Recombinant fusion protein of respiratory syncytial virus of prefusion conformation and preparation method and application of recombinant fusion protein

Abstract

The present invention relates to a respiratory syncytial virus subtype B (RSV-B) recombinant F protein, a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a host cell wherein the polynucleotide, the nucleic acid construct or the expression vector is transformed or transfected, a stabilized trimer formed therefrom, and a method for preparing the same. An immunogenic composition comprising any of the foregoing, and its use in the preparation of a vaccine for the prevention and / or treatment of respiratory syncytial virus infection. The RSV-B recombinant F protein comprises at least one specific epitope of the pre-fusion F protein, a stable F protein tripolymer with pre-fusion conformation can be formed, the expression is stable, the form is uniform, and the yield is greatly improved; the formed F protein trimer has good immunogenicity, can stimulate an organism to generate high-level antibody titer, and has great significance in clinical treatment and prevention and control of the respiratory syncytial virus.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202311261041.1, entitled "A Recombinant Fusion Protein of Respiratory Syncytial Virus in Pre-Fusion Conformation, Its Preparation Method and Use".

[0002] Cross-referencing

[0003] This application claims priority to Chinese Patent Application No. 202211199603.X, filed on September 29, 2022, entitled "A Recombinant Fusion Protein of Respiratory Syncytial Virus in Pre-Fusion Conformation, Preparation Method Thereof and Use Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0004] This invention relates to the field of vaccine technology, specifically to a recombinant F protein of respiratory syncytial virus subtype B (RSV-B), a stable trimer formed therefrom, an immunogenic composition containing therefrom, and methods for preparing and using the same. Background Technology

[0005] Respiratory syncytial virus (RSV) is the most common pathogen causing acute respiratory infections in infants. Bronchiolitis caused by RSV infection is a leading cause of hospitalization in children under 2 years of age and a significant factor contributing to increased infant mortality. The WHO estimates that 64 million children worldwide are infected with RSV annually, and 160,000 die from it. In 2020, the number of children under 5 years of age with severe RSV infection globally reached 34.6 million. Another high-risk group for RSV is the elderly. Several prospective studies have analyzed the incidence of RSV in community-dwelling elderly individuals, revealing an annual incidence rate between 2% and 10%. Large-scale epidemiological studies have shown that RSV causes hospitalizations and mortality rates comparable to influenza in the elderly population.

[0006] RSV has a single serotype with two major antigenic subgroups, A and B. Strains of both subtypes often co-circulate, but only one subtype typically dominates during epidemic periods. In temperate regions, RSV infection exhibits a pronounced seasonality, peaking between mid-December and early February, and declining in late spring, with outbreaks occurring in late autumn and early winter. There is an urgent global need for safe, effective, and inexpensive preventative treatments for RSV infection.

[0007] RSV belongs to the genus *Pneumovirus* of the family Paramyxoviridae. It is a polymorphic enveloped virus with a diameter of approximately 120–300 nm, possessing segmentless negative-sense single-stranded RNA (15–16 kb) encoding 11 proteins. The viral envelope contains three proteins: attachment glycoprotein (G), fusion glycoprotein (F), and small hydrophobic (SH) protein. The G protein plays a role in host cell attachment, while the F protein is responsible for fusion and cell entry; neither of these processes requires the SH protein. Two RSV surface glycoproteins are the main neutralizing antigens: the G protein exhibits high sequence diversity and determines the viral antigenic subtypes (A and B); the fusion protein (F) is highly conserved between the two subtypes and is recognized by a wide range of cross-neutralizing antibodies. Due to its crucial role in RSV invasion and its highly conserved protein sequence, the RSV F protein is a target for neutralizing antibodies and a major antigen in vaccine development. The F protein is a type I transmembrane protein. The F protein first synthesizes a 574-amino acid precursor protein, F0, which has 5–6 post-translational N-linked glycosylation modifications. To become a functional fusion protein, F0 is treated by a furin-like protease at two multibase sites in the trans-Golgi apparatus, producing F1 (amino acids 137-574), F2 (amino acids 26-109), and Pep27 polypeptides. This results in two fragments, F1 and F2, linked by disulfide bonds, with sizes of 55 kDa and 15 kDa, respectively. The homologous F protein trimer contains two conformations: pre-fusion and post-fusion. The pre-fusion F protein is a metastable structure; when the virus fuses with a cell, the pre-fusion F protein transforms into the stable post-fusion F protein, a process that can also occur spontaneously. Since highly active neutralizing antibody epitopes against the F protein are primarily located on the pre-fusion F protein, maintaining the F protein in its pre-fusion conformation is crucial for RSV vaccine development. To date, no RSV vaccine has been marketed. Summary of the Invention

[0008] To overcome the problems existing in the prior art, the present invention provides a recombinant F protein of respiratory syncytial virus subtype B (RSV-B) (hereinafter referred to as "RSV-B-Fm" or "Fm"), a polynucleotide encoding the polynucleotide, a nucleic acid construct containing the polynucleotide, an expression vector containing the nucleic acid construct, a host cell transformed or transfected with the polynucleotide, the nucleic acid construct or the expression vector, a stable trimer formed by the recombinant F protein, an immunogenic composition comprising any one of the foregoing, and its use in the preparation of a vaccine for the prevention and / or treatment of respiratory syncytial virus infection.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a recombinant F protein of respiratory syncytial virus subtype B (RSV-B), which is a recombinant F protein based on respiratory syncytial virus subtype B, also referred to herein as RSV-B recombinant protein or RSV-B recombinant F protein. The RSV-B recombinant F protein comprises an RSV-B-F1 peptide and an RSV-B-F2 peptide, wherein the RSV-B-F1 peptide and / or the RSV-B-F2 peptide contain at least one proline mutation relative to the corresponding peptide of the wild-type RSV-B F protein.

[0011] In a specific embodiment, the RSV-B-F1 peptide corresponds to amino acid segments at positions 26-98, 26-108, or 26-97 in the amino acid sequence of the wild-type RSV-BF protein as shown in SEQ ID NO:1, and the RSV-B-F2 peptide corresponds to amino acid segments at positions 136-513, 145-513, 138-513, or 137-513 in the amino acid sequence of the wild-type RSV-B F protein as shown in SEQ ID NO:1. Furthermore, the RSV-B-F1 peptide and / or the corresponding peptide in the RSV-B-F2 peptide relative to the wild-type RSV-B F protein contains one or more proline mutations.

[0012] Preferably, the one or more proline mutations are selected from the following mutations in the amino acid sequence of the wild-type RSV-BF protein as shown in SEQ ID NO:1:

[0013] K65P, T67P, D73P, L138P, G139P, L141P, E161P, Q210P, I214P, S215P, N216P, Q279P, S377P;

[0014] Preferably, the one or more proline mutations are selected from the following mutations or combinations thereof:

[0015] T67P; for example, in RSV-B recombinant F protein Fm1 as shown in SEQ ID NO:29;

[0016] L141P; for example, in RSV-B recombinant F protein Fm 2 as shown in SEQ ID NO:30;

[0017] Q279P; for example, in RSV-B recombinant F protein Fm3 as shown in SEQ ID NO:31;

[0018] S377P; for example, in RSV-B recombinant F protein Fm 4 as shown in SEQ ID NO:32;

[0019] T67P+L141P; for example, in RSV-B recombinant F protein Fm 5 as shown in SEQ ID NO:33;

[0020] L141P+Q279P; for example, in RSV-B recombinant F protein Fm6 as shown in SEQ ID NO:34;

[0021] L141P+S377P; for example, in RSV-B recombinant F protein Fm 7 as shown in SEQ ID NO:35;

[0022] T67P+Q279P; for example, in RSV-B recombinant F protein Fm8 as shown in SEQ ID NO:36;

[0023] T67P+S377P; for example, in RSV-B recombinant F protein Fm9 as shown in SEQ ID NO:37;

[0024] Q279P+S377P; for example, in RSV-B recombinant F protein Fm 10 as shown in SEQ ID NO:38;

[0025] T67P+L141P+Q279P; for example, in RSV-B recombinant F protein Fm 11 as shown in SEQ ID NO:39;

[0026] T67P+L141P+S377P; for example, in RSV-B recombinant F protein Fm 12 as shown in SEQ ID NO:40;

[0027] L141P+Q279P+S377P; for example, in RSV-B recombinant F protein Fm 13 as shown in SEQ ID NO:41;

[0028] T67P+Q279P+S377P; for example, in RSV-B recombinant F protein Fm 14 as shown in SEQ ID NO:42;

[0029] T67P+L138P+G139P; for example, in RSV-B recombinant F protein Fm 15 as shown in SEQ ID NO:43;

[0030] L138P+G139P+Q279P; for example, in RSV-B recombinant F protein Fm 16 as shown in SEQ ID NO:44;

[0031] L138P+G139P+S377P; for example, in RSV-B recombinant F protein Fm 17 as shown in SEQ ID NO:45;

[0032] T67P+L138P+G139P+Q279P; for example, in RSV-B recombinant F protein Fm18 as shown in SEQ ID NO:46;

[0033] T67P+L138P+G139P+S377P; for example, in RSV-B recombinant F protein Fm19 as shown in SEQ ID NO:47;

[0034] L138P+G139P+Q279P+S377P; for example, in RSV-B recombinant F protein Fm20 as shown in SEQ ID NO:48;

[0035] T67P+L141P+Q279P+S377P; for example, in RSV-B recombinant F protein Fm21 as shown in SEQ ID NO:49;

[0036] T67P+L138P+G139P+Q279P+S377P; for example, in RSV-B recombinant F protein Fm 22 as shown in SEQ ID NO:50;

[0037] T67P+L138P+G139P+L141P+Q279P+S377P; for example, in RSV-B recombinant F protein Fm 23 as shown in SEQ ID NO:51;

[0038] Optionally, the T67P mutation in the above mutations or combinations of mutations can be replaced by a K65P mutation (e.g., RSV-B recombinant F protein Fm24 as shown in SEQ ID NO:52) or a D73P mutation (e.g., RSV-B recombinant F protein Fm25 as shown in SEQ ID NO:53);

[0039] Optionally, the L141P mutation in the above mutations or combinations of mutations can be replaced by the L138P mutation or the G139P mutation;

[0040] Optionally, the above mutations or combinations of mutations may also include one or more mutations selected from the following: N216P mutation, Q210P mutation, I214P mutation, S215P mutation, and E161P mutation.

[0041] For example, in some embodiments, the above mutation is further introduced with the N216P mutation, for example, in the RSV-B recombinant F protein Fm 26-34 as shown in any one of SEQ ID NO:54-62; in other embodiments, the above mutation is further introduced with the Q210P mutation, for example, in the RSV-B recombinant F protein Fm 35 as shown in SEQ ID NO:63; in other embodiments, the above mutation is further introduced with the I214P mutation, for example, in the RSV-B recombinant F protein Fm 36 as shown in SEQ ID NO:64; in other embodiments, the above mutation is further introduced with the S215P mutation, for example, in the RSV-B recombinant F protein Fm 37-38 as shown in SEQ ID NO:65-66; in other embodiments, the above mutation is further introduced with the E161P mutation, for example, in the RSV-B recombinant F protein Fm 39-55 as shown in any one of SEQ ID NO:67-83.

[0042] In some embodiments, the RSV-B-F1 peptide and the RSV-B-F2 peptide are directly linked, for example, in RSV-B recombinant F protein Fm 74-76 (as shown in SEQ ID NO:102-104).

[0043] In other embodiments, the RSV-B-F1 peptide and the RSV-B-F2 peptide are linked by a connecting bridge;

[0044] Preferably, the connecting bridge is selected from:

[0045] (i) (GS)m linker, wherein m = 1-5, preferably 1-3; optionally, it contains one or more amino acid mutations; for example, as shown in SEQ ID NO:9, for example in RSV-B recombinant F protein Fm 69-71 (as shown in SEQ ID NO:97-99), or as shown in SEQ ID NO:10, for example in RSV-B recombinant F protein Fm 72-73 (as shown in SEQ ID NO:100-101);

[0046] (ii) (GGGGS)n linker bridges, wherein n = 1-5, preferably 1-3; optionally, comprising one or more amino acid mutations, such as mutations to proline, addition of amino acids, etc.; in specific embodiments, this type of linker bridge is shown in the following sequences: SEQ ID NO:2 (e.g., in RSV-B recombinant F protein Fm 1-55 (as shown in SEQ ID NO:29-83)), SEQ ID NO:3 (e.g., in RSV-B recombinant F protein Fm 56-59 (as shown in SEQ ID NO:84-87)), SEQ ID NO:4 (e.g., in RSV-B recombinant F protein Fm 60 (as shown in SEQ ID NO:88)), SEQ ID NO:5 (e.g., in RSV-B recombinant F protein Fm 61 (as shown in SEQ ID NO:89)) or SEQ ID NO:6 (e.g., in RSV-B recombinant F protein Fm 62 (as shown in SEQ ID NO:90));

[0047] (iii) The linking sequence between wild-type F1 and F2 peptides, and F0 itself, optionally, is the sequence shown in SEQ ID NO:7 (e.g., in RSV-B recombinant F protein Fm 63-65 (e.g., in SEQ ID NO:91-93));

[0048] (iv) The sequence obtained by mutating the furin restriction site on the linker (iii), optionally, is the sequence shown in SEQ ID NO:8 (e.g., in RSV-B recombinant F protein Fm 66-68 (e.g., SEQ ID NO:94-96)).

[0049] Preferably, the linker comprises, or is composed of, an amino acid sequence selected from, the following: as shown in SEQ ID NO:2-10.

[0050] In a preferred embodiment, the RSV-B recombinant F protein comprises, or consists of, an amino acid sequence selected from the group consisting of, SEQ ID NO:29-104, or an amino acid sequence having the same or substantially the same immunogenicity as the amino acid sequence shown in any of SEQ ID NO:29-104 obtained by substituting, deleting or adding one or more amino acids.

[0051] Furthermore, in some embodiments, the RSV-B recombinant F protein also includes a trimer tag;

[0052] Preferably, the trimer tag is located at the C-terminus and has an amino acid sequence selected from the group consisting of: SEQ ID NO:24-27;

[0053] Optionally, a His tag, as shown in SEQ ID NO:28, can be added to the C-terminus to facilitate subsequent protein isolation and purification.

[0054] Furthermore, in some embodiments, the RSV-B recombinant F protein also includes a signal peptide;

[0055] Preferably, the signal peptide is located at the N-terminus and has an amino acid sequence selected from the group consisting of: SEQ ID NO: 11-23.

[0056] In a second aspect, the present invention provides a polynucleotide that encodes the RSV-B recombinant F protein as described in the first aspect above.

[0057] In a specific implementation, the polynucleotide is a nucleotide sequence optimized with human codons, and can be DNA or mRNA;

[0058] In some embodiments, the polynucleotide is a DNA molecule, preferably comprising, or consisting of, a DNA sequence as shown in one of SEQ ID NO: 105-180.

[0059] In other embodiments, the polynucleotide is an mRNA molecule, preferably comprising, or consisting of, an RNA sequence corresponding to, or composed of, a DNA sequence as shown in one of SEQ ID NO: 105-180.

[0060] Thirdly, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, and optionally, at least one expression regulatory element operatively linked to the polynucleotide.

[0061] Fourthly, the present invention provides an expression vector comprising the nucleic acid construct as described in the third aspect above.

[0062] Fifthly, the present invention provides a host cell wherein the cell is transformed or transfected with the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, or the expression vector as described in the fourth aspect above.

[0063] Optionally, the host cell is a mammalian cell, insect cell, yeast cell, or bacterial cell;

[0064] Further optionally, the mammalian cells are 293T cells, 293F cells, or CHO cells;

[0065] Alternatively, the bacterial cells may be Escherichia coli cells.

[0066] In a sixth aspect, the present invention provides a recombinant F protein trimer of respiratory syncytial virus subtype B (RSV-B), which is composed of three recombinant F proteins of RSV-B as described in the first aspect above.

[0067] In a seventh aspect, the present invention provides the use of the RSV-B recombinant F protein as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above, or the RSV-B recombinant F protein trimer as described in the sixth aspect above in the preparation of a vaccine for the prevention and / or treatment of respiratory syncytial virus infection.

[0068] Eighthly, the present invention provides a vaccine or immunogenic composition comprising the RSV-B recombinant F protein as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above, or the RSV-B recombinant F protein trimer as described in the sixth aspect above, and physiologically acceptable mediators, adjuvants, excipients, carriers, and / or diluents.

[0069] In some preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus recombinant protein vaccine, comprising the RSV-B recombinant F protein as described in the first aspect above or the RSV-B recombinant F protein trimer as described in the sixth aspect above, and an adjuvant;

[0070] Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant, and AS-like adjuvant.

[0071] In some other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus DNA vaccine, comprising:

[0072] (1) Eukaryotic expression vectors; and

[0073] (2) Constructing a DNA sequence encoding the RSV-B recombinant F protein as described in the first aspect above into the eukaryotic expression vector, preferably a DNA sequence as shown in any one of SEQ ID NO:105-180;

[0074] Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

[0075] In some other preferred embodiments, the vaccine or immunogenic composition is a respiratory syncytial virus mRNA vaccine, the mRNA vaccine comprising:

[0076] (I) An mRNA sequence encoding the RSV-B recombinant F protein as described in the first aspect above, preferably an mRNA sequence corresponding to the DNA sequence shown in any one of SEQ ID NO: 105-180; and

[0077] (II) Lipid nanoparticles.

[0078] In another preferred embodiment, the vaccine or immunogenic composition is a respiratory syncytial virus-viral vector vaccine, comprising:

[0079] (1) Viral backbone vector; and

[0080] (2) A DNA sequence encoding the RSV-B recombinant F protein as described in the first aspect above, which is constructed into the viral backbone vector, preferably a DNA sequence as shown in any one of SEQ ID NO:105-180;

[0081] Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

[0082] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;

[0083] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0084] Preferably, the oral preparation is selected from tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated preparations, pellets, sublingual tablets, and ointments;

[0085] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, injectable or bolus-applied preparation.

[0086] Ninthly, the present invention provides a method for preparing recombinant RSV-B F protein as described in the first aspect above, characterized in that the preparation method comprises:

[0087] The codon-optimized nucleotide sequence encoding the RSV-B recombinant F protein as described in the first aspect above is coupled with a nucleotide sequence encoding a signal peptide at the 5' end and a nucleotide sequence encoding a trimer tag and a histidine tag and a stop codon at the 3' end. The recombinant protein is then cloned and expressed. Correct recombinants are screened, and then transfected into expression system cells for expression. The cell culture supernatant is collected, and the RSV-B recombinant F protein is isolated from it.

[0088] In one possible implementation of the above method, the expression system cells are mammalian cells, insect cells, yeast cells, or bacterial cells; optionally, the mammalian cells are 293T cells, 293F cells, or CHO cells; optionally, the bacterial cells are Escherichia coli cells.

[0089] Beneficial effects

[0090] When natural RSV-B virus F protein antigen is recombinantly expressed in vitro, the pre-fusion conformation of the F antigen protein is unstable, making it impossible to obtain the pre-fusion conformation F antigen protein. In this invention, by introducing one or more proline mutations into the F1 and / or F2 peptide segments of the respiratory syncytial virus type B F protein, a stable pre-fusion conformation RSV-B recombinant F protein of this invention is formed. Experiments show that the formed RSV-B recombinant F protein contains at least one specific epitope of the pre-fusion F protein, and its expression is stable, uniform, and its yield is greatly improved. Furthermore, the RSV-B recombinant F protein of this invention has excellent immunogenicity, capable of stimulating the body to produce high levels of antibody titers (after booster immunization with the recombinant protein without adjuvant, the induced antigen-specific antibody titers all reach over 10,000, while with the addition of AddaVax adjuvant, the antigen-specific antibody titers can be further increased by 10-100 times), which is of great significance for the clinical treatment and prevention of respiratory syncytial virus. Attached Figure Description

[0091] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0092] Figure 1 In Example 3, the expression of RSV-B recombinant F protein in the supernatant was detected by ELISA using the D25 monoclonal antibody. The x-axis represents the experimental group (i.e., the RSV-B recombinant F protein detected), and the y-axis represents the OD450 nm absorbance value.

[0093] Figure 2 In Example 3, palizumab was used to detect the expression of RSV-B recombinant F protein in the supernatant using ELISA. The x-axis represents the experimental group (i.e., the RSV-B recombinant F protein detected), and the y-axis represents the absorbance at OD450nm.

[0094] Figure 3In Example 3, the expression of some RSV-B recombinant F protein in the diluted supernatant was detected by ELISA using the D25 monoclonal antibody. The horizontal axis represents the Log10 value of the dilution factor of the cell supernatant, and the vertical axis represents the absorbance value at OD450 nm. The right-hand caption shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0095] Figure 4 In Example 3, palizumab was used to detect the expression of some RSV-B recombinant F protein in the diluted supernatant by ELISA. The horizontal axis represents the Log10 value of the dilution factor of the cell supernatant, and the vertical axis represents the absorbance value at OD450nm. The right-hand caption shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0096] Figure 5 The image shows the results of ultracentrifugation analysis of the purified RSV-B recombinant F protein Fm41 expression supernatant by molecular sieve chromatography and the purified protein in Example 4. The left image is the UV absorption spectrum of the RSV-B recombinant F protein Fm41 expression supernatant during molecular sieve chromatography purification, and the right image is the ultracentrifugation analysis results of the purified RSV-B recombinant F protein Fm41.

[0097] Figure 6 The image shows the UV absorption spectrum of the expression supernatant of RSV-B recombinant F protein Fm23 during purification by molecular sieve chromatography in Example 4.

[0098] Figure 7 The image shows the UV absorption spectrum of the expression supernatant of RSV-B recombinant F protein Fm54 during purification by molecular sieve chromatography in Example 4.

[0099] Figure 8 In Example 6, the titer level of specific antibody IgG in the serum of mice after primary and booster immunization using RSV-B recombinant F protein Fm 41 as an immunogen, as described in Example 5, was detected by ELISA. The horizontal axis represents the grouping in Table 1, and the vertical axis represents the antibody titer.

[0100] Figure 9 In Example 8, D25, AM14, parizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm1-10 and WT recombinant F proteins in diluted supernatants and the binding of each recombinant F protein to the aforementioned antibodies using ELISA. The horizontal axis represents the Log10 value of the cell supernatant dilution factor, and the vertical axis represents the OD450 nm absorbance value. The right-hand figure shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0101] Figure 10 In Example 8, D25, AM14, parizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 11-20 recombinant F protein in diluted supernatants and the binding of each recombinant F protein to the aforementioned antibodies using ELISA. The horizontal axis represents the Log10 value of the cell supernatant dilution factor, and the vertical axis represents the OD450 nm absorbance value. The right-hand figure shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0102] Figure 11 In Example 8, D25, AM14, parizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 21-30 recombinant F protein in diluted supernatants and the binding of each recombinant F protein to the aforementioned antibodies using ELISA. The horizontal axis represents the Log10 value of the cell supernatant dilution factor, and the vertical axis represents the OD450 nm absorbance value. The right-hand figure shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0103] Figure 12 In Example 8, D25, AM14, parizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 31-40 recombinant F protein in diluted supernatants and the binding of each recombinant F protein to the aforementioned antibodies using ELISA. The horizontal axis represents the Log10 value of the cell supernatant dilution factor, and the vertical axis represents the OD450 nm absorbance value. The right-hand figure shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0104] Figure 13 In Example 8, D25, AM14, parizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 41-50 recombinant F protein in diluted supernatants and the binding of each recombinant F protein to the aforementioned antibodies using ELISA. The horizontal axis represents the Log10 value of the cell supernatant dilution factor, and the vertical axis represents the OD450 nm absorbance value. The right-hand figure shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0105] Figure 14In Example 8, D25, AM14, parizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 51-60 recombinant F protein in diluted supernatants and the binding of each recombinant F protein to the aforementioned antibodies using ELISA. The horizontal axis represents the Log10 value of the cell supernatant dilution factor, and the vertical axis represents the OD450 nm absorbance value. The right-hand figure shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0106] Figure 15 In Example 8, D25, AM14, parizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 61-70 recombinant F protein in diluted supernatants and the binding of each recombinant F protein to the aforementioned antibodies using ELISA. The horizontal axis represents the Log10 value of the cell supernatant dilution factor, and the vertical axis represents the OD450 nm absorbance value. The right-hand figure shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0107] Figure 16 In Example 8, D25, AM14, parizumab, MPE8, 101F, and hRSV90 monoclonal antibodies were used to detect the expression of RSV-B-Fm 71-76 recombinant F protein in diluted supernatants and the binding of each recombinant F protein to the aforementioned antibodies using ELISA. The horizontal axis represents the Log10 value of the cell supernatant dilution factor, and the vertical axis represents the OD450 nm absorbance value. The right-hand figure shows the experimental group (i.e., the RSV-B recombinant F protein detected).

[0108] Figure 17 The SDS-PAGE results of the isolated and purified RSV-B-Fm recombinant F proteins 36, 37, 38, 52, 53, 70, 75, and 76 in Example 9 are shown.

[0109] Figure 18 The Western Blot results of the isolated and purified RSV-B-Fm recombinant F proteins 36, 37, 38, 52, 53, 70, 75, and 76 are shown in Example 9.

[0110] Figure 19 In Example 10, D25 monoclonal antibody was used to detect the binding of RSV-B-Fm 36, 37, 52, 53, 75, and 76 recombinant F proteins to the antibody before and after storage at 30°C for 4 weeks using ELISA. The x-axis represents protein concentration, and the y-axis represents the absorbance at OD450 nm.

[0111] Figure 20In Example 10, the binding of RSV-B-Fm 36, 37, 52, 53, 75, and 76 recombinant F proteins to the antibody was detected by ELISA before and after storage at 30°C for 4 weeks using parizumab. The x-axis represents protein concentration, and the y-axis represents the absorbance at OD450 nm.

[0112] Figure 21 In Example 12, the titer level of specific binding antibody IgG in the serum of mice after primary immunization (A) and booster immunization (B) using RSV-B-Fm23, 36, 37, 38, 41, 52, 53, 54, 70, 75, and 76 recombinant F proteins as immunogens, as described in Example 11, was detected by ELISA. The horizontal axis represents the vaccine group, and the vertical axis represents the antibody titer.

[0113] Figure 22 In Example 13, the viral load in the lung tissue of mice after immunization with the vaccine described in Example 11 and challenged with respiratory syncytial virus via intranasal droplets was detected by plaque assay. The horizontal axis represents the vaccine group, and the vertical axis represents the viral load. Detailed Implementation

[0114] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0115] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0116] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0117] Example 1: Construction of expression plasmid for RSV-B recombinant F protein of the present invention

[0118] In this embodiment, 76 RSV-B recombinant F proteins of the present invention were designed and constructed, which were named RSV-B-Fm 1-76, and their amino acid sequences are shown in SEQ ID NO:29-104, respectively.

[0119] Based on the codon preference of mammalian cells, the nucleic acid sequence encoding the RSV-B recombinant F protein—RSV-B-Fm 1-76 of the present invention was optimized to obtain optimized nucleic acid coding sequences, as shown in SEQ ID NO:105-180. An EcoRI restriction site sequence, a Kozak sequence, and a signal peptide (as shown in SEQ ID NO:11-23) were added to the 5' end of the nucleic acid coding sequence shown in SEQ ID NO:105-180. A trimer tag (as shown in SEQ ID NO:24-27), a His tag (as shown in SEQ ID NO:28), a stop codon, and an XhoI restriction site sequence were added to the 3' end of the sequence. The gene was then synthesized by Genscript Biotech Co., Ltd., and ligated into the pCAGGS vector via the EcoRI and XhoI restriction sites to obtain an expression plasmid expressing the recombinant protein RSV-B-Fm 1-76.

[0120] The signal peptides and trimer tags used in each RSV-B recombinant F protein are as follows:

[0121] Fm 1-37 (amino acid sequences as shown in SEQ ID NO:29-65, and optimized nucleic acid coding sequences as shown in SEQ ID NO:105-141) and Fm 39-76 (amino acid sequences as shown in SEQ ID NO:67-104, and optimized nucleic acid coding sequences as shown in SEQ ID NO:143-180) both use the signal peptide shown in SEQ ID NO:14 and the trimer tag shown in SEQ ID NO:24;

[0122] Fm 38 (amino acid sequence as shown in SEQ ID NO:66, optimized nucleic acid coding sequence as shown in SEQ ID NO:142) uses the signal peptide shown in SEQ ID NO:12 and the trimer tag shown in SEQ ID NO:25.

[0123] Example 2: Detection of antibody expression and isolation / purification

[0124] In this embodiment, the heavy and light chain sequences of antibodies and their expression plasmids were constructed according to the methods disclosed in the literature (Z.Wei et al., Analytical Chemistry 79, 2797-2805 (2007); Q.Zhu et al., Science Translational Medicine 9, (2017)).

[0125] antibody expression

[0126] 14-16 hours before transfection, 293T cells with high density were divided into plates (e.g., a 10cm culture dish 100% filled with 293T cells was passaged at a 1:3 ratio). After 14-16 hours, when the cell density reached more than 70%, transfection could be performed. During transfection, the heavy chain and light chain plasmids of the antibody were co-transfected into 293T cells at a ratio of 2:3. 4-6 hours after transfection, the cells were washed twice with PBS and cultured in serum-free DMEM medium. Cell supernatants were collected on days 3 and 7 post-transfection, centrifuged to remove cell debris, and the antibody supernatants obtained from the two transfections were mixed for subsequent antibody protein purification.

[0127] Antibody purification

[0128] Connect a Protein A (5 ml) HP affinity column (GE) to an AKTAPurifier / Explorer / FPLC / START (GE). The procedure on the instrument is as follows: First, flush the column with water to remove 20% ethanol. Then, equilibrate the column with 20 mM Na3PO4, pH 7.0 buffer. Once the conductivity on the instrument is stable, inject the antibody supernatant through a 10 ml loop at a flow rate of 2 ml / min to bind with Protein A. After the UV stabilizes, add approximately 0.8 ml of 1 M Tris pH 9.0 buffer (collection volume approximately 3.2 ml) to the subsequent collection tube. Then, change the program to 100% 0.1 M Gly pH 3.0 to elute the antibody bound to the column. Collect the eluent and then replace the antibody buffer with PBS using a concentration and buffer replacement method. The resulting antibody solution can be used directly or aliquoted and stored at -80°C for later use.

[0129] Example 3: Expression and conformational identification of RSV-B-Fm recombinant protein

[0130] In this embodiment, HEK293T was transfected with the partial RSV-B-Fm recombinant protein (Fm 12-76) expression plasmid constructed in Example 1 to express RSV-B-Fm recombinant protein. The expression of the recombinant protein was then detected by ELISA using the detection antibodies D25 monoclonal antibody and palizumab prepared in Example 2 to determine the conformation of the expressed recombinant protein.

[0131] Specifically, HEK293T cells were cultured in DMEM medium containing 10% FBS, and the cell density was increased to over 70% before transfection. HEK293T cells were transfected with the RSV-B-Fm recombinant protein Fm 12-76 expression plasmid constructed in Example 1. Four to six hours after transfection, the cell culture medium was replaced with serum-free DMEM, and the cells were cultured for another 3 days. The cell culture supernatant was collected, and antigen protein expression was detected by ELISA using D25 monoclonal antibody and palizumab, respectively. The specific detection methods are as follows:

[0132] (1) The purified detection antibodies (D25 monoclonal antibody and palizumab) obtained in Example 2 were diluted to 1 μg / ml with ELISA coating solution (Solepro, C1050), and 100 μl was added to each well of a 96-well ELISA plate (Coring, 3590) and incubated at 4°C for 12 hours.

[0133] (2) Discard the coating solution, add PBS, and wash once; add 200 μl of 5% skim milk prepared with PBS as the blocking solution to the 96-well plate, block, and let stand at room temperature for 1 hour; after blocking, wash once with PBS solution.

[0134] (3) During the blocking period described in step (2), the cell culture supernatant was diluted with blocking solution, starting from 5-fold and then diluted in 3-fold increments; then, 100 μl of the original culture supernatant of each RSV-B recombinant F protein to be tested or a portion of the culture supernatant of each dilution of RSV-B recombinant F protein (Fm 22, 23, 41, 51, 52, 53, 54, 57) was added to each well of the ELISA plate. The negative control was to add blocking solution and incubate at 37 degrees for 2 hours, and then wash 4 times with PBST.

[0135] (4) Add HRP-labeled Anti-His antibody (purchased from MBL), incubate at 37°C for 1.5 hours, then wash with PBST 5-6 times; then add TMB chromogenic solution for color development, and after an appropriate reaction time, add 2M hydrochloric acid to terminate the reaction, and detect the OD450 value on an ELISA reader.

[0136] The expression results of each RSV-B recombinant F protein in the supernatant stock solution are as follows: Figure 1 and 2 As shown, from Figure 1 and 2The results show that, compared with NC and wild-type RSV-B F proteins, the expression of all tested RSV-B-Fm recombinant proteins was significantly increased; furthermore, all tested RSV-B-Fm recombinant proteins could bind to both palizumab (which recognizes both the pre-fusion and post-fusion conformations of the F protein) and D25 monoclonal antibody (which only recognizes the pre-fusion conformation of the F protein), indicating that the RSV-B-Fm recombinant protein of the present invention is in the pre-fusion conformation.

[0137] The expression results of RSV-B recombinant F protein Fm 22, 23, 41, 51, 52, 53, 54, and 57 in the serially diluted supernatant are as follows: Figure 3 and 4 As shown, from Figure 3 and 4 The results show that when the supernatant of RSV-B-Fm cells 22, 23, 41, 51, 52, 53, 54, and 57 was diluted 1000-fold, the protein could still be detected by either D25 monoclonal antibody or palizumab, indicating that RSV-B-Fm cells 22, 23, 41, 51, 52, 53, 54, and 57 have high expression levels.

[0138] Example 4: Expression, purification and molecular weight identification of RSV-B-Fm recombinant protein

[0139] In this embodiment, the RSV-B-Fm 41, 23, and 54 recombinant protein expression plasmid constructed in Example 1 was transfected into HEK293T to express the RSV-B-Fm 41, 23, and 54 recombinant proteins. The proteins were purified by His affinity chromatography and gel filtration chromatography (also known as "molecular sieve chromatography"). The target proteins were collected and analyzed by ultracentrifugation to determine the molecular weight of the expressed recombinant proteins.

[0140] Specifically, HEK293T cells were cultured in DMEM medium containing 10% FBS, and the cell density was increased to over 70% before transfection. HEK293T cells were transfected with the RSV-B-Fm 41, 23, and 54 recombinant protein expression plasmids constructed in Example 1. 4-6 hours after transfection, the cell culture medium was replaced with serum-free DMEM, and the cells were cultured for another 3 days. After collecting the supernatant, DMEM medium was added again, and the cells were cultured for another 4 days. The supernatant was collected again. The cell culture supernatants collected twice were mixed and centrifuged at 5000 rpm for 30 minutes. The supernatant was filtered through a 0.22 μm filter membrane to bind the target protein to a HisTrap excel column (5 mL, GE Healthcare). Non-specifically bound proteins were then eluted with an elution buffer containing 20 mM Tris, 150 mM NaCl, pH 8.0, and 30 mM imidazole. The target protein was then eluted again with an elution buffer containing 20 mM Tris, 150 mM NaCl, pH 8.0, and 400 mM imidazole. The fraction containing the target protein was collected, concentrated, and subjected to molecular sieve chromatography (Superdex 200 Increase 10 / 300GL or Superdex 200 Hiload 16 / 60, GE Healthcare) to obtain purified RSV-B-Fm recombinant protein antigen. Simultaneously, the target protein peak was collected for analytical ultracentrifugation to determine the molecular weight of the expressed recombinant protein.

[0141] The UV absorption spectrum of molecular sieve chromatography for recombinant protein RSV-B-Fm 41 is shown below. Figure 5 As shown in the left figure, the elution peak of recombinant protein RSV-B-Fm 41 contains not only the target protein peak but also a small amount of multimer impurity peaks. The target protein peak was collected and analyzed by ultracentrifugation, and the results are as follows. Figure 5 As shown in the right figure; by Figure 5 As shown in the right figure, the measured molecular weight of RSV-B-Fm 41 is 170 kDa, which is consistent with its theoretical molecular weight as a trimer, indicating that the expressed RSV-B-Fm 41 recombinant protein is in trimer form.

[0142] The UV absorption spectra of RSV-B-Fm 23 and 54 molecular sieve chromatography are as follows: Figure 6 , 7 As shown, by Figure 6 , 7 It can be seen that the elution peak of RSV-B-Fm 23 contains not only the target protein peak but also a small amount of polymer impurity peaks, while the elution peak of RSV-B-Fm 54 is almost a single peak with virtually no impurity peaks.

[0143] Example 5: RSV-B-Fm recombinant protein immunization experiment in mice

[0144] In this embodiment, mice were immunized with the recombinant protein RSV-B-Fm 41 obtained in Example 4; the experimental mice were 4-6 week old BALB / c mice with an average weight of 15-20g.

[0145] Specifically, with or without adjuvant, the BALB / c mice were immunized with the recombinant protein RSV-B-Fm41 obtained in Example 4, with the adjuvant being an MF59-like adjuvant—AddaVax; at the same time, the mice were immunized with the same dose of physiological saline as a negative control.

[0146] The immunization groupings, immunogens used in each group, immunogen dosages used in each group, and adjuvant information are shown in Table 1. Blank cells indicate "none". The RSV-B-Fm 41 recombinant protein was diluted in physiological saline to the required concentration. The adjuvant-added groups required further emulsification with the adjuvant in separate groups. Each group consisted of 6 mice.

[0147] Table 1

[0148]

[0149] Mice in each group were injected intramuscularly with recombinant protein vaccine or saline on days 0 and 14, with each immunization volume being 100 μl. Blood was collected from the tail vein on days 13 and 28. After the mouse blood was allowed to stand, it was centrifuged at 3000 rpm for 10 minutes to obtain serum, which was further inactivated (incubated at 56℃ for 30 minutes) and then aliquoted and stored at -80℃.

[0150] Example 6: ELISA assay for detecting vaccine-induced specific antibody titers

[0151] In this embodiment, the titer of specific IgG antibodies in the serum of mice immunized with RSV-B-Fm 41 recombinant protein in Example 5 was detected by ELISA.

[0152] Specifically, the following procedure is adopted:

[0153] (1) The RSV-B-Fm 41 recombinant protein prepared in Example 4 was diluted to 3 μg / ml with ELISA coating buffer (Solepro, C1050), and 100 μl was added to each well of a 96-well ELISA plate (Coring, 3590) and incubated at 4°C for 12 hours.

[0154] (2) Discard the coating solution, add PBS, and wash once; add 5% skim milk prepared with PBS as the blocking solution to the 96-well plate, 100 μl per well, and let it stand at room temperature for 1 hour to block; after blocking, wash once with PBS solution.

[0155] (3) During the blocking period described in step (2), mouse serum samples were diluted with blocking solution, starting from 10-fold and then diluted in 2-fold increments; then, 100 μl of immune serum dilution solution was added to each well of the ELISA plate, and the negative control was to add blocking solution. The plates were incubated at 37 degrees for 2 hours, and then washed 4 times with PBST.

[0156] (4) Add HRP-conjugated goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:2000 with blocking buffer, incubate at 37°C for 1 hour, then wash 5-6 times with PBST; add TMB chromogenic solution for color development, and after an appropriate reaction time, add 2M hydrochloric acid to terminate the reaction, and detect the OD450 value on an ELISA reader.

[0157] Antibody titer is defined as the highest dilution of serum with a reaction value greater than 2.1 times the negative control value. When the reaction value at the lowest dilution (limit of detection) is still less than 2.1 times the background value, the titer of the sample is defined as half of the lowest dilution, i.e., 1:5.

[0158] The results are as follows Figure 8 As shown, Figure 8 The results showed that the RSV-B-Fm 41 recombinant protein had good immunogenicity. The antigen-specific antibody titers induced by booster immunization with the recombinant protein without adjuvant reached over 10,000, while the antigen-specific antibody titers could be further increased by 10-100 times after the addition of AddaVax adjuvant.

[0159] Example 7: Detection of antibody expression and isolation / purification

[0160] Following the antibody heavy chain and light chain sequences and their expression plasmid construction methods disclosed in the literature (Jones HG., et al. PLoS Pathog 15(7):e1007944(2019); Harshbarger, W., et al. Mabs 13(1):1955812(2021); Wen, X., et al. Nat Microbiol 2,16272(2017); Fabian Sesterhenn., et al. Science 368,eaay5051(2020); Mousa, J., et al. Nat Microbiol 2,16271(2017)), heavy chain and light chain expression plasmids of the detection antibodies for RSV F protein—AM14, MPE8, 101F and hRSV90 monoclonal antibodies—were constructed respectively.

[0161] The specific methods for antibody expression, separation and purification are as described in Example 2. The final antibody solution can be used directly or aliquoted and stored at -80°C for later use.

[0162] Example 8: Expression and conformational identification of RSV-B-Fm recombinant protein

[0163] In this embodiment, all RSV-B-Fm recombinant protein expression plasmids (Fm 1-76) constructed in Example 1 were transfected into HEK293T to express RSV-B-Fm recombinant proteins. The expression levels and conformations of the recombinant proteins were determined by ELISA using the detection antibodies prepared in Examples 2 and 7. The specific method is as described in Example 3.

[0164] The expression results of each RSV-B recombinant F protein in the supernatant stock solution are as follows: Figure 9-16 As shown, by Figure 9-16 It was found that, compared with wild-type RSV-B F protein, the expression of recombinant RSV-B-Fm 2, 6, 11-20, 21-24, 29, 31, 33-37, 39, 40, 41-45, 51-53, 55-60, 61-65, 69, 70, and 71-76 F proteins was significantly increased. Furthermore, these recombinant F proteins could bind to monoclonal antibodies corresponding to the pre-fusion and post-fusion common epitope II (i.e., palizumab) and the common epitope IV (i.e., 101F monoclonal antibody), as well as to pre-fusion conformation-specific epitopes such as epitope 2. The corresponding monoclonal antibody (i.e., D25 monoclonal antibody), the corresponding monoclonal antibody for the trimer-dependent spatial conformation epitope (i.e., AM14 monoclonal antibody), the corresponding monoclonal antibody for epitope V (i.e., hRSV90 monoclonal antibody), or the corresponding monoclonal antibody for epitope III (i.e., MPE8 monoclonal antibody) indicates that the RSV-B-Fm 2, 6, 11-20, 21-24, 29, 31, 33-40, 41-45, 51-60, 61-65, 69, 70, 71-76 recombinant F proteins of the present invention are in their pre-fusion conformation.

[0165] Example 9: Expression, purification and SDS-PAGE identification of RSV-B-Fm recombinant protein

[0166] In this embodiment, the RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, 76 recombinant protein expression plasmid constructed in Example 1 was transfected into HEK293T to express the RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, 76 recombinant proteins. The proteins were then purified by His affinity chromatography and gel filtration chromatography (also known as "molecular sieve chromatography"). The specific operation steps are as described in Example 4.

[0167] The target protein peak was collected after gel filtration chromatography and analyzed by SDS-PAGE. The results are as follows: Figure 17 As shown, Figure 17The results showed that the recombinant proteins RSV-B-Fm 36, 37, 38, 52, 53, 70, 75, and 76 all exhibited a distinct protein band, indicating that they all have high purity and molecular weights between 55 kDa and 70 kDa, which is consistent with the theoretical molecular weight.

[0168] Furthermore, since each RSV-B-Fm recombinant F protein is a histidine-tagged recombinant protein, this embodiment also used an anti-histidine-tagged antibody conjugated with horseradish peroxidase for Western blotting identification, and the results are as follows. Figure 18 As shown; Figure 18 The results showed that histidine tag expression was detected in the collected solutions of each RSV-B-Fm recombinant F protein, indicating that the expression of these RSV-B-Fm recombinant F proteins was consistent with expectations and was correctly expressed.

[0169] Example 10: Stability detection of RSV-B-Fm recombinant protein

[0170] The RSV-B-Fm recombinant proteins 36, 37, 52, 53, 75, and 76 obtained in Examples 4 and 9 were sterile filtered and stored at 30°C. After 4 weeks, they were analyzed by ELISA using the D25 monoclonal antibody (targeting the epitope). The binding of antigen and antibody (pelizumab targeting epitope II) to the protein was determined by ELISA to assess the long-term stability of the protein during storage. The specific detection method is as follows:

[0171] (1) The purified antibodies (palizumab and D25 monoclonal antibody) obtained in Examples 2 and 8 were diluted to 1 μg / ml with ELISA coating solution (Solepro, C1050), and 100 μl was added to each well of a 96-well ELISA plate (Corning, 3590) and incubated at 4°C for 12 hours.

[0172] (2) Discard the coating solution, add PBS, and wash once; add 200 μl of 5% skim milk prepared with PBS as the blocking solution to the 96-well plate, block, and let stand at room temperature for 1 hour; after blocking, wash once with PBS solution.

[0173] (3) During the blocking process described in step (2), RSV-B-Fm recombinant protein was diluted with blocking buffer, starting from 300 ng / ml and then diluted in a 3-fold gradient. After that, 100 μl of serially diluted RSV-B-Fm recombinant protein was added to each well of the ELISA plate. The negative control was to add blocking buffer and incubate at 37 degrees for 2 hours, followed by washing with PBST 4 times.

[0174] (4) Add HRP-labeled Anti-His antibody (purchased from MBL), incubate at 37°C for 1.5 hours, then wash with PBST 5-6 times; then add TMB chromogenic solution for color development, and after an appropriate reaction time, add 2M hydrochloric acid to terminate the reaction, and detect the OD450 value on an ELISA reader.

[0175] The ELISA results using D25 monoclonal antibody and palizumab are as follows: Figure 19 and 20 As shown, Figure 19 and 20 The results showed that, compared with the initial proteins at week 0, the binding of each RSV-B-Fm recombinant protein (36, 37, 52, 53, 75, 76) to D25 antibody and parizumab did not change significantly after being stored at 30°C for 4 weeks, indicating that these RSV-B-Fm recombinant proteins have good stability.

[0176] Example 11: RSV-B-Fm recombinant protein immunization experiment in mice

[0177] Mice were immunized with the recombinant RSV-B-Fm proteins 23, 36, 37, 38, 41, 52, 53, 70, 75, and 76 obtained in Examples 4 and 10. The experimental mice were 6-8 week old BALB / c mice with an average weight of 15-20g.

[0178] Specifically, mice were immunized with the aforementioned RSV-B-Fm recombinant F protein at a dose of 12 μg each time. The adjuvant was a combination of aluminum hydroxide adjuvant (purchased from Croda) and CpG (purchased from Invivogen). The vaccine preparation method was as follows: each RSV-B-Fm recombinant protein was diluted in physiological saline to the required concentration. The aluminum hydroxide adjuvant was first mixed with the antigen protein, and then mixed with the CpG adjuvant. The placebo group was given physiological saline. Four 6-8 week old BALB / c mice (average weight 15-20g) were used in each group.

[0179] Mice in each group were injected intramuscularly with recombinant protein vaccine or saline on days 0 and 14, with each immunization volume being 100 μl. Blood was collected from the tail vein on days 13 and 28. After the mouse blood was allowed to stand, it was centrifuged at 3000 rpm for 10 minutes to obtain serum, which was further inactivated (incubated at 56℃ for 30 minutes) and then aliquoted and stored at -80℃.

[0180] Example 12: ELISA assay for detecting antigen-specific antibody titers induced by vaccine

[0181] In this embodiment, the titer (Log antibody titer) of specific IgG antibodies in the serum of mice after primary and secondary immunization with the RSV-B-Fm 23, 36, 37, 38, 41, 52, 53, 70, 75, 76 recombinant protein vaccines from Example 11 was detected by ELISA. The specific method and the definition of antibody titer values ​​are the same as in Example 6.

[0182] The specific IgG antibody titers in the serum of mice after primary and secondary immunizations are as follows: Figure 21 A and Figure 21 As shown in B, Figure 21 Figures A and 21B show that all RSV-B-Fm recombinant F protein vaccines induced antigen-specific antibody titers of over 10,000 after the first immunization, and the titers induced after the second immunization could be further increased to over 1,000,000, indicating that RSV-B-Fm recombinant F protein vaccines 23, 36, 37, 38, 41, 52, 53, 70, 75, and 76 all have excellent immunogenicity.

[0183] Example 13: Evaluation of the protective effect of vaccine against viral challenge

[0184] For mice immunized with the vaccines in Example 11, respiratory syncytial virus (RSV) Long strain was challenged via nasal drops in week 4 following the second immunization, at a dose of 10^4 PFU / 50 μl / mouse. On day 5 post-challenge, the mice were euthanized, and their lungs were harvested. The lungs of four mice in each group were weighed and recorded, then added to DMEM medium and homogenized using a tissue homogenizer. The lung tissue supernatant was obtained after centrifugation at 5000 g / min for 10 minutes. The viral load of RSV in the lung tissue supernatant was detected using a plaque assay, and the viral copy number per gram of lung tissue was calculated.

[0185] The specific method for the plaque assay is as follows: Prepare BHK cells in a 12-well plate one day in advance, with 1*10 cells per well. 5For each cell, the starting well was a 10-fold dilution of the original tissue solution, i.e., 50 μl of lung tissue supernatant was added to 450 μl of DMEM medium and mixed well. This was followed by six 10-fold serial dilutions. Cells were washed twice with PBS. 400 μl of the diluted lung tissue supernatant was used to infect cells per well and incubated at 37°C for 2 hours. Cells were then washed with PBS, and 1 ml of a mixture of 2% sodium carboxymethyl cellulose and 2X DMEM was added to each well. The cells were incubated at 37°C for 4 days. Cells were washed once with PBS, fixed with methanol at room temperature for 10 minutes, washed once with PBS, and then blocked at 37°C for 1 hour with PBST containing 5% skim milk. Palizumab diluted with 5% skim milk was added and incubated at 37°C for 1 hour. Cells were washed three times with PBST (PBS with 0.05% Tween 20 added), and then horseradish peroxidase-labeled goat anti-human IgG secondary antibody (purchased from Beyotime Biotechnology) was added and incubated at 37°C for 1 hour. Cells were washed three times with PBST (PBS with 0.05% Tween 20 added), and then AEC substrate (purchased from BD Biosciences) was added for reaction. Brown spots were identified as positive spots, and the PFU value of each sample was counted.

[0186] The results are as follows Figure 22 As shown, Figure 22 The results showed that high titers of respiratory syncytial virus (RSV) could be detected in the lung tissue of the placebo immunization group after vaccine challenge, while no virus could be detected in any of the vaccine immunization groups. This indicates that the RSV-B-Fm 23, 36, 37, 38, 41, 52, 53, 70, 75, and 76 recombinant protein vaccines can all protect the body from RSV infection and produce a good protective effect.

[0187] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A recombinant F protein of respiratory syncytial virus subtype B, characterized in that, The amino acid sequence of the recombinant F protein of respiratory syncytial virus subtype B is selected from: SEQ ID NO:29-50, 52-63, 67-68, 70-79, 82-97, 99-102.

2. The recombinant F protein of respiratory syncytial virus subtype B according to claim 1, characterized in that, The recombinant F protein of respiratory syncytial virus subtype B also includes a trimer tag; Preferably, the trimer tag is located at the C-terminus and has an amino acid sequence selected from the group consisting of: SEQ ID NO: 24-27; And / or, the recombinant F protein of respiratory syncytial virus subtype B further includes a signal peptide; Preferably, the signal peptide is located at the N-terminus and has an amino acid sequence selected from the group consisting of: SEQ ID NO:11-23.

3. A polynucleotide encoding the recombinant F protein of respiratory syncytial virus subtype B as described in claim 1 or 2.

4. The polynucleotide according to claim 3, characterized in that, The polynucleotide is a DNA molecule or an mRNA molecule; Preferably, the DNA molecule comprises, or is composed of, a DNA sequence as shown in one of SEQ ID NO: 105-126, 128-139, 143-144, 146-155, 158-173, 175-178; Preferably, the mRNA molecule comprises, or is composed of, an RNA sequence corresponding to, or consisting of, a DNA sequence as shown in one of SEQ ID NO: 105-126, 128-139, 143-144, 146-155, 158-173, or 175-178.

5. A nucleic acid construct comprising a polynucleotide as described in claim 3 or 4, and optionally, at least one expression regulatory element operatively linked to said polynucleotide.

6. An expression vector comprising the nucleic acid construct as described in claim 5.

7. A host cell wherein the cell is transformed or transfected with the polynucleotide of claim 3 or 4, the nucleic acid construct of claim 5, or the expression vector of claim 6; Optionally, the host cell is a mammalian cell, insect cell, yeast cell, or bacterial cell; Further optionally, the mammalian cells are 293T cells, 293F cells, or CHO cells; Alternatively, the bacterial cells may be Escherichia coli cells.

8. A type B respiratory syncytial virus (RSV) recombinant F protein trimer, which is composed of three recombinant F proteins of RSV subtype B as described in claim 1 or 2.

9. The use of the recombinant F protein of respiratory syncytial virus subtype B as described in claim 1 or 2, the polynucleotide as described in claim 3 or 4, the nucleic acid construct as described in claim 5, the expression vector as described in claim 6, the host cell as described in claim 7, or the recombinant F protein trimer of respiratory syncytial virus type B as described in claim 8 in the preparation of a vaccine for the prevention and / or treatment of respiratory syncytial virus infection.

10. A vaccine or immunogenic composition comprising the recombinant F protein of respiratory syncytial virus subtype B as claimed in claim 1 or 2, the polynucleotide as claimed in claim 3 or 4, the nucleic acid construct as claimed in claim 5, the expression vector as claimed in claim 6, the host cell as claimed in claim 7 or the recombinant F protein trimer of respiratory syncytial virus type B as claimed in claim 8, and physiologically acceptable mediators, adjuvants, excipients, carriers and / or diluents.

11. The vaccine or immunogenic composition according to claim 10, which is a recombinant respiratory syncytial virus (RSV) protein vaccine, comprising the recombinant F protein of RSV subtype B as described in claim 1 or 2 or the recombinant F protein trimer of RSV type B as described in claim 8 and an adjuvant. Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant, and AS-like adjuvant.

12. The vaccine or immunogenic composition according to claim 10, wherein it is a respiratory syncytial virus DNA vaccine, the DNA vaccine comprising: (i) Eukaryotic expression vectors; and (ii) Constructing a DNA sequence encoding the recombinant F protein of respiratory syncytial virus subtype B as described in claim 1 or 2 into the eukaryotic expression vector, preferably a DNA sequence as shown in one of SEQ ID NO: 105-126, 128-139, 143-144, 146-155, 158-173, 175-178; Preferably, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

13. The vaccine or immunogenic composition according to claim 10, wherein it is a respiratory syncytial virus mRNA vaccine, the mRNA vaccine comprising: (I) An mRNA sequence encoding the recombinant F protein of respiratory syncytial virus subtype B as described in claim 1 or 2, preferably an mRNA sequence corresponding to a DNA sequence as shown in any one of SEQ ID NO: 105-126, 128-139, 143-144, 146-155, 158-173, 175-178; and (II) Lipid nanoparticles.

14. The vaccine or immunogenic composition according to claim 10, which is a respiratory syncytial virus viral vector vaccine, comprising: (1) Viral backbone vector; and (2) A DNA sequence encoding the recombinant F protein of respiratory syncytial virus subtype B as described in claim 1 or 2, which is constructed into the viral backbone vector, preferably a DNA sequence as shown in one of SEQ ID NO: 105-126, 128-139, 143-144, 146-155, 158-173, 175-178; Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

15. The vaccine or immunogenic composition according to any one of claims 10-14, characterized in that, The vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation; Preferably, the nasal spray is selected from aerosols, sprays, and powders; Preferably, the oral preparation is selected from tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated preparations, pellets, sublingual tablets, and ointments; Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, injectable or bolus-applied preparation.

16. The method for preparing recombinant F protein of respiratory syncytial virus subtype B according to claim 1, characterized in that, The preparation method includes: The codon-optimized nucleotide sequence encoding the recombinant F protein of respiratory syncytial virus subtype B as described in claim 1 is coupled with a nucleotide sequence encoding a signal peptide at the 5' end and a nucleotide sequence encoding a trimer tag and a histidine tag and a stop codon at the 3' end. The recombinant protein is then cloned and expressed. Correct recombinants are screened, and then transfected into expression system cells for expression. The cell culture supernatant is collected, and the RSV-B recombinant F protein is isolated from it. Optionally, the expression system cells are mammalian cells, insect cells, yeast cells, or bacterial cells; Further optionally, the mammalian cells are 293T cells, 293F cells, or CHO cells; Alternatively, the bacterial cells may be Escherichia coli cells.

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