蛋白和PreF分别表达后自组装
在铁蛋白和PreF突变蛋白的序列上分别添加标签，1：1通过标签进行自组装。本申请在铁蛋白的N端通过linker添加Catcher(GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT，SEQ ID No.28)；在PreF蛋白的C端通过linker添加Spy(RGVPHIVMVDAYKRYK，SEQ ID No.29)标签。表达后通过Spy-Catcher相互作用，将两者组装成纳米颗粒。
mRNA质粒构建
通过HindIII和XbaI，将需要构建mRNA的序列插入pVAX1(南京金斯瑞生物科技有限公司提供，质粒图谱如图)中。相关质粒由南京金斯瑞生物科技有限公司进行合成、提供。
实施例7 DNA电导入小鼠实验-体内筛选
免疫：6-8周雌性Balb/c小鼠(斯贝福(北京)生物技术有限公司，货号：B201-02)。
实验方法：选取64只6-8周Balb/c小鼠，随机分成8组，每组8只，每组处理方法如下：
向BALB/c小鼠电导入包含上述8组DNA序列质粒(SEQ ID No.20-27)。将50μg载体质粒注射到大腿后侧肌肉中，然后使用设备(Terasa Healthcare Sci-Tech，中国)施加六个电脉冲(60V，20ms)。 通过EP接种50μg pKS的小鼠作为阴性对照。每隔两周1次，共对小鼠进行2次免疫。取二免血清进行中和实验，对比中和保护效率。
中和实验：
使用Hep-2细胞在10％牛血清的DMEM培养基中培养RSV病毒A型Long株(RSV病毒A型Long株记载于文献：Cultures of HEp-2 cells persistently infected by human respiratory syncytial virus differ in chemokine expression and resistance to apoptosis as compared to lytic infections of the same cell type中)的TCID50为2.81E+07。选取上述各组小鼠血清各8份，使用2％牛血清的DMEM培养基进行稀释。与等体积200TCID50的病毒液混合，37℃放置1小时，每孔200ul铺到Hep-2细胞板，每只小鼠血清设置3个复孔，37℃培养5-7天，观察细胞病变情况。
结果如表5所示：
表5：不同DNA质粒的中和效价
可见，DNA的8个序列均可以产生中和效价，其值均大于100，而DNA-A、DNA-B、DNA-C、DNA-D的效果更佳(分别编码RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP)，其值大于700，这可能与其编码的融合蛋白的突变相关，它们的突变均具有在第88位的突变。
在8个序列中选出4个中和表现最优的，进行mRNA疫苗的制备。
其中,编码RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP的mRNA序列为将SEQ ID No.20-23中的T替换为U而获得。
其中，SEQ ID No.20-23的Pre-F突变蛋白的编码区和铁蛋白突变体的编码区如表8所限定。
编码RSV-PreF-E-NP、RSV-PreF-F-NP、RSV-PreF-G-NP、RSV-PreF-H-NP的mRNA序列可将SEQ ID No.24-27中的T替换为U而获得。
实施例8 mRNA疫苗的制备
如实施例7所描述，人工合成质粒DNA序列，DNA序列含RNA转录相关的元件。质粒转化入大肠杆菌中进行扩增。发酵纯化后的质粒用限制性核酸内切酶BspQ1线性化。用T7体外转录试剂盒进行转录，获得的未加帽的mRNA。分别用DNaseI消化转录模板，并用沉淀法纯化mRNA。用Cap1加帽试剂盒给mRNA加帽，并分别用mRNA纯化试剂盒对加帽后的mRNA进行纯化(分别为编码RSV-PreF-A-NP、RSV-PreF-B-NP、RSV-PreF-C-NP、RSV-PreF-D-NP的mRNA序列)。将纯化后的mRNA溶解于酸性柠檬酸钠缓冲液中，待用。
通过现有技术中已知的方法将每条mRNA序列用含阳离子脂质的脂质纳米颗粒包封获得mRNA疫苗(mRNA-LNP)，具体如下：将DLin-MC3-DMA：DSPC：胆固醇：PEG2000-DMG按摩尔比为45:10:43:2在乙醇中溶解、混合。设定纳米药物制造设备总流速12ml/min。将mRNA溶液与脂质纳米颗粒混合溶液分别按流速比3：1包封，包封完成后，切向流过滤系统超滤换液收集样品，并加入蔗糖溶液，得到mRNA疫苗(mRNA-LNP)。取样测得包封率均>91％，平均粒径约为82nm，PDI<0.1,Zeta电位约为-8.20mV。
实施例9体内免疫原性研究
免疫：在第0天，将BALB/c小鼠皮内(i.d.)注射实施例8制备的mRNA疫苗。
中和效价检测：采用和实施例7相同的方法进行检测。
中和效价结果：
结果如表6：
表6：mRNA疫苗中和效价结果
可见，本发明的纳米颗粒形态mRNA在小鼠体内引起的中和保护抗体滴度超过了我们同步制作的MRK-12(序列和方法来自Moderna RSV mRNA专利US20230114180A1的SEQ ID No.21)，以编码RSV-PreF-D-NP、RSV-PreF-B-NP、RSV-PreF-C-NP的mRNA疫苗效果尤其好。
血清Elispot分析：
通过细胞内细胞因子染色流式细胞术(ICS)方法检测受到mRNA疫苗刺激(每组5个样本)后分泌IFN-γ细胞的频数。
结果如图8所示，编码RSV-PreF-D-NP的mRNA疫苗(RSV-PreF-D-NP组)免疫刺激后，每2×105的脾细胞分泌IFN-γ的频率均值可达到73.4个(5个样本分别为58.33、51、79、97.7和81)，而对照组MRK-12仅为6.4个(5个样本分别为0、0.66、27、2.33和2)，两者有显著性的差异。以上Elispot细胞免疫结果说明，本发明的mRNA疫苗诱导的细胞免疫显著高于MRK-12,说明有明显的细胞免疫保护效果，而细胞免疫能带来更长效和全面的保护。
实施例10自组装纳米颗粒的体内免疫原性研究
将在铁蛋白和PreF突变蛋白上分别添加Catcher和Spy标签(参见实施例6)，按照现有技术的包封方法，将两者以质量比1：1的mRNA序列用含阳离子脂质的脂质纳米颗粒包封，从而获得mRNA疫苗(mRNA-LNP)。
免疫：在第0天，将BALB/c小鼠皮内(i.d.)注射实施例8制备的mRNA疫苗。
中和效价检测：采用和实施例7相同的方法进行检测。
中和效价结果如表7.
表7：自组装mRNA疫苗中和效价结果
从中和结果可见，自组装的mRNA疫苗与融合表达的结果相似，都能实现获得较好的中和效价，尤其以RSV-PreF-D-NP和RSV-PreF-B-NP更佳，与其他两组有显著性的差异。
实施例11、铁蛋白-PreF融合蛋白的佐剂筛选
将实施例1纯化得到的铁蛋白-PreF(RSV-PreF-D-NP，SEQ ID No.15)融合蛋白(下述表格中以RSV-NP表示)。选择使用溶剂为20mM磷酸缓冲液，0.15M NaCl,pH 4.5)，得到RSV铁蛋白-PreF融合蛋白抗原。
一、分组
疫苗组：
1：铝佐剂疫苗配置
室温条件下将0.4mg/ml的稀释抗原与氢氧化铝佐剂混悬液或磷酸铝佐剂混悬液(其中铝含量10mg/ml，溶质为氢氧化铝或磷酸铝，溶剂为PBS；购自长春生物制品研究所)按照不同体积置于加有转子的玻璃瓶中80rpm/min混合均匀，得到不同铝盐佐剂疫苗，其中抗原与佐剂的质量比如表9或表10所示。
2：CpG佐剂疫苗配置
室温条件下将0.4mg/ml的稀释抗原和10mg/ml表11所示各个CpG1018的溶液(溶剂为生理盐水， 溶质为CpG1018，表11中以CpG表示)按照不同体积置于加有转子的玻璃瓶中80rpm/min混合均匀，得到不同配比的CpG佐剂疫苗，其中抗原与佐剂的质量比如表11所示。
3：双佐剂疫苗配置
室温条件下将0.4mg/ml的稀释抗原、铝佐剂(氢氧化铝混悬液(其中铝含量10mg/ml)、10mg/ml浓度CpG的溶液按照不同体积置于加有转子的玻璃瓶中低速混合均匀，得到不同双佐剂疫苗，其中抗原与佐剂的质量比如表12或表13所示。
将上述制备的疫苗，无菌条件下分装入2ml西林瓶内(或预填充玻璃注射器内)，每瓶0.5ml(或1.0ml)，密封后放置于2～8℃避光保存。
佐剂组：
4、纯佐剂组制备
同上3、双佐剂疫苗配置的方法，仅是不含有抗原。
蛋白组：
5、无佐剂组制备
仅包含抗原。
6、PBS对照组
PBS对照组配制：取出PBS powder(购自索莱宝，货号：P1003)，每袋用2L无菌蒸馏水溶解。
二、免疫
取出上述一制备的各组，以C57BL/6小鼠(购自斯贝福(北京)生物技术有限公司)为动物模型开展了免疫原性研究。
选择6-8周龄C57BL/6小鼠随机分组，每组8只小鼠，大腿肌肉注射上述制备的各组，并设置疫苗组、无佐剂组和佐剂组，在0d、21d(以第一次免疫当天记作第0天，免疫当天起的一周，记作免疫的第1周)进行二次免疫(免疫剂量和方式如下述四中所示)，二次免疫间隔21天。
在免疫第3、5周采血，第5周取脾脏。
三、ELISA法检测血清中的抗体
免疫后第28天(约5周)采取小鼠血清用于ELISA分析，ELISA分析具体步骤如下：使用每孔200ng的RSV的F蛋白(义翘神州，货号：11049-V08B)进行包被，小鼠血清作为一抗，按照250倍、1250倍、6250倍、31250倍、156250倍、781250倍、3906250倍进行梯度稀释，二抗为鼠二抗(Cell Signaling Technology，货号：7076S)，使用酶标仪(上海科华，货号：RD-SH-012)进行信号读取。
经过2次免疫后的小鼠血清在ELISA中产生的效价检测结果：
A：筛选最佳的铝佐剂
表9不同铝盐佐剂配制的重组疫苗免疫小鼠的ELISA效价
从表9可以看出，相对于不添加铝佐剂组，两种常用的铝佐剂均可以显著性增加抗体效价，氢氧化铝佐剂与RSV-NP的组合产生更高的血清ELISA。
B：筛选最佳的铝佐剂剂量
表10筛选最佳的铝佐剂剂量的ELISA效价
结果如表10所示，30-70μg的铝佐剂均可提高抗体效价，以50μg铝佐剂的免疫效果最佳。
C：筛选到最佳的CpG佐剂剂量
表11筛选到最佳的CpG佐剂剂量的ELISA效价
结果如表11所示，25-100μg均可提高抗体效价，以50μg CpG1018佐剂的免疫效果最佳
D：筛选到最佳的双佐剂剂量
表12筛选到最佳的双佐剂剂量的ELISA效价
结果如表12所示，CpG佐剂的剂量在50μg时达到了一个平台期；相对于25μg的剂量，免疫效价显著性的提高，其后增加CpG佐剂剂量，免疫效价维持在稳定的水平。
D：探索最佳的抗原剂量
表13小鼠上探索最佳的抗原剂量的ELISA效价
结果如表13所示，抗原剂量在1μg左右达到平台期(24组与23组的P<0.01，24组与25组、26组的P>0.05)，更高的抗原剂量并没有显著提升ELISA效价值。
结论：疫苗制剂处方检测中发现，抗原含量1μg最佳；氢氧化铝佐剂50μg，CpG佐剂50μg最佳。
四、小鼠血清CPE中和试验
使用Hep-2细胞在10％牛血清的DMEM培养基中培养RSV病毒A型Long株(RSV病毒A型Long株记载于文献：Cultures of HEp-2 cells persistently infected by human respiratory syncytial virus differ in chemokine expression and resistance to apoptosis as compared to lytic infections of the same cell type中)的TCID50为2.81E+07。选取表13中的第24组，参照表14删除相应的组分，制备各实验组，各组小 鼠血清各8份，使用2％牛血清的DMEM培养基进行稀释。从40倍稀释开始，按照3倍稀释梯度稀释至174960倍，与等体积200TCID50的病毒液混合，37℃放置1小时，每孔200ul铺到Hep-2细胞板，每只小鼠血清设置3个复孔，37℃培养5-7天，观察细胞病变情况。
结果如表14所示。结果表明：本发明中的双佐剂组中和效价远超出单佐剂组，达到73152，Log2为16.5(表14)。
表14 RSV病毒A型中和效价均值
RSV病毒的主要流行株分A型和B型。按照上述实验方法，将RSV病毒A型Long株替换为RSV病毒B型株(记载于文献：Genetic Diversity and Molecular Epidemiology of Circulating Respiratory Syncytial Virus in Central Taiwan,2008–2017中)，使用上述各组小鼠血清进行同样程序的效价分析，得出表15数据。
表15 RSV病毒B型中和效价均值
如表15所示，双佐剂组的中和效价相对于单佐剂组具有显著性的提高，两者具有协同作用。
采用其他PreF突变体的融合蛋白(例如RSV-PreF-A-NP(SEQ ID No.12)、RSV-PreF-B-NP(SEQ ID No.13)、RSV-PreF-C-NP(SEQ ID No.14)、RSV-PreF-E-NP(SEQ ID No.16)等等)观察到类似的效价和效价趋势。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。

Claims (23)

  1. 一种蛋白，其为将Pre-F蛋白氨基酸序列进行如下a1)-a18)中至少一种突变后得到的蛋白：
    a1)将Pre-F蛋白氨基酸序列第67位的异亮氨酸突变为天冬酰胺；
    a2)将Pre-F蛋白氨基酸序列第88位的丝氨酸突变为天冬酰胺；
    a3)将Pre-F蛋白氨基酸序列第110位的半胱氨酸突变为丙氨酸；
    a4)将Pre-F蛋白氨基酸序列第144位的天冬酰胺突变为甘氨酸，且在第144位和第145位氨基酸之间插入半胱氨酸；
    a5)将Pre-F蛋白氨基酸序列第159位的酪氨酸突变为半胱氨酸；
    a6)将Pre-F蛋白氨基酸序列第173位的半胱氨酸缺失；
    a7)将Pre-F蛋白氨基酸序列第202位的丙氨酸突变为半胱氨酸；
    a8)将Pre-F蛋白氨基酸序列第227位的异亮氨酸突变为天冬酰胺；
    a9)将Pre-F蛋白氨基酸序列第236位的丝氨酸突变为精氨酸；
    a10)将Pre-F蛋白氨基酸序列第248位的丝氨酸突变为半胱氨酸；
    a11)将Pre-F蛋白氨基酸序列第289位的谷氨酸突变为天冬酰胺；
    a12)将Pre-F蛋白氨基酸序列第309位的丝氨酸突变为天冬酰胺；
    a13)将Pre-F蛋白氨基酸序列第334位的精氨酸突变为酪氨酸；
    a14)将Pre-F蛋白氨基酸序列第344位的天冬酰胺突变为谷氨酸；
    a15)将Pre-F蛋白氨基酸序列第370位的丝氨酸突变为甘氨酸；
    a16)将Pre-F蛋白氨基酸序列第389位的天冬酰胺突变为半胱氨酸；
    a17)将Pre-F蛋白氨基酸序列第420位的半胱氨酸突变为酪氨酸；
    a18)将Pre-F蛋白氨基酸序列第469位的精氨酸突变为天冬酰胺；
    所述Pre-F蛋白为如下任一种：
    (A1)SEQ ID No.1所示的蛋白；
    (A2)在(A1)所述蛋白的N端或/和C端连接标签得到的融合蛋白；
    (A3)将(A1)-(A2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白；
    (A4)与(A1)-(A2)任一种具有98％以上同一性且具有相同功能的蛋白。
  2. 根据权利要求1所述的蛋白，其特征在于：所述蛋白为如下任一种：
    (M1)SEQ ID No.2或SEQ ID No.3或SEQ ID No.4或SEQ ID No.5或SEQ ID No.6或SEQ ID No.7或SEQ ID No.8或SEQ ID No.9所示的蛋白；
    (M2)在(M1)所述蛋白的N端或/和C端连接标签得到的融合蛋白；
    (M3)将(M1)-(M2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白；
    (M4)与(M1)-(M2)任一种具有98％以上同一性且具有相同功能的蛋白。
  3. 一种融合蛋白，其包括权利要求1所述的蛋白和铁蛋白突变体；
    所述铁蛋白突变体为将铁蛋白氨基酸序列进行如下b1)-b3)中至少一种突变后得到的蛋白：
    b1)将铁蛋白氨基酸序列第15位的天冬酰胺突变为谷氨酰胺；
    b2)将铁蛋白氨基酸序列第96位的丝氨酸突变为天冬酰胺；
    b3)将铁蛋白氨基酸序列第119位的酪氨酸突变为精氨酸；
    所述铁蛋白为如下任一种：
    (B1)SEQ ID No.10所示的蛋白；
    (B2)在(B1)所述蛋白的N端或/和C端连接标签得到的融合蛋白；
    (B3)将(B1)-(B2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白；
    (B4)与(B1)-(B2)任一种具有98％以上同一性且具有相同功能的蛋白。
  4. 根据权利要求3所述的融合蛋白，其特征在于：所述铁蛋白突变体为如下任一种：
    (N1)SEQ ID No.11所示的蛋白；
    (N2)在(N1)所述蛋白的N端或/和C端连接标签得到的融合蛋白；
    (N3)将(N1)-(N2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白；
    (N4)与(N1)-(N2)任一种具有98％以上同一性且具有相同功能的蛋白；
    或，所述融合蛋白为如下任一种：
    (C1)SEQ ID No.12或SEQ ID No.13或SEQ ID No.14或SEQ ID No.15或SEQ ID No.16或SEQ ID No.17或SEQ ID No.18或SEQ ID No.19所示的蛋白；
    (C2)在(C1)所述蛋白的N端或/和C端连接标签得到的融合蛋白；
    (C3)将(C1)-(C2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白；
    (C4)与(C1)-(C2)任一种具有98％以上同一性且具有相同功能的蛋白。
  5. 生物材料，为下述D1)-D5)中至少一种：
    D1)编码权利要求1或2所述蛋白或权利要求3或4所述融合蛋白的核酸分子；
    D2)含有D1)所述核酸分子的表达盒；
    D3)含有D1)所述核酸分子的重组载体、或含有D2)所述表达盒的重组载体；
    D4)含有D1)所述核酸分子的重组微生物、含有D2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
    D5)含有D1)所述核酸分子的重组细胞系、含有D2)所述表达盒的重组细胞系、或含有D3)所述重组载体的重组细胞系。
  6. 根据权利要求5所述的生物材料，其特征在于，所述核酸分子包括DNA或者mRNA。
  7. 权利要求1或2所述的蛋白或权利要求3或4所述的融合蛋白的制备方法，包括如下步骤：使编码权利要求1或2所述蛋白的核酸分子或编码权利要求3或4所述融合蛋白的核酸分子在生物或生物细胞中进行表达，得到所述蛋白或融合蛋白。
  8. 根据权利要求7所述的方法，其特征在于：所述方法包括如下步骤：将编码权利要求1或2所述蛋白的核酸分子或编码权利要求3或4所述融合蛋白的核酸分子导入CHO K1Q细胞，得到重组细胞；培养所述重组细胞，得到所述蛋白或融合蛋白。
  9. 一种mRNA，其特征在于，所述mRNA包含第一开放阅读框，所述第一开放阅读框包含编码单体亚基蛋白和来自RSV的至少一个免疫原性部分的核酸：所述单体亚基蛋白选自下组的单体铁蛋白亚基，单体encapsulin蛋白，单体03-33蛋白，单体硫加氧酶还原酶蛋白，单体二氧四氢蝶啶合酶蛋白，和/或单体丙酮酸脱氢酶复合物二氢硫辛酰胺乙酰转移酶蛋白，所述第一开放阅读框表达的单体亚基蛋白自组装为纳米颗粒，且在所述纳米颗粒的表面上展示所述来自RSV的至少一个免疫原性部分。
  10. 一种mRNA，其特征在于，所述mRNA至少包含两个开放阅读框，其中，第一开放阅读框包括编码选自下组的单体亚基蛋白的核酸：单体铁蛋白亚基，单体encapsulin蛋白，单体03-33蛋白，单体硫加氧酶还原酶蛋白，单体二氧四氢蝶啶合酶蛋白，和/或单体丙酮酸脱氢酶复合物二氢硫辛酰胺乙酰转移酶蛋白，第二开放阅读框包括编码来自RSV的至少一个免疫原性部分的核酸，所述第一开放阅读框表达的单体亚基蛋白能自组装为纳米颗粒，且第二开放阅读框表达的所述至少一个免疫原性部分能够与第一开放阅读框表达的所述纳米颗粒结合。
  11. 根据权利要求9或10所述的mRNA，其特征在于，来自RSV的至少一个免疫原性部分选自权利要求1-2任一所述的蛋白或者权利要求3-4任一所述的融合蛋白。
  12. 根据权利要求11所述的mRNA，其特征在于，所述mRNA包括如下任一种：
    (H1)、SEQ ID No.30-33所示的mRNA的部分或者全部；
    (H2)、(H1)的简并或者互补序列；
    (H3)、与(H1)或(H2)具有80％以上同一性且具有编码相同功能的蛋白的mRNA。
  13. 权利要求9-12任一所述的mRNA的制备方法，其特征在于，所述制备方法包括根据mRNA编码的单体亚基蛋白和来自RSV的至少一个免疫原性部分设计DNA序列，构建包含所述DNA的载体，将载 体转录表达mRNA，
    优选的，所述来自RSV的至少一个免疫原性部分包括pre-F蛋白的突变蛋白，所述单体亚基蛋白包括铁蛋白突变体，所述pre-F蛋白的突变蛋白与铁蛋白突变体融合表达或自组装。
  14. 权利要求1或2所述的蛋白、权利要求3或4所述的融合蛋白、权利要求5或6所述的生物材料、按照权利要求7或8所述方法制备得到的蛋白或融合蛋白、或权利要求9-12任一所述的mRNA在如下X1)-X4)任一种中的应用；
    X1)作为免疫原；
    X2)制备抗呼吸道合胞病毒的产品；
    X3)制备预防和/或治疗呼吸道合胞病毒感染的产品；
    X4)制备预防和/或治疗呼吸道合胞病毒所致疾病的产品。
  15. 一种疫苗，所述疫苗包括权利要求1或2所述的蛋白、权利要求3或4所述的融合蛋白、权利要求5或6所述的生物材料、按照权利要求7或8所述方法制备得到的蛋白或融合蛋白或权利要求9-12任一所述的mRNA。
  16. 根据权利要求15所述的疫苗，其特征在于，所述疫苗还包括佐剂，优选的，所述疫苗包括双佐剂，所述双佐剂包括铝佐剂和CpG佐剂。
  17. 根据权利要求16所述的RSV疫苗，其特征在于，所述铝佐剂包括氢氧化铝、磷酸铝中的一种或多种，优选的，所述蛋白与所述铝佐剂的质量比可为1：(0.5-300)。
  18. 根据权利要求16-17任一所述的RSV疫苗，其特征在于，所述CpG佐剂包括任一的CpG佐剂，例如CpG1018、CpG-cjx等等，优选的，所述蛋白与所述CpG佐剂的质量比可为1：(0.5-300)。
  19. 根据权利要求16-18任一所述的RSV疫苗，其特征在于，所述双佐剂中铝佐剂与CpG佐剂的质量比为(1-5)：(5-1)
  20. 权利要求16-19任一所述的疫苗在如下Y1)-Y3)任一种中的应用；
    Y1)制备抗呼吸道合胞病毒的产品；
    Y2)制备预防和/或治疗呼吸道合胞病毒感染的产品；
    Y3)制备预防和/或治疗呼吸道合胞病毒所致疾病的产品。
  21. 一种药物组合物，其特征在于，所述药物组合物包括权利要求9-12任一所述的mRNA，
    优选的，所述mRNA包封于所述脂质纳米颗粒中，
    更优选的，所述脂质纳米颗粒包括阳离子脂质、中性磷脂、甾族脂质和聚乙二醇-脂质。
  22. 权利要求21所述的药物组合物的制备方法，其特征在于，所述制备方法包括制备mRNA的步骤。
  23. 根据权利要求22所述的制备方法，其特征在于，所述制备方法包括将脂质纳米颗粒溶解至溶剂后与mRNA混合后制得药物组合物。
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